All

Various Locations Across the United States on Various times

july10-1	DR. KINCAID: Good morning, everyone, and thank you for coming. As the agency host for this meeting, I want to welcome you all. Dr. Peter Marks, the Center Director of Biologics at the FDA, will be giving some more in-depth opening remarks, but I just as a representative of NIAID, one of the co-sponsors, I just wanted to welcome you all and thank you for attending.
july10-2	DR. KINCAID: At NIAID in particular, our programmatic efforts are driven by a combination of recognition of unmet medical needs and scientific opportunity. I think we all can recognize that antimicrobial resistance is a present and growing unmet medical need that needs attention, and this meeting and you coming here today, you are bringing us scientific opportunities that we wish to capitalize on and programmatically develop projects to move forward, and so I'm very hopeful for this meeting to give us some ideas and concepts that we, in conjunction with our sister agency at the FDA, can move forward to better prepare the ground for regulatory science that will enable the development in this whole category of countermeasures.So I wish you a very productive and pleasant and entertaining and educational meeting, and I'll turn it over to Peter.
july10-3	DR. MARKS: Okay. So welcome, everyone. Thank you all for coming to this workshop on bacteriophage therapy that's covering both scientific and regulatory issues.
july10-4	DR. MARKS: The workshop's co-sponsored by the Center for Biologics Evaluation and Research at the Food and Drug Administration, in collaboration with the National Institute of Allergy and Infectious Diseases at the National Institutes of Health, and before I go much further I'd like to take a moment to thank those at both institutions for all of their work and their efforts putting together what should be a very engaging and informative program over the next two days.
july10-5	DR. MARKS: I also want to take the opportunity to thank all of those who will be presenting and serving on panel discussions for making the time to travel here and to do so, all of those who have braved Metro to make it here, thank you, even those coming from locally.
july10-6	DR. MARKS: Antibiotic development got underway seriously in the 1940s and reached its heyday in the1950s and 1960s. Although initially it was the source of miraculous cures for a variety of infections that were previously difficult or impossible to treat, it was not long before the problem of resistance to antibiotics that had been developed occurred.
july10-7	DR. MARKS: Over the past two decades, such antibiotic resistance has really escalated to crisis-level proportions, and we now have the development of Methicillin-resistant Staphylococcus aureus that's present in many communities at high levels, vancomycin-resistant enterococci, and a variety of gram-negative organisms, such as Klebsiella and Pseudomonas species, that are resistant to multiple different antibiotics. In fact, some are remarkably resistant.
july10-8	DR. MARKS: Ironically, though it was discovered a bit over a century ago before the modern antibiotic era, bacteriophage may turn out to be important therapeutics in combatting antibiotic-resistant infections.
july10-9	DR. MARKS: First discovered by the English microbiologist Twort in 1915 and then characterized further by the French-Canadian scientist FÃ©lix d'Herelle, it was not until decades later the details of the lytic mode of action of phage were understood.Such limitations in mechanistic understanding, combined with the ready availability of antibiotics following the Second World War, led to the development of phage therapy for the treatment of human infections in the United States and Western Europe to really slow down and to be shelved for a number of decades. The work on phage therapy continued in some Eastern European countries, including Poland and Russia.
july10-10	DR. MARKS: Over the past decade, however, the investigation of potential phage therapy has seen a renaissance globally as certain infections have proven to be quite resistant to our existing complement of antibiotics and the discovery of novel antibiotics to combat such resistance have become increasingly challenging from a practical perspective.
july10-11	DR. MARKS: On the other hand, phage therapy appears to be non-toxic in humans and in animals, and phage have the benefit that their bacterial specificity allows sparing of the remainder of the beneficial microbiota. In addition, there's the potential to either select or engineer phage to target bacteria that develop resistance to these agents.
july10-12	DR. MARKS: Over the next two days there will be talks and discussions covering the scientific, manufacturing, clinical, and regulatory issuessurrounding the use of phage therapy. Though challenges clearly remain in the development of phage therapy for the prevention or treatment of infections in humans, their potential clinical utility seems quite promising in a time when other options seem much less so, and that's particularly in certain circumstances, and thus -- let's see -- the adage 'What's old is new again' seems quite appropriate when describing the situation with phage, or said more modernly, 'Old is the new new,' and we look forward to a very informative and productive workshop over the next days.
july10-13	DR. MARKS: Thank you very much, and we really appreciate your coming today.
july10-14	DR. MARKS: (Applause.)
july10-15	DR. KINCAID: Good morning, everyone. My name is Randall Kincaid, and I'll be serving as moderator in the first session today. Just so that you're aware of it, we have an additional room, a companion room which is connected by VTC, and it is possible that if questions arise from those in that room, we will be entertaining sort of alternate questions if that comes up.
july10-16	DR. KINCAID: Before we begin, I was asked to make a couple brief statements. First of all, that eachspeaker outside of the government was asked to submit documentation outlining their financial interests. This is an important matter, and these records are all in place. And secondly, that the proceedings of the workshop are being recorded for purposes of transcription and these will be made available at the end of our sessions. Well, it'll actually be made available after we've compiled them and all of that.
july10-17	DR. KINCAID: So, without further ado, I'd like to introduce our first speaker, Dr. Ry Young from the Center for Phage Technology at Texas A&M, who will provide us an overview of bacteriophage then and now.
july10-18	DR. YOUNG: So thanks for putting me in a position of leading off this, I think, historic conference. Since I work on phage lysis, I usually am the last talk in a phage conference, and that's because most people don't care about lysis and a lot of people have to leave early to catch flights. I couldn't figure out why I was chosen as the first one in this case. There's lots of other people who could have been more appropriate choices for the first speaker, but given the Metro problems and the security problems, I think Randy thought a lot of people might be late, and so it's better to have a buffer in the front.So we're going to -- this is going to be largely historical. I'm going to -- I'm not sure exactly why I chose to do it that way. I guess I saw the title 'Phage Then and Now,' and I felt pretty intimidated by that because I actually did not know much about how phage therapy had been conducted in the Phage Therapy 1.0 in the first part of the 20th Century, so I spent a lot of the last two weeks or more boning up on it, and I felt like I needed to pass what I learned or at least what I think I learned on.
july10-19	DR. YOUNG: But then I also got this stern email from Randy saying I had to not only identify my conflicts of interest but also make it clear at the beginning of the talk. I think that makes sense. So I started thinking about my conflicts of interest, and I think really you're asking for bias, and I wanted to show this slide because I am a direct descendant of the DelbrÃ¼ck-Luria-Stent school. I was a Ph.D. student
july10-20	DR. YOUNG: with -- where's the pointer? Is that the pointer right here?
july10-21	DR. YOUNG: Okay, I was Ph.D. student with this man, Hans Bremer, who was the first American post-doc brought to the United States by Gunther Stent, who was, of course, kind of the Luca Brasi if you view the phage group as a gang, Gunther was the Luca Brasi ofthat gang, and he went around recruiting talent in Germany and devastated Western Europe and brought them and seated them all over the United States, including in Texas, where I ended up after my Navy service. This is me in 1972 as a Ph.D. student. I had a little more hair. As you can see, the receding hairline was already going back, but I was imbued with the philosophy of the phage group. The place I did my training was a hotbed of phage biology; essentially, all 20 scientists worked on phages of different types.
july10-22	DR. YOUNG: And this is a quote from, a famous quote at the end of the introduction to a textbook that essentially everybody in my generation used as the textbook for phage genetics, and that was, 'The strange bacteriophage therapy chapter of the history of medicine may now be fairly considered as closed.' So, if there was ever a -- and this was in the 1960s, right. So, if there was ever an attempt to put something to sleep, this was it. Phage therapy sleeps with the fishes.
july10-23	DR. YOUNG: So, before I go on, I want you to notice, by the way, this was 1972 that this picture was taken. I want to acknowledge that there are -- I know two of these people, Betty Kutter and Carl Merril are here. I thought Shankar was going to be here. Is he not?Menaj is not here. Well, it's too bad. So I want to point out that all three of these people have a much deeper background in the applications of phage therapy. They were all committed to convincing the scientific public that there was a future in this 20 years before or 10 years at least before I even thought of it.
july10-24	DR. YOUNG: In fact, since I was up until quite recently biased by my training in the phage group, I was actually very anti-phage therapy, and if you want to notice here, these are publications in very respected journals published in 1968 and 1969 when I was still in the Navy. And, in fact, that's three years before I was back in graduate school, so these people have a much better background and perspective and should be giving this talk.
july10-25	DR. YOUNG: And, in fact, this is the way they all looked in 1972. The fact that they're so well-preserved.
july10-26	DR. YOUNG: (Laughter.)
july10-27	DR. YOUNG: Dr. YOUNG: All right. So the real conflict of interest in terms of, I guess, finances came when I was recruited to GangaGen. So, up to 2002, at the ASM convention where I was giving a lecture, I met Janakiraman or J. Ramachandran, who is the founder andCEO of the company GangaGen, a company at that time based in Bangalore, and those of you who know him, he has a charismatic personality and was very eloquent in convincing me that there was a future for phage therapy.
july10-28	DR. YOUNG: He first recruited me to the Scientific Advisory Board, and I've been active on that even to about 2009. I actually served as a research director for this company in my one year of biotech, and the most important thing to note is the company actually survived that spectacularly incompetent year of mine. And, in fact, they are still in business. They have products in clinical trials, and they have said hello to me, but they haven't asked me for any further input from me for about eight years, which shows that his acumen was even better.
july10-29	DR. YOUNG: But it did lead very importantly to me being able to convince the Texas A&M Board of Regents to establish a Center for Phage Technology, and I think we're the only state institution, more than $5 million was in setup money, and four faculty positions and an annual budget have been committed to the notion that phage biology can be put to important translational uses.
july10-30	DR. YOUNG: So, overall, I have three biases, threeconflicts. I have the original. By training, I was very anti-phage therapy. I still am a stockholder and member of the SAB of GangaGen, so there is a second bias. And then the third bias is I'm actually running a center that is meant to promulgate the use of phage translation. So that's the truth.
july10-31	DR. YOUNG: So the main sources for this talk are these here. I won't go through reading them. I'm primarily going to be focusing on the Eaton and Bayne-Jones Journal of American -- this is a review commissioned by the American Medical Association back in 1934, and it's the one that's regularly cited as being the death knell of phage therapy, of Phage Therapy 1.0, and some others. I'm going to leave these for people in the PDF file, those that can look these up.
july10-32	DR. YOUNG: I am not going to be talking about, I think, a much larger group of data about phage therapy in Eastern Europe and in Europe and in France. I've only recently become aware of how deep and scientifically complex all of that record is. It was explicitly ignored in the 1934 report, and since I'm trying to bring you up to speed on what happened to phage therapy in the United States, I really couldn't, and I don't have the expertise really, and Dr. GÃ´rski is going to be following up and I'm sure he'll be talkingabout that. This is the outline of the talk and I'm going to -- first, I'm going to do basically a review of phage therapy in the United States, I call it Phage Therapy 1.0, a brief word about the methods and formulations that were used. I'll spend the bulk of the talk reviewing the Eaton and Bayne-Jones report. These are the conclusions. They concluded the scientific basis of phage biology was in dispute, the commercialization was premature, and I think, I hope, I should be able to convince you that there was actual bias against both phage theory and d'Herelle in this report, and then I have a one slide 20/20 hindsight on what went wrong, and then a brief segment at the end about Phage Therapy 2.0 and how it's different and proposed standards.
july10-33	DR. YOUNG: This is the first paper. This is the beginning of it all. There's d'Herelle himself. This paper was read in September 1917. It was in the French National Academy of Sciences. I believe it's the first time where bacteriophage is actually written, and even here d'Herelle is already citing some of the properties of phages that we know are critical in our attempt to use them. That is, they're very often highly specific, and he also noted theycould be acclimatized. In a lot of ways, that means you could, by passaging them through target strains, you could adapt them to grow more efficiently on those strains.
july10-34	DR. YOUNG: I should point out that Gunther Stent even did -- so d'Herelle passed away in the mid-1940s, and phage therapy was really not active in the United States, at least in major publications, for the entire period of the '50s, '60s and '70s. Even then, Gunther Stent found it necessary in a review of a biography of d'Herelle to vilify him if you read these statements. This was written for a review in Science Magazine, and he couldn't say things like widely reviled, he had nothing to do with the conceptual ideological origins of molecular biology, which was ridiculous because he invented the plaque assay, without which we would know nothing about what goes on. He dearly enjoyed accepting undeserved credit. Well, we all do that.
july10-35	DR. YOUNG: (Laughter.)
july10-36	DR. YOUNG: So you can see that Luca Brasi still had his knives out for this guy. But at the time -- this was long after d'Herelle, d'Herelle didn't know that Gunther Stent was going to try to trash his science and his reputation. d'Herelle, he had contemporary enemies. It's always bad when youhave an enemy whose face is on a stamp. (Laughter.)
july10-37	DR. YOUNG: And so it turns out Jules Bordet, who was high up in the Pasteur organization and a Nobel Prize winner, and I'm pretty sure that Bordetella is named for him. So he's a Nobel Prize winner who really despised d'Herelle and, in fact, was the major mover in pointing out that Twort had discovered phages two years earlier than d'Herelle. And John Northrop, who was a later Nobel Prize winner, was very much an opponent of the so-called d'Herelle-Twort Theory that phages were viruses, and then Albert Krueger, who was a prominent bacteriologist at Berkeley, was a protege of Northrop and basically his hit man, and wrote many, many anti-phage and anti-d'Herelle tracks. And it didn't help that both Northrop and Krueger were editors of major scientific journals at the time.
july10-38	DR. YOUNG: So this is their theory. They had a completely -- a theory they view was an Occam's Razor Theory, that is, a much simpler idea, and that is that basically that what phage is was a self-replicating endolytic enzyme, so that there's an enzyme that would degrade a bacteria and then in the process of degrading that bacteria to create more enzymes fromdegrading the large molecules in the cell wall. So, effectively, it's analogous to the autocatalytic formation of trypsin from trypsinogen.
july10-39	DR. YOUNG: So, basically, their idea was lysozyme is a prion. This is the first prion theory from many, many years ago, and this was very -- because these people were so prominent and because their academic credentials and circles of contacts and editorships had a lot of sway, whereas d'Herelle didn't even have a college degree, it was difficult for d'Herelle to compete with this.
july10-40	DR. YOUNG: So d'Herelle went on to aggressively promulgate his ideas for using phage as a tool against bacterial infections. He was widely successful. In 1928, he sold his company that he'd set up to make phages for a million francs, and that was the same year that d'Herelle also took his job, full professorship at Yale, and apparently he did not tell his dean about this conflict of interest in terms
july10-41	DR. YOUNG: of -- so I actually think -- somebody may correct me here, but the company was run very badly for a while. Then he eventually took it over again. But in any case, the laboratory bacteriophage, which was the company that was making up to 10,000 doses per day in the late 1930s, including a phage Phi X174, whichturned out to be a really key experimental phage. So they sold what they called polyvalent phage capsules against dysentery, carbuncles, sinusitus, et cetera, and they supplied other companies, including Eli Lilly in the United States.
july10-42	DR. YOUNG: There were a lot of problems with these things, as you'll -- that basically we couldn't -- you expect for a premature commercialization. All of their so-called phages were simply filter sterilized lysates. There was no purification at all. There was no quality control, no standards. Assays were usually yes or no in terms of whether they worked or not.
july10-43	DR. YOUNG: The so-called polyvalent phage mixtures, which by their definition had multiple different phages, each targeted against a different bacterial potential pathogen. Sometimes these were grown together in mixed culture, and that led to simplification of the mixtures, and then the companies that distributed often put in disinfectants to preserve the phage lysates, but, of course, it does preserve them dead.
july10-44	DR. YOUNG: And this is a famous advertisement that I always showed in my phage class every year. This is the actual -- this is the advertisement for different polyvalent phage cocktails, including intesti-phageand pyo-phage, and I think these are at least ancestrally related to the -- Betty, isn't that right? They're ancestrally related to the ones --
july10-45	DR. KUTTER: In the 1930s.
july10-46	DR. YOUNG: -- to Billeci. Yeah.
july10-47	DR. KUTTER: We still have some of the vials.
july10-48	DR. YOUNG: Right. And the intesti-phage and pyo-phage mixtures have been developed and matured and also sequenced, I think, now, so we now know what was in them, at least in some of them.
july10-49	DR. YOUNG: Anyway. So this is a phage for whatever ails you. I notice here that this is 1936. It says it's under the control of Dr. d'Herelle. So this might be after he re-took control of the company.
july10-50	DR. YOUNG: But one thing I noticed in reviewing this literature was that there was a corruption of the word 'polyvalent.' So polyvalent was originally meant to mean multiple different phages, each targeted against a different strain, a different bacterial genus, so the pyo-phage would have phages against all possible or as many possible important bacterial enteric pathogens.
july10-51	DR. YOUNG: But polyvalent come to mean as in general usage as any phage preparation that would attackmultiple different 'races' or strains of the same species. So you have to be careful when you're reading the literature. They say polyvalent phage X, and you realize it's not really a polyvalent phage, it's a phage that plays against several host strains.
july10-52	DR. YOUNG: Okay. So there was, because of this lack of standardization and the other practices, for about a decade, there were many, many clinical studies done or treatments done and reported using these commercial preparations. There were at least four companies involved, and it led Margaret Straub and Martha Applebaum to publish in 1933 in Journal of American Medical Association their standardization. They went out and bought three samples. I think they had three of the four companies represented: Eli Lilly, Squibb, and Swan-Myers. Squibb called their preparation polyvalent phage for staph. Eli Lilly called it staphyl gel.
july10-53	DR. YOUNG: And they basically tested these off the shelf. They didn't ask the -- they bought them and tested their activity, and their findings were that the Lilly products contained an antiseptic that simply killed the phage and all the bacteria they mixed it with. The Squibb phage were highly variable. The one batch they bought had virtually no phage activity init. The next had high phage activity. And Swan-Myers had a potent staph phage in it, but it had, even though it was reputed to be effective against B. coli, it had no activity against any kind of colon bacteria.
july10-54	DR. YOUNG: So this was kind of a warning shot that the stuff that was being used at least from commercial sources wasn't reliable. It is interesting that they showed their bias a little bit here because they wanted to say that the reason why they're doing this is is they wanted to protect the reputation of genuine bacteriophage. So I thought that was -- it made me feel good that they actually cared.
july10-55	DR. YOUNG: So I actually ran -- I encountered a 1930 sales manual for Eli Lilly which I thought was interesting. There are industry representatives here, and Lilly arguably is one of the most certainly prominent bio and pharmaceutical company. So this is a manual that each new salesman was given in I think it was a week-long training course. So it's organized by lessons, and the next to last lesson, Lesson 38, is bacteriophage because Eli Lilly was selling bacteriophage, and so I highlighted some of the things here.
july10-56	DR. YOUNG: They were very cagey about what they said these things were. They wanted their salespeople tobe able to talk to doctors and be able to answer questions, and they carefully note here that the Twort-d'Herelle hypothesis that these are basically viral living particles is not accepted yet by other investigators, and then very explicitly in italics, this is their italics, 'It is too soon to evaluate phagotherapy,' even though they're selling these lysates.
july10-57	DR. YOUNG: So these are the lysates that were sold and I believe at least two of these were directly from the bacteriophage company in Paris that d'Herelle had founded. So here it shows how they're prepared and, in fact, merthiolate is added to preserve, and I'm sure, although merthiolate's not hugely bad for phage, if you have it around for a month, I think it probably will kill it. It's very important to note that under what name are the bacteriophage filtrates licensed by NIH, and they're licensed as bacterial antigens, never as bacteriophages. So that was something that each salesperson had to say. These are not phages, these are antigenic lysates.
july10-58	DR. YOUNG: But they did come to the same conclusion that when you had failures with phage it was because there was a mismatch between the phage and the targeted bacteria. So they were of obviously a verymixed mind about the whole process. Okay. So this is the report. It was commissioned by the AMA, the so-called Council on Pharmacy and Chemistry. It was published over three issues of the Journal of American Medical Association with the explicit endorsement of the council, and I'm just going to highlight the things that are important.
july10-59	DR. YOUNG: They set out at the top to evaluate two things: the bacteriophage phenomenon, which I was a little surprised by, I didn't realize that they were going to evaluate the basic science; and then the therapeutic usefulness of bacteriophage.
july10-60	DR. YOUNG: And I did a little work looking into the background of the people who wrote this, and it turns out that Stanhope Bayne-Jones may have had a conflict of interest or at least a little bit of a question mark about him being assigned to do this. He had just taken the bacteriology position at Yale that had been vacated under very hostile circumstances by d'Herelle in 1933. The dean at Yale had basically invited d'Herelle to leave. He had first become afoul with him when he found out about his commercial connections, but also because he essentially never stayed there for more than a month at a time, and he also had a superb talent for pissing people off.So Stanhope Bayne-Jones was already -- at least you could argue that he might have been a little bit -- he may have tended more to find fault with d'Herelle than if he wanted to make his new dean happy.
july10-61	DR. YOUNG: He actually is a very stand-up guy, ended up a brigadier general. He was a multi-decorated war hero from World War I. He was the first person to study phage lysates with millisecond imaging, so he's a hero of mine, and he was actually a major mover in the 1964 Surgeon General's report that started the anti-smoking campaign. So, I mean, I don't want to cast any bad -- he certainly was a very consequential figure.
july10-62	DR. YOUNG: And so they really point out that they are summarizing about 100 studies, and so the first thing that hit me was, my gosh, that's already selected. There were more than -- it turns out there were more than 100 studies out there, and they only wanted to review the ones that they felt were most significant. But I now worry that this meant that they didn't review the Eastern and French literature for this very reason. And it also might be a language problem.
july10-63	DR. YOUNG: Okay. So this is the organization. I'm not going to go through it all, but basically the firstpart of it is evaluating the theory and the rest of it is evaluating the actual on a disease-by-disease basis, and, finally, there are summary and conclusions. Up to now I had never read anything but No. 6.
july10-64	DR. YOUNG: So I'll just quickly summarize what -- the first part where they are looking at the mode of action, the theories, the origin, it was all kind of a hodgepodge. Its literature is of 1933, and when I read this, I was quite pleased because they were very, very measured. They came to conclusions that were perfectly reasonable in terms of specificity, the fact that you could adapt them by passage and phages. They even had the size of particles approximately right, and the fact that they're antigenic themselves, and that they were in most part robust in storage but were sensitive to antiseptics.
july10-65	DR. YOUNG: And then they pointed out that you could get phage-resistant variants after treatment, and those variants could have either increased or decreased pathogenicity. So up to that -- when I finished reading that, I thought, wow, these guys were really on top of it. And so then you read this.
july10-66	DR. YOUNG: The last sentence, 'It's obvious there is great significance and importance. However, it's beenexploited detrimentally by manufacturers.' So I thought, uh-oh, that doesn't sound good. And then I moved on, and then they end this section by saying in the composition that the phage preparations are just lysates and that part, it was accurate, at least at that time.
july10-67	DR. YOUNG: So the dagger came in the start of the section of a review of phage therapy in terms of practice. The first dagger comes in the first section about in vitro experiments and they cite, there were 12 studies cited. Other than d'Herelle, there were 12 extant studies, and they find uniformly that serum or blood and also bile either eliminated phage activity or greatly inhibited it, and by name they explicitly refute d'Herelle's experiments that had been published 10 years before as being unreproducible. And, first of all, I had never heard this. I never heard that serum inactivates phage.
july10-68	DR. YOUNG: And then the second section was in vitro bacteriophage therapy in experimental animals. There were 21 cases of animal experimentations cited. All of them were negative. In each case, it was done the right way. They were active on the bacterial strain and infection model using a variety of animals and a variety of disease-causing bacteria, and theirconclusion was uniformly negative that in no case where animal model experiments had been successful, and explicitly again named d'Herelle's S. pullorum experience in chickens as being unreproducible.
july10-69	DR. YOUNG: So, bottom line, phages are inhibited by blood and have not been shown to have efficacy in animal models, and at that point, the game was over because for the rest of the report they're simply going through and trying to explain away any positive results because, as far as they're concerned, if it didn't work in experimental animals where you could do controlled experiments, the rest of it was just confirmation bias.
july10-70	DR. YOUNG: So here's the next section, there's the eight different disease things, and we obviously can't go through that in a very short time, but the whole experimental evaluation in human infection starts with this: 'The many good reports make it difficult to assert that lytic filtrates are without effect.' So they're complaining about the fact that they can't just say there's no effect.
july10-71	DR. YOUNG: But then they go through and find for each disease system here why the positive results that were reported were not significant, and so basically they were so biased by their finding that animal tests hadbeen uniform failures, they concluded phage therapy had undemonstrated validity.
july10-72	DR. YOUNG: They do point out that in the cases where negative they did the right thing. They said in most of the negative cases it's very likely due to the way the experimenter or the physician was using them, that the phages simply didn't work in vitro on the bacterium, and they, in fact, concluded in this very negative diatribe that for this reason in vitro lysis should always be demonstrated. So flash forward to Phage Therapy 2.0 now, that's sort of the way we're working, that we make sure that the disease bug is sensitive to the phages that are used.
july10-73	DR. YOUNG: So I'll skip down here. There were more than 80 citations, including 70 studies involving thousands of patients. I've said already that they're essentially either inconclusive or negative value. They make a big point to refute d'Herelle's success, the most highly publicized success with both cholera and plague, and in all these cases which appear to be well controlled, including double-blind experiments, they said they were unable to reproduce d'Herelle's results, and mentioned him by name. The one exception is local infections, boils, furunculosis, staphylococcus.So they had so many and apparently well done studies with staph infections and boils and skin eruptions that they were unable to come up with any saying that they were possibly efficacious. Nearly uniformly positive. And in this case, they used both commercial phages and home brewed phages where the physician just went and isolated phages from sewage and used them directly.
july10-74	DR. YOUNG: Okay. So there were 11 summaries and conclusions after this long thing. I'll just show a few of them here.
july10-75	DR. YOUNG: First of all, and I think the tell tale of the bias in this, their very first statement was, 'd'Herelle's bacteriophage is probably an inanimate enzyme, not a virus parasite.' So the Prion Theory. So there was not a shred of evidence evaluated in this entire long review, and they started off saying that it was undemonstrated. Now they're coming down on the side of the Northropites and the Prion stories. I just find that amazing.
july10-76	DR. YOUNG: It says it repeated their finding that lytic action was inhibited by blood, and they find positive results only for local staphylococcus infections and possibly cystitis. They just say they would be convincing. Importantly, there's no evidence thatphage lysis or phage killing or propagation occurs in vivo at all, and then favorable results may have been due to immunizing action of the bacterial proteins in the broth filtrates.
july10-77	DR. YOUNG: So this is my sort of 20/20 hindsight version of this. First of all, I think the fix was in. I think if you read this, you know, it's not an airplane read, but if you ever sit down and read it, I've read the whole thing and I've read about a third of the cited English language studies, they never miss an attempt to specifically denigrate d'Herelle or d'Herelle's hypothesis, and as I said, they ignored the Eastern European and French studies that were far more positive and had, I think, irrefutable anecdotal data in terms of statistics.
july10-78	DR. YOUNG: In almost every case that I've read so far, no matter what the physician said they used, actually, they would often claim to use polyvalent phage, but, in fact, in almost every case I think you can presume that they used a single phage, and in most cases, they did not test that single phage against the bacterium in every case of infection that they tested.
july10-79	DR. YOUNG: So very likely these were simply -- many of the failures were due to specific mismatches or you could certainly get -- with a single phage, you willalmost certainly get rapid rise of resistant bacteria. And the frequent successes with staph, I think, can be due to the fact of the omnipresence of phage K. So what's phage K?
july10-80	DR. YOUNG: So phage K is I call the mother of all polyvalent phages. Phage K is actually closely related or related to the very first phage that Twort identified back in 1915. It's a large 130 kb DNA myophage, which means there's a contractile tail. There are 30 whole genomes that are greater than 90 percent identical in RefSeek and Genvac. All right. So 99 percent of the -- the phage K is 99 percent identical to Team One, which is a staph phage in the Tbilisi cocktail, and it was actually first described in the Vurnet Laboratory in Australia in 1935. Based on just a very quick survey, this phage has been patented many times.
july10-81	DR. YOUNG: (Laughter.)
july10-82	DR. YOUNG: And so why are phage K and its relatives polyvalent phages in itself? The main reason is that the receptor for phage K is an N-Acetyl glucosamine in the cell wall of teichoic acid, and the key thing to that is that that is essential for the viability of -- so this phage has found a receptor that can -- essentially very difficult to change it orlose it because the bacteria will be inviable. The other thing that's unique about it is that there's not a single instance of GATC in the 130 kb genome. If there's evidence for intelligent or at least sadistic design, this is one of them. So by random statistics you expect 300 of these in that sequence and there's not a single one, which gives us automatically immune to restriction by the major restriction enzyme of the staph aureus.
july10-83	DR. YOUNG: And finally there's what I call type dominance. If you go out and look for virulent phages in any sewage or environmental sample that plate on a large collection of staph, you'll always get phage K. There's another type, a small photophage that you can get, but they're usually a very narrow host range. Phage K is recognized in wall teichoic acid. It's what you get and it doesn't matter where you isolate it, in Japan or United States or anywhere else. So just somehow for some reason there's been a bottleneck in the history of staph aureus, and it completely went to phage K and its friends.
july10-84	DR. YOUNG: Okay. So I think in the interest of time I'm going to skip down to the next section. I do say I am still confused and I would love to have somebody help me think through this, why the serum results wereso uniformly negative and surely there are modern data that somebody's published that can refute this, and why were the animal experiments so uniformly successful.
july10-85	DR. YOUNG: The simplest Occam's Razor argument is that they were careful in what they cited and that there was anti-confirmation bias, but I don't want to conclude that until I know more about what was actually going on at that time.
july10-86	DR. YOUNG: So, in the last few years, obviously, there's been -- so that Phage Therapy 1.0 died and was put to death basically mainly by that 1934 report which I find was highly biased. In the last few years, there's been an acceleration because of the onset of multiple drug resistance, as you all well know. We had this wonderful meeting like this just two years ago, in July. It already feels like a decade has passed in terms of what's going on, and just this last year there have been successful emergency IND phage treatments, which you're going to be hearing about in detail.
july10-87	DR. YOUNG: And so I had no role in this except as somebody who had emails come in and sent emails out. That's basically what I did. So this is a picture of the -- and this is my total experience with phagetherapy, so I don't have to do a meta analysis. So I'm just going to state the facts, and most of you have read about this. Tom Patterson contracted a Acinetobacter baumannii infection, spent months in the ICU, and then the physician, Chip Schooley, who will be taking to you very soon this morning, obtained permission via the eIND mechanism for attempting phage therapy. The strain which we will call TP1, Tom Patterson 1 was isolated and it was shown to be multiple drug resistant and was shipped to -- at the end of February 2016, the Patterson team contacted two agencies, our agency, Center for Phage Technology, and the BDRD at the U.S. Naval Medical Research Center for phages having obtained, I guess, the eIND permission. So this was the last week in February.
july10-88	DR. YOUNG: So what we did was we solicited phages from everybody we could think of because we didn't have any, and we got a bunch of phages from AmpliPhi. I have to say that it was a uniformly yes answer, forget the paperwork, don't worry about IP. Instantly people ran to the mailboxes and sent their phages, and we tested, I think, 40 phages from around the world and one of them worked against this strain and that was the one from AmpliPhi, and we spent about a month isolating new phages.The Navy already had a wonderful, large, complex collection of AB phages, and they have a very efficient plate reader-based semi-automated liquid growth testing system. So they were contacted later, and they were able to identify phages much faster.
july10-89	DR. YOUNG: And then the two sources prepared phage cocktails. It was a nightmare. It shut our laboratory and center and academic activities down for two full months. We eventually provided four phages each as cocktails and mixed the phages together and made phage cocktails. They were eventually shipped to UCSD Hospital mid-March 2016, and some of the cocktails were actually purified by organic solvent extraction to lower the endotoxin levels, and that was primarily done by people in Forest Rohwer's lab at San Diego State University.
july10-90	DR. YOUNG: By mid-March 2016, the CPT cocktail was administered through an abdominal drain, and the Navy cocktail was administered IV, and I think the data suggests it was the most important component. Within a week or so, there was a bacterium isolated from -- was that from the blood or from the drain? I can't remember. It was a drain bug, and the strain TP3 was resistant to all the phages that were used, all eight phages that were used in the original cocktail.The Navy then used their rapid system to isolate another phage that grew against TP3, and was able to then use that to modify the IV cocktail. Phage therapy continued for eight weeks, and the patient recovered, and there he is. I think the Superman designation should be on her chest because she is a super woman, and she and Dr. Chip Schooley need to have -- in fact, I think there's going to be a movie about them. Isn't that right? Who's playing you, Chip? Is it Tom?
july10-91	DR. SCHOOLEY: Jack Black.
july10-92	DR. YOUNG: Oh, Jack.
july10-93	DR. YOUNG: (Laughter.)
july10-94	DR. YOUNG: All right. So that's Phage Therapy 2.0 from my point of view. There are some others and we'll hear about them here.
july10-95	DR. YOUNG: So I have questions for you. I know that half, more than half of the people here are regulators or people involved in this.
july10-96	DR. YOUNG: The single patient eIND pathway, is there a limit to the application of this mechanism? We'd like to know what happens when there's a negative outcome. From our point of view being non-regulators, it looks like it's going to continue. I get daily, I get appeals for phage therapy. There are alwaysemergencies. There's more MDR bacterial infections that are occurring and phage therapy is likely to work, so it seems like we sort of fit the bill for eINDs.
july10-97	DR. YOUNG: However, the only propagation that we used because of the urgency of our task was whether or not the phages would grow in liquid culture. We did no characterization of the phages. Of all the nine phages at the viral or molecular level, all eight phages used in the first strain were all very similar. We think they are all large myophages and probably use the same and/or linked receptors, probably an outer membrane lipopolysaccharide. And so, in this case, Phage Therapy 2.0 was the same as Phage Therapy 1.0.
july10-98	DR. YOUNG: So I'll just come to the bottom line here. I think we ought to have -- since we have this now 100 years of advanced -- there's no longer any doubt about the molecular and biological nature of phages. We have detailed knowledge of the moving parts of phages and how they work, although I think a lot more needs to be done on phages not of E. coli and B. subtilis, and we have or can develop rapidly tools for determining receptors, affinities, DNA modifications, et cetera. These are my suggestions.
july10-99	DR. YOUNG: I think we should have available phages foreIND applications that have complete annotated genome sequences beforehand. They should have EM images sufficiently accurate to determine tails, tail fibers, and other attachment appendages. We should know the nature of their DNA modifications. There should be established purification and titer requirements, and I think in the long run the cocktails, that if we had that as a starting material, when the balloon goes up and an eIND is in motion, the cocktails that are assembled will have a much better likelihood of efficacy and redundancy so to avoid resistance, and the long-term, if we had hundreds or thousands of these cases, the retrospective value of having the genomic information raises an enhancing possibility that we might be able to predict efficacy strictly from genomic information.
july10-100	DR. YOUNG: So thanks for that. I hope we have -- I hope I haven't bored you too much with this retrospective, and I appreciate your patience. Forty-eight seconds left.
july10-101	DR. YOUNG: (Applause.)
july10-102	DR. KINCAID: Well, first, I'd just like to point out that the choice of you as the first speaker was not because we expected people to be hung up on Metro or anything like that. I think you wereselected primarily for the strength of your intellect and, as only a true Texan could, to cover a century of events and in a story-telling manner.
july10-103	DR. KINCAID: I don't know if there's anyone who would like to ask Dr. Young a question. I think we have time for one as it turns out.
july10-104	DR. KINCAID: All right. Well -- oops.
july10-105	AUDIENCE MEMBER: One place in your slide you mention that the company who are making this lysate indicated that this is a bacterial antigen. Did the title mention that these are actually a vaccine type of agents?
july10-106	DR. YOUNG: So Lilly was very carefully telling their salespeople to not claim that these were phages that would lyse bacteria. But instead, these were lysates produced by phage lysis that would immunize the host. That was the official.
july10-107	DR. YOUNG: Yet, on the other hand, all of their warnings and everything suggested that the phage had to be targeted properly and should be shown to grow in vivo -- in vitro beforehand, so they were clearly confused, and I don't think in these days a company would go to the market with that much confusion built into the products.
july10-108	AUDIENCE MEMBER: Thank you.
july10-109	DR. KINCAID: All right. So we're going to move on and we'll learn from a very practical point of view the experience that has taken place in Poland over many decades, and so we're privileged to have Dr. Andrzej GÃ´rski from the Institute on Immunology and Experimental Therapy and the Polish Academy of Sciences. Andrzej.
july10-110	DR. GÃ“RSKI: I wish to thank my colleagues from FDA and NIH for inviting me here.
july10-111	DR. GÃ“RSKI: Well, in our work, we sometimes refer not only to FÃ©lix d'Herelle, and I don't need to waste your time by going into details which were already covered by Dr. Young, and you know it from your own knowledge, but also to another famous, eminent Canadian doctor and scientist whom you know, Sir William Osler, and his accomplishments are well known in terms of his medical achievements. He's been also recognized as a philosopher and ethicist, and some of his profound statements are listed here. And we believe that our purpose in treating patients with antibiotic-resistant infection was not merely to eradicate infection but to treat the patient who has antibiotic-resistant infections, so do not treat -- do not eradicate the infection at all costs.
july10-112	DR. GÃ“RSKI: This is the institute where our center islocated in Wroclaw. The center, the institute is more than 50 years old, and our therapy center has been opened 12 years ago, and we operate under the umbrella of the Declaration of Helsinki and respective regulations of European Medicines Agency, as well as Polish regulations which are contained in the legislation of medical profession and the Polish Constitution and so on.
july10-113	DR. GÃ“RSKI: And I wish to emphasize again that what we are doing is experimental therapy, which is also known in Europe as compassionate use, and in America, it's often referred to as expanded access.
july10-114	DR. GÃ“RSKI: This is a kind of summary of our thinking about why experimental therapy is so important for further progress in phage therapy even though it does not formally yield the data which, let's say, could be considered as fully scientifically relevant according to the standards of evidence-based medicine.
july10-115	DR. GÃ“RSKI: Why? First of all, we have already achieved the data which support the notion that phage therapy is safe. We know that side effects are not very frequent and we know the side effects. We know what we can expect when giving the patients phages orally, indirectly, or topically.
july10-116	DR. GÃ“RSKI: We also learned a very interesting lessonabout the relationship between phage administration and antibody production. As you well know, there has been a common belief that antibody production to phage should be a limiting factor in the success of phage therapy, which we know it's not that simple.
july10-117	DR. GÃ“RSKI: Of course, experimental therapy can provide the idea and planning for optimal clinical trials which we have not been able to accomplish yet simply because of lack of funding.
july10-118	DR. GÃ“RSKI: Another very interesting area, and we have been deeply engaged in this area for the past 10 years, is the relationship between phage and immune response, how immune system reacts to phages, but also how phages act in immune system. We have published an interesting paper recently which is available online, 'Phage and Immunomodulation,' and I suggest that perhaps you will find a minute to go over this paper.
july10-119	DR. GÃ“RSKI: So phage and immunomodulation is something we believe which may be also a future application in phage therapy which is not directly related to eradication of infection. And, of course, the experimental therapy is important for our promoting knowledge and fund raising.
july10-120	DR. GÃ“RSKI: I know from my own experience that the average understanding of what phages are at least inPolish medical and lay communities around the globe, many doctors do not know what are phages. Medical students have very limited knowledge. So I believe by engaging in experimental therapy we also serve this very important purpose of raising awareness of what phages are, what is their current possible use, and perhaps what are the hopes for the future.
july10-121	DR. GÃ“RSKI: In my younger years, I've been facing the development of organ transplantation in my country, and sometimes I believe that the current development in phage therapy are quite similar. I remember how it was tough at the beginning to transplant a kidney. In Poland, you had to ask the prosecutor for personal approval of each transplant; otherwise it would be considered illegal. And so now we have a very active, very fruitful organ transplantation like everywhere else. Hopefully, the phage therapy should follow the same route.
july10-122	DR. GÃ“RSKI: For those of you who might be interested in this quite interesting field of ethics review of compassionate use, I would refer you to our paper which is now in press in BMC Medicine, which addresses specific aspects of the ethical review and dilemmas of experimental therapy.
july10-123	DR. GÃ“RSKI: Now there's nothing exceptional in ourapproach in terms of the let's say production of phage lysates. The scheme is presented here. We also use purified phage preparations, but they are, of course, much more costly, so it's quite controversial whether or not you should charge a patient for a product which is much more expensive, yet today we don't have formal proof of efficacy. So, of course, for clinical trials, that's another issue. But for experimental therapy today, it's kind of difficult dilemma.
july10-124	DR. GÃ“RSKI: This is the current bacteriophage collection of our institute. As you may see, it's quite rich. We have more than 800 of total phages, and the specificity and range is shown on the slide.
july10-125	DR. GÃ“RSKI: And the spectra, yeah, we are very glad of our anti-staph phages, including MRSA. We cover almost 100 percent of the Polish strains, and quite high coverage for other bacteriophages, except perhaps Pseudomonas aeruginosa. We are not happy. As you see, we can cover slightly more than 50 percent of the spectrum.
july10-126	DR. GÃ“RSKI: Quite recently, we wanted to increase the efficacy and the range of our bacteriophage preparations by propagating our phages on the bacteria that were rendered plasmid- and prophage-free, and without going into the details for which I do not havetime, you may see that when you propagate the phage on such strains, the phage titer and positive lytic reaction may increase. So this is the initial phage titer on a host bacterium and then on a bacterium that was cleaned of PPE, and you see the increased titer and the increased positive reaction.
july10-127	DR. GÃ“RSKI: In fact, we have a short abstract describing this phenomenon, and the text of the abstract is presented here, and the main information is highlighted in this fragment of the text. Probably in summary we can increase the efficiency of our phage preparations in future by working on purified strains rather than standard initial strains.
july10-128	DR. GÃ“RSKI: The specification of our final phage preparation is depicted here. Activity, stability, degree of purification, and -- well, that's kind of our local pharmacy.
july10-129	DR. GÃ“RSKI: Now the philosophy regarding phage therapy indications, contra-indications, and termination. This has been presented many times already and, in fact, if I remember well, I presented this data in this room two years ago. They have been published, so I don't know if I should go into the details. For those of you who are interested in those details, already five years ago they were presented in ourpaper published in Advances in Bioresearch. But generally speaking, the philosophy and the practice is straightforward. There's nothing exceptional here, maybe except that so far most of our work has been done on monotherapy. We've been using monovalent phage preparations rather than cocktails, although we have some preliminary data on cocktails which I show you later on, but again I beg for your understanding because they are really preliminary.
july10-130	DR. GÃ“RSKI: Now, of course, what about phage therapy patient monitoring? What we do when we watch our patients? Of course, we perform clinical detail, clinical evaluation, and as you probably know, we don't have many patients because we have to spend at least one hour, and probably more, with each patient explaining to him. Sometimes there are patients from abroad, from Germany, also from U.S., so you need to explain everything starting from scratch. What are the phages? What are the pros and cons and so on and so on. So really, it's a kind of really hard work, like in our profession, of course, that's nothing exceptional, except that you really need to have to spend a substantial time to satisfy your patient and yourself.
july10-131	DR. GÃ“RSKI: And, of course, very frequently, becausemost of them are very complicated cases, we must seek external consultant opinion. Then pathogen isolation and testing of use, and then we perform detailed lab monitoring, including CRP, blood analysis, organ function.
july10-132	DR. GÃ“RSKI: Well, regarding our patients with chronic bacterial prostatitis, we perform old 4-glass test, which enables urologists to localize where is the infection located in urinary tract.
july10-133	DR. GÃ“RSKI: Now it's a kind of historical test. I don't think it's performed in United States, but it still has its value, but we live in times when doctors have little time, so it will be probably unrealistic to recommend the testing, but it has its value and, of course, you can gain important scientific information by obtaining fluids from data and other part of the urinary tract.
july10-134	DR. GÃ“RSKI: Immune monitoring. There is, of course, an important question whether or not the effects that we are observing in response to phage therapy are not simply let's say the immunostimulatory effects of bacteria that remains on phages themselves, and according to our experience, and the experience has been published and we have quite a few papers addressing this issue, although there are, of course,reactions of immune system to phages, we believe that we cannot ascribe the beneficial effects of phage therapy to mere upgrading, upregulation of immune system. We don't think it's that simple.
july10-135	DR. GÃ“RSKI: We categorize the results of our treatment into seven major categories. They are listed here, and again I will not go into details because I believe they are straightforward. Of course, we hope to obtain the Category A pathogen eradication and full recovery, but it happens rather rarely in about 10 percent of cases. Overall, we consider Categories A through C as good responses to phage therapy, and D-G are considered as not a great response to phage therapy.
july10-136	DR. GÃ“RSKI: What I already mentioned and there's nothing unusual in this statement. We in our material, and this is not only in material from our phage therapy center but also historical material of the past because phage therapy in Poland is almost as old as discovery of phages. What is notable is the remarkable safety of phage therapy. Here, you see that on our material of almost 160 patients we observed good tolerance in almost 80 percent of cases, and the lack tolerance in less than 4 percent of patients, which force us to terminate treatment.And we also have in press another paper which includes more recent data from the past three years. This is the characteristic of these almost 150 patients, the indications for phage therapy, and the routes of administration, type of applied phages. Maybe we don't have time now to go into details but just to give you the general idea of the patient types and the way we administer our phages.
july10-137	DR. GÃ“RSKI: And those results, which have been published, so there's nothing new, in fact, in this data which I present except that we have also results from the most recent cohort of patients which have been treated in years 2011 through 2013, and you may see that it's amazingly close. The results are amazingly similar. Almost 40 percent of good responses in years 2008 to 2010, and almost the same result obtained with most recent patients.
july10-138	DR. GÃ“RSKI: So, in summary, in about 40 percent of cases we obtain something which we categorize as a good response according to the description I showed you in an earlier slide.
july10-139	DR. GÃ“RSKI: And now this good response translates to quite interesting and promising results in patients with genital and urinary tract infections. Most of those patients, although not all of them, are thosewho had chronic bacterial prostatitis, and they have been treated using indirect administration. In this group of patients, our results seem to be quite promising, and other responses shown in other clinical settings.
july10-140	DR. GÃ“RSKI: Now I would like to stop for a moment on this slide because here again I ask for your understanding that the data are very limited and the number of patients is small. But I think it's something new and something potentially, not perhaps relevant today but potentially relevant, and it may give food for thought for future work.
july10-141	DR. GÃ“RSKI: Here, we compared the efficacy of the monovalent phage lysis versus phage cocktail. The description of the content of this preparation is provided here. Right, non-purified monovalent phage lysates, and non-purified phage cocktail, which contain a mixture of three staph phages.
july10-142	DR. GÃ“RSKI: So you see here that again using this very limited material there is very little difference between the monovalent and cocktail. However, when we used the purified cocktails, this difference was significantly higher. The results achieved with the purified phage cocktail were much better.
july10-143	DR. GÃ“RSKI: However, for that and other reasons, thetiter of the phage preparation was also higher, so it's difficult for me to say whether or not those better results is due to the purity or higher phage content or both. One way or another I decided to present to you this data simply because of the fact that you can achieve more than 50 percent of success using purified phage cocktail.
july10-144	DR. GÃ“RSKI: Of course, using purified phage cocktails versus monovalent cocktails, you can easily organize a conference to discuss this controversial issue, and, again, we don't have time probably to go into this philosophy today, I know. But one word of the caution, I can cite the most recent paper by Oechslin regarding this issue because there is a kind of over-enthusiasm in recommending cocktails. We'll see.
july10-145	DR. GÃ“RSKI: Now this slide shows you the changes in phage profile and acquisition of phage resistance. What is probably most important is how muted we are regarding the application of phages once the resistance develop. This resistance develops, of course, here, and these two panels show you the percentage, but still we are able to identify a phage in our phage collections to continue the treatment if it's necessary, and the percentage of this success is shown in this slide.Now antiphage activity of sera from patients receiving phage therapy, we have published four or five papers already, so again I will not go into details, but what is probably most important is following oral administration the level of antibody is very low. You may also find quite low antibody production using inter-rectal administration, which is maybe unexpected, but that's the fact.
july10-146	DR. GÃ“RSKI: And most importantly, there appears to be no clear association between the level of antibody -- antiphage-producing antibody in serum and the outcome of therapy, which is shown here, right, with a patient with a high level of antibodies, yet the result of treatment was good, good response, clinical improvement. In another patient who had high responses -- by the way, you may see that this level of antibodies may drop subsequently, and again we had good response to therapy.
july10-147	DR. GÃ“RSKI: So phage therapy and antibody responses is a complex story. You cannot simply say that antibody response is limited because it probably depends on a variety of factors. Some of them are listed here. Certainly, patients in neurological status, we cannot forget that at least in our material 50 percent of patients which come to our center are immunodeficient.Then there is a question of route of phage administration, which I've shown. We don't get high level of antibodies using oral therapy, phage immunogenicity, phage immunogenicity varies, purified preparations versus lysates, cocktails versus monovalent phages, and type of antibody involved in phage binding.
july10-148	DR. GÃ“RSKI: Now the question how long the phage antibody persists, we show that they can eventually drop, and a kind of provocative statement because who knows whether the good antibody response to phage therapy is a good prognostic sign. Maybe. Who knows? Maybe it simply signals that the immune system recovers. It's able to provide, to offer, to mount a good immune response which does not inactivate phage, at least not at the level of periphery. Right? Who knows? We need more data.
july10-149	DR. GÃ“RSKI: Another interesting aspect is phage interactions with phagocytes. We just published a review on this, so, again, I will not go into details, but I'll show you the results from one patient, how phage therapy can contribute to, and this is one sign of let's say improvement of immune system following phage therapy regarding ability to kill bacteria by polymorphonuclear cells and monocytes.So, during the successful phage therapy, as you can see here, the patient could recover -- the patient's ability to kill bacteria could significantly increase, and another, I mention phage and immunomodulation, and we have quite interesting data regarding the potential effects on phages on the indices of inflammation. Here, you see the CRP levels initially and following nine the use of therapy the value drop from 35 to 14, and then after second round of therapy to almost normal, even though the eradication has not been achieved.
july10-150	DR. GÃ“RSKI: So there is a -- again, I repeat -- a very interesting area, how phage interact with immune system, and we have published data showing that they can inhibit a reactive flux against phages.
july10-151	DR. GÃ“RSKI: We published a book on phage therapy which received a good opinion in Lancet Infectious Diseases, in Clinical Infectious Diseases, and a number of papers. One of them presents the present and future of phage therapy, and maybe I'm kind of selfish because I was the first author, but to some extent this is a kind of visionary paper.
july10-152	DR. GÃ“RSKI: And, of course, we realize that observational studies are not the evidence-based medicine. Yet I would like to cite here the statementmade by Dr. Califf about the value, potential value of such work, and I will in the end, I will refer again to this paper published recently in Future Microbiology about phage translocation from the lumen of intestine through surrounding tissue and gut-associated lymphoid tissue, and this has in our philosophy, in our thinking, not only -- this is not the only mechanistic let's say movement of phages, but on the way they interact with cells in immune system, which is shown here, and they may beget powerful, powerful immune reactions. We were very glad to see that this theory of phage translocation is already cited.
july10-153	DR. GÃ“RSKI: Well, what is the future? It's difficult to tell the future, but I'm sure you know the report prepared by Wellcome Trust and the projections. What are the alternatives to antibiotics for using wild type phages according to this forum only 9 percent of success? If this is so, if this is true, what is the future? What we should do in parallel to developing clinical trials?
july10-154	DR. GÃ“RSKI: This is a letter in Polish, but also it has an English portion. This is a letter which was sent by Minister of Health of Belgium to the Minister of Health of Poland. Of course, it went through thebureaucracy of our Ministry of Health and this is the result, so I apologize. You don't need to read the Polish text, but you can concentrate on this portion which tells you what is most important.
july10-155	DR. GÃ“RSKI: And what is most important, and this is probably my message, I find the message that we need to develop clinical trials, but I believe, and I'm glad to see the Minister of Health of Belgium agrees, that we also need to expand the existing programs of experimental therapy. Thank you.
july10-156	DR. GÃ“RSKI: (Applause.)
july10-157	DR. KINCAID: Do we have any questions for Dr. GÃ´rski? Could you use the microphone, please?
july10-158	AUDIENCE MEMBER: Thank you, Dr. GÃ´rski. I have a question about your source of your phages for your institute. Are you constantly going out to environmental sources like waste water treatment plants to isolate new phages?
july10-159	DR. GÃ“RSKI: Yes.
july10-160	AUDIENCE MEMBER: Or what's your procedure about that?
july10-161	DR. GÃ“RSKI: Well, the procedure is standard. I don't think I will go into technical there in details. You know, it's standard procedure for phage procurement which has been described indetails in a series of papers published by Frontiers in Microbiology recently. There is a paper by Beata Weber-Dabrowska, et al. under the title 'Phage Procurement for Therapeutic Purposes.' I think it gives you the most updated information.
july10-162	AUDIENCE MEMBER: That's the primary source for your phages is waste water untreated sewage?
july10-163	DR. GÃ“RSKI: We have also historical phages which are very old, which we inherit even from the time of Ludwik Hirszfeld, who worked, who founded our institute and before we were here, he was already working also, and he's been already engaged in the collection of the strains of phages, so part of our phages are historically related.
july10-164	AUDIENCE MEMBER: You mentioned that you have seen a specific -- a phage-specific antibody response in about 17 percent of patients treated with single phages and about 43 percent of patients treated with cocktails. Since phages can differ in their immunogenicity, were the same phages used in the cocktails that were used individually? Is that a direct comparison?
july10-165	DR. GÃ“RSKI: Yes. Yes. Yes. So it's a kind of learning. If you believe that the peripheral, peripheral antibody are peripheral -- and in thepresence of antibodies peripherally may be a little bit impacted for the success of phage therapy.
july10-166	AUDIENCE MEMBER: Dr. GÃ´rski, thank you so much for very useful data on clinical use. It's very impressive. And my question about the allergy testing, what allergy --
july10-167	DR. GÃ“RSKI: Allergy?
july10-168	AUDIENCE MEMBER: Allergy. You mentioned that you test your patients.
july10-169	DR. GÃ“RSKI: Very good question.
july10-170	AUDIENCE MEMBER: And my question actually has two parts. What allergen do you use? Is it like phage, something from phage, and what method do you use for this?
july10-171	DR. GÃ“RSKI: As far as allergy, we did not prohibit IgE responses, but interestingly enough, when you monitor the leukocytosis in our patients, in none of these patients we have increased value of eosinophils, which was striking. In no patient, I repeat, we had increased value of eosinophils.
july10-172	DR. GÃ“RSKI: And I'm also aware of the work of my associate, Krystyna Dabrowska, a very bright molecular biologist, and I know that she just presented a very impressive poster which she will be presenting in Evergreen, that in experimental animals, when youinject phages, when you administer phages, I think she had some data, if I remember well, that there is no specific allergy to phages in mice.
july10-173	DR. GÃ“RSKI: So, very unexpectedly, there appears to be no strong allergic reaction as measured by the data. In contrast, you have a decreased CRP value, and clinically relevant allergic reactions, they can appear, but they are relatively very rare. As you mentioned, less than 4 percent patients develop such reactions that cause us to terminate the treatment.
july10-174	AUDIENCE MEMBER: So you did not use test allergen in --
july10-175	DR. GÃ“RSKI: Sorry? I did not hear you.
july10-176	AUDIENCE MEMBER: You did not use test allergen made of phages, right? You just used indirect methods to see the allergy.
july10-177	DR. GÃ“RSKI: Yeah.
july10-178	AUDIENCE MEMBER: Okay. Thank you.
july10-179	DR. KINCAID: I think what we're going to do is we're going to go to a break right now, and for those of you who do have questions, I encourage you to speak with Dr. GÃ´rski or Dr. Young, and we will return at 10:15. Thanks a lot.
july10-180	DR. KINCAID: (Whereupon, a short recess was taken.)
july10-181	DR. KINCAID: Thank you very much. I'm alsogoing to take this moment to mention to all the speakers and to those asking questions, all of those good thoughts will go to waste, certainly as relates to the ability to be transcribed, if you don't speak into the microphone, and I would suggest to the speakers that they listen for the resonance because that's what really allows you to know that you're being heard.
july10-182	DR. KINCAID: So, without further ado, I'd like to introduce Chip Schooley from the University of California, San Diego, the Department of Infectious Diseases, who has an absolutely stunning story, one that many of you are already familiar with. It's a story that was given its initial promotion by Ry Young earlier in the day. So, without further ado, Chip.
july10-183	DR. SCHOOLEY: Thanks very much, Randy. I'd like to thank Randy and the rest of the organizers for the opportunity to talk to you a bit about Tom Patterson, who you heard a bit about this morning from Ry Young. This is going to be a very clinical talk. I was asked to kind of give people a sense of how these cases play out in the context of an emergency IND and to talk about some of the strengths and weaknesses of this approach.
july10-184	DR. SCHOOLEY: I am going to show pictures of the patientand of his wife. They both are not just giving consent, they're actually quite enthusiastic about this.
july10-185	DR. SCHOOLEY: So the story began in Egypt in November of 2015 when they took a vacation during Thanksgiving down the Nile, and this is the inside of the boat that they were on.
july10-186	DR. SCHOOLEY: (Laughter.)
july10-187	DR. SCHOOLEY: Let's see if we can get this to go here.
july10-188	DR. SCHOOLEY: Okay, we'll get this back in a second. We'll have to stop the timer here.
july10-189	DR. SCHOOLEY: In any case, Steffanie Strathdee, the patient's wife, and Tom took a vacation over Thanksgiving 2015 to Egypt and decided to take a barge down the Nile. As they're pulling into Luxor, he developed abdominal pain and fever. Tom is a friend of mine, as is Steffanie, and they texted me and said they were concerned about Tom and wondered whether this could be gastroenteritis or something else.
july10-190	DR. SCHOOLEY: As this played out, it became more and more clear it was something else, and it was suggested that they get to a hospital. He was a 68-year-old diabetic, a little bit overweight, and it sounded more and more like gallstone hepatitis, gallstonecholecystitis and pancreatitis. They went to the hospital in Luxor. They had been seen by a ship physician who saw him and gave him Gentamicin. This always works. In this case, it didn't. He showed up in the hospital and the same physician actually was running the ICU and this time gave him some fourth generation Cephalosporin, some fluids, stabilized him, and then he was evacuated to the university hospital in Frankfurt, where Stefan Zeuzem and his colleagues took care of him.
july10-191	DR. SCHOOLEY: When he got to Frankfurt, it was found that he had a large pancreatic pseudocyst, shown here with the green tags behind his stomach, both in this plane and in this, a relatively large fluid collection. On the second hospital day in Frankfurt, they threw an endoscope into his stomach. They then used an endoscope to put two stents through the wall of his stomach into this fluid cavity to drain the abscess cavity into his stomach.
july10-192	DR. SCHOOLEY: In this first acquisition of fluid, they grew Candida glabrata and Acinetobacter baumannii that was resistant to most antibiotics except for colistin, tigecycline and meropenem.
july10-193	DR. SCHOOLEY: He continued to be febrile, was on pressors, was delirious. The following two days later they didan ERCP, noted that his biliary tree was partially obstructed by a pre-papillary stone which was extracted, a stent was placed to establish drainage of his biliary tree. These cultures also grew Acinetobacter baumannii. Colistin was added. Imipenem was changed to meropenem based on some of the sensitivities that were emerging, and arrangements were made to transfer him to San Diego.
july10-194	DR. SCHOOLEY: By the time he got to San Diego on
july10-195	DR. SCHOOLEY: December 12, the organism was meropenem-resistant. The GI consultants felt that he needed surgery. We felt he needed surgery. The only people who didn't were the surgeons. They were concerned about the fact that he was quite unstable, and not being able to do the surgery ourselves, we had to go along with their plan.
july10-196	DR. SCHOOLEY: So the plan was to try to manage him medically and to drain the abscessed cavities as they evolved as you'll see percutaneously using interventional radiology throughout the course of this. He developed a pancreatic cyst. This too was drained. The Acinetobacter continued to develop increasing resistance as we treated him with rounds of antibiotics.
july10-197	DR. SCHOOLEY: He then began by the middle of January togrow B. fragilis from a number of these drains. We didn't have good control of the source of his necrotic pancreas. He had an episode of septic shock and was transferred to the ICU, and at this point in time was found to have B. fragilis bacteremia.
july10-198	DR. SCHOOLEY: He shortly thereafter developed emphysematous cholecystitis. Another interventional radiology drainage tube was placed. One of our colleagues in the Department of Pediatrics did some synergy studies in vitro and showed that if you squinted, that if you used azithromycin, colistin and rifampin together, there was some evidence of synergy, so these were added to his antibiotic regimen. Developed some renal failure. The colistin was held and then restarted later on. He then developed increasing abdominal distension. Paracentesis revealed that Acinetobacter was now in his peritoneal fluid.
july10-199	DR. SCHOOLEY: By middle of March, he had the multiple intra-abdominal collections being drained through five IR drains. We continued to try to convince the surgeons to go in and do a definitive drainage procedure, and the cycle we got into was that when he was sick they said he's too sick to operate on. Call us back when he's better. We called them back when hewas better. They would say he's getting better. You need to just leave him alone, let him continue to get better. So we got into this cycle through February and March in which we were not able to get him, in our view, adequately drained.
july10-200	DR. SCHOOLEY: As time went on, though, he continued to deteriorate. Additional organ systems began to fail. He became stuporous. He ended up on two pressors. He ended up intubated on a ventilator and began to develop hepatic dysfunction in addition to his renal dysfunction.
july10-201	DR. SCHOOLEY: This kind of gives you a sense of his course. You can see his fever curve with multiple fever spikes throughout. White count kind of bouncing around between 10- and 25,000, often in conjunction with isolations of organisms from his bloodstream and peritoneal fluid in other places.
july10-202	DR. SCHOOLEY: His wife, Steffanie Strathdee, is really the hero of this story. She had kind of been watching this and continued to read. She is an infectious disease epidemiologist, trained as a Ph.D., not as an MD, but she is really quite versatile in microbiology and continued to read about Acinetobacter, and came across a paper that was published in PubMed about the use of Acinetobacter baumannii in the treatmentof -- I'm sorry -- of phages in the treatment of Acinetobacter baumannii, and she sent me this paper by email.
july10-203	DR. SCHOOLEY: This was about mid-March, and my response to her at the time was, you know, it's not like we're really knocking the socks off this infection, we're willing to try anything at this point, and certainly there's been a lot of history of phage therapy in other places, as we've heard today. Very little evidence that it will do any harm, and certainly we're not getting where we need to go with our current approach to therapy.
july10-204	DR. SCHOOLEY: My inward thought, however, was that the chance that we're going to find somebody to make us phages in time to be able to use them and to deal with all the bureaucracy both in terms of the local bureaucracy at UCSD and the regulatory bureaucracy outside UCSD was slim, but given the fact that we had really very little to offer and Steffanie needed some hope, we decided to go ahead and go full steam ahead.
july10-205	DR. SCHOOLEY: She got in touch by email with the group in Georgia. They referred her to Jean-Paul Pirnay in Brussels because they had been collaborating. Dr. Pirnay said he'd love to help. In fact, he had some phages that were active against Middle East-derivedAcinetobacter and that they were in the hands of Ry Young's laboratory at Texas A&M, and she suggested that the organism be sent to Ry.
july10-206	DR. SCHOOLEY: She telephoned him, caught him in his lab and talked to him for a little over an hour, and over the phone he decided to go ahead and try this out, and said he would commit his laboratory for the next couple of weeks to see if he could come up with something that might be used to treat her husband.
july10-207	DR. SCHOOLEY: The organism was sent to Texas A&M, and the phage search began. Unfortunately, at the same time the phage was being -- that Dr. Pirnay had was being shipped from Belgium to UCSD, preparations were being made and it was found that that particular phage did not have activity against our patient's organism.
july10-208	DR. SCHOOLEY: In the library at Texas A&M, one of the phage from AmpliPhi was found to have activity. Dr. Young got in touch with AmpliPhi and they very quickly said, 'Of course, we'd be happy to let you use that phage.' And then he looked for environmental sources of additional phage that could be used in a cocktail against this patient's organism.
july10-209	DR. SCHOOLEY: At that point, I called the FDA to get in touch with them to tell them that we would likely be asking for an eIND to give a home brew cocktail ofbacteriophage to a patient with a multidrug-resistant Acinetobacter. The FDA reviewer, who will be running a panel tomorrow afternoon, Cara Fiore, was very supportive, in fact, and said she wanted to organize a conference call with CBER to talk about some of the issues that they had been thinking about in terms of phage therapy. That was on March 1.
july10-210	DR. SCHOOLEY: By March 4, she had made some internal discussions and said they really only need to know about if endotoxin assays had been done, what we knew about that, and what was being done about sterility. Otherwise, they were ready to go and they didn't want these stipulations to get in the way of starting phage therapy, and we provided that information a couple of days later and had approval from the FDA to proceed relatively quickly.
july10-211	DR. SCHOOLEY: Meanwhile, back at UCSD, there was a lot of back and forth about what this was all about. Luckily, the patient's wife was one of the deans at UCSD and she got in touch with the chancellor, who was anxious to keep her happy, and he instructed the lawyers to make sure this worked. And so we had the university attorneys on our side very early on, which was very helpful, and actually a very positive interaction with the attorneys at Texas A&M.I then had to talk to the investigational drug pharmacy that would be administering the phage. They independently then contacted the Institutional Biosafety Committee, who told me that they would be meeting in three weeks to discuss the application to use -- to allow the phage into UCSD, and in the meantime, an MTA was being worked out between UCSD and Texas A&M.
july10-212	DR. SCHOOLEY: And then, at that time, the FDA reviewer said that she also heard that there was some additional places that Acinetobacter-directed phage were being developed and suggested that if we wanted to, it might be useful to talk to the combined program that the Army and the Navy had been leading down the road, and she provided me with a couple of phone numbers and we decided to go ahead and do this. This was at a time when we're still full speed ahead down at Texas A&M, but we didn't yet have a phage cocktail that we could use.
july10-213	DR. SCHOOLEY: So I got in touch with the two programs. The Navy was willing to have us ship the patient isolate to them, and we did. In the meantime -- this actually was actually sent by Ry's lab to the Navy, the first one, and they began to screen for phage as well.Fairly soon, this is -- you'll see more of this tomorrow, but this is just using the Navy's Biolog approach on the Texas A&M phage. You can see that compared to the control conditions, the phages individually or in a cocktail were able to suppress the Acinetobacter that the patient was growing. The Navy also were developing phage, as I'll show you in just a minute.
july10-214	DR. SCHOOLEY: The phage that -- the cocktail that was constructed were the AmpliPhi phage and three environmental phages that Ry's lab had come up with after they started the screening.
july10-215	DR. SCHOOLEY: The plan was to go ahead and ship this cocktail to us and to do the endotoxin testing kind of as the phage were in transit. This is the Navy cocktail showing the four Navy phages and the cocktail together with the expression, 'This work being done by Biswajit Biswas,' who will be talking to you tomorrow about some of the issues related to preparation of the phages and selection for resistance.
july10-216	DR. SCHOOLEY: We were back in touch with the FDA and one of the concerns we had was that each of the phages would have to be treated as an individual product, which would have required eight INDs. We were very pleased to hear that they were willing to consider thetherapeutic approach to this patient's organism as a single -- would be covered under a single IND and that we would only need to submit another one if we decided to switch and treat a different organism. Luckily, he didn't have a different organism that needed to be treated, so this entire process was carried out under a single eIND.
july10-217	DR. SCHOOLEY: Then we began to run into trouble with the endotoxin. About the time the phage cocktail arrived from Texas A&M the initial endotoxin assay showed quite a bit of endotoxin in the phage preps. We weren't sure whether this was an issue related to the endotoxin itself or whether there was an artifact in the assay. The assay repeated at San Diego State again showed quite a bit of endotoxin in the preps.
july10-218	DR. SCHOOLEY: Forest Rohwer's lab at San Diego State then was engaged to try to scrub these preps and did that with an octanol extraction that, as I'll show you in a minute, was quite successful.
july10-219	DR. SCHOOLEY: The first batch of Navy phages arrived shortly thereafter. They too had an unacceptably high level of endotoxin in them and, when measured at San Diego State, in fact, even a log higher than was seen in the assay done at the Navy.
july10-220	DR. SCHOOLEY: The Texas A&M phage were scrubbed at SanDiego State with an octanol extraction and tangential centrifugation. The Navy then made a second batch of phage and scrubbed their phage using a cesium chloride gradient, which is their approach to phage purification. And at this point, we actually had phage that were really quite clean, giving you a
july10-221	DR. SCHOOLEY: sense -- so this is the Navy cocktail, all of them together, showing you the endotoxin concentration per milliliter. These are the individual phage from Texas A&M. Again, much improved over the previous batches.
july10-222	DR. SCHOOLEY: So, at this point, we had two sets of four-phage cocktails. The Navy cocktail is shown here. Each of these were environmentally obtained Myoviridae, as was suggested. These may well have been very similar. We really didn't have time to try to look for phage that were quite different in terms of their tropism and mechanisms of action. Same thing happened from Texas A&M for other Myoviridae. Again, environmental samples and shipped simultaneously.
july10-223	DR. SCHOOLEY: The Texas A&M cocktail happened to arrive several days earlier than the Navy cocktail, and we decided that -- and then a second generation cocktail that Dr. Biswas will talk about tomorrow included a phage that was active against one of the organisms, the Acinetobacter that was selected for resistance toall eight of the initially employed phage. The phage arrive on March 15 and we have the data related to the endotoxin scrubbing, and this is the interventional radiologist, Andrew Picel, who was giving the phage into the three cavities that at this point were still being drained. The approach taken was to irrigate the cavities, then to introduce ten to the ninth plaque-forming units into each of the abscessed cavities, cross-clamped the cavity for about half an hour and then allowed the cavities to drain.
july10-224	DR. SCHOOLEY: We saw a change in the characteristics of the drainage fluid, the pseudocyst, for example, before and after the phage were administered. Whether this is causal or not, we don't know, but certainly we did see a change in the characteristics of the drainage fluid.
july10-225	DR. SCHOOLEY: About two days later the Navy phage cocktail were ready to go. The patient had in the meantime stabilized but hadn't gotten demonstrably better. We had been seeing over the previous four to five weeks kind of each day things were gradually worse. In the two days between the administration of the intracavitary phage and the time the Navy phage were ready, there were no changes in his clinical status. Whether or not it was causal or just happenstance,hard to know, but we still felt that we were not where we needed to be in terms of getting him better, and we knew that we had isolated Acinetobacter from his peritoneal cavity, which would be outside the field of the phage being introduced into the abscessed cavities. Every sputum that we obtained was full of Acinetobacter. Acinetobacter had been isolated from his urine and from time to time later on from his bloodstream. So we felt that if we were going to make any headway that we needed to switch to a parenteral administration approach and decided to give the Navy phage cocktail intravenously.
july10-226	DR. SCHOOLEY: This is the ID fellow, Melanie McCauley, giving the first dose intravenously. He tolerated both phage administration routes quite well. He gradually saw fewer and fewer pressors over the course of the next 24 hours. Got gradually better, and then on Sunday evening actually woke up and recognized his daughter, who was sitting by the bedside.
july10-227	DR. SCHOOLEY: At that point, we found that the Acinetobacter was now sensitive to minocycline. That was added to have maximal benefit from antibiotics as well. But Sunday morning things got worse again, and he again began to require pressors. His mental status declined and by 8 in the morning he was on threepressors and unarousable again. I was very concerned we'd done something with the phage. We stopped the phage therapy, cultured the bags to make sure that there were no bacterial contaminations, but also aware that he was someone who could certainly have intervening complications of being on the assay use, so we broadened his antibiotics and lo and behold found that he was now -- the next day he was growing anaerobic gram negative rods from his blood, probably again from his necrotic pancreatic bed, turned out to be a Bacteroides thetaiotaomicron.
july10-228	DR. SCHOOLEY: He got better by Monday night, but again Tuesday morning he was in shock, requiring three pressors. This time he was noted to be in atrial fibrillation. The pulmonary attendings and fellows were sure it was the phage therapy again, and when we looked more carefully and it was found that he was in atrial fibrillation, I said did he -- I asked them whether he had become hypotensive before or after the atrial fibrillation. They said it was after. I said why don't you correct his rhythm and I bet you'll find that his blood pressure improves, and it turned out he got much better and it was really because they had diuresed him and had potassium depleted him that hehad flipped into atrial fibrillation. The only reason I'm getting into this is that patients like this have multiple complications that always get blamed on the new therapeutic, and that's what was going on throughout this first week of therapy.
july10-229	DR. SCHOOLEY: Here he was by that evening, the next evening awake and interacting again with his family. He began to grow Acinetobacter less frequently, but we didn't have really good quantitative cultures, over the course of the next several days developed phage-resistant Acinetobacter that you'll hear about tomorrow from Dr. Biswas. We did some phage PK to give a sense of how this is just phage being given intravenously at time zero and then monitored in his bloodstream you can see cleared by 60 minutes.
july10-230	DR. SCHOOLEY: His course after that was relatively chaotic. He was a sick guy. He had another bout of Acinetobacter sepsis associated with the drain that was in his biliary tree, migrating into his liver, but he gradually got better and was discharged in August.
july10-231	DR. SCHOOLEY: Here he is leaving Las Vegas, and here he is in May just before returning to work with his wife, Steffanie Strathdee, both big fans of phage at this point in their homes. I'm sorry they can't be with ustoday. It would be much more fun to have had them present this case than me.
july10-232	DR. SCHOOLEY: So lessons learned. It's feasible to develop a strain-specific bacteriophage cocktail if you have two academic groups turn over everything they're doing for several months for a single patient. The therapy was well tolerated and I'm convinced really turned his course around given where he'd been going over the course of that period of time, and that people like this seem to be very complicated to both treat and assess in the context of eIND therapy.
july10-233	DR. SCHOOLEY: So, to finish, the strengths of eIND therapy, you benefit patients individually. Certainly, Dr. Patterson was benefitted by this therapy as far as I could tell as his physician. The eIND is very flexible. You can treat many different kinds of patients if they require therapy based on eIND considerations. And from the regulatory perspective, it's relatively straightforward.
july10-234	DR. SCHOOLEY: The weaknesses are, however, that every patient's different and it's very had to aggregate patients and make sense of the data you collect prospectively. Every time you do this at a new place it's very complicated. The nurses were sure we were going to kill him with the phage. The pharmacy wassure that we were going to contaminate the pharmacy. The Institutional Biosafety Committee had my name on a list for a while, and I had been the previous chair of that committee. And the other issue is that none of the regimens are going to be standardized if you're treating one patient at a time.
july10-235	DR. SCHOOLEY: You don't collect the data in a standard way. You don't have standardized end points, and it's not sustainable. You can't have academic laboratories doing this over and over again, and we had no resources to do this. We were shipping things back and forth using personal accounts. So there needs to be a more sustainable way to approach this.
july10-236	DR. SCHOOLEY: So I'll stop there. We'll talk about more of this, I hope, on the panel and just say this was really a village that did this. Multiple people engaged in the laboratories that were making the phage. Scott Salka at AmpliPhi made the phage available. Quickly needed advice from Dr. Merril was extremely helpful in terms of the overall therapeutic approach. We were very fortunate to have the help of the FDA in approaching this. We had helpful lawyers, which is like an oxymoron sometimes, and very helpful people in our administration, which is also an oxymoron, and finally, a bunch of extremelyenthusiastic physicians that made all of the difference and allowed this to go forward. So thanks very much.
july10-237	DR. SCHOOLEY: (Applause.)
july10-238	DR. KINCAID: That's an impressive number of stars that were aligned at the right time for that gentleman. A very interesting story.
july10-239	DR. KINCAID: I think what we'll do now is we'll go on to Dr. Narayan from Yale University, who will provide another example of treatment under eIND, which is obviously an important tool available under such desperate conditions.
july10-240	DR. KINCAID: Did you have something, Marcus, for me? Okay.
july10-241	DR. KINCAID: All right. At any rate, I'd like to introduce Dr. Deepak Narayan from Yale University, School of Medicine, and he'll give us an overview of another case, quite a different case involving a Pseudomonas infection. Deepak.
july10-242	DR. NARAYAN: Good morning, everyone. Before I get started, I'd like to thank a whole host of people who have enabled me to be here. Randy, Roger, and most importantly Peter Marks, whose connection with Yale enabled me to move things along, as you will see.So just a brief description of my talk. This is not going to be laden with scientific data, as Dr. GÃ´rski's was. It is a mixture of an apology for surgeons, some clinical storytelling, and some history which I think you'll find interesting.
july10-243	DR. NARAYAN: So, as someone pointed out, paraphrasing Halliday, God must have really loved viruses because they are the most numerous replicating entities on this earth. The phage structure that we all learned in high school pretty much holds true now, and this is my first contact with phages when I learned about Twort and d'Herelle when I was in the 11th grade.
july10-244	DR. NARAYAN: Coming from India as I do, I have personal experience with phage generation from the Ganges where you see sights like these where people drink directly out of the Ganges and never seem to suffer any ill effects, but it is when we drink water from New Haven, we seem to work up a huge host of gastroenteritis regardless of what else we do.
july10-245	DR. NARAYAN: So the funny thing was that Hankin reported that a substance in the Ganges River prevented cholera and this was remarked upon by a fellow in New England named Mark Twain in his Tramps Abroad book. But the real hero of all this, as has been pointed out multiple times this morning, is d'Herelle, who wasalso the model for Arrowsmith by Sinclair Lewis, and interestingly was brought to New Haven by Dean Winternitz, which was pointed out by an earlier speaker, and owing to sharp practices that Gunther Stent wrote about in his review of Bill Summer's biography was asked to leave.
july10-246	DR. NARAYAN: The most interesting coincidence, in fact, this whole episode has been a list of multiple coincidences, sort of like Swiss cheese holes lining up, but in a good sort of a way. His office was right next to mine when it was created about 100 years ago. He didn't really do too well at New Haven, probably drank the same tap water that I did, malaria -- phrenic nerve palsy and sort of moved on to France.
july10-247	DR. NARAYAN: So the big issue is why am I as a plastic surgeon talking about all this stuff, and what might not be admittedly obvious is that we deal with a whole host of infectious problems, including necrotizing fasciitis, abscesses in the head and neck, infected craniotomy plates and so forth, as well as dealing with infected prostheses on a fairly regular basis. This is an example, and I apologize for the goriness of the pictures. You cannot have a surgeon talk without gory pictures.
july10-248	DR. NARAYAN: For instance, this case of an infectedprosthesis which was referred from a local hospital. As you can see, a fairly large volume of foreign material, open wound ring, pus, and a standard treatment for this is to wash it out a few times, move what's called a muscle flap, in this case a gastrocnemius muscle, close it, and with a full expectation that it will heal, which it does most of the time.
july10-249	DR. NARAYAN: We have other problems that we deal with, sort of more appropriate to the case that we're discussing. This is a veteran who presented with basically pus around the Dacron graft, an aortic valve replacement going all the way down, and this whole thing smelled like a sewer, and for a few days we were concerned that he had a colonic fistula.
july10-250	DR. NARAYAN: Again, the treatment is to wash it out repeatedly, make sure you get good by-fill control, flip the pectoralis major muscle into the wound, close it using a wound vac, especially because it's infected, and then have him present with a well-healed wound approximately two months after the procedure.
july10-251	DR. NARAYAN: And one final note. Another area where Dacron grafts are often used, vascular bypass grafts for lower extremity ischemia, and in this picture you see the graft right about there. That little line isto show the radiograph is where the graft actually is, and the groin is probably the most commonly contaminated site of vascular graft infections.
july10-252	DR. NARAYAN: So the treatment, once again, as with the chest, is to wash it out, get as much control locally as you can, and flip a nearby muscle in order to deliver antibiotics appropriately, with the antibiotic regimen continuing for about six weeks after the procedure.
july10-253	DR. NARAYAN: We discussed these traditional approaches approximately 10 years ago now, and the reason for doing that is, again, because of the aging population we're beginning to see a greater number of these patients presenting with graft infections, and for the most part, and I want to emphasize this, is that we don't really need to resort to out-of-the-box thinking, such as phage therapy, in terms of treatment.
july10-254	DR. NARAYAN: In fact, over the years, over the last 15 years we've dealt with approximately 150 graft infections, only one has been lost because of an infection with Pseudomonas as a matter of fact of the anastomotic site.
july10-255	DR. NARAYAN: As you all know, there's a dramatic decrease in antibiotic drug approvals, an increase inantibiotic resistance, and I will not belabor the issue, and despite encouraging slogans such as 'Bad bugs need new drugs,' really not much has been forthcoming.
july10-256	DR. NARAYAN: Pseudomonas aeruginosa is of particular interest to surgeons because it's responsible for a significant number of nosocomial pneumonias, burn, wound infections, which is of particular relevance to plastic surgeons, as well as other immunocompromised populations, as you know. And this is a really clever bug which has evolved multiple mechanisms by which to thwart the efforts of surgeons and ID specialists and most importantly has been associated with the formation of biofilm, especially with prosthetic material.
july10-257	DR. NARAYAN: Now there are a whole host of mechanisms by which Pseudomonas survives, which I will, again, not belabor this crowd with, and a partial list of the drugs effluxed by the -- multiple drug efflux mechanisms are listed here for you to see.
july10-258	DR. NARAYAN: So the story again begins as another coincidence whereby I was contacted by Dr. Paul Turner and his post-doc, Benjamin Chan, to set up a treatment for diabetic wound infections, and part of this discussion centered around the isolation of a newphage, the OMK01, which has been written about, which is the outer membrane polar knockout one, and this was isolated funnily enough from Dodge Pond in Connecticut, a site of Navy testing and apparently so toxic that none of the residents eat the fish from this pond anymore, and this is from a direct quote from a patient.
july10-259	DR. NARAYAN: So the phage therapy approach, as you all know, can mimic the use of antibiotics. You treat it with a phage. You kill as many as you can. Resistance to phage is developed, and essentially what ends up happening is that you basically cannot use the phage to treat them anymore.
july10-260	DR. NARAYAN: So these resistance-targeting antibiotics, which are basically a combination of these two, might actually help to deal with this problem. So the amazing thing about this OMK01 was that it actually latched on to the drug efflux pumps. And so the thought was that maybe we can use evolution to help us, and I want to emphasize that all this was done in Paul Turner's and Ben Chan's lab, and they were kind enough to lend me these slides.
july10-261	DR. NARAYAN: So the thought was that if the resistant organism was forced to make a choice between resistance to antibiotics and resistance to phage, itwould lose because it was so closely intertwined that it could only be resistant to one, and, in fact, an in vitro test did demonstrate that this actually happened.
july10-262	DR. NARAYAN: So I'm just going to briefly talk to you about the patient in question. This was a 75-year-old male, had a coronary artery bypass graft, was done in a neighboring hospital, along with an aortic arch replacement, similar to the picture that I showed you in the infected thoracic cavity.
july10-263	DR. NARAYAN: Following surgery, he developed empyema and became extremely sick, requiring four pressors, at which point he was transferred over to Yale-New Haven Hospital. My boss happened to be on call, and he was leaving town, and so being the most junior on the totem pole, I was given the enviable task of taking care of this gentleman who -- which is now a matter of public record -- who was a faculty member and thereby obviously raised the strain involved in treating him.
july10-264	DR. NARAYAN: So the patient was placed on antibiotics appropriately since he grew Pseudomonas, as depicted in these green shadows that you see on the cartoon here. So, after -- and I'm going to try to see if this actually works -- washing him a few times, you end up with this sort of a scenario where you have apus pocket, and I'm sorry if you can't see this back here, sort of the heart of the matter, if you will, which was the cause of the problems, as we'll talk about it. You can actually see the greenish block.
july10-265	DR. NARAYAN: And so, again, in keeping with the previous treatment, keep washing him out as many times as we could. In this case, we did it about three times, and then used a muscle flap to close it. So, as a part of increasing the immune delivery in this area, we harvested the omentum through a laparoscope, which is the yellow plat-like substance that you see here, and during the course of this harvest we found that the field was filling up with blood, and, to my absolute horror, found out that he had actually ruptured his ventricle on the table, which, of course, prompted a repair with the help of the cardiothoracic surgeons, which you see out here. We used a piece of lung to patch that defect, and fortunately for all of us he survived.
july10-266	DR. NARAYAN: So, despite actually having been discharged from the hospital, he was admitted at least four times for episodes of sepsis requiring IV antibiotics. He was placed on ciprofloxacin as a suppressive measure. He did present with one episode of bleeding which I thought was from the outer -- it turned out it wasjust a rib poking through an intercostal artery, which we cleaned up.
july10-267	DR. NARAYAN: He was then asked to follow up and multiple requests from his son, who had a Ph.D. from Yale in immunology, urged me to find other places to treat this gentleman. Again, as Dr. Schooley pointed out, cardiothoracic surgeons refused to operate on him, saying that he was doing well and should be left alone. We contacted surgeons in Tokyo, Zurich, as well as in Texas, asking if this aortic arch could be replaced, and all of them basically declined to operate.
july10-268	DR. NARAYAN: His son, who was pushing for experimental treatment, you know, suggested new antibiotics, and during the course of this whole business, I met with Drs. Paul Turner and Chan, who, as I said, presented a project for treatment of chronic wounds, and a few days after the meeting I realized that this gentleman is a perfect treatment choice for phages.
july10-269	DR. NARAYAN: We organized an eIND, and thanks to Peter Marks and Cara Fiore, who were extremely helpful in moving this along, we did get initial permission to proceed, but the patient was lost to follow up since he left the country and was not heard of until January of 2016. Apparently, by report, had been gettingintravenous ceftazidime as the patient could afford it, for over a year and a half intravenously.
july10-270	DR. NARAYAN: So, when they presented again, this was due to bleeding from a fistula site that had never healed, and the Pseudomonas that was repeatedly cultured from this obviously was a potential source of a problem. And so the FDA obviously gave us permission to proceed. And one of the happiest emails that I ever received in my life was that I did not need to go through the HIC for approval.
july10-271	DR. NARAYAN: So, when I sent this to the HIC and they promptly approved it, we decided to go ahead and treat this gentleman, and a whole host of bacteriophages were tested by Drs. Turner and Chan, and we created a three-phage cocktail. The endotoxin business was also of concern, but we had it independently verified by a laboratory in Cape Cod to meet EU standards. So the thought was that potentially we could use three of these phages, one to weaken the biofilm, one to potentially remove the colonists, and then use ceftazidime to finish the job off.
july10-272	DR. NARAYAN: So, as it turns out, we ended up using just the OMK01, and with the help of our interventional radiologist, Dr. Mojibian, we accessed -- tried to access the abscessed cavity, which you'll see theneedle trying to go into that space that I showed you on the video.
july10-273	DR. NARAYAN: Now, in a further twist to the whole thing, the day we were doing this when we organized the OR, the emergency and anesthesia teams, the interventional radiologist, who is from Iran, received a call from his wife urging him not to do the procedure. As it turns out, the patient in question was a legend in Persian medicine, Iranian medicine, if you will, and the thought -- the wife was worried that they would not be able to go back to Iran if something happened to the gentleman on the table.
july10-274	DR. NARAYAN: So there was a hasty discussion about all this prior to the injection, but thanks to the fortitude of Dr. Mojibian, we decided to proceed. Despite that, trying to access this for over an hour, we were able to inject just a few milliliters of solution, so we decided instead to actually push the phages in through the fistula site, seal it off, and let the patient be.
july10-275	DR. NARAYAN: So the patient was sealed off with this thing in place for over 48 hours, and he immediately left town to go back to his home country. It turns out that six weeks later he suffered a perforation of the aortic arch from a bony spicule which resultedfrom a re-growth of the bone from the debridement that we'd done earlier. So the graft was partially replaced by surgeons in Iran, and the cultures just revealed Candida, not Pseudomonas. He was treated for the Candida and has been free of all antibiotics now for about 15 months.
july10-276	DR. NARAYAN: So, in conclusion, obviously, this case with an N of 1 is hardly the basis of treatment of all vascular graft infection, but the important thing I want to point out is that there are conventional methods of treating these infections, as I mentioned earlier, over 150 infections treated fairly successfully, with the exception of one who burst his graft due to Pseudomonas infection, and the scope of phage treatment for these highly resistant infections remains to be explored. Thank you.
july10-277	DR. NARAYAN: (Applause.)
july10-278	DR. KINCAID: I think so that we can move more quickly to the panel discussion we will have an opportunity for Dr. Narayan, Dr. Schooley, and others to field a few questions at the beginning of the panel discussion.
july10-279	DR. KINCAID: At this point, I'd like to invite Dr. Gabard to the podium. This project that he is responsible for directing is a noteworthy project in the historyof phage therapy because it is a randomized multi- center clinical trial and, as we'll probably learn, not without its challenges. So I think this is a very important step going forward as it lays the foundation for a more rational data-based approach towards using phage for medical interventions. Dr. Gabard.
july10-280	DR. GABARD: Good morning, everybody. Thank you for the organizers for inviting me to talk about what we do at Pherecydes Pharma. Of course, this is the usual statement.
july10-281	DR. GABARD: So, first, I'd like to introduce my talk by explaining what we have been doing in the company for several years, the different types of approach we have been using for phage therapy. So the first thing you probably heard quite a lot about is the Phagoburn project. Actually, we entered phage therapy by starting through the standard regulatory routes with a fixed product like an antibiotic, and actually we were testing two products, each of them with more than 10 phages, so we call that complex product, and they were really specific to either E. coli or Pseudomonas aeruginosa.
july10-282	DR. GABARD: Then the second category of products we have been developing are two other cocktails, and I'm just giving you the example of one of them, which are muchsmaller. I guess the experience of handling the manufacturing of products with more than 10 phages has been a good experience, and we decided to go with smaller cocktails of four phages.
july10-283	DR. GABARD: Very recently I show some data that probably we have not been showing yet. We have been also involved in two compassionate treatments with tailored products.
july10-284	DR. GABARD: So, regarding the product of the Phagoburn study, from the point of view of the regulatory route, it was considered as a frozen cocktail with no possibilities of evolution, so of changing the phages within the product and, of course, it was quite unmanageable to be able to adapt the phage of that product with so many phages in the composition.
july10-285	DR. GABARD: It's important to understand also what this definition is about of an active pharmaceutical ingredient and a drug product. If I take the example of an antibiotic, an antibiotic has usually a single API. Here, we are talking about drug products that were made of 12 and 13 API, which is a very, very big challenge on the manufacturing side and the regulatory side.
july10-286	DR. GABARD: So that has been a challenge and we have had some issues regarding shelf life of the managing ofthe APIs in the drug product. When you do GMP manufacturing for any types of products, you are supposed to provide the guarantee that your active ingredients are stable over time. We have not been capable of finding ways, technical ways to demonstrate that each active was being always at the same concentration within the product's shelf life.
july10-287	DR. GABARD: We have been capable of doing that, of course, for each individual phage, but not for the phage inside the drug product, and if anybody in this room has a way to do that, has a technical solution to do that in the complex product of 10 phages, I would be happy to learn from that person.
july10-288	DR. GABARD: Of course, when we started the first process, we heard about the endotoxin content. Our first manufacturing process was too high in endotoxins. We were not in the range of 45,000, but in the range of probably 30,000, and in order to be able to use that product in the patients, we had to go through a dilution, a dilution at the point of care so that the clinicians were doing a 1,000 full dilution before to use the treatment.
july10-289	DR. GABARD: Now, if we move to the second category of cocktails we have been developing, I have been mentioning two of those here. The first one fallsagainst Staphylococcus aureus, and the second one is against Pseudomonas aeruginosa. Here, we have really severely reduced the number of phages, trying to isolate phages with broader spectrum of activity in order to have less APIs because, on the CMC point of view, on the manufacturing point of view, it makes your life much, much easier.
july10-290	DR. GABARD: Of course, with such a low number of bacteriophages, evolution is possible, but under which registration frame, and I think this is very important that we address that issue during this workshop. If you want to make an evolution of a phage in a product, what is the status of that new phage? This is important.
july10-291	DR. GABARD: And then, of course, we have been working a lot on improving the endotoxin content, the purification of the products, the GMP process overall, and now we can say that development of toxins we have is at about two in units in a range and that we have improved the yield through a new purification by a factor of 10 to 100 according to each phage. So we usually routinely yield phages at about 10 to the 11th, 10 to the 12th pfu per milliliter in the GMP process.
july10-292	DR. GABARD: Now, if we move to the next very recentchange in the treatment approach, we have been doing very recently, early 2017, two treatments that I will detail a bit more after that, using product prepared, really tailored for the patient. For that product, the goal would be to use GMP products, but I will explain that we didn't have any GMP phages. We had GMP-like phages, and after discussion with the pharmacists from the hospitals, as well as the clinicians, we got the authorization to apply these products.
july10-293	DR. GABARD: Of course, here, we are not talking about product evolution because, by essence, you are doing a diagnostic and you are just delivering the phages that are active against the infection. But then the regulatory status of these products is really something that we need to address during this workshop.
july10-294	DR. GABARD: If you use no GMP phages like we have been doing but produced like GMP phages, we enter more or less in what we call in Europe or in France the magisterial formula, which is usually done in the pharmacy hospital. Then, of course, if you do GMP phage for single patients, it requires a regulatory frame, and what kind of regulatory frame can we use for that? I think this is the type of questions weneed to address. So a few words on Phagoburn, you're going to be disappointed because I'm not going to deliver the data today. I'm going to provide some preliminary information. You know that the study was performed in 11 burn units across Europe. Actually, only six of them recruited patients, so it was a challenge, and I have been listing the most -- the major recruiters, of course, is the Percy hospital close to Paris, the military hospital, and the Queen Astrid Military Hospital in Belgium. We were talking about Jean-Paul Pirnay in a previous presentation, who is coming from that hospital. So they were the biggest recruiters in the study.
july10-295	DR. GABARD: The time frame of the trial, of the project is explained above, and I think we were very much too optimistic, especially on the CMC manufacturing. We thought we would do GMP phages within 12 months, and we ended up doing GMP phages within 24 months, and you see that the results -- I cannot show -- I cannot talk about the data because, as you can see, the consortium met about 10 days ago and the preliminary clinical data have been shown for the first time to the consortium only 10 days ago. We have not time, we did not have time yet to review all the data and toanalyze all the -- especially the biological data. So we expect to publish all this probably before the end of the year. But anyway, you have some issues, as we have been getting through this clinical trial, and I can give some information about what we see.
july10-296	DR. GABARD: When we started to do the study, we were doing two cocktails, one against E. coli, the other one against Pseudomonas aeruginosa, and the epidemiological infection data that we got from all the hospitals were in a way not realistic, and we know that only today.
july10-297	DR. GABARD: Why was that not realistic? Because when we check in detail the epidemiological data for, let's say, checking how many E. coli cases the hospitals got, actually, they usually count an E. coli infection as a case when the E. coli infection is the major bug that the patient got. But in most of these cases actually the patients gets poly-infections.
july10-298	DR. GABARD: So you're going to get data that says that is a patient that has an E. coli infection, but it doesn't tell you that on top of that the patient also had Klebsiella or maybe a Staphylococcus aureus at the level of the colonization, but when you have a product which is mono-specific, when it comes to the time to deliver treatment, you cannot include that patientbecause it's not a mono-infection. So be very, very careful if you do start clinical studies with phage with monovalent product to really check the value of the epidemiological data, so which ended up for us adding only one patient included with the product against E. coli, which is called PP0121, and we decided last January to stop the arms of the study with that product. So all the data we are generating now and that we are analyzing today at this moment are really for the patients that have been treated with a cocktail against Pseudomonas aeruginosa only.
july10-299	DR. GABARD: Of course, one other thing you need to understand is that it's the same case, by the way, for the compassionate use treatments with the type of patients we have been handling. We are talking about people that are severely burned. Some of them were burned up to 90 percent of their skin surface. In the red, it was probably in the range of 20 to 30 percent deep burn, infected deep burns. It's impossible to avoid using antibiotics. It's simply impossible. The ethic committees would not agree anything about that.
july10-300	DR. GABARD: So you really have to think very early in your clinical process in your stratification that you're going to have to analyze against antibiotic, and not only against the antibiotic that might beprescribed before you even include the patient because this is the case. The guy who was going to be treated may already be under antibiotics because of a respiratory tract infection.
july10-301	DR. GABARD: You have also to stratify on the fact that these patients may get this respiratory tract infections when he is being treated by the local treatment, during the course of the treatment, and that makes your stratification even more complex.
july10-302	DR. GABARD: Okay. Of course, the severity of the patients of the cases. When we started the trial, we didn't get too much in consideration and we had data safety monitoring board, an independent data safety monitoring board which was reviewing the ethical treatment of the patients during all the clinical trial. And after they met the first time, they said that we should really check the severity of the patient before deciding to recruit or not -- include this patient into the trial, and we decided to implement something which is called the SOFA test, which is some kind of a monitoring process that checks how bad the patient is, in which situation it is, and if he is really in a very bad situation, then the rule was not to include the patient because the chances that the patient die anyway are so high that you wouldnot be able to generate any data. And then, on top of that, when we reviewed all the literature about the primary end points that have been tested for checking antibiotics, I'm not sure that primary end points that have been tailored for antibiotic checkings are exactly the same for phages because these primary end points have been set up for fixed molecules, and here we are talking about living organism, so that's also something we'll discuss when we show the data.
july10-303	DR. GABARD: Okay. So here I'm going to switch now to the two patient cases we got early this year. First of all, maybe a few things about the regulatory status in France. With the Phagoburn studies, there was a special committee who met last year organized by the French regulatory agency. It's called a CSST, and this special committee agreed that the phages from our Phagoburn study can be provided to the patients for treating them.
july10-304	DR. GABARD: So there is a possibility in France to now treat patients with GMP-produced phages from our company, and these treatments are only reserved to patients that are either critically ill, they may die from the infections, or that patients that have a functional risk of losing let's say one hand, onefoot, something like that. So really serious cases. Unfortunately, we didn't have any more stocks of these products in the company and we ended up having the request from the hospital. I mean, we have requests all the time, but serious requests from the Center of Reference on Staphylococcus aureus infections in France, which is based in Lyon. And they asked us very recently, in February, if we could provide bacteriophages to treat one of their patients. And in that case, we didn't have any GMP product left.
july10-305	DR. GABARD: So we talked with the agency and we said we can provide GMP-like products, and when I say 'GMP-like,' I mean they are really produced exactly the same way. There is not all the paperwork for the GMP, but all the quality control tests are exactly the same. And we said we can provide this. Is that acceptable to the French agency?
july10-306	DR. GABARD: And the French agency said, I think this is not our responsibility because we are not anymore in the GMP stages. This is the responsibility of the pharmacist and the clinicians to agree or not, and especially the pharmacists to agree or not about the quality of the products you can provide. And the pharmacists providing the data we provided said that it was fine, that we could do the treatment, but Imust insist that these products were not GMP. They were GMP-like, and the process is really for us now that we have been experimenting that a couple of times the clinician makes a request that goes to the regulatory agency. It takes about a few hours.
july10-307	DR. GABARD: Their agency asks us if we want to do the treatment. If we say yes, we just ask in a rush process to receive the strain, which is about half a day to get it in the lab. We do preliminary screening. We check what phages are available in our collection that are active against the strain, and then we send back those bacteriophages, which basically it take about 48 hours, and then we have to provide all the data, quality data that we have already for the phage in collections, and then we can provide the phages. Let's say in less than a week the treatment can start.
july10-308	DR. GABARD: Here are the data. So this is the first patient which was the one probably to help us to set up the process with the hospital and the agency. In that phage, we got no more phage of the GMP produced through the Phagoburn project, so we used some phages that we have against respiratory tract infections, Pseudomonas aeruginosa respiratory tract infections in the normal phage project, and you see the efficacy ofthe phages here, without treatment here, and the four phages here, or three phages, and this is the product, and we send the product not prepared in a cocktail but independently.
july10-309	DR. GABARD: Then we check for the titer, which was in that old fixed -- they were higher in titer, but we put the titer exactly the same for each phage, so we dropped it from 10 to the 12th to 10 to the 10. Of course, for these phages, because they were in collection, they were fully sequenced, fully analyzed, the genome was fully characterized, and we know they were confirmed by sequence analysis without any lysogenic behavior. They were checked for sterility, pH, and contaminant, and as I said, contaminant endotoxin content was about two units of enzyme per whatever you need. The host cell DNA was undetectable and the host cell proteins were below 20 microgram per milliliter.
july10-310	DR. GABARD: And this is the case that I'm talking about. This is a man who got cancer and had to have cement put into -- to replace -- how do you say that in English? Metastasize, is that correct? Yeah. Bone metastasize, it was removed, replaced by a cement, and the cement ended up bringing Pseudomonas aeruginosa infection, which was total resistant except a littlebit to colistin. It was still a little bit sensitive to colistin.
july10-311	DR. GABARD: So we got the approval for sending the product, I think it was on a Tuesday. They did the treatment on a Wednesday, and we decided to go for four applications because, in that case, the wound was still accessible during several days and we decided to go for four treatments of four phages each time.
july10-312	DR. GABARD: So they applied the treatment on the Friday. They did the first wound sampling because we have been doing monitoring in the wounds to see if we were getting any resistant. So they did the sampling after the first application. It was a Monday. We got the data on the Tuesday, and the Tuesday itself after the first application the wound was sterile.
july10-313	DR. GABARD: So we still maintained the three other treatments and the patient was cured and saved, except that a few weeks later, a few months later he died from his cancer because he has a general cancer. So that was a success, but, I mean, you save the -- the guy die from not being infected anymore, which is a partial success I should say.
july10-314	DR. GABARD: So this is the type of things that we have been doing. I will not explain too much. The debridement, the administration of the phage. I thinkit was about 20 milliliters in the wound, and then you see what's happening after that.
july10-315	DR. GABARD: And this one has not been described yet because it's more recent. In that case, it was interesting because it was contamination where we had to prepare two mix of phages against two bacterial species, so it was not a mono-specific product, it was a product against two species, Pseudomonas aeruginosa and Staphylococcus aureus, and you see this lady had a serious infection where you can see what's happening, and here in the infection we were detecting Pseudomonas aeruginosa and Staphylococcus aureus just a few days before the administration of the product.
july10-316	DR. GABARD: So this is what the patient has been receiving. It was three phages against each bacterial strain and they were mixed just before the use at the hospital facility by the pharmacist under a laminar sterile hood.
july10-317	DR. GABARD: Well, the conclusion of all that is that today she has been treated for about three months now, and she is fine. We still have recently got the information that she has a Staphylococcus lugdunensis available, so we are going to check if the phages we have is efficient against that bacteria, but she's in good shape.Okay. Maybe to expand on the discussion this afternoon, here I have been choosing a process that we have been through the company from standard fixed cocktail to precise precision medicine to just a little bit challenge the regulatory environment.
july10-318	DR. GABARD: If we talk about the complex cocktail or cocktail which is described as an antibiotic, the regulatory frame is available and is ready for you to go through a standard process of market authorization.
july10-319	DR. GABARD: But if we go to tailored preparation, you have seen that this type of magisterial preparation does not go, at least in Europe, through a market authorization or a registration process. It's an individual treatment for an individual person.
july10-320	DR. GABARD: If we end up going for these types of personalized treatments, there is no real framework for approving that type of treatment. So I have been putting in that arrow the personalized drug product with viable evolutive phages that should be GMP produced under which type of registration, and I have been showing some examples that we are going to face in the future.
july10-321	DR. GABARD: If you take the, for instance, the target bacteria A, which could be E. coli, you have a bank of bacteria phages, and the patients, you do a diagnosticbecause we believe too that the preliminary diagnostic is essential and it's going to be something that is going to be requested anyway by all the antibiotic-resistant plan that all requests, without any exception, to do a preliminary diagnostic before applying the antibiotic. So it's going to be the same for the phages.
july10-322	DR. GABARD: So the patient might have one bacterial species and you do a treatment with three bacteriophages, and another patient, which is more or less the case, the second case I was describing, has two -- here, I'm talking about three bacterial infections and you're providing phages against two or three bacterial infections.
july10-323	DR. GABARD: And the third case is that patient number three which has maybe one bacterial infection which is being treated by a first treatment, but that treatment is not efficient enough and you have to go back with a variant of the phage you have been using for preparing the first treatment, and what is the status of that variant?
july10-324	DR. GABARD: So I think the questions we need to really address today are more or less described into this slide. There is no process today, if we have let's say develop a data package, for getting theauthorization to treat the patient with a certain number of phages, and you generate new phages that could be either variant phages or that could be new-found phages into a sewage system but belonging to the same category, what kind of data package are we going to provide against these phages?
july10-325	DR. GABARD: So there is the authority to say that we could start from a homologous group saying that you have some kind of a group which is representative of the phage family and that that needs to be fully characterized, but the new phages that belong to that group can get a short data package without all the treatments, testing pre-clinical studies in animals and all these things, and be eligible to get into manufacturing, or the same for the -- now, if we talk about the bacteria, the manufacturing process, there are ideas to go for validation of a manufacturing process that would be eligible to any phage being produced, but would that be the case for a manufacturing process which is defined for one bacterial species, or would that be eligible to any type of bacterial species even if you talk about making a gram-negative or a gram-positive bacteria, because then the manufacturing process is not exactly the same.In one case, you're going to check for endotoxin content if it's a gram-negative bacteria. In the other case, you're going to check about hemolysins, for instance, which is not something that you're going to check eventually for endotoxin -- for a gram-negative bacteria.
july10-326	DR. GABARD: And then what type of quality control level do you want to get for the set of reference phages? Everybody agrees that it should be a full set of data that has been trained to summarize with identity, toxicity, pre-clinical data, PK, PD, efficacy, sterility, but the phage that gets into this category of homologous group, can we just -- is it sufficient to do identity and sterility?
july10-327	DR. GABARD: And I want also to bring some ideas of that. I'm not the father of these ideas. There is somebody in Belgium, Dr. Fauconnier, who has really some good ideas about the -- from the regulatory agencies in Belgium, that has pretty good ideas on how we can take bits and pieces from different regulatory process to build up a process for the phage therapy.
july10-328	DR. GABARD: For instance, we are talking about banking. When we prepare the banks of bacteria that are going to produce the phages or the banks of bacteriophages that are going to be administered to the patient,there is a process which is called the -- for the allergen extract prepared for a single individual where the source of material can be very diverse: pollen, molds, animal epidermals, insect, food, environmental, et cetera, and the extraction materials vary a lot according to the material you want to use.
july10-329	DR. GABARD: Well, there is a process here which is available today in our countries for approving such products. So maybe for doing the banking of the bacteria and the phages we could get inspired from that process.
july10-330	DR. GABARD: On the production process, you have something in the U.S., I believe, which is called the drug master file, which is something that is some kind of, if I am correct, some kind of a design, pre-design process of manufacturing which is not going to change and that you enter on one side your material and at the end you get your products out, and this is a fixed product. This is a fixed process.
july10-331	DR. GABARD: If you use always the same process, you can refer to that drug master file number and not have to explain each time how you are going to manufacture your product. That also is maybe a good idea for manufacturing of the phages.
july10-332	DR. GABARD: And now regarding the third point of phagetherapy is product evolution. Product evolution, as you know, is fully agreed when you make a vaccine. Well, there is a multi-strain dossier that we have in Europe where you can change the component of a vaccine very easily without years to wait, just to adapt the treatment to the evolution of the threat. Here, it could be the same thing.
july10-333	DR. GABARD: I mean, if you have an homologous group of bacteriophages and you want to change, make an evolution of one of that phage in that homologous group, maybe you have a process that we can copy to just adapt our regulatory process to a quick evolution with only a limited number of tests for getting approval of that modified phage.
july10-334	DR. GABARD: So this is it. Thank you.
july10-335	DR. GABARD: (Applause.)
july10-336	DR. KINCAID: I think at this point it would probably be a good idea to have all of our speakers come up, and I'd also like to invite Dr. Doran Fink from FDA and Dr. Betty Kutter so that we can first field some questions because I realize there hasn't been an opportunity for all of you who might have questions, but we also have some topics that might be stimulating in terms of their potential consequence to development of phage therapy in the future.So, if I could have the speakers come up here, please.
july10-337	DR. KINCAID: (Pause.)
july10-338	DR. KINCAID: So, before we begin, I'd just like to invite anyone who has questions, who may have questions in particular for the last three speakers, to use this as an opportunity to ask those, and then we will move on to some of the topics that have been selected for this. Dr. Stibitz.
july10-339	DR. STIBITZ: Yes. I just wanted to ask about something in Ry's talk. You stated, I think, in one of your last slides that you think there is value in characterizing base modifications for phage. Could you elaborate a little bit on what you think the value of that is for phage that we want to vet prior to using for therapy?
july10-340	DR. YOUNG: Hello, can you hear me? There we go. Is it working? I can't tell from that.
july10-341	DR. YOUNG: So DNA modification is a major way in which phages can become insensitive to or can overcome host defenses beyond the resistance, classical resistance. So many virulent phages that especially have unusual DNA, some of them have, for example, no thiamine, only uracil as their DNA base.
july10-342	DR. YOUNG: But the methods for, high-tech new methodsfor looking at protein and nucleic acids don't really work very well, the ones we have for assessing the modifications in these phages, but I think there are approaches that can be developed that are more based on classical nucleic acid chemistry that could be very informative.
july10-343	DR. YOUNG: If we had a way of rapidly checking a new promising phage for its DNA content and how much of it is modified and how much of it is normal, I think you could then eventually index that against many species.
july10-344	DR. STIBITZ: So I'm just wondering to what degree. I mean, if it's being used as a -- by the phage as a resistance mechanism to host defenses, wouldn't that be captured just in the normal screening for in vitro activity?
july10-345	DR. YOUNG: Well, yeah, but you wouldn't know what was causing it, right?
july10-346	DR. STIBITZ: Sure.
july10-347	DR. YOUNG: And so you could have a phage, you could have one gene change and then you would have a gain or loss of the ability to survive in that organism. I mean, I think having the -- sort of the classic way of just checking the pattern of resistance is certainly the thing you want to do, but we have the ability and I think the incentive to go beyond that tothe molecular level. If we had more and more data, even if it wasn't absolutely required for --
july10-348	DR. STIBITZ: Right.
july10-349	DR. YOUNG: -- the emergency application, we would be able to look back and start cross-indexing these molecular features with efficacy and with redundancy.
july10-350	DR. KUTTER: Well, maybe I'm the person also to say something about that since I've been working since 1963 on the question of the role of hydroxymethylcytosine in T4 phage, and one thing that came out this past year emphasizes something that may be relevant in terms of thinking particularly about phages to be used in the gastrointestinal tract, and that was something that Sankar Adhya and a student from Florida had done, finding something called super spreader phages.
july10-351	DR. KUTTER: They found them when they isolated them from nature, that there were a couple of phages that tended to under rather -- under conditions that really looked for them -- to be able to spread plasmids for antibiotic resistance to all sorts of different kinds of bacteria, not just ones where it could be through phages carrying those.
july10-352	DR. KUTTER: And the way they figured out an idea of whatwas going on is they went back and used phage that we had made about 40 years ago that were T4 that are able to make phage that are purely cytosine in their DNA, and they are missing a variety of different genes, including the genes to make the hydroxymethylcytosine but also the genes to shut off transcription of host DNA and the genes to degrade the host DNA that are cytosine-specific. And they found when they used that strain that was missing all of those, suddenly they could generate something that was not a full super spreader thing but that the T4 by itself showed none of that property, and they had three orders of magnitude more spreading when they were using those.
july10-353	DR. KUTTER: Now what hasn't been looked at at very many phage at all is the degree to which they degrade host DNA, and often you don't even know whether they have the nucleases to do it. There are other things that need to be sorted out more, like the ability to infect stationary phase cells and things like that.
july10-354	DR. KUTTER: So what's really needed, I think, is for NIH and USDA and so forth to fund a lot more of these really basic kinds of things, and what we have now is a few undergrads are working in my lab to try to look at some of the other standard phages and to see whether they can get the super spreader phenotype anddoing something like phage hunters, and getting undergraduate schools all over the country to be looking at some of these properties.
july10-355	DR. KUTTER: I teach at a -- for those of you who
july10-356	DR. KUTTER: don't -- I'm Betty Kutter, by the way, and for those of you who don't know, I've been teaching for very long at a school where almost all of my work is done by undergraduates since 1972, and I'd like all of you to invite you to our Evergreen international phage meeting, our 22nd one of which will be, biennial meeting, will be in August, but bringing people from a lot of different backgrounds and really getting more young people involved in asking a lot of these questions that will never be done, I think, if we only have the major labs to follow them up.
july10-357	DR. KUTTER: Thank you for the opportunity to make an ad.
july10-358	DR. KUTTER: (Laughter.)
july10-359	DR. KINCAID: Please.
july10-360	MR. McCLAIN: Yeah, Bruce McClain, United States Army. You know, most of the applications that we've heard today were irrigations of a wound or irrigations of an infected body surface. I mean, I think there was only the single intravenous administration. And I know that in your manufacturing you're concentrating on endotoxin levels and stuff,and yet these wounds are swimming with endotoxin. It may be that the endotoxin concentrations from a manufacturing standpoint is a minor component and you may want to propose to your regulatory, you know, colleagues that the endotoxin concentration may be really irrelevant for that type of therapy.
july10-361	DR. GABARD: Yeah, we would have loved to be able to do that. Unfortunately, you have something called the pharmacopeia, and the pharmacopeia has some standards regarding endotoxin content and there is not so many standards, but there is at least one of them which is giving figures about the amount of endotoxins you may have when you do an IV administration, and then there are a case -- although the treatment was topical, the agencies considered, because these were seriously burned patients, that they asked us simply could the bacteria become septic. I mean, could it get into the bloodstream? And we said yes.
july10-362	DR. GABARD: So they asked, and what about the bacteriophages? Can they go in the blood as well? And we said yes, they are going to follow the bacteria. So they said then the standard for your product needs to be for IV administration, and the endotoxin content must be about that level.
july10-363	DR. SCHOOLEY: This even came up in thepatient that I discussed. As I mentioned, we had this very nice improvement on Saturday night. By Sunday morning, he was looking as if he was headed in the wrong direction again.
july10-364	DR. SCHOOLEY: I called one of my colleagues from University of Colorado, Charles Dinarello, who has done a bit of work in this area, because I was concerned that this was endotoxin-related and was trying to talk to him about some ways to try to block this if this was what we had done by escalating the dose of phage, which I didn't get into today. His comment was, you know, with endotoxin there is tachyphylaxis anyway. Why are you worried about this?
july10-365	DR. SCHOOLEY: So, you know, again, I think it's something to consider, but I also think it's technically feasible to scrub it anyway, so why not. You know, in the context of most situations, now that there are several ways to purify the phage, I don't see any real reason not to unless you're trying to do it kind of on the end of a hood in the back of your car.
july10-366	DR. FINK: So, from a regulatory perspective, I agree with Skip's point entirely. From a safety perspective, what we worry about is the product characteristics and the intended use, and if someone comes to us with a well thought out scientificrationale for why worrying about a particular impurity is not important and why trying to get rid of that impurity would be overly burdensome, then we would certainly take that argument into consideration. But I haven't heard such an argument yet for endotoxin.
july10-367	DR. KINCAID: Next question, please.
july10-368	MR. TURNER: Hi. I'm Paul Turner from Yale University. I had a question for JÃ©rÃ´me about -- and maybe Ry or others want to chime in for this. You mentioned the challenge of the evolution of the phage, but what about the more proximate issue of the competition among phages in a cocktail, how much of that have you studied and, you know, there's a possibility that it could actually negate each other's success during the treatment because they'll compete?
july10-369	DR. GABARD: Very good question. We have not been doing that with the phages of the Phagoburn study, but we have been doing some other studies with some other phages from the other projects, and we have seen -- it's very preliminary, but I think there is some good work done in California. We have seen that if you have -- how can I put it? If you want to use four phages to fight a bacteria infection, and only one is active, and you put the three others, you may lose some activity, clearly. So it's better to usephages that are only active against your strain and to limit the number.
july10-370	MR. TURNER: Yeah, we were fortunate. We could only go with -- that we could go with only one phage in the case that Deepak talked about. But, okay, that's good. I think it's an interesting problem that needs follow-up. Thanks.
july10-371	DR. KUTTER: We've done some looking at various individual phages, like three different kinds of Pseudomonas phages or T4 with several other kinds of phages, and you certainly find some cases where you wind up with a complete blocking of production over the short term of at least one of them.
july10-372	DR. KUTTER: Now, with the T4, for example, even though it would block all of the T5 and some of the other kinds of phages when they were simultaneously there, if you had a low enough MOI that there were a few percent of the cells that were only infected with one of them, then 24 hours later the T5-like phage was doing better because what happens is that when you're affecting T4 at high multiplicity, it has lysis inhibition and instead of lysing at 30 minutes it lyses at six or seven hours, and that allows the other phages to catch up.
july10-373	DR. KUTTER: So we found that there really wereadvantages, but there are a lot of reasons why, for example, in Georgia with the cocktails, and they say to infect with a relatively low multiplicity so that you're looking at them having to expand and having to grow, and we had the same kinds of results that we saw with some more work with using phage and treating sheep, that, again, you found out that the optimal multiplicity was significantly lower than throwing lots and lots of phage at all the bacteria.
july10-374	DR. GÃ“RSKI: I mentioned in my talk that we made such a preliminary observation which may suggest that patients and cocktails may have higher phage antibody levels than those receiving single preparations. Regardless of the outcome of the story what is the role of peripheral-blocked anti-phage antibody in phage therapy outcome, this is kind of information which is interesting because, in our work, we have found also that phages differ in their immunogenicity. It may well be that some phages that are present in a phage cocktail may act as adjuvants. This is something we need to consider in the future.
july10-375	AUDIENCE MEMBER: I have a question for Dr. GÃ³rski and the French company about propagation of phages once you -- larger propagation in terms of actually using it in the clinic.Do you find it more useful to transfer the phage to a different bacterial host, or do you tend to keep it in the original strain that you fished it out with? And I'm just curious if there's any usefulness in transferring it somewhere else for either higher phage production or something like that.
july10-376	DR. GABARD: Well, the selection of the bacteria for production is important, clearly. Usually we tend to try to find phages that are being produced into a single bacterial strain just for manufacturing cost reasons. We couldn't do that for the first E. coli product for the Phagoburn study where we had to use, if my memory is right, seven bacterial strains for manufacturing, which was very expensive because then you have seven working -- well, a master and working banks.
july10-377	DR. GABARD: So, in the solution process, when you have the choice, it's always better to go for one strain, and sometime the surprises of this manufacturing strain cannot be used as the titration strain, so you have to go from the one manufacturing strain, and you may have to go for a different strain for titrating your phage during the manufacturing process.
july10-378	DR. GÃ“RSKI: Well, I think we have the similar policy. I also mentioned preliminary datawhich, again, are very, very preliminary but interesting that when you propagate phages on a strain that is freed of plasmid and prophage you may get increased titer and broader host range. This is something good. It's very promising but again requires further study.
july10-379	DR. BISWAS: Hi. My name is Biswajit Biswas. I am from BRD Navy. So I have a question for Dr. JÃ©rÃ´me Gabard. This is a technical question.
july10-380	DR. BISWAS: I saw in one of your slides that you are monitoring phage bacterial interaction by lysis method and you are monitoring it through the spectrophotometer reading. My question is when phage lyse the bacteria it produce debris also. So how relevant is this one for your monitoring system for phage efficacy?
july10-381	DR. GABARD: I cannot answer that. I'm sorry. It's not my expertise. It's too technical. Send me the question and I will ask to our team because it's beyond my knowledge.
july10-382	DR. BISWAS: Thank you.
july10-383	DR. KUTTER: Actually, when you do lyse phages, lyse bacteria with phages, we often monitor it by OD because the OD goes way down at least for E. coli and Pseudomonas and Staph. At the time when yourburst of phage is complete, the OD almost totally vanishes, so it has to do with the way the bacteria interact with the light and the concentrations in the bacteria rather than just the three.
july10-384	DR. BISWAS: Yeah, I understood your point, but we see in many bacteria and many phage, we have lot of clinical isolate. We see those clinical isolate when we lyse, not all the time they go through the complete lysis, sometimes they lyse but produce the debris which is targeted, and that is the problem because when you compare one phage to other and the phage lyse differently in the same bacteria, that is a problem. So that is my point. Thanks.
july10-385	DR. KUTTER: Yeah, it doesn't lyse it totally but like eight-fold or something like that usually with the standard ones, but that's a good point, yeah.
july10-386	LT REGEIMBAL: Good morning. My name is Lt. Jimmy Regeimbal. I'm from -- actually, I'm from NAMRU-6 now in Peru, but I was previously at NMRC here. And my question actually is much more general to actually the entire panel. Is it possible to take a step back and to actually not think about the product being tested as an individual cocktail, but instead your product is a library of phages from whichyou have differentially compounded cocktails that are personalized or individualized as long as that main library has been characterized and deemed safe, and whatever that means, and whatever you think you need to find a safe library?
july10-387	LT REGEIMBAL: And so you're really personalizing everything because I found it interesting, Dr. Gabard, that you started with fixed cocktails. My guess would be that if you have good coverage in those Phagoburn trials it'll probably work fairly well, and if it doesn't have any coverage, you're probably not going to see much efficacy.
july10-388	LT REGEIMBAL: And so what if the whole point is to take a step back and go this isn't our product? Our product is a library, and maybe 20 years from now, like Dr. Young was saying, that you might find that every time you compound a cocktail against baumannii you find the three -- the same three phages are in it.
july10-389	LT REGEIMBAL: And so it's like okay, then we'll just start with those three. But to say we understand that now might be very premature. And so is there a framework from which you can say our product in our clinical trials need to test a library, not a cocktail in any stretch of the word, and so what you're really doing is much more -- like we're starting a new way ofregulating phages, not like antibiotics or drugs or anything like it, but it's completely new. Is that even possible?
july10-390	DR. KUTTER: That's exactly what GÃ³rski does in Poland.
july10-391	LT REGEIMBAL: That's what I understand, but I guess the point is, is that -- what's the point? You have to understand that the problem with specificity, the problems of resistance like, for example, rather than going and make a new variant to phage the rule has provided 10 to the 31st. Like why don't you just go find another one?
july10-392	LT REGEIMBAL: And so instead, if you have an iterative library that's constantly being updated over time like a flu shot or something else, you won't need to constantly -- but it will also change your CMC, it will change the characterizations that are required if it's in the same field, you know what I mean, so, just generally speaking, is that possible to do in the West or in the U.S.?
july10-393	DR. FINK: Yeah. So, you know, what you describe is certainly different than the way that antibiotics have been regulated and licensed by FDA to date, but it isn't necessarily new. It doesn't necessarily require a new regulatory framework. Youknow, the key question, and I'm going to talk about this a little bit more in my presentation this afternoon, is that if you have a large library of phages, you know, what are the data that you need to ensure that any phage that you pick out of that library is going to be both safe and effective for the intended use? And there may be some, you know, data that you can derive from a subset of phages in that library that will allow you to make that type of determination, but, you know, we're not there yet, and that's, you know, that's where the field, you know, really needs to get together and do some thinking.
july10-394	DR. GABARD: And then, in addition to that, what kind of data package do you provide? If I go back to Phagoburn, we were lucky enough to initiate our clinical trials with cocktails. I think we had 16 phages in the first E. coli cocktail, so all of them characterized, sequenced and data package, at least technical package already available for these collections.
july10-395	DR. GABARD: So, at the time being when we knew that we didn't think about this bank issue, and now we have some -- you know, when we have banks, small banks of bacteriophages. But if tomorrow you want to do that, a recommendation of let's say 50 phages, you're goingto do 50 phages in a cocktail and to do that in two pre-clinical testing in animal models. So that's where the threshold is. Can we just define a group in which when you do all these necessary data for toxicity safety, pharmacokinetics and so on, and how do we define that group, and how do we expand that group with phages that belong to the same group with a limited number of data?
july10-396	DR. YOUNG: So that ultimately, if we do genomics correctly in a large enough set, we should be able to do it essentially by genomic analysis period, which is becoming ridiculously cheap, but we have to collect the data now for over a very large number of applications so we can start making those correlations. That's my feeling. Everything eventually is determined by the genome.
july10-397	MR. CHEN: Yeah, my name is Rong Chen from Phagelux. I have a question to Dr. JÃ©rÃ´me Gabard actually similar to the previous question, but it's a more practical, real. You select a cocktail which is fixed number of phages, and they only target a certain strain of the bacteria. Now, when you do the clinical trials, multi-site clinical trials, especially multi-country, you will actually face the problem, likely you can have a different strain of the bacterialinfection. So, therefore, when the trial -- when you have such a situation, I'm wondering in the Phagoburn study how did you manage such an issue?
july10-398	DR. GABARD: At the beginning, we decided to collect strains from all around Europe and USA so that we had some kind of a pretty big collection that could represent the genetic diversity of the bacterial species, but I think it's important for the next studies that may be run and conducted that a preliminary diagnostic is done before planning the treatment, and in our case, it was impossible to do it, but I think a diagnostic before preliminary treatment is a good idea so that you make sure that you recruit a patient that is really sensitive to your treatment.
july10-399	MR. CHEN: And that's what you did in the study?
july10-400	DR. GABARD: We didn't do that in the study for Phagoburn. For Phagoburn, we tried to make a wide spectrum cocktail based on selecting phages against a wide collection of bacteria from the same species.
july10-401	AUDIENCE MEMBER: Could I ask about that? Did you do retrospective when the Phago -- if something didn't go right? Did they check to see whether the cocktail worked against the isolatedbacteria?
july10-402	DR. GABARD: I think for phage therapy, it's important to remember you have a low number of recruitment of patients. If your number is already low and you don't check at the beginning of the strain the sensitivity to the treatment, then you end up with potentially reducing the number of efficient patients.
july10-403	AUDIENCE MEMBER: But did you check?
july10-404	DR. GABARD: No, we didn't.
july10-405	AUDIENCE MEMBER: I mean afterwards.
july10-406	DR. GABARD: Afterward, of course, we did.
july10-407	AUDIENCE MEMBER: And so you could correlate failures with absence of --
july10-408	DR. GABARD: This is going to come in the paper.
july10-409	AUDIENCE MEMBER: Yes, nice try.
july10-410	DR. GABARD: Good try.
july10-411	AUDIENCE MEMBER: Yeah.
july10-412	AUDIENCE MEMBER: (Laughter.)
july10-413	DR. KINCAID: Please.
july10-414	AUDIENCE MEMBER: So I'm going to be a little bit of a heretic, I guess. I've seen a lot of examples of compassionate use for phage. Makes sense. I've heard about the banks being formed. The question I want to ask and it may be both transnational. Isthis going to be more than an academic national effort to develop phage therapy? In other words, what are the economic incentives for industrial development? Part of the issue with antibiotics has been, of course, in the early days, a lot of big companies were involved in antibiotic development, but as that become less lucrative they all dropped out. Many of them have dropped out. Any new antibiotics are basically reserved for, you know, third-line use when it's absolutely necessary to use it.
july10-415	AUDIENCE MEMBER: So what's the economic incentives for developing phage therapy at an industrial scale for, you know, the vast population as opposed to compassionate use?
july10-416	DR. KUTTER: I can answer. One piece of that, when we first got involved in this back in 1997, two people came from Tbilisi and brought their phages, and we got a bunch of bacteria from cystic fibrosis patients from Children's Hospital in Seattle, and theirs had been used in wound care, and what we found was that all but one of those was very effectively hit by the group of phages in both Pyophage and Intestiphage, and the one that wasn't hit later on when we did the genomic analysis of the 16S RNA turned out not to be aeruginosa even though it had beendiagnosed as such. That's not true with all kinds of bacteria. There certainly are some, but that's something that companies need to think about as they're developing it.
july10-417	DR. KUTTER: Similarly, against E. coli, a bunch of similar ones have been isolated against O157 from countries in every part of the world, from Iran to Korea to Australia to Evergreen, and some of those between Evergreen and Belgium were very similar. So it seems like most of the phages wind up going to a lot of different countries.
july10-418	DR. SCHOOLEY: I was just going to say I think, you know, we have to be a little careful about trying to get so general that you can't get to specifics about could it ever be used. I think there are some clinical indications that you could think about that might be first pegs in the board.
july10-419	DR. SCHOOLEY: For example, if you find that you can more reliably sterilize prosthetic joint infections by adding a phage to an antibiotic directed at an organism that doesn't require 16 phages to cover it, like Staph, you may be able to find a product there that has a much more traditional paradigm, development paradigm.
july10-420	DR. SCHOOLEY: As you begin to do that, then you can startfilling the blanks around that as people develop a bit more comfort with the general therapeutic approach and as more of the molecular data that Ry is talking about evolves and you can start thinking about how to extrapolate from that situation.
july10-421	DR. SCHOOLEY: So I think it would be a big mistake to shoot our feet off before we start trying to walk by saying it'll never be scalable and why would pharma ever do this. So I think it's great to raise it, but I hope nobody outside the room heard it.
july10-422	DR. SCHOOLEY: (Laughter.)
july10-423	DR. KINCAID: I'm going to take a small prerogative and just put a footnote on our first phage workshop that was held two years ago. We did receive interest from major providers of solution sets for surgical intervention. So it's one of those cases, as Chip just pointed out, where there are people always who are looking for an opportunity if they feel that it's going to make their products better or improve or, in a contrary sense, to reduce the liabilities associated with their products. They'll probably in a very measured way take whatever measures are necessary to consider phage as potential, you know, adjunctive elements to their products.
july10-424	DR. KINCAID: So I think I agree with Chip that we have towait and see how these things play out as more and more people become familiar with the nature of the potential for the product.
july10-425	MS. EMRICK: Good morning, afternoon. I am Robin Emrick, and just a member of the interested public. And listening to you guys this morning got me thinking about something and, actually, Dr. Schooley, you sort of hinted at it. I hear about the problems of the specificity and nailing it down, and I thought I wonder if somebody isn't already looking at and solved the idea of having a less acute circumstance where like, okay, you're going to have this kind of surgery in three weeks and we're going to start you on some kind of a, I'll say generalized phage therapy that's going to knock down the prevalence of resistance plasmids that may or may not be present in your system. Just kind of prime your body to already be a little more responsive to the antibiotics they already have.
july10-426	DR. KINCAID: So that turns out to be a rephrasing of one of the topics that we had talked about discussing here. We've had such a good response I didn't want to break that flow, but in a more general sense, it would be useful for the panel to weigh in on the sort of scope of phage use that couldbe considered in a preemptive way as a prophylaxis. Is this something that one might consider, whether it be surgical intervention or decolonization of at-risk patient populations? I mean, quite apart from the business model. What's the feeling from surgeons and others?
july10-427	DR. NARAYAN: So that's a thought that I brought up with Randy earlier on. The problem, though, is that if, as we pointed out earlier, you start administering these phages way ahead of surgery, then you can potentially build up other resistant organisms, as we've seen with antibiotics. It seems to make complete sense to sort of rid your body of all antibiotics before you proceed to interventional procedures which implant large volume of foreign substances, but it's never been shown to be effective since antibiotics are useful if you give them immediately before surgery and doses during surgery.
july10-428	DR. NARAYAN: And so the question is could we do that with phages as well, as opposed to doing it a priori and then building up resistant organisms?
july10-429	DR. SCHOOLEY: I'd like to invite Dr. Narayan to join our Department of Surgery because our surgeons often are not that concrete in their thinking because they put antibiotic beads in everything, andthe data supporting that is relatively modest. If you think about it, there are situations, though, where phage are not going to be -- as far as we can tell aren't a big problem. If you have a prosthesis you're putting in, you do get periodically Staph epi infections, for example, of hips. But the difficulty in clinical development is if you have a good surgeon and you have good antibiotic and you have good antiseptic conditions, the instance of that is low enough that showing that by sprinkling some phage in that you've decreased the instance of that complication is complicated, is very difficult.
july10-430	DR. SCHOOLEY: And so I think the clinical development paradigm is complicated even though theoretically it makes a lot of sense as long as you do it at the time and don't get way ahead of yourself and allow for second and third generation organisms to populate, which is what Darwin's all about.
july10-431	DR. KUTTER: I think one other thing that we've been thinking about, and we've done some work and published one paper on working with treating diabetic toe ulcers with phage, and we started by using just pure Staph phage. We did that even though we knew there probably are other bacteria besides the ones that are coming out and what they're saying, butthe podiatrists see Staph as the head of the snake in these kinds of particularly very poorly aerated toe situations and so forth, and what we found is even though we had Pyophage available with others if we needed it, the staph alone has been enough to treat all 11 patients we've tried before whose only other possibility was amputation, which normally then within five years even if it's just a toe to start with leads to death.
july10-432	DR. KUTTER: And so I'd really like to see some of those kinds of situations. I mean, I've seen Staph and diabetic foot being a logical target since I saw my first experiment in 1996. It wasn't an experiment, it was a treatment by the leading surgeon in Tbilisi, and what amazed me was not only that it worked to treat a foot that had come in for amputation, but he was 95 percent sure it would work.
july10-433	DR. KUTTER: In other words, you're talking there with a situation where it may be that Staph is indeed the head of the snake and simply making a bed that allows other bacteria to grow as well, but the wound's all healed. That's the final thing that we've used so far even though half of them have very obvious osteomyelitis. There's clearly bone infection and the Staph is getting into the bone.And I would really like to see, and I think several groups, both AmpliPhi and Pherecydes are talking about really going to that model, and what we're just starting to do now is to do metagenomics and look at the wounds before and after and see in that case what bacteria are really present and to what degree our model is true.
july10-434	DR. KUTTER: I think we need to choose some of those simple situations and make it possible without it costing a million dollars right away for trials to be run that are simply adding something to a standard treatment that's happening and not being an expensive clinical trial or even very expensive processes. He's just been doing it in his office, and it's simply something that we add without any extra expense. And I think we need a lot more of that kind of data and not just the data that's come from things that are what will be necessary for the companies wanting to make a lot of bucks about it.
july10-435	DR. KUTTER: I'd like to see a simple Staph phage thing be in effect like the -- like it's used with vaccines or even with aspirin where it's -- you know, what we use is about $5 worth of phage from Tbilisi to treat, and I'd like there to be situations where we can get a lot of this kind of data that's done very generallyand for relatively little money and be able to really build up an understanding better of what's going on.
july10-436	DR. NARAYAN: I'm going to circle back to the commercial question as well as reply to the previous question. So a good scenario, for instance, is ventral hernias are a very common surgical problem. You have a big operation. A significant number, especially in this day and age with obesity being so high, develop ventral hernias. And so you treat ventral hernias by putting in prosthetic mesh, and then we see this sort of cycle of when it gets infected you have this core population that cannot get rid of an infection.
july10-437	DR. NARAYAN: So, to Randy's point, that might be a situation where you can actually apply certain phages specific to the bug that you've sort of identified. In fact, there are matrices available now, rifampin-coated prosthetic meshes which sort of address the issue of MRSA, for instance, and that might actually be a commercially viable proposition given the increasing number of surgeries that you see, as well as addressing the issue of, you know, prophylactically giving phage even though in clean cases the incidence of infection is really low, say, maybe on the order of 1 to 2 percent. These cases represent a fairly largenumber that can actually be both commercially viable as well as treatable by phages specific to the particular bacteria.
july10-438	DR. FINK: One last point, and I see we have another question. So, just to get back to the issue of preventative use of phages or use for decolonization, there's no a priori reason why from a regulatory standpoint a phage therapy product couldn't be developed for preventative use, and, in fact, it's serendipitous that the regulatory review of bacteriophage products is housed in the Office of Vaccines at CBER, so we have a lot of experience regulating preventative products, and, of course, the devil is always in the details of clinical trial design and selection of end points.
july10-439	DR. KINCAID: Okay. We have time for one question according to my timer. So, Carl.
july10-440	DR. MERRIL: I'd like the panel, if they could, to amplify a little bit more about the economics. I want to just make a comment. I'm carrying this because, in 2003, I was invited to give the Harold Neu Infectious Disease Conference lecture on phage. This was when I had just done a study that we published in PNAS showing that phage could be highly efficacious and we could even make specialphage that were long-circulating, and so they invited me to give this lecture.
july10-441	DR. MERRIL: But, in fact, the people from the companies, this was Glaxo and some other companies, said exactly what the previous questioner had brought up, that there just wasn't money in infectious diseases and they were cutting back on their antibiotic production.
july10-442	DR. MERRIL: But the reason I'm bringing it up as a question now is that there are factors that are affecting the economics, and I wonder if you could comment on them. For instance, the fact that hospitals are now responsible for hospital-acquired infections, number one; and number two, the time spent in an ICU can be far greater than anything anybody spends on any of these therapies we're talking about.
july10-443	DR. MERRIL: I'm sure with Dr. Patterson his ICU time was immense.
july10-444	DR. SCHOOLEY: He was lucky he was sick during a period when there was no cap on lifetime costs from insurance companies.
july10-445	DR. SCHOOLEY: There are all kinds of costs that can be calculated into how this all comes back to us as a society. The problem is that they're all in different buckets, and that is where, you know, we have to try to figure out how to rationalize it so that we as a society can realize that the investment's worth it,but the individual funders themselves don't see it in it for them.
july10-446	DR. SCHOOLEY: Companies want something that you give people for life, so they don't like antibiotics that work. They like antiretrovirals because you have to give them for life, but they don't like anti-HCV drugs, for example. Phage that work and sterilize, they're not going to like any more than they like these fourth generation antibiotics that they give to six people twice a year who have bacteria you can't treat with anybody else.
july10-447	DR. SCHOOLEY: As an infectious disease physician, I'm always arguing with the hospital that the reason we're there is to reduce antibiotic use so you have fewer people in the ICU with multidrug-resistant antibiotics, and therefore I need to find a way to pay the faculty in my division, and the department in the hospital always says, well, just bill the patients. We're the ones who collect the money for the hospital. Don't you worry about that.
july10-448	DR. SCHOOLEY: So you end up -- I think it's really a multiple bucket issue more than it is an issue of is it worth it as a society to do this. I think it is worth it as a society, but I think we need to be more creative in terms of how we cost account it. That'sthe extent of my physician/country doctor statement. DR. GABARD: I'm going to take on that physician/country doctor statement role for a second. So, if you look at it from a strictly commercial standpoint, all you have to do is look around. The Epi-Pen cost has gone up 400 percent. Martin Shkreli is on trial now for increasing costs for generics over 7,000 percent. So there may be something in the market forces itself drives the economics of potentially using this in a commercially viable fashion, and so that's never been addressed and I hope it never gets out of this room that, you know, phages can be sort of overpriced, if you will, to make economic sense for the companies.
july10-449	DR. GÃ“RSKI: One thing that surprises me always when we have this type of discussion is that, let's say someone gets cancer, you're going to increase his life by 12 months, and you're going to spend 20,000 bucks while doing that in treatment. So maybe he's going to lose his leg and you're going to spend 5,000 euros or dollars to preventative treatment, and this is too expensive? There is a problem.
july10-450	DR. KINCAID: Well, I wanted to take this moment, first of all, it's an interesting note to endon, so philosophical but also so real, and this is a classical concern for infectious disease generally speaking, not just phage.
july10-451	DR. KINCAID: But anyway, I want to take a moment to thank all of our speakers and our panelists for a very stimulating morning session, and I'm not exactly sure when we return. Do you have the number? One-oh-five according to Roger. Okay. Thank you very much.
july10-452	DR. KINCAID: (Applause.)
july10-453	DR. KINCAID: (Whereupon, at 12:15 p.m., the workshop in the above-entitled matter recessed, to reconvene at 1:05 p.m. this same day, Monday, July 10, 2017.)
july10-454	DR. KINCAID: A F T E R N O O N S E S S I O N (1:10 p.m.)
july10-455	DR. CARLSON: The rest of the people who are still at lunch will filter in briefly in the next few minutes. So we're going to get started back up with Session 2, and you can see on the agenda that we're really talking about regulatory considerations for phage products in this session. So we're going to start with some phage characterization and CMC and then get some talks from FDA folks who are actively involved in regulating these things.
july10-456	DR. CARLSON: So, with that, I will start off by introducing Jason Gill from the Center for Phage Technology. He's going to talk to us about phage characterization.
july10-457	DR. GILL: So good afternoon. I'd like to thank the organizers for inviting me here to give this talk.
july10-458	DR. GILL: So I was asked to talk about phage characterization, which is a relatively large topic, so I'm obviously not going to be able to cover everything, all aspects of all things that you can characterize about phages. Some of the stuff I'm going to be talking about was touched on this morning when we were talking about the development of phagetherapeutic products. But really I want to kind of give an overview of where I think we are now and what I see as some obvious paths for the future, and I don't want -- we don't want to intend this to be like some kind of, you know, dictat that I'm giving to the audience about the things that everybody has to do, but these are things that we've been doing in our program and I think they've been helpful for us.
july10-459	DR. GILL: All right. So, when we talk about phage characterization, really, there's -- you know, we're doing this for a reason, right, so the reason is really you want to increase the efficacy, right, so you want to try to pick phages that hopefully have highest efficacy or at the very least get rid of phages that you think are not going to have any efficacy.
july10-460	DR. GILL: We want to increase safety, right. So we want to obviously eliminate phages that might have any deleterious features, at least ones that we can identify now with our understanding of phage biology, and also to increase efficiency, which is not something which is often talked about, but really this has to do with keeping yourself sane and trying to pick phages that actually are going to hopefully be better behaved in the lab and ones you can actuallydeal with, and to try to reduce your workload. So, whenever you want to try to characterize a phage, I think especially when we're talking about phage applications here for some kind of therapeutic, you really want to have a reason, right. So, for example, if you characterize a phage for thermal stability, you know, unless you're planning on putting the phage in a 70 degree environment, it doesn't really help you that much.
july10-461	DR. GILL: So, really, you should have an end point and you should have an actionable outcome for the characterization. Are you going to use this information to make a decision about using the phage or are you going to be able to rank the phages or are you going to be able to, you know, make some kind of rational combination of phages?
july10-462	DR. GILL: So host range obviously is probably the oldest character of a phage that people have looked at. It's still, I think, one of the most important characteristics. It really is kind of the minimum requirement for any kind of efficacy, right? I think the basic requirement for a phage you want to use for any kind of antibacterial is that it has to actually infect the strain that you're attempting to treat, right? That's kind of the basic.But the needs of host range will vary depending on the application you're looking for. So, for the more kind of company approach where you're trying to have like a mass produced and distributed product like, for example, Phagoburn, or the company approach, you know, you're really going to have a bias towards finding phages that have broader host range as much as possible. But if you're going for kind of the experimental, you know, personalized medicine eIND approach, having broad host range phages in a collection is convenient, but it's not necessarily required because if you have a phage that only infects that strain that you're trying to treat, but if it works well, that's really all you need.
july10-463	DR. GILL: But even then host range doesn't nearly give you all the information that you need, right. So you want to have all the phages that infect one target strain, and so this is going to work. Yes.
july10-464	DR. GILL: So, if you can imagine, here this is a KPC1. This is actually the NIH clinical center outbreak strain here. So we have a lot of phages that infect this strain. These are six phages that are isolated in our group over the last few years, and they all infect this strain. So, if this is the strain you want to treat, you have a lot of phage options. Andso these tells you these are phages you could use, but you may not necessarily want to use all six. That might be redundant. And you probably want to use more than one. And even if it does infect the strain, you don't have any guarantee it'll actually have any efficacy in vivo.
july10-465	DR. GILL: So another assay that you can -- factor that you can characterize is, you know, virulence, and so this is basically the ability of the phage to inhibit bacterial growth in liquid culture. This is probably one of the -- this is probably the second oldest character that's used for phage therapy. This is done in test tube, so we have very, of course, sophisticated ways of measuring it now. This is done in an automated plate reader where you can take a time point every 10 minutes. But really the result you're looking at is the integration of the absorption rate, latent period, and burst size of the phage altogether in this liquid culture.
july10-466	DR. GILL: So you can have this high throughput, but the principle is really the same as it was in the 1930s, but if you optimize the method a little bit, you can get some distinguishing between different phages. So here on the left these are all phages that infect -- these are KPC positive Klebsiellapneumoniae, a clinical strain. So, on the left, you have a high input MOI of phage to bacteria. All the phages look roughly the same, so you can see they're all what you would call virulent in liquid culture.
july10-467	DR. GILL: But if you, you know, tweak your inputs a little bit and you have less phage going in, you can see and you can start to separate these phages, and then this may give you some kind of indication of efficacy of these phages, and you can see this one phage down here, which is called 'Pharr,' suppresses growth better than the other phages, and so this might give you some indication that maybe this phage would be -- if you have to pick one of these phages that all infect the same strain, maybe Pharr would be one that you'd want to look at more closely as opposed to other phages.
july10-468	DR. GILL: So, when you're determining host range, I mean, the classic, the good-old spot assay, which I'm sure a lot of people here have done, it gives you a basic measure of phage sensitivity, but you can actually couple your virulence screening with your host strain, which is something we've started doing and Dr. Biswas has also started doing this as well in his nice -- the Omnilog System where you can kind of just do the virulence assays in a high throughput way and you can get anidea of the host strains and the virulence of the phage in one go.
july10-469	DR. GILL: So here are two different Salmonella strains with the same phage. You can see this strain here, 3003 is insensitive to this phage, right. You don't see any difference in growth when you add the phage, whereas here you can see an inflection of growth and so this tells you that this strain is sensitive to the phage, and also, if you have a number of phages that are sensitive, that give you some growth inflection, you get some idea about how sensitive they are or how active they are against that strain.
july10-470	DR. GILL: So that's kind of where we are now and that's really how we're selecting phages. You have to remember these are really criteria that were developed, you know, in the '30s, and what we do now is much more sophisticated ways of measuring it, but the principle is about the same, and so we think this is kind of the minimum for being able to deploy a phage, but we really should be able to try to get more information about the phages now, like the genome sequence and phenotypes of phage insensitivity and receptor use.
july10-471	DR. GILL: So one thing I didn't mention before is detection of temperate phages. So this is somethingthat the early phage therapists that were talked about this morning in the '30s didn't really have to deal with because they didn't know it existed yet.
july10-472	DR. GILL: One of the kind of classic things you see sometimes in the literature is that you can just look at a plaque morphology and turbid plaques mean temperate phages, and that is really not true. There are a lot of virulent phages that will make turbid plaques, and we have temperate phages that will make very nice and clear plaques.
july10-473	DR. GILL: So, in our experience, the virulence assays, like I showed you earlier, one way to -- the temperate phages will tend to fall out in those kinds of assays because you'll have really rapid growth of lysogens that will come up very quickly and they will look just bad in the virulence assay.
july10-474	DR. GILL: Another method that we use is to just isolate phage insensitive, you know, mutants, what we call air quote 'mutants' of a bacterial strain, and then we just look in the culture supernatant for the presence of that phage because, if it was actually insensitive to the phage because it formed a lysogen, most temperate phages will spontaneously induce from the lysogen stage at a low rate, and so just overnight culture supernatants will have phage that you can thenspot them back on the parent, and so if you have that phage there, that's a good indication that it actually formed a lysogen, and also you can use PCR to screen as well if you have the genome sequence.
july10-475	DR. GILL: So this is one way to look to see if you have a temperate phage or a couple ways you can use.
july10-476	DR. GILL: So I'll talk a little bit about receptor use. So, if you know what the phageâ€™s receptor is, that really can help predict the interaction and also maybe help you plan ahead to overcome bacterial resistance in the future. So our experience with the Patterson case, of course, is that we didn't have any of this information, so it's not essential for use, but it is nice to have that information, I think. So, if you have the opportunity to get that kind of information, I think it would be beneficial to get, and there's a couple ways you can get at this. You can get it on a purely phenotypic level, which is usually looking at cross-resistance, and you can get at it on the more genetic level.
july10-477	DR. GILL: So, if we look at just doing this straight up kind of classic cross-resistance, so these are -- this is a panel of phages against Salmonella anatum from my lab, and here we just want to look at what's boxed out here.So we have a bunch of phages here on the left, and we isolated phage-resistant mutants against all these various phages here, 6, 9, 12, 15, and 27A, and so you can see that, for example, the Mut15 phage is now insensitive to phage 15, as you'd expect. But we want to look here at phages 6 and 9, so if we have a mutant that's insensitive to phage 6, it's still sensitive to phage 9, and we have vice-versa. Mutant that's sensitive to phage 9 or that's become resistant to phage 9 is still sensitive to phage 6.
july10-478	DR. GILL: All right, because it's kind of reciprocal cross-resistance here, so when you look at these kinds of phenotypes, this can help you design a phage cocktail that'll help you maybe overcome phage resistance because we know that if the cell becomes resistant to one phage it will still be sensitive to the other one and vice-versa.
july10-479	DR. GILL: It's a pretty low-tech method, all right, so you're really just, you know, you're just plating stuff out on agar plates and picking surviving colonies. It's not super-complicated, and it doesn't really tell you what the receptor is, but it just tells you what the phenotypes are, and you can see this is borne out in these kinds of, you know, liquid virulence assays again. You can see phage 6 and phage9. If you expose them to the bacteria, you get resistance, survivors come up after about eight to 10 hours, will start growing up, but if you mix the two of them together, you suppress the arrival of that resistance, at least for the 12 hours that we ran the experiment.
july10-480	DR. GILL: You can see that if you were worried about phage resistance you can get around that. You can kind of get ahead of the game by rationally designing the phage cocktail if you know what the resistant phenotypes are.
july10-481	DR. GILL: Sometimes it's not super-cooperative, so this is a whole bunch of phages we isolated against KPC K. pneumoniae, and we found that pretty much all of the phages here -- so here we have the bacterial strain, here it is on the left this time. These are all resistant to each of these phages and the phages here are on the top.
july10-482	DR. GILL: So you can see of this whole phage collection here every strain that became resistant to one phage became resistant to every other phage in this panel, which is a little disappointing, but it means that all these phages are probably using the same receptor and one thing it does help us out with is it means that there's probably not much point inmixing a bunch of these phages together because, if the host becomes resistant to one phage, it can become resistant to all the other ones, and really that then guides us to actually, you know, doing a new phage hunt and taking one of these phage-resistant mutants, then finding new phages that will infect this strain from the environment or generating a mutant in the lab, and this is what we have done. We were able to go out into the environment using one of these hosts and we can find phages that infect these strains just fine to overcome that resistance.
july10-483	DR. GILL: So actually genetically determining the phage receptor is a little more arduous to do, but it can definitely be worthwhile. So phages can really -- they can recognize pretty much anything on the cell surface. That can be carbohydrates like a capsule or LPS. It can be an outer membrane protein or membrane protein or any kind of cell surface extension like a flagella or a pili.
july10-484	DR. GILL: So there's a few ways you can get at genetically what the actual receptor feature is. If you have existing knockout libraries, for example, of a convenient host that are already knocked out in all your known surface features, you can just spot the phages against those on a plate and you'll find itsresistant to some of them and you can get to the receptor that way if you happen to have that.
july10-485	DR. GILL: You can do Tn5 mutagenesis or isolate spontaneous knockouts or spontaneous phages with the mutants, then just re-sequence them to get to the nature of the phage receptor, right. It's a little more work to do, but if you actually know what actual surface feature of the phages you're going after, you can do some neat tricks, right.
july10-486	DR. GILL: So one is that if you know what the receptor is, you can maybe predict if the resistance is linked to other phenotypes, like reduced virulence or sensitivity to antibiotics. So this is a table from a paper from a few years ago, and these are phages against a Klebsiella pneumoniae strain, and you can see the phage-sensitive strain here. We're looking at the LD50, right, so this is how, basically how virulent that strain is.
july10-487	DR. GILL: There's a wild type strain here as 1.5 times 10 to the 8th, and when they made phage-resistant mutants against the phage here called phage NK5, you see that the LD50 went up by a lot, right. So, basically, this strain became 50 to 100 times less virulent once it became resistant to the phage, and they hypothesize that the receptor is probably thecapsule or the LPS, which are important for virulence in Klebsiella, but if you know what the receptor is, then you can actually maybe pick phages that are going to go after known virulence determinants on the cell surface.
july10-488	DR. GILL: And another, you know, great trick from -- this is much more recently, is looking at this phage which is actually specific for this outer membrane protein which is an efflux pump that caused antibiotic resistance. So, if you have a phage specific for that particular efflux pump, right, so when the -- and this is the strain here, and when it becomes resistant to the phage, you can see that it becomes sensitive to tetracycline, right. So, again, you have a huge fitness cost associated with the strain becoming resistant to the phage. And so, if you knew beforehand that that's what this phage does, then you can actually look at doing co-treatment with the phage and an antibiotic.
july10-489	DR. GILL: So we like to do phage genomics. That's one of our fun hobbies that we do at the center. There are about 2200 phages, phage genomes in INSDC right now, which is really actually a small fraction when you compare that to how many bacterial genomes there are in the database, so they're really prettyunderrepresented still. So right now you can have some predictive ability using phage genomics to pick out a phage that you might want to use. So you can increase efficacy and safety. So one thing you can -- by looking at the genome, it can also help tell you if the phages can be virulent or temperate, right. So we know enough about phage biology now that if you find a phage which looks exactly like T7, that's going to be a virulent phage, right. So we have some understanding on phage biology to that level.
july10-490	DR. GILL: You can look for toxins, virulence factors, at least ones that you might know of, and you can exclude phages that you might expect to perform poorly. For example, if you had a phage and it turned out to be an F-specific phage, it's very easy for the host to become resistant to those, and you might want to then put those farther down the list in terms of when you want to use for therapeutics, and it can also really increase your efficiency, and this is -- it's kind of a selfish reason for doing it, but still it really saves you a lot of work because if you have the genome and you can see that you have a bunch of phages that are almost exactly the same as each other, you may not want to necessarily continue developing all ofthem, right. You might just want to pick a few representatives and go with that to prevent, you know, duplicating work, and you can also make some predictions on how the phage is going to function based on the type.
july10-491	DR. GILL: So I want to show you a couple examples from our own work of really how helpful phage genomics is. This is a genome of a phage called BcepIL02. When we sequenced it, it really had no homologs in the database. It's kind of a novel type. It's 63 kb genome, circularly permuted. Here is the map. So what's important here to notice is that where this red arrow is pointing, it has tyrosine recombinase, and actually next to it it has cI and Cro-like transcription regulators, so it really looks like maybe a temperate phage.
july10-492	DR. GILL: But before we had done the genome we had actually used this in a mouse model of Burkholderia cenocepacia lung infection, and it was therapeutically effective, right. This phage gave us about a 100-fold, two log reduction in bacterial load in the mouse lung, and, you know, I thought it was a successful study. But then, once we sequenced the genome, we found it looked like a temperate phage.
july10-493	DR. GILL: In the past in the previous assay I told youabout where you take your phage-resistant mutants and you look to see if they're lysogens. So it turns out that this phage is what we call a cryptic-temperate phage, so it is a temperate phage, but it's unable to actually form a stable lysogen in the host that we were using in the mouse model, and the reasons for that we think is because the att sites that this phage recognizes was not -- it didn't happen to be present in that strain, so it wasn't able to stably integrate, but it was nonetheless a temperate phage.
july10-494	DR. GILL: So we broke like one of our rules right off the bat here and accidently used a temperate phage for therapeutic use and while it worked well in the mouse model we would really have to think long and hard about it if we actually wanted to try to deploy something like this in the world.
july10-495	DR. GILL: So another -- this is, of course, the clinical intervention that Chip talked about this morning. So we were involved in this in February and March and April of last year. So the CPT got the call in and we received the strain from UCSD, as Chip had told us about earlier, and we isolated de novo three new phages and we had a fourth called AC4 which was supplied by AmpliPhi, and the U.S. Navy isolated four phages, and this was turned around in about 15 daysfrom the time we received the strain, which was, I thought, pretty heroic. But the infecting strain became resistant to all the phages after eight days, and as Ry talked about earlier, this whole venture really kind of turned the whole lab upside down for two, three months.
july10-496	DR. GILL: I'm showing these two people here. This is Jacob and Adriana. These are the two people that actually did most of the work in the lab, and I want to show you this picture because they haven't slept in about three days when this picture was taken. This is actually the first shipment of phage that was ready to be shipped in the FedEx, like same-day delivery pick-up guy was about to come. And so it was a huge really heroic effort, right, to generate these phages.
july10-497	DR. GILL: And it turns out that after the dust had settled and then over the summer and the fall of last year we sequenced the genomes of all the phages that we had and it turns out they're all almost exactly the same, right. They're all T4-like large myophages that infect Acinetobacter baumannii. There are some differences. They are not exactly the same. There is some variability here, but I wouldn't say the diversity is very high, and this is one reason probably why the strain became resistant to all thephages at more or less the same time, was because all the phages were pretty close to each other.
july10-498	DR. GILL: So, if we had had this knowledge beforehand, this here is a map actually showing two of our phages. This is C2P24 and C1P12. You can see of the three phages we isolated they were almost 100 percent identical. They were very, very close to each other. Only a few SNPs different between them.
july10-499	DR. GILL: You can see here AC4 is somewhat different. This is showing this protein-protein similarity here. But if we had known this beforehand, we really could have saved ourselves, you know, a lot of stress and headache and heartache preparing four phages when we probably could have just prepared two or maybe even one, right. So that would have actually really streamlined the efforts a lot better and we could have had a much more efficient deployment of the treatment. We could have maybe anticipated that we would have this cross-resistant would come up if we had foreknowledge of what the receptors are, and so we're currently working on determining what the phage receptors are. I think Dr. Biswas is going to talk more about that this afternoon, so I'm going to leave it there.
july10-500	DR. GILL: And also the in vivo screening, right. So,this is something that we still do. I think screening phages in an animal model is still the standard way to de-risk new treatments, but really I'm thinking about trying to do this kind of in vivo work in a
july10-501	DR. GILL: different -- maybe with a different strategy, right. Most of the stuff that has been published to date is about seeing -- to show that the phages can work, right.
july10-502	DR. GILL: So it's like proof-of-concept type of thing, and you want to show the phages are able to control infections in vivo, but you could maybe start looking at trying to -- if you have a bunch of phages that you're pretty sure are going to work, then try to figure out which ones are going to work best, right. And so while you're doing this you also will be able to get data on dynamics and administration routes and also the phenotypes of phage-resistant bacteria and also maybe the immunogenicity of the phages as well.
july10-503	DR. GILL: So this won't be able to be done for all phages, right, so anyone who's ever done animal model development, it's pretty arduous. Not all the past strains you're going to be working with are going to probably work in the mouse model. So you're going to have to have a few kind of go-to strains and use phages that infect those strains to get some of thisinformation. So you won't be able to screen necessarily every phage, but you'll probably be able to screen at least kind of a subset of the phages you have or at least a representative of all the different types of phages that you have.
july10-504	DR. GILL: This is an example of some data from a paper from Debarbieux's group at Pasteur from a few years ago where they tested the same kind of idea. They tested a bunch of phages here. These phages up here that are color in the top are phages they isolated against this particular Pseudomonas aeruginosa challenge strain, and then down here they have other phages, and you can see some of these work better than others, right. So the ones that are up here in the colors work better. You had better survival of the mice following a bacterial challenge, and the gray ones down here worked worse.
july10-505	DR. GILL: And, you know, this also does kind of go back to they did those in vitro virulence assays and you can see the ones that did worse in the in vitro assays also did worse in this in vivo screen. So this can give you also some information about maybe what phages you want to use and not.
july10-506	DR. GILL: So PhiKZ is actually an old, well-known, very virulent in vitro phage of Pseudomonasaeruginosa. It's one of these giant phages, giant myophages. But if you had this kind of data, you might think, well, I'm not sure if I want to bother giving this to a person or to try to use this for treatment because it doesn't do very well in the mouse model, right. So it's again another way to try to increase your efficacy.
july10-507	DR. GILL: So the last thing I want to talk about is phage immunogenicity, right. So this is a classic Merril & Biswas, 1996 paper, right. So this showed that if you had phages, that you could select for phages that had longer circulating half-life, as shown up here as opposed to the wild type, and they had improved efficiency, as you see here. These are the long-circulating phages down here, and these are the wild type, and you can see the long-circulating phages perform better in a mouse model.
july10-508	DR. GILL: So maybe you could screen for phages that have longer half-life. You know, short duration half-life might be kind of a generic feature of phages, but maybe it would be worthwhile to actually select for phages that, you know, you could select -- these are all mutants, right, of the long-circulating phages. So you could just select for these perhaps and have these in your arsenal, long-circulating versions ofother phages you know that have already worked. And another issue that was brought up was the antibody response to phages. So this might be an issue that if you do repeated treatments that you're going to maybe lose efficacy or perhaps you'll need to switch phages, so if one person becomes immune to one phage and you want to treat them again, you have to use a different phage. So perhaps you want to have some idea about what the cross-reactivity, the cross- antigenicity of the phages is before you do the treatment, right, so you'll know you have serogroups A, B, C, D phages, and if you treated somebody with A and B the first time, that if you're going to treat them again, you want to use C and D types the next time.
july10-509	DR. GILL: So this is just a proposed work flow that we use in our lab. So we start with a bunch of phages and we use restriction digestion actually to de-duplicate. So anyone that has identical restriction patterns, we just eliminate those or just keep one. We then do these assays, and then from here you'll pick some and then do -- say such as phage cross-receptor use and then perhaps other types of characterization as well.
july10-510	DR. GILL: So the current outlook. So recentindividual cases have shown that emergency use of phages is a viable treatment option, and really the near future -- almost all phage treatment that we can see until some of the company products come online, which will probably take some time, it's all going to be these kinds of emergency basis, and so the rapid turnaround time is really going to limit what characterization you can do. But if you have a standing collection you can characterize in the background, you can be prepared, right, and you can have the phages that you're already able to turn around relatively quickly, and then, as we gather more data, we'll be able to interpret that data better, I think, as well.
july10-511	DR. GILL: So, with that, I'd like to thank you for your attention and take any questions.
july10-512	DR. GILL: (Applause.)
july10-513	DR. CARLSON: We have a couple minutes for questions if anyone has any for Dr. Gill.
july10-514	AUDIENCE MEMBER: Hi. Nice presentation. I have one simple question. So sometimes we see when we do selective pressure using phage the bacteria start, you know, under selective pressure, there are receptors which reduce number expressed on the surface of the bacteria and that can, you know, it's a phagebacteria is also growth competition, so in that case, bacteria will start over-dominating the culture or media. So how you predict those type of situations in the actual clinical scenario?
july10-515	DR. GILL: This is like a physiological response, right, you're talking about? Yeah, so those are really -- I mean, those are the hardest ones to get at because they're not stable, right. So we have had phages where, you know, either their resistance is physiological or they just revert really quickly as soon as you remove the phage, and it's tricky. What we've had to do -- I can tell you at least the experience of Klebsiella, that we've had to kind of abuse them a lot, so we'll really kind of co-culture them with the phage for a long time so that hopefully that phenotype becomes permanent, or if it's something that reverts really quickly, they can accumulate some kind of compensatory mutations that allows them to maintain it without wanting to revert instantly.
july10-516	DR. GILL: But, yeah, phenotypic changes, they're tough. They're tough to deal with, and I think there's still a lot about the phage host interaction that we don't know. This is kind of the Rumsfeld style like unknown unknowns, right? There are still a lot of those out there.AUDIENCE MEMBER: So sometimes it's just a simple phase variation --
july10-517	DR. GILL: Yeah.
july10-518	AUDIENCE MEMBER: -- sometimes.
july10-519	DR. GILL: Yeah.
july10-520	AUDIENCE MEMBER: Okay, thanks.
july10-521	AUDIENCE MEMBER: I have a question about if you have a preexisting library or a set of phages that have been characterized and you get a new strain that comes in from the clinic no one's ever seen before, and you figure out that, you know, five different phages from all over your library work, do you envision a need to characterize how that specific cocktail works in the context of that specific strain in order to see efficacy? Like do you -- because if you do that every single time, you could imagine that that would be prohibitive if you're --
july10-522	DR. GILL: Yeah.
july10-523	AUDIENCE MEMBER: Do you understand what I mean?
july10-524	DR. GILL: Yeah. Well, I think, you know, it's hard to say when the rubber hits the road, right, how this will actually play out. I think the hope is that if you have the phage -- the phage collection is relatively well characterized, then you could do somekind of simple experiments just to see if that new strain behaves like it does against the other strains you've already tested, like, for example, if it's the simple kind of virulence assays, it gives you the same kind of phenotype as it does, and maybe you could do cross-resistance assays because you can generate those mutants, it doesn't take too long, right. It's really an overnight, and you can get those and you can test them within a few days, but, again, it depends on how much of an emergency it is, right.
july10-525	DR. GILL: So, if you have the time, then you could do a few more confirmatory or follow-up experiments, but if you don't, I think you just -- the idea is that having that library characterized already will give you at least some assurance that it's likely to work, more likely to work than if you just picked a random phage off the street.
july10-526	DR. KINCAID: Yeah, I was just curious whether or not you'd developed any assays that assess biofilm disruption and/or whether or not that's worth any effort?
july10-527	DR. GILL: Yeah, that's something -- it looks like we can talk about everything. So that is another issue, biofilm disruption. So there are these kinds of standard, you know, in vitro biofilm assaysyou can use and we've used those. I mean, it's just in a 96-well plate, you know, for like crystal violet staining, and so we have some phages, for example, that have the capsular depolymerase enzymes on them that will degrade the capsule, and those are a little more effective at removing biofilm, but, yeah, that could be another aspect, though, of the characterization in addition to say a standard virulence assay is to look at biofilm reduction.
july10-528	DR. CARLSON: Okay, great. All right. Thank you.
july10-529	DR. GILL: Thank you.
july10-530	DR. CARLSON: So, obviously, we can have more questions at the panel later. So up next we're going to have Susan Lehman from AmpliPhi Biosciences, who's going to talk to us about CMC and other considerations for phage products.
july10-531	DR. LEHMAN: Good afternoon. I'm going to talk about this topic today in the context of what I think is a bit of a gap between everything we know about phage biology and what we need to get us to the point of having phage therapeutic products that are accessible for a large number of people.
july10-532	DR. LEHMAN: One of the biggest challenges for phage therapy apart from maybe money, I think, isintegrating all of the R&D expertise that exists and all of the CMC expertise that exists. Jason talked a lot about phage characterization among the data that we can collect on the research side. What's the key data coming from that R&D environment that we need to collect in order to build a robust manufacturing program and put together a CMC package that can be taken to the relevant regulatory agency to get permission to proceed into clinical trials in humans?
july10-533	DR. LEHMAN: There is a ton of fantastic phage biology knowledge throughout the world, and there's also a really well-developed infrastructure for commercial antibacterial development. I think bridging that gap between those two fields of expertise is a particular challenge for phage therapy. I think the gap exists partly because there's such a long history of human phage use, and that happened largely outside of the drug development sphere, and that's put phage therapy in a bit of an unusual position relative to other novel antimicrobial classes that come to the FDA or the EMA or any of the other regulatory bodies in the early stages of development because we just simply have so much other experience and there's an underlying belief that it works, and so I think that's led to some historical tension between the knowledgethat we have about phages and phage therapy and the traditional drug development pathway that an antibiotic would go through.
july10-534	DR. LEHMAN: But I also hope that I can finish convincing a number of people who may not be convinced already that phage therapy absolutely can fit into a traditionally regulated drug development pathway and that also the established antimicrobial development community can adapt to all the strange little ways that phages don't quite fit in. They don't quite act the same way as a small molecule drug does, and they don't even really act the same way as a non-replicating biologic. There's always some extra complications when you've got something that's self-replicating.
july10-535	DR. LEHMAN: When I make that statement that phages can fit into a traditional drug development pathway, I'm basing that on AmpliPhi's experiences. We are engaged in traditional drug development programs. We have two lead products, a cocktail for Staph aureus and a cocktail for Pseudomonas aeruginosa. Both of them have very high coverage across the various isolates of both species that we've collected, and that's been true as we've continued to collect new isolates over time.Last year, we completed two Phase I safety trials with the Staph aureus product, one in chronic sinusitis patients in Australia and one in healthy volunteers in the U.S. toward the skin and soft tissue infection. In both cases, the product was safe and well-tolerated in the subjects, and following those trials and some additional conversations that we've had, we are well-positioned to move that product forward into Phase II clinical trials.
july10-536	DR. LEHMAN: For our Pseudomonas product, we are hoping to enter Phase I trials next year. We've had a successful consultation with the MHRA because our partner is there in the U.K. and are well positioned to move forward in that, with that product as well.
july10-537	DR. LEHMAN: There's also been some talk about compassionate use cases. AmpliPhi has responded to physician requests to use our products and our phages under expanded use schemes, and in those scenarios, we've had interest in a number of other indications besides the infections that are listed here and things like IV administration, and we are investigating those as well.
july10-538	DR. LEHMAN: So you've done all this great work with the phage characterization that Jason was talking about. You've designed a great product and what do you needto get from that data into a clinical trial to a product that hopefully works and you can get regulatory approval to take to market?
july10-539	DR. LEHMAN: As you move out of that R&D environment into a more regulated manufacturing, testing, and clinical environment, everything becomes quite a bit more structured. Broadly speaking, you need to scale up fermentation and purification. You need assays that you can use to monitor your process and also to monitor the final product that you get out of it. You need to figure out how you're going to formulate and deliver your drug. You need to make sure that it's going to be shelf-stable for long enough that you have a useful product, and there are industry standards that govern all of these things: things like how your biological stocks are maintained and used; how your process validation is done; the quality systems that govern all of these; what kind of claims that you can make about drug delivery and drug dosing; how you store and test stability for your products; and how you test safety before you can get permission to move forward into humans; and then, of course, once you enter clinical trials, there are a number of regulations that govern how those trials are conducted and how the data is analyzed.I'm going to start by talking a little bit about the first two stones on my little stepping stone pathway. The overall message for GMP manufacturing, the underlying principle is process consistency. You need to manufacture the same thing every time, doing the same thing every time.
july10-540	DR. LEHMAN: I've put up an example schematic for a hypothetical four-phage product where there are two bacteriophages that are grown in one manufacturing host and two phages grown in a second manufacturing host. There was a comment made this morning about whether you pick a manufacturing host early in development or later in development. I definitely agree with the statement that it's better to do it early.
july10-541	DR. LEHMAN: You don't -- as was said this morning, bacteriophages just can behave a little bit differently based on what you've grown them in, and you certainly wouldn't want to get to the stage of manufacturing and find out, you know, switch a host and find out that all that characterization data you've so carefully collected is no longer entirely relevant to what you're about to manufacture.
july10-542	DR. LEHMAN: So, having said that, you've done all that work, you've transferred your phages and manufacturinghosts to two of the GMP manufacturing environment, and set up a two-tier manufacturing system. You've got master stocks that are really well-characterized, working stocks that you're going to use to make each batch, and when you've used up all your working stocks, you can go back to your master stock and make a new set of working stocks.
july10-543	DR. LEHMAN: Every time you make a new batch of phage you take one of those working manufacturing host vials, one of those working viral seed vials, put them together, do your fermentation and purification processes, get your drug substance out, and mix your purified phages in known ratios to generate your drug product.
july10-544	DR. LEHMAN: There are a couple of things that this does. Two of the most important ones are that it limits serial passage, so you can maintain vertical consistency between those master stocks and every batch of drug substance that you make. It's particularly important for phages because there's a replicating genome.
july10-545	DR. LEHMAN: The other really important thing that isn't entirely captured by three little boxes on a screen is how important your process consistency is through this step. Every time you make a new batch of phage youfollow the exact same process through the fermentation, through the purification. The process might differ for different phages in your product, but for a given phage, same thing every time, and it's extensively documented.
july10-546	DR. LEHMAN: Having gone through the process of setting up that manufacturing system so that you are making the same thing every time, you need analytical methods so that you can demonstrate that consistency both to yourself and to others. You can't test everything. You have to rely on the processes you've set up and you have to have designed good processes, but you can test a number of key characteristics against established criteria to give yourself the confidence that everything has gone the way you've set it up, and a lot of those assays are going to be heavily dependent on the phage characterization that you developed early and the process development data that you gathered early on.
july10-547	DR. LEHMAN: So it's important to keep that in mind during the early R&D phase. I think it's really easy in a research environment to get very caught up in screening phages, picking the best phages. If you're in the genetic engineering side of things, making the best phages. But everything that you pick iseventually have go through the, you know, process of controlled manufacturing and analysis, and so you do yourself a lot of favors if you think ahead to these analytical processes while you're earlier in development.
july10-548	DR. LEHMAN: Part of that thinking ahead is thinking about the difference between good research analytical methods and good analytical methods for GMP manufacturing. There are a lot of good research assays that have controls, standards, they work the same way when you run them again, they measure what you think they're measuring.
july10-549	DR. LEHMAN: But when you move into a GMP manufacturing environment, you have to know a lot more. You have to have much more in-depth knowledge about the assay performance both in terms of trends over time and the way that the assay functions every individual time that you run it. You don't get to decide that you're going to run a gel, an agarose gel at a slightly higher voltage for a shorter period of time because you've got somewhere to go. It's got to be the same every time.
july10-550	DR. LEHMAN: I don't think I've ever worked in an academic research lab that made everyone in the lab who did plaque assays take the same tube and test itmultiple different days and make sure that everybody in the lab got the same number within a pretty tight margin of variability and then said, oh, the lab next door that we collaborate with or the lab two universities over that we collaborate with, we're going to give you the same samples and make sure that you get exactly the same results.
july10-551	DR. LEHMAN: We make an effort in those environments to make sure that our results aren't wildly different, but we don't really put hard numbers on those kinds of things, and in a GMP manufacturing environment, we have to.
july10-552	DR. LEHMAN: There are also a number of scenarios in a research and development environment where what we're doing is somewhat exploratory. You're not just interested in things that have a yes or no output. In a GMP manufacturing environment, in a quality control testing environment, you don't want that fuzzy middle. You need something that has a clear readout and it's clearly interpretable as a yes or no answer.
july10-553	DR. LEHMAN: And, finally, you -- well, not finally, there are a lot of examples I haven't given, but finally for this slide, is the assay actually helpful? Can it be run in a reasonable amount of time for what you need? Are the tolerances that you put on thatassay tight enough to detect problems when they exist, or are they so tight that you start flagging things that you shouldn't?
july10-554	DR. LEHMAN: All of that assay development and process development occurs in stages. It matures over time, and the validation level of those assays also matures over time. Some of your assays are going to be industry standards. Some of them are going to be product-specific, and the product-specific ones are likely to be the biggest challenges.
july10-555	DR. LEHMAN: I'm not going to talk about all of these. I want to highlight a few. Phage concentration, obviously, a key parameter for a phage product.
july10-556	DR. LEHMAN: What host are you going to test it in? There was some conversation this morning about manufacturing hosts and assay hosts that people have found were not -- they didn't work if you use the same one, so it might work if you use the same one, it might not.
july10-557	DR. LEHMAN: Are you testing a single phage? Testing the concentration of a single phage is fairly straightforward. What about if you're trying to test the concentration of three or four or five or 12 individual phages within a cocktail? That's a much bigger challenge. Identity and purity of your phagesare also obviously key. You want to make sure you've grown the phage you think you have. You haven't cross-contaminated any of your stocks.
july10-558	DR. LEHMAN: How informative are the assays that you have? There are a lot of different methods available to assess phage identity and phage purity. They each have different strengths and weaknesses. Obviously, you need something that's going to differentiate among different phages. You also need something that's got the right capacity to detect a problem. PCR is great, it's fast, it has a nice, clear readout, but it's only based on a tiny section of the genome, so it's going to be useful for some kinds of assays, but it may not be useful if you're looking for changes that may occur outside of that section of the genome.
july10-559	DR. LEHMAN: When it comes to removing impurities, are you at a phase of development where you have a really good idea of a couple of specific host cell proteins that your process -- that you can test for to tell if your process is working well and you've gotten good purification, or are you at a stage of development where you need to look more generally at bigger picture of what's going on in your purification?
july10-560	DR. LEHMAN: If you're talking about endotoxin, how you plan to administer the phage product is going todetermine what your acceptable endotoxin limits are. With microbial contamination, there are established test methods for bacterial burden and for sterility. That's good when you need to test that, but you also should think about where in the process you might have contamination occurring. Maybe there are points in your manufacturing process where it would be beneficial to do a risk-based assessment of what the likely problems are to arise, and so you can test as you go along for some of those most likely events and reduce the risk that you're going to get to the end of your process and have something that fails, fails a quality assessment, and you could have found out before you spent the time and money going through the whole thing. You could have found out early on that there was a problem with that batch.
july10-561	DR. LEHMAN: The next three stepping stones on my little graphic are here. There's a ton of things to think about just in terms of the practicalities of developing phage products. There are guidelines that govern a lot of these, things like stability testing and device qualification if you're going to use a delivery device. I want to use this to give you a specific example of the ways in which a lot of these things can get more complicated than you initiallypredict. Our Phase I trial on rhinosinusitis patients administered the phage as part of a sinus wash. It was a sinus wash that's used as part of the standard of care for CRS patients, and they have about a cup of water and they dissolve a little saline pouch into it and run it through their sinuses in both directions, and we thought we'll put the phage product in the sinus wash. It seems really simple, and you think, okay, let's make sure that the saline, you know, components don't interfere with phage viability.
july10-562	DR. LEHMAN: What about the water? Most patients use boiled tap water, municipal tap water to prepare that saline wash. The municipal tap water where we were going to run the trial, boy, even after you boil it, it's not so good for the viability of that phage product.
july10-563	DR. LEHMAN: So, in that trial, we ended up providing every subject with a case of water that we knew would have no problems and they took that home with them. We did all the testing in advance, so we knew that we needed to do that, but it's a level of -- it's that extra level of testing for compatibility that you need to think of throughout these processes, and it takes time.Clinical trials and clinical development in general are hard. They get harder when you add phages. Assuming you've picked a good infection target that matches phage biology really well, you've got a plan for your clinical trial progression that's going to get you to the indication that you're targeting, and you have a sufficient patient population to enroll those trials in a reasonable amount of time, what are you going to measure? Is your primary end point clinical? Is it microbiological? If it's microbiological, do you have a threshold for success versus failure?
july10-564	DR. LEHMAN: Do you need new assays? Sometimes the infrastructure that's available in clinical microbiology labs aren't going to be equipped to handle phage-specific assays particularly well because they simply don't work with it. They're not used to it. So, if you have to qualify a new assay and potentially qualify a new lab, if it's a multi-center trial, are you going to try and qualify multiple labs at all of the sites, or are you going to look into centralized testing?
july10-565	DR. LEHMAN: You made it through all of this. Before you can get permission that it's safe to proceed into humans, you need to submit all of that collected datato the relevant regulatory authority. In the U.S., this is done under an Investigational New Drug application, or an IND. Different countries have different requirements, but the general structure is similar. You present your overall plan. An investigator's brochure communicates your core product and quality characteristics to the physicians and also to the institutional committees that are responsible for approving the trial, and all of the non-clinical data and all of the quality information from your manufacturing come together in that as well.
july10-566	DR. LEHMAN: The structure of these elements will change over time. As you move through clinical development, you obviously acquire more clinical data, but the general structure is there, and the FDA, the elements to this aren't a secret. The FDA and the ICH have lots of really detailed information online about exactly what goes into all of them.
july10-567	DR. LEHMAN: So, to end, I think my message here is that you can -- I mean, our experience so far certainly has been that you can fit phage development into industry standard processes, and a lot of times through this I was making reference to the fact that there are industry standards for X. I think a lot of times in the phage therapy community we tend to view thoseindustry standards and requirements as a hurdle, and I don't believe we need to see them that way.
july10-568	DR. LEHMAN: Certainly, requirements that need to be met, and that takes time and effort and money. They're also the things that are going to let us treat a number of people to make phage therapy available to a large number of people.
july10-569	DR. LEHMAN: There was a comment earlier this morning that the kind of really intensive, focused effort that's required for a compassionate use case isn't necessarily sustainable. The way we can make phage treatment sustainable is through this kind of approval pathway, to fit them into the drug development pathways that exist, and the good news in a lot of ways is that all of this, all of the infrastructure for this, does exist from the antibiotic world. There are ways that we need to fit phages into that and there are some specific challenges associated with that, but there's also a lot of CMC expertise out there that we can use and we can use to our benefit.
july10-570	DR. LEHMAN: I promised I'd talk a little bit about some of the expanded access use for products as well. Our tendency so far has been to use GMP products when possible, and because the products that we have have such high coverage across different strains of thespecies that they target, we think that it's quite feasible to use GMP products in a number of cases or at least a GMP-like version of those products.
july10-571	DR. LEHMAN: In situations where GMP material isn't possible or isn't available, we still believe that non-GMP material can and should meet very high standards. When it comes to key attributes, such as understanding that, you know, phages are lytic, having really well-documented evidence of your product quality, using purification methods that are appropriate to develop material for human use, doing microbiological testing, handling things in a controlled way, a lot of the way that these attributes get handled in a non-GMP product may be a little bit different from the way they're handled in a GMP product, but we can still meet high standards of quality for those.
july10-572	DR. LEHMAN: My message for today has been that the phage biology expertise that exists in the phage community is absolutely compatible with a drug development pathway. There are a lot of exciting conversations that I think are being had about additional ways to look at this. The concept of phage libraries is a really good one to talk about, and I think that having some development through some of the traditionalpathways is going to help move those conversations forward, which was mentioned this morning.
july10-573	DR. LEHMAN: We have a lot to learn as we all move forward, and I'm very grateful to the FDA and the NIH for continuing to organize this workshop because I think we've gotten a lot of useful discussion and will continue to over the rest of today and tomorrow.
july10-574	DR. LEHMAN: I think I have time for one question.
july10-575	DR. LEHMAN: (Applause.)
july10-576	DR. CARLSON: So we can take a few questions. We started a couple minutes late.
july10-577	AUDIENCE MEMBER: So a nice presentation. So my question, specific question is that whatever you presented here is good for a fixed cocktail model. But if you are a dynamic, you know, phage library and you want to make a product out of it because we know from my experience a fixed cocktail model is not going to work all the time because the resistance for the phage is pretty often because the phage-bacterial interaction happen more randomly than the antibiotic, mainly the fungal products, and the bacteria.
july10-578	AUDIENCE MEMBER: So, in that environment, today's fixed cocktail will not work tomorrow's bacteria. So how you address this question, how you?
july10-579	DR. LEHMAN: I think there are a couple ofpieces to that answer. One of the things that we've found with the cocktails that are predesigned that we have is that they have been very broadly active, and in terms of, you know, when you go and collect clinical isolates every couple of years, new clinical isolates every couple of years, they've maintained pretty broad activity.
july10-580	DR. LEHMAN: The inherent frequency of resistance is pretty low, and you also have -- so you have a lot of cases where those are going to be useful. In cases when they're not, we are open to -- we want to have the discussions about whether having very well-characterized libraries of phages has a -- basically whether there's a way to fit that into an FDA-regulated system, and certainly, as I've indicated, we've been involved in some compassionate use cases where there's -- where permission to use something that's less well-characterized is easy to get.
july10-581	DR. LEHMAN: I think there's also some difference to keep in mind between species. Staph aureus is probably a lot easier to hit with a defined cocktail in more cases because the organism itself is more homogenous. Pseudomonas aeruginosa, you start to get into an organism that's a little more genetically diverse, and you get to something like Acinetobacter baumannii andthat's even harder. So I wouldn't want to get scared off by the challenges of Acinetobacter when we're looking at something like Staph. I think we need to talk in a very context-dependent way about the need for additional -- the need for, I guess, the relative utility of those pre-defined cocktails because there are a lot of cases where they think they are going to be very useful. At least that's been our experience with the data that we have.
july10-582	AUDIENCE MEMBER: I basically wanted to ask the same question as him, but the data you have where you continue to see efficacy even when there's new clinical isolates emerge, are you doing that in animal models or are you doing that in vitro?
july10-583	DR. LEHMAN: The bulk of it is in vitro because you can simply test so much more.
july10-584	AUDIENCE MEMBER: But one would have to -- not to be belligerent, but one would have to consider that in vivo -- I mean, even manufacturing strains matter. So, if you shift the center of your population away from optimization to the target, it could be that in vivo that's -- or, sorry -- in vitro it's undetectable because diffusion is controlled in a liquid broth, for example.But when you put that into an organism with an active immune system and CRPs and three dimensions, you might not see the same efficacy downstream and you might get different kinds of resistance emerging in an animal that's also adding selection pressures of its own vice a context where you're just in the broth. Does that make sense to you?
july10-585	DR. LEHMAN: Yeah, yeah, and I think everyone agrees you can't do quite as much testing in an in vivo system as you can do in an in vitro system, and I think the testing that gets done in an in vitro system in that capacity needs to be chosen pretty carefully. For example, a mouse model is only going to support a certain size bacterial population, so there's only so much of that that may be informative in a mouse. But as you get to bigger and bigger animals, you can do even less.
july10-586	DR. LEHMAN: So that's one of the areas that compassionate use cases may help us to understand a little better. It may help us to define the questions a little better because a human has -- it's -- ultimately our question is what is it going to do in a human being, and as we collect some data from a lot of these cases, we're going to have a better sense of what those questions are, which may let us go back anddo some more focused in vivo testing in animal systems as well.
july10-587	AUDIENCE MEMBER: Coming to the in vivo question, one of the biggest challenges that we have in antimicrobial development, and certainly it'll apply to bacteriophage is in our patients how much to give and how long to give it. So, while in antimicrobial development we have very recently well-defined strategies and pathways to arrive at the PK/PD, what pre-clinical animal model systems PK/PD analysis do you do to be able to know how much to give and how long to give?
july10-588	DR. LEHMAN: That's a real challenge. A lot of traditional PK/PD is done in uninfected animals, and that's not going to tell you the same things in phages that it will tell you with a small molecule drug because the dosing changes and the way that the phage can hide from biological clearance mechanisms changes when a susceptible bacterial population is present.
july10-589	AUDIENCE MEMBER: For example, in the wound models that you have and in the implant, we do have animal model systems for those. Were they developed in order to -- and explored in order to know again how much to give, how long to give?
july10-590	DR. LEHMAN: In most cases not. Figuring out how to do proper PK/PD for phage therapy is a huge unknown and I think it's something that's understudied in the field in general, and we've got a lot to learn when it comes to figuring that out, and some of the work that Dr. GÃ³rski has been doing and his colleagues in Poland looking at what kind of antibody responses exist and how those antibody responses correlate to clinical outcomes has been valuable, and I think, you know, as companies go through controlled clinical trials, we have a lot to build on in that specific area. Or, I'm sorry, we have a lot to build up in that specific area.
july10-591	DR. CARLSON: So we can continue with questions along these lines in the discussion session later and I guarantee we'll talk about phage, personalized phage therapy there. It keeps coming up in, I think, just about every talk, so I promise we'll come back to it at the panel discussion.
july10-592	DR. CARLSON: So up next we have Scott Stibitz from FDA CBER, who's going to talk to us about regulatory pathways and CMC considerations for bacteriophage products.
july10-593	DR. STIBITZ: Great. Thank you. This is the point where I would normally thank the organizersfor inviting me, but instead I will thank all the other organizers for their hard work and for the CBER staff and NIAID staff, who have just done a fantastic job getting this all together.
july10-594	DR. STIBITZ: So this talk constitutes what we kind of sometimes refer to as regulatory outreach, and when I was younger and more foolish, I said I would never ever give a regulatory talk, but I've now given quite a few and I actually really like them because it gives us a chance to sort of set the record straight in a way to really give accurate information. We often hear people talking about the FDA and what we think and what we'll allow and what we won't allow, and sometimes it takes us by surprise.
july10-595	DR. STIBITZ: In terms of phage therapy, the most pervasive is -- which is stated over and over again -- FDA will never allow phage therapy. So I also have to just throw this disclaimer up here to let you know that my comments will not bind or obligate the FDA.
july10-596	DR. STIBITZ: So, just to kind of address off the starting block some of these misconceptions, I put together a few points that address some of these issues we've heard.
july10-597	DR. STIBITZ: So one is, you know, does CBER FDA have a history of regulating novel products and treatmentmodalities, in other words, weird stuff? And the fact is, yes, we do. I think the most recent example of that would be fecal transplants.
july10-598	DR. STIBITZ: Are clinical trials on phage therapy proceeding under FDA oversight? Well, I can give the positive answer yes, but only because those have been publicized by the companies involved. Otherwise, I would not be able to make that statement.
july10-599	DR. STIBITZ: Similarly, we do allow compassionate use of phage therapy. Our preferred term is 'expanded access,' but I can kind of tell it's a losing battle.
july10-600	DR. STIBITZ: (Laughter.)
july10-601	DR. STIBITZ: So are there new and challenging aspects to clinical trial design? Absolutely. Are there novel CMC challenges or considerations? Yes. Does the FDA actually in-house have research projects on phage therapy? That is also true. A story for another day.
july10-602	DR. STIBITZ: And so what I think all this adds up to is I hope that you are convinced and I think everything I've heard so far today and what you'll hear tomorrow is that the FDA does not have a preexisting negative position towards phage therapy.
july10-603	DR. STIBITZ: So a brief outline. I want to talk about who at FDA is responsible for regulating phagetherapy, if we could put a face on the monolith, a brief overview of regulatory procedures, and then talk about CMC issues that may be special relative to phage.
july10-604	DR. STIBITZ: So to the first. FDA contains many centers. I've thrown up some here. This is not a comprehensive list, but we are in the Center for Biologics Evaluation and Research. It occurred to me as I was looking at my slides today actually each of these other centers probably will have some interaction with us or something to say about phage therapy as well.
july10-605	DR. STIBITZ: So we're the Center for Biologics. It's natural to ask the question what's a biologic. In almost all cases, a biologic is also a drug, but here's a definition from the Public Health Service Act, but the most critical statement in here is, and this echoes the definition of a drug, is that it's applicable to the prevention, treatment, or cure of a disease or condition of human beings.
july10-606	DR. STIBITZ: So, within CBER, we are within the Office of Vaccines Research and Review, as has already been pointed out, and there are three divisions. The division in the middle here is Vaccine and Related Product Applications. They are the people who really manage the files, communicate with the sponsors, andcoordinate the reviews. The reviews include in almost all cases -- I mean, sorry -- in all cases I would have to say product review, and that's done in the divisions that actually do laboratory research. So those of us in those divisions, we have labs, we do experiments, and then we also do product review.
july10-607	DR. STIBITZ: And within DVRPA we have clinical review and, as needed, toxicology reviews and also, as needed, statistical reviews or quite often consults with other divisions that might have, for example, clinical expertise.
july10-608	DR. STIBITZ: Okay. So when in product development does the FDA get involved? As many of you are aware, this is first in-human use. When that happens, it's supposed to be done under IND. That has at least two effects. One is that it's done under our supervision with our advice, but it has a legal ramification, is that it allows use of an otherwise illegal product in interstate commerce or in clinical trials. So the IND is basically an exemption from having to use a licensed product. But it's also important to remember that not all INDs are for product development. We get some that would be called research-only studies, I think, by most people.
july10-609	DR. STIBITZ: So this is kind of a summary of the wholepicture. I want to spend a little time talking about it. We have the phases of IND research here, and the boundaries between these can sometimes be somewhat mobile. Certainly, many studies would span Phase I, Phase II or Phase II, Phase III, but the important aspect here is that through the IND process it's basically progressive implementation.
july10-610	DR. STIBITZ: I have broken it down into three aspects here. One is effectiveness. So the trials that you're doing, Phase I, for example, could be simply safety. Phase II could be preliminary evidence of efficacy, and then Phase III, of course, is your pivotal clinical trial, collecting the data to support a license application.
july10-611	DR. STIBITZ: Similarly, manufacturing consistency. In the beginning, it could be quite simple. During this process, there may be changes made to the product, dosing might be altered, dosing or formulation, but by the time you get to Phase III and are ready for your clinical trial this should be basically set.
july10-612	DR. STIBITZ: Similarly for assay developments. The, you know, assays in Phase I should be basically scientifically sound. By Phase II, you start to think about qualifying, and by Phase III, assays to be used in the pivotal trial should be fully validated.And then, if all goes well, license application leads to an FDA license, and, of course, that's not set in stone. BLA supplements after the license can be used to change things as long as it's not too terribly drastic, but there can be some changes to the product. There can be changes in manufacturing. There could be clinical studies to support a different dose or a different indication, changes in the manufacturing equipment, et cetera, et cetera.
july10-613	DR. STIBITZ: And then I'll talk again about this more, but leading into all this, especially for people that don't have a lot of experience, we have opportunities to meet in what's called a pre-IND meeting.
july10-614	DR. STIBITZ: So one of the things that I like to tell people because the term 'CGMP' strikes fear in the heart of many would-be IND sponsors is that GMP is not GMP, is not GMP. In other words, what people think of as a GMP lot fully validated, you know, all the things that you think of when people say, oh, well, we need to get a GMP lot to begin studies. In fact, GMP in Phase I is not as rigorous, and so I've quoted this guidance which is out there. The approach described in this guidance reflects the fact that some manufacturing controls and the extent of manufacturingcontrols needed to achieve appropriate product quality differ not only between investigational and commercial manufacture but also among the various phases of clinical trials, and boiled down, this means that for Phase I, CGMP is not required to be as extensive as for later phases or for an approved product.
july10-615	DR. STIBITZ: Who sponsors biologics INDs? Big companies, small companies, individual bench researchers, individual clinical investigators, and other agency. And so the point that I'm trying to make here is that the regulatory expertise and regulatory support that's available to sponsors varies greatly, and this is why at critical points the opportunity exists and it's highly recommended to meet with us, for example, prior to submission as in a pre-IND or prior to pivotal studies, license application, et cetera, and this is where we really work out a lot of the details.
july10-616	DR. STIBITZ: And just to reprise this slide to make this point that because biologics are so different from one another we can't have, you know, kind of clearly prospective milestones that will apply to all products and that, you know, the sliding scale or progressive implementation, the milestones on that are arrived at through conversations between the sponsor and the FDA.
july10-617	DR. STIBITZ: Again, pre-IND meetings. The stated purposeof these as per this guidance that's shown here is to discuss CMC issues as they relate to primarily safety of an investigational new drug proposed for use in clinical studies, and the way this works, some of you will have done this and are familiar, but the sponsor poses questions, provides a description of the CMC.
july10-618	DR. STIBITZ: This is a case where you get out of it what you put into it. So the more detail that you can give us about where you are in product development, how much information you have, and what your real questions are to us, the more you will get out of it.
july10-619	DR. STIBITZ: And so what happens is that CBER assembles a full review team, so that would be product, clinical, toxicology if indicated, statistically possibly at an early stage, and we do a full review of what you have submitted. This is good for everybody. It's good for the sponsor because they get a much better and clearer idea of what we're asking for. It's good for us because, when the IND actually comes in, the review is much more straightforward, and it's good for both of us because fewer studies go on clinical hold.
july10-620	DR. STIBITZ: And so, if all goes well, the ultimate goal is an FDA license. For a product to be licensed, that requires three things: that it be shown to be safe and effective and able to be manufacturedconsistently. And, again, the details of what a safety database would look like or what the demonstration of effectiveness is in any particular case will be based on the nature of the product and our discussions.
july10-621	DR. STIBITZ: So, finally, just to get to some more phage-specific stuff, I tried to organize this around unique, as I see them, aspects of bacteriophage, all of which have been touched on already and will be. But phage are incredibly diverse. They're highly specific. They mediate genetic transfer. They're antigenic. They're generally assumed not to interact with human cells, and as part of that, there's basically a high expectation of safety.
july10-622	DR. STIBITZ: So just to go into some of these one by one. Now some of these have positive aspects and some have not so positive aspects. In terms of diversity for many bacterial hosts, and again I think the dogma that there are billions of phage out there for any bug may be not so true for all bugs. I mean, certainly, Ry talked this morning about how Staph phage seem to have been dominated by the K-like phages, and I think Andy Camilli's work has shown that there is really a very small number of cholera phage out there, but this is a concept that we work with, that there are lots ofphage out there and it should be possible to find new phage. So I put inexhaustible in quotes because maybe it's not inexhaustible, but it's a big supply of natural products.
july10-623	DR. STIBITZ: But the downside of diversity is that every bacteriophage-bacterial host pair is unique, and so it is, in fact, problematic to draw a priority or general conclusions, either good or bad, about phage in general from specific examples.
july10-624	DR. STIBITZ: Specificity of phage, these will generally be pathogen-specific treatments, which raises a whole new issue when it comes to clinical trial design, which our next speaker will be speaking of. Now the good thing is that we expect less disruption to the microbiota, as has been stated. But it also, as has been stated, will usually require identification or diagnosis of the agent prior to treatment.
july10-625	DR. STIBITZ: We generally talk only in terms of receptor actions, interactions, as dictating specificity, and in that regard, as was presented very nicely by Jason, there's a future for using receptor identification to inform cocktail composition, to avoid the issue of resistance to all phage in a cocktail simultaneously. But I just want to plant a seed that receptor is not the only source of specificity, and in work in ourlab, it looks like both gene expression and replication may be playing a role in that as well.
july10-626	DR. STIBITZ: Bacteriophage are immunogenic. An adaptive response is likely. This may limit the length of use or re-use, but not that much is really known about this in clearly defined studies. I was extremely interested to hear all that Dr. GÃ³rski had to say on this, and I think that they're really getting at a lot of these issues, but I took away from that it's not as dire as it seems. I mean, in some cases, it does not appear to limit their therapeutic use. But it's also unclear at this point, I think, what safety concerns arise from immunogenicity. I'll leave it at that.
july10-627	DR. STIBITZ: Okay. And so one of the most unique aspects of bacteriophage is the fact that they mediate genetic transfer. So those genes can be transferred. Transfer may be part of the phage genome itself. This is what's called lysogenic conversion, and the phage itself contains genes for antibiotic resistance, virulent factors, what have you. There are many, many examples of this out there.
july10-628	DR. STIBITZ: So that by integrating its genome into the genome of the host, those genes now become part of the genome of the host, so special abilities often in terms of resistance or pathogenicity, should I sayvirulence are bestowed. The other way is by what's called transduction, and this is where phage particles move genes between hosts. There are generally recognized to be two kinds: generalized, in which all chromosomal markers of the host organism are transduced with equal frequency, and the other is specialized, and this refers only to lysogenic phage where once having integrated, imprecise excision will lead to phage particles that now carry genes that were close to the insertion site. And so, by restricting our use to non-lysogenic phage, we really get rid of two of these concerns and are left with that of generalized transduction.
july10-629	DR. STIBITZ: So, just to make sure that we're all on the same page, generalized transduction refers to the fact that some bacteriophage when they infect a cell create new copies of themselves and then start to package that into phage heads, will sometimes pick up a copy or a hunk of host DNA. Those particles, so this lysate coming out of this infection would have infectious particles, wild type phage, but also these transducing particles, which will contain a hunk of the host genome. Those particles are not infectious, but they can adsorb and inject their DNA and have itincorporated into the genome recipient bacteria. So what are some ways that you can look at this? A microbiology approach would be to do a transduction assay. Simply take your phage, you propagate it on a bacterial strain that contains a selectable marker, such as antibiotic-resistance. You just take that transducing lysate or potentially transducing lysate, apply it to an antibiotic-sensitive strain and plate on media containing antibiotics. If the phage is capable of transducing that marker, you should be able to detect it.
july10-630	DR. STIBITZ: From a molecular biology standpoint, one can just take your phage lysate and examine it using more sensitive techniques perhaps for the presence of host genes. So PCR can be used for that or you could even deep sequence.
july10-631	DR. STIBITZ: For lysogeny, and Jason made basically the same points, look at your plaques. Dogma has it if they're turbid it's lysogenic, but if they're clear they're virulent. He mentioned exceptions that are turbid yet virulent, and we have examples of ones that are clear yet lysogenic. But what you do is you pick bacteria from the center. You see if any bacteria in that battleground are still alive. One of the ways they could be alive is if they're lysogens andtherefore phage-resistant, and then you test those for release of phage, either spontaneous or after chemical induction.
july10-632	DR. STIBITZ: Most people that I ask how do you decide your phage is lysogenic or not, they say just look at the sequence. So they've obviously done the genomic sequence and analyzed for the presence of repressors, homology to other known lysogenic phage, and any indicators of lysogenic lifestyle, and you could have a hybrid approach where you take some of these survivors and then you determine that DNA sequence of a putative lysogen and see the phage as an insertion. That's an approach we've taken recently.
july10-633	DR. STIBITZ: So the current consensus, I think I'm safe in saying that, I hear these echoed over and over, of what type of characterization do we want for phage for therapy. In terms of the phage phenotypes itself, non-lysogenic, non-transducing. For the phage genotypes which could be assessed by DNA sequence analysis, free of undesirable genes, such as antibiotic-resistance and virulence factors, and the phage preparations should be pure and sterile and, we believe, low endotoxin, although we're having an interesting discussion about that.
july10-634	DR. STIBITZ: Phage cocktails have generally been proposedto increase the spectrum of treatment, in other words, against more strains of a given organism, but also to avoid resistance, and regulatory implications are that each phage should have relevant activity. In other words, you don't just throw the stuff in there for good measure. Potency tests should address each phage in the cocktail, and stability testing, likewise, should assess each phage in a cocktail. Future inclusion of additional or replacement phage should be supported by CMC information.
july10-635	DR. STIBITZ: And then, finally, other things that would be nice to have but I don't think are going to be requirements, and all of these have been referred to, so I'll just mention them. The idea of stealth, this is from the Merril and Adhya work showing that mutants could exist in circulation longer; from Andy Camilli's work, a nice example of using virulence factors as receptors so that resistant mutants are less virulent; and then, of course, the nice story about the MDR Pseudomonas aeruginosa treated under compassionate use, which actually used antibiotic-resistant proteins as a receptor. And, again, this is something I think we have an ongoing discussion about, but possibly being able to propagate on non-lysogenic, non-pathogenic, non-antibiotic-resistant hosts.So, finally, last side, almost on time, this is a feel good slide. FDA is committed to facilitating the testing of phage therapy in clinical trials. We do not feel that, and hope that you do not, the FDA regulatory review presents an obstacle to the assessment of safety and efficacy of phage therapy. We believe that regulatory officials, scientists, and product development developers have shared goals and need to work together and to do that communication is vital, and as investigations begin, meeting with the FDA early is highly recommended.
july10-636	DR. STIBITZ: Some resources which I will distribute because you can't write them down. Just wanted to thank my group and two people there in particular, Sheila Dreher-Lesnick and Roger Plaut, who helped me the most with the slides, and Roger with many, many other aspects of this workshop, and then all the other folks who were involved in practice sessions. So thank you very much.
july10-637	DR. STIBITZ: (Applause.)
july10-638	DR. CARLSON: We can take a couple questions for Scott before we have a break. You're on.
july10-639	AUDIENCE MEMBER: Thanks, that was really nice. I agree we're all in it together and we should try the best we can.You know, we haven't heard anything about RNA phages, and I'm wondering from your standpoint are they just off the table because you're worried about high mutation rate or just what you're thinking on that?
july10-640	DR. STIBITZ: I don't think anything's off the table. They haven't come up that I'm aware of as people isolate phage that they think have the characteristics for phage therapy.
july10-641	AUDIENCE MEMBER: Yeah, I think it has a lot of potential and people just don't bother to look.
july10-642	DR. STIBITZ: Right. I mean, we will if those come on the scene and people are proposing to use them, we'll look at them.
july10-643	AUDIENCE MEMBER: Yeah, your statement about the transduction is what reminded me because you don't really have to worry about the transduction if you're dealing with an RNA phage. But, okay. Thanks.
july10-644	DR. STIBITZ: Fair point. Do you know of any that are in the running? Really? Okay. Can't wait to hear about that. Yes?
july10-645	AUDIENCE MEMBER: All right. Simple question.
july10-646	DR. STIBITZ: Sure.
july10-647	AUDIENCE MEMBER: Does OVRR regulategenetically modified bacteriophages, or will that go over to the gene therapy group?
july10-648	DR. STIBITZ: Sure. I mean, in the extent that there will be among the phage that we will be regulating I'm certain genetically engineered phage.
july10-649	AUDIENCE MEMBER: But will you share it with the gene therapy reviewers in the other office? They changed their name.
july10-650	DR. STIBITZ: Well, I think we have ongoing discussions about who best to perform those reviews.
july10-651	AUDIENCE MEMBER: Okay. So --
july10-652	DR. STIBITZ: I thought you were going to ask the question, which is does a flag go up because they're genetically modified.
july10-653	AUDIENCE MEMBER: Well, that is the indirect question, yes. I mean, this is the primary lead.
july10-654	DR. STIBITZ: So we don't review them from the aspect of being GMOs or genetically engineered. I think we review based on the unique characteristics that that modification provides, not because as a class they're genetically modified --
july10-655	AUDIENCE MEMBER: Okay.
july10-656	DR. STIBITZ: -- if that makes sense. I'm not going to say they're the same as the wild type because clearly they've been modified, but we would bereviewing what the effect of those modifications was.
july10-657	AUDIENCE MEMBER: Okay. But who would review them from a clinical perspective then?
july10-658	DR. STIBITZ: Beg your pardon?
july10-659	AUDIENCE MEMBER: From a clinical, the clinical study perspective.
july10-660	DR. STIBITZ: Are you asking if they have been used?
july10-661	AUDIENCE MEMBER: No. I'm asking would OVRR take the lead or would the --
july10-662	DR. STIBITZ: Well, like I said, those are discussions that are ongoing as to --
july10-663	AUDIENCE MEMBER: Okay.
july10-664	DR. STIBITZ: Currently, OVRR is doing the phage therapy.
july10-665	AUDIENCE MEMBER: All right.
july10-666	DR. STIBITZ: If there are indications that we would expand that, that will come.
july10-667	AUDIENCE MEMBER: Okay. Thank you.
july10-668	AUDIENCE MEMBER: Hi, Scott, another clear, positive speech. I think all the talk is -- I think it's referring to let's say pharmaceutical products. Now, if phages used together with a device, how that going to be reviewed, and if phages is going to be used in the hospital for hard service disinfectant,how that going to be reviewed?
july10-669	DR. STIBITZ: So I believe what you're talking about is a combination product, for example, where phage might be embedded in a matrix of some sort.
july10-670	AUDIENCE MEMBER: Uh-huh.
july10-671	DR. STIBITZ: Right. So those aspects -- the matrix itself, which would probably be considered a medical device, I believe there are biocompatibility studies that has to be done as part of that, and then we would collaborate with CDRH, Center for Devices, on review of that product.
july10-672	AUDIENCE MEMBER: The last bit. If it's used as a disinfectant, how's that going to be reviewed?
july10-673	DR. STIBITZ: Disinfectant like on surfaces in a hospital?
july10-674	AUDIENCE MEMBER: Yeah.
july10-675	DR. STIBITZ: That's a good one. I'll have to think about that. We are almost exclusively concerned with human studies, so it's not clear -- I mean, clearly, that would not require human study. Exactly what part of FDA would deal with that, it's not clear to me. But you're aware, of course, that CFSAN, the Center for Food Safety and AppliedNutrition, has approved phage for use on meats and fish to decontaminate, but that's a little bit different.
july10-676	DR. CARLSON: All right. So that's all the time we have for this session. We're going to take a quick break. I'm going to say we'll come back at 3 because we're running a little behind. If you have questions for Scott, you can obviously come ask him now, but we'll get back to all these topics during the discussion panel later.
july10-677	DR. CARLSON: (Whereupon, a short recess was taken.)
july10-678	DR. CARLSON: Everybody, we're going to try and get started again, if you can take your seats.
july10-679	DR. CARLSON: (Pause.)
july10-680	DR. CARLSON: Okay. We're going to continue with the FDA presenters. Next up is Doran Fink, also from CBER, a clinical reviewer, is going to talk about regulatory considerations for clinical evaluation of phage products.
july10-681	DR. FINK: All right. Who's ready for some more regulatory talks?
july10-682	DR. FINK: (Chorus of no's and applause.)
july10-683	DR. FINK: Yeah. Is everyone recaffeinated? Good to go? Okay.
july10-684	DR. FINK: So the usual FDA disclaimer. My commentsare an informal communication and represent my own best judgment, not Scott's judgment, that was his talk. This is my own best judgment, and, of course, what I say does not bind or obligate the FDA.
july10-685	DR. FINK: And, actually, as I've been listening to the talks throughout the day today, I've realized that pretty much everything I'm going to talk about has already been covered, so I might as well just skip to the summary slides. No.
july10-686	DR. FINK: (Laughter.)
july10-687	DR. FINK: I'll throw in a few bits of wisdom or maybe not so much wisdom from the clinical regulatory perspective. So I'm going to start out just with a few introductory slides about key variables and overlying regulatory principles for phage therapy. The bulk of the talk will be about considerations for clinical development under IND and licensure of phage therapy products. I'll talk a little bit about personalized phage therapy in this section as well. And then I'll end with some discussion and some additional information about compassionate use of phage therapy products under our expanded access IND mechanism.
july10-688	DR. FINK: So these are not an exhaustive list of variables that are relevant to phage therapy productsthat have been discussed, some in great detail during previous talks, so you can think about phage therapy in terms of spectrum of activity, whether you're talking about defined products, either a single phage or a cocktail active against one or more bacterial strains or species versus a personalized phage therapy product that is selected for activity against a single clinical isolate.
july10-689	DR. FINK: You can think about route of administration, whether it's topical, interlesional, inhaled, intravenous, oral, or others, and then, if you throw in that the product is administered with a device or as part of a matrix, that adds a layer of complexity.
july10-690	DR. FINK: You can also think about whether the phage therapy product is intended for use as a stand-alone product, which we really haven't talked about at all, or whether it's to be used as an adjunct to antibiotics or as salvage therapy.
july10-691	DR. FINK: No matter which of these variables you need to consider, there are a number of regulatory principles that will apply to all phage therapy products. First and foremost, that phage therapy products are by definition, and Scott showed you the regulatory definition in his talk, phage therapy products are biological drugs. They are biologics andthey're drugs. And so, consequently, any clinical use of an unlicensed phage therapy product in the U.S. must be conducted under Investigational New Drug or IND regulations. So, for all intents and purposes at the current time, this applies to all phage therapy products. We don't have any that are licensed for use in the U.S.
july10-692	DR. FINK: And in order to get a phage therapy product licensed for use in the U.S., we will require a demonstration of safety, purity, potency, which is interpreted to mean effectiveness, and consistency of manufacture. And I have safety and potency underlined because those are the attributes that are the chief concerns of clinical development.
july10-693	DR. FINK: So Scott had a slide that was very much like this one in his talk. I'm not going to dwell on it too much, only to show you that, as Scott said, while effectiveness in manufacturing consistency are attributes that are demonstrated in greater detail and with greater certainty, as the various stages of clinical development progress, safety considerations predominate throughout the development process, beginning with pre-clinical development and extending all the way through licensure and beyond.
july10-694	DR. FINK: So what are the safety considerations thatare relevant for phage therapy products? Well, we've heard a lot today that phage are directly active only against specific target bacteria and are presumed for all intents and purposes to be inert with respect to human cells and tissues, and certainly we have a lot of accumulated clinical experience, mostly anecdotal, that would appear to corroborate this presumption. However, for any new investigational product, I think it's still important to consider a couple of safety items.
july10-695	DR. FINK: Number one, whether certain human tissues might be sensitive to components of phage material. So, as an example, you might imagine a patient who has a fulminant lower respiratory tract infection with compromised airway function and whether introduction of a large amount of phage antigen might somehow inflame that tissue and at least initially exacerbate the patient's condition. This is a theoretical concern. It's not something that's been described, but something to think about.
july10-696	DR. FINK: Similarly, one might worry about the potential toxic effects of product excipients or impurities. We've had some discussion today about residual endotoxin in phage preparations. One might also worry about the potential toxic effects of adevice or a matrix that's used to administer the phage therapy product.
july10-697	DR. FINK: And then, in addition to these potentially direct effect-related safety concerns, you might also want to consider indirect effects, such as effects of bacterial lysis at the site of infection, whether the phage might be able to transfer antibiotic resistance genes, and, finally, whether the phage might result in some changes to the microbiome. This would obviously be a greater concern for a cocktail that has a much wider spectrum of activity than it would for a single phage therapy product, but again just something to consider.
july10-698	DR. FINK: So, when one is thinking about initiating clinical development of a phage therapy product under IND, the antibiotic development model will naturally come to mind, and in that model, first-in-human Phase I studies of investigational drugs are typically conducted in healthy volunteers and they focus on safety and, if applicable, which is usually the case for antibiotics, pharmacokinetics.
july10-699	DR. FINK: However, for a phage therapy product, it's unclear how relevant safety and PK data generated in healthy volunteers would be to patients with active infections where the phage may interact with andmultiply in target bacteria. So, as alternatives, one might consider taking into account, of course, the potential risks and any pre-clinical data that are available, conducting first-in-human studies not in healthy subjects but in relatively healthy subjects who are colonized by target bacteria but who do not have active infections or, to take it even a step further, first-in-human studies in less severely ill patients with active infections caused by the target bacteria.
july10-700	DR. FINK: Once you've selected your patient population, then the question is, well, what is your starting dose? What is your regimen? How do you select these?
july10-701	DR. FINK: Well, one approach would be to rely on data from relevant animal models. If there is prior clinical experience with related phage therapy products, that experience might be informative. Alternatively, do you just go with the maximum achievable titer in preparation? These are all possibilities.
july10-702	DR. FINK: Then, once you've started your trial, how do you optimize the dose and regimen for later development? What data can you collect from this first-in-human study and later studies to arrive at adose and regimen that will be the most safe and most effective?
july10-703	DR. FINK: So here are some relevant questions. How informative are pharmacokinetic data for making dose and regimen adjustments? Is clearance from the bloodstream after IV infusion relevant? We've heard an argument that maybe it's not. Is pharmacokinetic data from the site of infection informative? Maybe, maybe not. And how informative are measures of phage activity that are not directly related to morbidity and mortality? For example, does quantitative culture of the target bacteria from the site of infection help you in any way in determining dose and regimen?
july10-704	DR. FINK: Ultimately, the specific data to support initiation of clinical development and the design of early phase studies for phage therapy products will be reviewed by us in the context of IND submissions, and as Scott mentioned, we encourage prospective developers of phage therapy products to request a pre-IND meeting with us to discuss these topics. I have a URL up here on official FDA guidance for requesting formal regulatory meetings.
july10-705	DR. FINK: So let's think a little bit more toward late-stage development and licensure, and the regulatory principles that will be important here arethat labeling requirements are that all indications for a licensed product must be supported by substantial evidence of effectiveness. This substantial evidence must come from demonstration of effectiveness based on adequate and well-controlled clinical studies using a product that is standardized as to identity, strength, quality, purity, and dosage form.
july10-706	DR. FINK: So, as you can imagine, this is going to be challenging enough for defined phage cocktails. It's going to be ever-more challenging for personalized phage therapy. We do have initiatives for development and licensure of personalized medicine products, and so, you know, you can rely on the FDA to exercise regulatory flexibility in its approach to these requirements, but regardless, the intended indication of the phage therapy product will guide the design of the clinical trials to demonstrate safety and effectiveness.
july10-707	DR. FINK: So there are, of course, a number of very important challenges for demonstrating effectiveness of phage therapy products, in particular, those that are intended for use against multidrug-resistant bacterial organisms. Some of the challenges are outlined here.For example, it can be challenging to recruit adequate numbers of subjects with the relevant disease process and pathogen to the intended use, even in larger multi-center trials. It can be challenging to identify and enroll eligible subjects in a timely manner relative to the course of illness. One related challenge is that the bacteria present at the site of infection at the onset of illness may be very different phenotypically from the bacteria that are present after antibiotic treatment has been ongoing for some time and even after phage therapy has been initiated and ongoing for some time.
july10-708	DR. FINK: And, finally, there is obviously the potential for confounding by non-uniformity of concomitant treatments, such as antibiotics and other therapies, especially for critically ill subjects where you cannot ethically withhold standard of care.
july10-709	DR. FINK: Fortunately, there are potential avenues available to address many of these challenges. One such avenue is the possibility of streamlined and/or adaptive trial designs. Joe Toerner will talk next after me and will discuss in some detail the CBER draft guidance on pathogen-focused antibacterial therapies, parts of which may be relevant to development of phage therapy products.Supportive efficacy data from relevant animal models may also be important for supporting licensure. And, finally, there's the availability of alternative licensure pathways, for example, accelerated approval in which approval can be based on a surrogate end point that is reasonably likely to predict clinical benefit, with the caveat, of course, that there's a post-licensure requirement to confirm this benefit.
july10-710	DR. FINK: I've heard a lot from people during the breaks about these and other challenges. Oh, my.
july10-711	DR. FINK: (Laughter.)
july10-712	DR. FINK: Okay. I'll soldier on. So, you know, one suggestion might be that developers who are just starting out on clinical trials for -- is my mike off now? My mike is off.
july10-713	DR. FINK: Okay. So developers who are just starting off on development of phage therapy products might first want to try to minimize the variables that are inherent to the intended use. So, you know, maybe start with disease processes and patient populations where you can minimize those variables. Maybe start with Staph infections, Staph wound infections, generate data that might be more broadly generalizable to other disease processes and build from there.So let's spend a few slides talking about personalized phage therapy, and by personalized phage therapy, in case anyone has not yet been hit over the head with it today, is the situation in which one or more phage selected from a library after screening for activity against specific clinical isolates from a specific patient. And then, on top of that, additional phage may be selected during the treatment course if evidence of decreasing effectiveness due to the development of bacterial resistance or immune clearance is found.
july10-714	DR. FINK: So the principal challenge is that the phage library may include a very large number of uncharacterized phage, so how then to provide this as a licensed product while ensuring safety and effectiveness.
july10-715	DR. FINK: Well, it turns out that we've encountered this type of situation in a regulatory manner in the past related to licensure of minimally manipulated allergenic placental or umbilical cord blood for use in specified hematopoietic disorders, and this situation is described in great detail in the FDA guidance cited below.
july10-716	DR. FINK: Just to boil it down to its pure essence, for these cellular products, each lot of the productis different, but the safety and effectiveness of each lot is ensured by, first of all, an established manufacturing process and, second of all, by specified product characteristics that are used as release criteria for the lot.
july10-717	DR. FINK: Now there's a huge caveat for using this as precedent for phage therapy, and the caveat is that the guidance above was based on a very large docket of data, accumulated over many years, together with advisory committee input on several occasions. So we're talking about a potentially long road ahead to arrive at a similar point for phage therapy products for personalized use.
july10-718	DR. FINK: So it's unclear at this time whether a similar approach would be feasible for personalized phage therapy products. Might be. Might not be. There may be other approaches that are feasible as well. But whether it will be feasible will really depend on this central question: Can safety and effectiveness of an entire phage library be inferred based on specified product characteristics and accumulated experience with a limited subset of phage from that library?
july10-719	DR. FINK: And to break that question down into a couple of different components, first of all, for agiven indication or disease process and usage or route of administration and dosing regimen, is it reasonable to extrapolate safety and effectiveness across different phages? I don't know. Someone out there please tell me.
july10-720	DR. FINK: For a given phage therapy product, is it reasonable to extrapolate effectiveness across different indications or usages? I think that's a higher bar to clear and typically is not the accepted paradigm for licensure of antibiotics.
july10-721	DR. FINK: And so the question that I would ask the field to weigh in on is what variables or product characteristics might be used to predict and prospectively address uncertainties with safety and/or effectiveness, and then how do you apply those predictions to ensuring safety and effectiveness of personalized phage therapy products?
july10-722	DR. FINK: So what does the road ahead look like? Well, right now, after many years of largely anecdotal experience, at least in the modern era, we currently have no licensed phage therapy products available in the U.S., and so while Jason correctly pointed out that at least in the very near term use of phage therapy is likely to be under expanded access, our challenge to you, to the phage therapy field, is toinitiate scientifically rigorous clinical development programs that include adequate and well-controlled clinical trials to support licensure of phage therapy products, and to continue the positive messaging that Scott started before the break, CBER is prepared to assist developers of phage therapy products in addressing this challenge.
july10-723	DR. FINK: Now that doesn't mean that we're going to have ready answers to all of your questions, including many of the big ones, but we will certainly evaluate your proposals and we will help you think about reasonable, feasible, and scientifically sound approaches to address these questions and to develop your products.
july10-724	DR. FINK: So, in the meantime, I'll end the talk with a little bit more information about compassionate use or use under expanded access IND. The regulations for this are outlined in 21 C.F.R. 312, subpart (i), and compassionate use is to facilitate the availability of investigational drugs for patients with serious or immediately life-threatening diseases or conditions. All compassionate use under expanded access is subject to the following requirements.
july10-725	DR. FINK: So, first of all, there is no available comparable or satisfactory alternative. Theenrollment of the patient in a clinical trial, presumably under IND, is not possible. The treating physician should judge that the potential benefit justifies the potential risks and that those potential risks are not unreasonable in the context of the patient's disease or condition. And, finally, providing the investigational drug will not interfere with the clinical development of the product for the expanded access use.
july10-726	DR. FINK: So I cannot overstate that the primary purpose of expanded access use is to provide access to investigational drugs for patients in need. We are happy to do so. However, expanded access use is not intended to facilitate systematic collection of safety or effectiveness data to support licensure, and therefore, expanded access use is not a substitute for adequate and well-controlled clinical trials.
july10-727	DR. FINK: There are several different categories of expanded access, each with its own criteria for initiating use. In general, the level of evidence required increases as the number of individuals to be treated increases. We've heard the most today about individual patient expanded access, which includes emergency use, and for this use, the probable risk from the drug should not be greater than the probablerisk from the disease, as I said a couple slides ago. The next step up would be an intermediate size population expanded access IND. Here, there needs to be evidence that the drug is safe at the dose and duration proposed for use to justify a clinical trial of approximately the same size as the number of patients intended to be treated. There should also be preliminary evidence of effectiveness.
july10-728	DR. FINK: I'd really like to steer the field away from intermediate-size expanded access use. I think whenever possible the use should be in the context of controlled clinical trials because that's where the most useful data is going to be generated.
july10-729	DR. FINK: The last category of expanded access which I'll just mention very briefly is treatment protocol or widespread use. Here, this use generally requires clinical data from Phase II or III trials, and usually there needs to be active pursuit of marketing approval for the investigational drug.
july10-730	DR. FINK: So here are the procedures for requesting expanded access use of a phage therapy product or any investigational drug for that matter. The request can be made as a new IND submission or as a new protocol in the context of an existing IND. The request needs to include applicable administrative CMC, pharm/tox,and clinical information, as outlined in the regulations.
july10-731	DR. FINK: The information that we would require for single patient emergency use is going to be much more limited than for more widespread expanded access use. Ideally, this information would include a clinical history and treatment plan and CMC information about the phage source and preparation, endotoxin content, and sterility, and activity against a clinical isolate.
july10-732	DR. FINK: As you saw from the examples this morning, these are not absolute requirements, and, you know, obviously, it would depend on the clinical status of the patient and the degree of the need. All expanded access use requires documentation of informed consent and IRB approval or, for emergency use, IRB notification after the fact.
july10-733	DR. FINK: CBER can authorize emergency use expanded access for single patients based on informal communication, for example, by telephone communication, oftentimes within hours. The authorization is given for a single treatment course. So what does that mean, single treatment course?
july10-734	DR. FINK: Well, ideally, this would be defined with respect to the duration and the number of doses, butwe recognize that for phage therapy this is not going to be possible a lot of the time, and there may even be some change to the product administered, as you saw in Chip Schooley's example as emergence of resistance develops over time.
july10-735	DR. FINK: So we'll work with you, you know. Come with a proposal, we'll work with you. If emergency expanded access use is authorized, a formal submission is required within 15 days after this authorization.
july10-736	DR. FINK: I have below some contact information for physicians who are considering emergency use expanded access of phage therapy for single patients. There are phone numbers for contacting our office during business hours as well as, after hours, the emergency line, or you can, if you prefer to contact us by email, there's a general address, and then Cara Fiore, who was mentioned in several of the morning talks, has graciously agreed to have her email address made public. She's really the focal point of phage therapy regulation in our office.
july10-737	DR. FINK: Okay. So I'll end with a couple of summary points. Clinical evaluation and use of unlicensed phage therapy products in the U.S. must be conducted under IND. Development and licensure of phage therapy products will depend on product characteristics andintended use. CBER is, of course, prepared to assist the phage therapy field in addressing scientific and regulatory challenges. And, finally, the expanded access IND mechanism is available for compassionate use of phage therapy products but is not a substitute for adequate and well-controlled clinical trials.
july10-738	DR. FINK: So I'd like to thank everyone at CBER who helped with preparation of this talk, and if we have time for any questions, I'm happy to take them.
july10-739	DR. FINK: (Applause.)
july10-740	DR. CARLSON: So we have just a little bit of time. We can take maybe two questions.
july10-741	AUDIENCE MEMBER: Yeah, thank you for that talk. I'm just kind of curious. I work at CDC and there's a real interest there, of course, in antimicrobial resistance and particularly in, you know, the use of fecal microbial transplants and probiotics in potentially treating -- you know, preventing antimicrobial resistance in the gut, in the gut flora or microbiome.
july10-742	AUDIENCE MEMBER: I'm kind of just curious. Do you see any sort of corollaries between your view of how you're going to deal with these issues with using probiotics and regulating those and phage?
july10-743	DR. FINK: Right. Well, I guess the biggestparallel is that, you know, while probiotics may be defined organisms, once you get into the realm of microbiota-based products, you're dealing with a, you know, largely uncharacterized product, and so that may vary from, you know, from batch to batch and lot to lot. And so there you're kind of running into the same questions about, you know, how do you ensure that a given lot of product is going to be safe and effective, you know, based on whatever data you've accumulated with that product to date.
july10-744	DR. FINK: So there are parallels and, of course, we have been working to address those very questions with microbiome-based products as well.
july10-745	MR. OUSTEROUT: Hi. Dave Ousterout from Locus Biosciences. Just curious what your thoughts are on making smaller data sets in terms of Phase II and, you know, the extreme is Animal Rule and how that might be more applicable in phage therapy, particularly for MDR.
july10-746	DR. FINK: Right. So, you know, we do recognize that, you know, powering trials for, you know, MDRO-related indications is going to be difficult. There are, you know, various ways that clinical trials can be structured that might be able to take advantage of smaller sample sizes while stillproviding substantial evidence of effectiveness. Joe might touch on that a little bit in his talk as well.
july10-747	DR. FINK: The issue of Animal Rule is an entirely different issue, you know, altogether.
july10-748	DR. CARLSON: Okay.
july10-749	AUDIENCE MEMBER: Hi, I'm just wondering about the safety of phage therapy. As you mentioned, you know, we have a lot of confidence on the safety of phages and your concern you highlighted will be the impurity and also endotoxin level.
july10-750	AUDIENCE MEMBER: So, if we follow the instruction and do the testing, eventual testing, do we still need to do the standard package for toxicity?
july10-751	DR. FINK: So, by toxicity are you referring to GLP?
july10-752	AUDIENCE MEMBER: Yeah.
july10-753	DR. FINK: General toxicology studies?
july10-754	AUDIENCE MEMBER: Yeah.
july10-755	DR. FINK: Right. So, you know, our position at this time is that based on the available data we do not see a requirement for GLP general toxicology studies for phage therapy products.
july10-756	DR. FINK: Now, there may be certain safety concerns that arise on a product-by-product basis that might be addressed with more focused safety studies that couldbe conducted in animals, but we don't at this time have a requirement for general GLP toxicology studies.
july10-757	AUDIENCE MEMBER: Okay, thank you. Can I ask another one? Okay.
july10-758	DR. CARLSON: Yes, go ahead.
july10-759	AUDIENCE MEMBER: You know, for the phage therapy you are dealing with the bacteria-resistance infections, so therefore it's not ethical to choose negative control, placebo control. And then if you choose the current, the standard treatment, now, if it's actually resistance do you think it's -- what's your suggestion like to choose the current standard when we know probably it's already resistant to it?
july10-760	DR. FINK: Right. So, I think Joe Toerner may have, you know, more to say about, you know, the control arm for some of these trials in his talk coming up. But generally you'd be looking to, you know, demonstrate statistical superiority of the combination of phage therapy plus whatever standard of care treatment is being given, whether it's actually effective or not in your trial.
july10-761	DR. CARLSON: So that is a good lead-in to introducing the next talk. So, next we're going to have Joe Toerner from the Center for Drugs who is going to talk to us about development of singlespecies antibacterial drugs.
july10-762	DR. TOERNER: Hi. Good afternoon. Thank you.
july10-763	DR. TOERNER: I spent six enjoyable years in the Division of Vaccines and Related Product Applications in CBER, and so I really appreciate being here and working and presenting again with friends and colleagues from CBER.
july10-764	DR. TOERNER: About 10 years ago I transferred to CDER in the Office of Antimicrobial Products, and it occurred to me putting this talk together that in the next half an hour I'm going to describe for you what was probably a decade of work in advancement of regulatory science to arrive at recommendations for sponsors interested in antibacterial drug development to have an achievable clinical development program, yet still falls within our statutory requirement that we establish safety and effectiveness of new drugs, and as part of the work that we've done we did include single species antibacterial drug development.
july10-765	DR. TOERNER: And so we recognized over the past couple of years that sponsors are more interested in clinical development programs in areas of unmet medical need, for example, patients with highly resistant bacterial infections. So, we did issue a draft guidancedocument in 2013, and that draft guidance is a guidance for unmet medical need more generally. I know Doran had mentioned a guidance that we have. It doesn't pertain specifically to single species drug development but the concepts in that guidance apply to single species antibacterial drug development.
july10-766	DR. TOERNER: Clinical trials have been completed and in fact some antibacterial drugs have been approved using the approaches in this draft guidance document. For example, ceftazidime-avibactam was an approval on the basis of some of the concepts that were described in this draft guidance document to help streamline antibacterial drugs for unmet medical need.
july10-767	DR. TOERNER: The types of drugs that we're seeing who are interested in this area of unmet medical need are generally drugs that have activity against Gram-negative bacterial, generally Enterobacteriaceae
july10-768	DR. TOERNER: that -- and some of which have anti-Pseudomonal activity, and the link below is the direct link to the draft guidance document.
july10-769	DR. TOERNER: So, in our guidance document we describe some clinical trial design options, and in the guidance we provided a discussion that a single trial in this area of unmet medical need can be adequate evidence of safety and effectiveness.For a more traditional development program we usually require two adequate and well-controlled trials, but a single non-inferiority trial in a body site of infection can be used as evidence of efficacy, and we have a number of different indication-specific guidance documents that clearly describe the end points and the justification for the non-inferiority margin to be used in those guidance documents, and of course a finding of superiority is always readily interpretable. But we have said that you can pool across different body sites of infection for a finding of superiority in a single trial, and to discuss with us what the end points would be for such a trial for superiority.
july10-770	DR. TOERNER: We also described what was a part of an Infectious Disease Society of America White Paper on drug development, and that's the nested trial design where from the beginning a trial was designed for non-inferiority, but as with any patient who enrolls into a clinical trial you subsequently obtain the in vitro susceptibility results and a patient in clinical practice as well may inadvertently have a bacterial infection that's resistant to the control antibacterial drug in such a time, and while you would never design a clinical trial where the comparatorgroup would be ineffective therapy, there does exist at least a potential for a few patients to have received ineffective therapy with a control drug, and it provides an opportunity then to pull that subgroup out and do a separate superiority analysis. So, we describe that type of trial design in the guidance.
july10-771	DR. TOERNER: And what's not specifically in the guidance but we have -- we have done this with the approval of ceftazidime-avibactam is for new beta-lactamase inhibitors where they're pairing with an approved beta-lactam drug, we can rely on our previous findings of safety and effectiveness from the approved antibacterial drug that's paired with a new beta-lactamase inhibitor and show a safety profile of the combination as well as providing evidence that the beta-lactamase is reversing the resistance.
july10-772	DR. TOERNER: And then, of course, a superiority trial design with adjunctive therapy plus standard of care showing superiority over standard of care.
july10-773	DR. TOERNER: So, these are some of the trial design options that we discussed in our draft guidance document, and they -- some of them are applicable to single species-specific drugs, but there is an increasing interest in this area, in particular, drugs that have activity against Pseudomonas aeruginosa orAcinetobacter baumannii, and designing scientifically sound and feasible development programs has been the focus of workshops and advisory committee discussion.
july10-774	DR. TOERNER: We do acknowledge that there are challenges with products that target a single species. They're not commonly identified in any one particular infection type. These patients are generally very ill, often in an intensive care unit setting, and you need to start effective therapy immediately, and the therapy should be empiric therapy because there's often diagnostic uncertainty at the time of presentation, and it's very difficult to identify patients in advance to even approach them for potential enrollment in a trial, and there's difficulty in maintaining a registry of such patients, but we do recognize that there is potential clinical utility of antibacterial drugs that target single species of bacteria, and we want to find feasible solutions to develop these products.
july10-775	DR. TOERNER: And so for the rest of my talk I'm going to be summarizing our discussions at two public workshops and then an advisory committee meeting that we held recently.
july10-776	DR. TOERNER: So, about this time last year we held a two-day workshop on facilitating antibacterial drugdevelopment for patients with unmet need, and we also discussed antibacterial drugs that target a single species of bacteria.
july10-777	DR. TOERNER: We then held another workshop in March of this year on animal model development; in particular, animal models for Acinetobacter baumannii and Pseudomonas aeruginosa, and then we brought all of this information that we gathered from these workshops and presented before an advisory committee meeting in a public discussion.
july10-778	DR. TOERNER: So, the workshop last year was a two-day workshop. The first day was on developing drugs for patients with unmet medical need in general. The second day was devoted to drugs that target a single bacterial species.
july10-779	DR. TOERNER: So, for the first day we did discuss the trial design considerations that were outlined in our unmet need guidance document, and an important issue emerged in that workshop was that there are truly significant challenges in pre-specifying a trial that's designed to show superiority in patients with multidrug-resistant bacteria.
july10-780	DR. TOERNER: And what was also emphasized at that workshop was the importance of obtaining good pharmacokinetic data in the target population ofpatients in the intensive care unit to ensure that you're offering patients the correct dose.
july10-781	DR. TOERNER: And again I provided the link for the meeting documents and the meeting transcript.
july10-782	DR. TOERNER: So, the second day was devoted to drugs that target a single bacterial species, and it was acknowledged that there are difficulties in conducting trials. We did provide a hypothetical case scenario of a drug that had antibacterial activity limited only to Pseudomonas aeruginosa, and so there were several clinical trial designs and topics that were discussed and all of them have challenges and limitations, and in the next four slides I'll go through each of these potential trial designs and clinical development considerations.
july10-783	DR. TOERNER: So, the first consideration was the non-inferiority clinical trial design. As I had mentioned, we have a number of indication-specific guidance documents that describe the end points and the justification for the non-inferiority margin, the treatment effect of a control antibacterial drug, and it was acknowledged that you can enroll in a single trial patients who have hospital-acquired pneumonia or ventilator-associated pneumonia, HABP/VABP.
july10-784	DR. TOERNER: You can enroll them in the same trial andinclude patients that have bacteremia regardless of the source of infection because those patients with bacteremia and multidrug-resistant organisms have the same mortality outcomes as patients with HABP and VABP, so you can enroll them in the same trial.
july10-785	DR. TOERNER: At the workshop discussion, the participants thought this could be a feasible option if we were to consider greater certainty in the efficacy findings.
july10-786	DR. TOERNER: So, for example, if we were to entertain a wider non-inferiority margin than what we describe for a traditional drug development program that you perhaps could have a smaller sample size and these would -- these non-inferiority trials could be a feasible option.
july10-787	DR. TOERNER: Enrollment wouldn't need to be limited to patients who have broadly-resistant organisms. You could enroll all comers, if you will, with these particular types of infections, and the availability of a rapid diagnostic would obviously help identify patients for enrollment but they wouldn't change the frequency with which these infections occur.
july10-788	DR. TOERNER: It was also acknowledged in any non-inferiority trial that you're going to have confounding by concomitant therapies and -- that are often used in this very sick patient population.The second option was superiority trials and, again, this is an obvious finding of efficacy, and here you would want to try to enroll patients who have evidence of broad resistance to available therapies, but these may be very difficult to enroll and identify into a trial. And you could enroll patients with one or more body sites of infection as we've outlined in our draft guidance document for unmet need, but the determination of superiority is difficult. And furthermore, to show superiority may be time-limited because it just depends on the available therapy and whether or not that therapy is considered to be sub-optimal, because once new therapies become available then your ability to demonstrate superiority becomes difficult.
july10-789	DR. TOERNER: So, the third option was to conduct a study in patients with a higher likelihood of having infections due to Pseudomonas aeruginosa such as patients with cystic fibrosis, and you'd need to clearly identify the clinical condition that you were treating whether it be, you know, pulmonary exacerbations caused by Pseudomonas aeruginosa, and then extrapolating the findings from a patient population with cystic fibrosis to a general population with other infections may be challenging.And then the fourth option was approval under the Animal Rule, so this is a setting where efficacy data is obtained from animal models of infection and this is generally done in settings where efficacy trials are not ethical and in the situation that we were considering that efficacy trials may not be feasible to conduct, and we acknowledged that animal efficacy data would likely be supported by at least some clinical data from patients with a variety of infections caused by the single species of bacteria.
july10-790	DR. TOERNER: So, it was this last option that led us to consider another workshop that we held in March. This was our animal models workshop, and we wanted to discuss in greater detail the current state of animal models of serious infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa and future directions in this area.
july10-791	DR. TOERNER: We did have participation from academia, industry, and other government agencies, and sponsors came to present their proposals for clinical development of two products. One had activity against, only against Acinetobacter baumannii. The other sponsor's product had activity only against Pseudomonas aeruginosa.So, the key topics that were discussed was an overview of the use of the Animal Rule to support approval for treatment of plague and treatment of anthrax. We discussed the current role of animal models, their attributes and shortcomings, and given the urgent need for these unmet medical need therapies we entertained what role the animal models would have that would accompany the limited clinical data that we would see in a clinical development program.
july10-792	DR. TOERNER: So, we approached this workshop with sort of some general achievable considerations that you could obtain at least some clinical data but it would be very limited. There would be evidence of activity and perhaps evidence of efficacy in a relevant animal model of infection. There would be robust pharmacokinetic and pharmacodynamic data that would be included in a clinical development program, and an acknowledgement that there would be limited human safety information, and of course we would have the required non-clinical safety data as any drug development program would have.
july10-793	DR. TOERNER: And so what was discussed at this workshop, again, was the concept of the non-inferiority trial, could this be done, and is this feasible, and, again, the use of prior and concomitant effective therapiescould confound the assessment of the treatment effect of the investigational drug.
july10-794	DR. TOERNER: But using a wide NI margin could potentially be feasible, and just to provide an example for HABP/VABP we allow, and we describe in our guidance document a non-inferiority margin of 10 percent for standard development programs, but for products that have and can address an unmet medical need, we consider a wider non-inferiority margin of 12.5 percent that has the effect of reducing the sample size in a trial that could be completed in a sooner period of time.
july10-795	DR. TOERNER: But in this -- in this discussion we did entertain the possibility of considering a non-inferiority margin that's even wider, more equal to the estimate of the treatment effect, and I think an entire talk could be designed on just how we approached and defined the non-inferiority margin for HABP/VABP. But as just a general consideration, and to try to summarize it as best as I can promptly, it took a number of published clinical trials over years to ascertain that ineffective therapy has probably the best mortality rate of about 60 percent.
july10-796	DR. TOERNER: Effective therapy has probably a worst mortality rate of about 30 percent. So that treatmentdifference then is a 30 percent difference of mortality.
july10-797	DR. TOERNER: So, that is a -- and because we're looking at older studies, there are cross-study comparisons, some are observational studies. So, in order to discount some of that uncertainty we define a treatment effect of approximately 20 percent, and so while for unmet need programs we're willing to go to 12.5 percent. Perhaps for single species product development we could consider a non-inferiority margin that approaches more towards 20 percent that would further reduce the sample size. And what Doran was mentioning could -- you know, could we work within the fact that we will have a limited trial size and could this potentially be feasible then for a sponsor to pre-specify this as a non-inferiority margin to move forward with clinical development.
july10-798	DR. TOERNER: Again, we discussed superiority trials, and as I had mentioned it's a time-sensitive approach. As new standard of care therapies become available it's not going to be possible to show superiority of an investigational drug, and so sponsors generally aren't willing to pre-specify a finding of superiority when they're planning their efficacy trials.
july10-799	DR. TOERNER: And so you'll see on this slide a lot ofthis language is very similar to language that's in the Animal Rule, but when considering even evidence of activity in an animal model we would want to know that the effect is demonstrated in more than one animal species, and that it's expected to react with the response predicted for humans; that the animal model infection is relevant to the clinical condition being studied in humans; and that the end point in the animal model is actually a -- is similar to the desired benefit in humans, which is generally survival or prevention of major morbidity.
july10-800	DR. TOERNER: And so, you know, at the conclusion of the two workshops that we had we thought, well, what
july10-801	DR. TOERNER: are -- what are potential outcomes of these types of programs that we talked about, and the best scenario is the first one, that we have a successful clinical trial with a finding of superiority or non-inferiority, acknowledging the limitations, and there are no major safety concerns.
july10-802	DR. TOERNER: The second possibility is that we just -- there's just no evidence to support a meaningful benefit, and similarly, the fourth scenario that the safety concerns do not allow a favorable risk/benefit assessment. You know, those are situations we don't like to see sponsors be in, but those would be moreclear scenarios of the findings of a clinical data package.
july10-803	DR. TOERNER: We have an interest in this third potential scenario which is that you can't really discern efficacy in the completed clinical trial that's small due to multiple confounders and to what extent then can we rely on the animal models of infection in such a scenario?
july10-804	DR. TOERNER: And just so I won't forget to mention, in our guidance document we do allow a very limited population at the dose and duration of therapy, approximately 300 patients is what we describe in our guidance document.
july10-805	DR. TOERNER: So, we took all of this information from the workshops and we presented summary information to our April 13th advisory committee meeting. We also presented information that was discussed in the public from the two sponsors who presented their proposals for clinical development scenarios, and the two key topics were development programs for single species-specific antibacterial drugs where the bacterial species is not commonly identified, and should a clinical development program not be feasible or the clinical data are not interpretable, what is the role of the animal models of infection.And so here on the slide is just a summary of our meeting -- of the discussion by our advisory committee, and the committee agreed there is an unmet medical need and that species-specific products are important for continued development.
july10-806	DR. TOERNER: But the next two bullet points were important for us to hear: that trials in humans can be conducted. They're complicated, they're difficult to do, but they can be conducted. And the third bullet point that there are limitations in the current animal models of infection and that the results of animal model studies should not be used as the sole source of efficacy. So, those two bullet points were important for us to hear.
july10-807	DR. TOERNER: They did find some interest in the presentations of the clinical development strategies in favor of the non-inferiority clinical trial design. For example, the investigational drug that has activity against Pseudomonas aeruginosa, if you pair that with ertapenem, that has a notable lack of activity against Pseudomonas aeruginosa but has broad coverage for many other bacterial pathogens that you'll be worried about empirically when starting therapy, you could design a non-inferiority trial with this as your test arm, compare that to a drug that hasefficacy against Pseudomonas aeruginosa and that would enable you then to show non-inferiority of the investigational drug for Pseudomonas in the patient population that has Pseudomonas aeruginosa.
july10-808	DR. TOERNER: Other comments from our Committee members were global clinical trials networks and it's noteworthy that in last week's New England Journal of Medicine Drs. Woodcock and LaVange from CDER described the concepts of having platform clinical trials, and described the antibacterial drug development as a potential area where having a platform clinical trial could -- could help industry, academia, and regulatory authorities to work together, and thought that rapid diagnostics would help enrollment in a clinical trial.
july10-809	DR. TOERNER: The Committee also talked about some post-marketing strategies. Is there a possibility for a drug distribution network? That was a question raised by our Committee members. Can we limit the indication to 'salvage,' for use only as a last option? And then our Committee members reminded us that we now have an operational Sentinel system where we can evaluate post-marketing safety and to make use of that.
july10-810	DR. TOERNER: And so I included the link here too for the Advisory Committee presentations and transcripts.So, the punchline is that in CEDR we are working with sponsors to design clinical trials that will establish safety and effectiveness of single species drugs, but we're willing to exercise flexibility and show greater uncertainty in that clinical development program that would allow a smaller clinical trial to be conducted.
july10-811	DR. TOERNER: We want sponsors to conduct robust pharmacokinetic analyses in the patient population that would use these drugs, and we're still
july10-812	DR. TOERNER: interested -- because of the uncertainty in the clinical development program, we're still interested in establishing animal models having greater certainty, greater understanding of the results of the animal models, so we're still interested in that component because that could still be supportive of the clinical trial findings in an overall data package.
july10-813	DR. TOERNER: So, I thank you for your attention and happy to answer any questions.
july10-814	DR. TOERNER: (Applause.)
july10-815	DR. CARLSON: Given the fact that we're a little bit behind time we're going to go to the panel in just a couple of minutes, I think, since I see one of our panelists. Brian, did you have a question?We're just going to go to the panel.
july10-816	AUDIENCE MEMBER: I have a question. On the panel? Okay, I follow you.
july10-817	DR. CARLSON: Yes, we'll just start the panel discussion now.
july10-818	AUDIENCE MEMBER: Okay.
july10-819	DR. CARLSON: And since you're going to be up here you can ask your question. So, I'll invite all the speakers and our two extra panelists to come up. The additional panelists are Cara Fiore who is a primary reviewer in CBER. You've seen her name on the screen a few times today, and Marion Gruber, the director of the Office of Vaccines.
july10-820	DR. CARLSON: And I'm told to remind all the panelists to speak directly into the microphones so that you are heard by the people in the overflow rooms.
july10-821	DR. CARLSON: So, we can go ahead and get started with your question.
july10-822	DR. GRUBER: Okay. Well, thank you. So, I had a question for Joe. Actually, I thought it was a very intriguing discussion here and I think we can -- we should really benefit from having further discussion with that division and to see, you know, if we can borrow from some of the approaches that they have mapped out.In that regard I wanted to ask Joe. So, you talked a bit about, you know, the value of doing non-inferiority or superiority trials for these, you know, single species, drugs or therapies. What I wanted to know is a bit in these clinical trial designs, the non-inferiority as well as superiority trials, the end points that you would be looking at. So, that's one question.
july10-823	DR. GRUBER: And the second perhaps related to this, you mentioned clinical trials and you mentioned Animal Rule approval, but you didn't really discuss the accelerated approval provisions that are also available to us, so I wondered if you could comment on that a bit.
july10-824	DR. TOERNER: Sure. Thanks, Marion, for the question.
july10-825	DR. TOERNER: So, we have -- we've done quite a bit of work to establish the end points for our indication-specific guidances, and many of the end points are different. So, for example, for HABP/VABP we found a strong treatment effect on the end point of all-cause mortality. So, clinical trials are being designed and conducted in HABP/VABP, and the primary efficacy end point is an end point of survival. And so that's one example.Another example is complicated urinary tract infection where we found a strong treatment effect on an end point of -- it's a responder end point where patients have to have microbiologic eradication on a urine culture after treatment, and they have to show evidence that their symptoms of urinary tract infection are gone, are resolved. And so that responder end point was found to have a very strong treatment effect and, you know, a third example is complicated intra-abdominal infection where we expect 28 days after completion of -- 28 days after enrollment we expect the patient to be free of symptoms from their complicated intra-abdominal infection, and so those are three very different types of end points.
july10-826	DR. TOERNER: And so if you're entertaining a clinical trial where you're enrolling lots of different infections, that's where we say in the guidance come and talk to us about how to approach this, and we've already had a strong discussion that allows patients with bacteremia at any site, any body site infection because their survival rate is identical to the survival rate in HABP/VABP, you can enroll those patients in the same trial and have the end point of survival.But how to approach a clinical trial where you're enrolling patients with complicated urinary tract infection, complicated intra-abdominal infection, HABP/VABP, you know, what's the end point to be used. We think for a finding of superiority you could probably use a combination of end points, but it may take some work to sort out how to approach this. Are there statistical concerns that we have to think about? Should we give more weight to survival end point and give less weight to a complicated urinary tract infection end point? You know, is there a way to weight the different end points in the patients?
july10-827	DR. TOERNER: So, those are some considerations that we have. We have thought about accelerated approval and in fact there is a brief paragraph about it in our draft guidance document, but because our clinical trial end points always occur within a couple of days or weeks with therapy, you know, the course of therapy is short, your clinical benefit is achieved -- is known in a very short period of time, we're finding it very difficult to apply the principles of accelerated approval where you have a surrogate end point.
july10-828	DR. TOERNER: But in the case of, you know, these infections there's really not a need for a surrogate. You know the clinical outcome at a very short periodof time during or after completion of therapy. So, we've found it challenging to apply the principles of accelerated approval to antibacterial drug development.
july10-829	DR. YOUNG: I don't have a question but I do have a suggestion for Doran and Scott. I'm getting inundated by phone calls and emails requesting information about clinical trials for phage therapy. And so I would hope that you would consider taking a subset of those beautiful slides the two of you showed and putting together a website at the FDA that we could send people to explain there are no clinical trials in the United States, and outline the other procedures that are open to them.
july10-830	DR. YOUNG: For example, having their physician explore eIND and the mechanism, because this is going to get worse and the publicity is increasing and this is a very unusual situation as somebody pointed out; it's something that we -- you know, lots of people think it's going to work but it's years away from any type of clinical approval. Just a suggestion.
july10-831	DR. FIORE: I actually have a question for Joe. In terms of the platform approach could you, for those of us who are not familiar with the platform clinical trial approach could you tell us what thatwould look like?
july10-832	DR. TOERNER: No, it's actually I would -- I'd refer you to the July 6 New England Journal of Medicine. There's a review article by Dr. Woodcock and Dr. LaVange, and as you know Dr. Woodcock is center director and Dr. LaVange is the office director for biostatistics. And in their review they discuss, it's mainly trials in cancer research where they have platform trials where you're enrolling -- it's just a way to -- it's just a way to enhance clinical development in cancer therapies.
july10-833	DR. TOERNER: One example is the I-SPY trial and another example is the Lung-MAP trial, but you're enrolling patients with different phenotypes of cancer because drug -- you know, oncology is getting more focused on, you know, what -- focused direct development that pertains to the expression of, you know, tumor expression factors, and so they want to capture a large number of potential patients into a trial, and so it's a way of having one trial, and so it's actually -- master protocol, is the title of the -- so, having a continuously running functional master protocol it's -- you know, you continue to enroll patients in a protocol, and if you don't have an antibacterial drug ready to go you're gathering dataon patients enrolled in the protocol on standard of care therapies, and then once you get an investigational drug that's ready to go you plug that into the master protocol, have it randomized controlled.
july10-834	DR. TOERNER: You can rely on -- I mean, to some extent it's a -- it's a historical control but when you plug in the new investigational drug into a master protocol you then can randomize so you have a component of a randomized concurrent control study, but you can also rely on some of the information you've obtained from your previously enrolled patients who have standard of care therapy and you can consider a three-to-one or four-to-one randomization, and it's just a way of efficiently doing a clinical development and multiple sponsors can then use the master protocol, so you could have two or three different investigational drugs that are entering and exiting the master protocol.
july10-835	DR. GABARD: Maybe a couple of comments. For all these products, phage therapy products that are going to target a single bacterial species when we are going to do comparisons with the standard of care and antibiotic, and if you want to show superiority and non-inferiority, the only segment where we canreally show that, because most of these antibiotics are fairly efficient, is on a subgroup of bacteria which are resistant to the antibiotics.
july10-836	DR. GABARD: And if the subgroup is the only choice to show the superiority or the non-inferiority then you need to recruit forever because you know that these cases are not so frequent, and then the level of recruitment is so low that it will take you maybe five years to get the proper number of patients to show the superiority. What can we do to avoid this problem?
july10-837	DR. TOERNER: I guess that question is -- I mean, it is a question of antibacterial drug resistance, and you are -- in a rough analogy you are comparing this to the Infectious Diseases Society of America and their nested clinical trial design. It just depends on how you set up your clinical trial. If what the Infectious Diseases Society of America
july10-838	DR. TOERNER: is -- and our guidance -- what we're saying is you set up your trial for non-inferiority and you seek out to establish non-inferiority.
july10-839	DR. TOERNER: It's only at the end of the day that you come to recognize that some of the patients may a have resistance phenotype. You can then pull those patients out and do a superiority, but you still have the clinical trial to show evidence of efficacy bynon-inferiority in the patients who have fully susceptible bacterial pathogens.
july10-840	DR. GABARD: Have you been thinking about expanding the therapeutic area to several therapeutic areas with one treatment? So, in other words, would it be possible to, instead of treating only with infection with a single product which is targeting one bacterial species, that you take in account in a trial several therapeutic areas. For instance, with infection and maybe ulcers and maybe something that are fairly comparable so that we expand the number of cases where we can provide the treatment to patients and then at the same time expand the number of cases where you might have resistance to antibiotics and then the frequency of the cases.
july10-841	DR. FINK: I think, Joe, you mentioned, you know, a strategy similar to that where you have enrollment of patients with multiple disease processes in the same trial, although in your scenario the unifying principle is that all patients get the same antibiotic against the same bacteria, so that would have to translate.
july10-842	DR. FINK: I think, going back to your first question, I think we do acknowledge, you know, for phage therapy there is an added complexity or challenge with respectto demonstrating even non-inferiority which is that, you know, unless you're willing to remove all concomitant antibacterial therapy with activity against the organism of interest like you might do with the, you know, investigational product plus your ertapenem strategy, then you really can't demonstrate non-inferiority.
july10-843	DR. FINK: And so I don't think that the phage therapy field is at a stage yet where we have the confidence of, you know, going it alone with phages for, you know, an infection of interest in covering, you know, everything else with empiric antibiotics, but I think you have to definitely think about that more.
july10-844	DR. LEHMAN: This is not a broad answer to that because this is a special case, but one of the things that -- one of the scenarios where that -- where we might not have some of the same problems. We found with chronic rhinosinusitis the patient population that really has that unmet medical need is a population that has already been through rhinoplasty and multiple rounds of antibiotics, and they still are experiencing symptoms that are not life threatening, but make their lives fairly miserable, and it's an unusual situation. That's why I say this is not a broad answer.But it is one indication where we may have an easier time collecting some of that data because, you know, standard of care for the patient population that we used in our clinical trial was a sinus wash. It's a saline wash. It relives some symptoms, but doesn't provide a long-term decolonization or eradication of the infection, and that's a scenario where there is an option to look at a placebo-controlled situation where the standard of care is not that great because it's not life threatening, not dealing with that same problem.
july10-845	AUDIENCE MEMBER: So, we've talked a great deal about sort of the clinical development side. I was wondering if we could take a moment for the non-clinical. So, I guess the question is what are the additional considerations or perhaps notable exceptions for non-clinical data and in particular just to get you to an IND, and in light of some of the things you may run into in the clinic, a more robust IND package?
july10-846	DR. FINK: So, you know, proof of principle in a relevant animal model is always nice. I don't know that it's an absolute requirement to initiate, you know, clinical trials, but it's certainly nice to have. PK data, to the extent that it might be usefuland, you know, that's an open-ended question, is -- you know, it's non-clinical information that could help guide and support the initial clinical, you know, trial design.
july10-847	DR. FINK: I don't know if there is any, you know, in vitro information that --
july10-848	AUDIENCE MEMBER: I think you're kind of nailing it in the sense that these parameters aren't exactly well defined as they are for a small molecule brethren, right. So, you know, your tox study, is that done in an infected animal or is a healthy animal okay? What does that signal really mean? You know, it's these types of situations for phage therapy as an active therapy that don't seem that clear.
july10-849	MR. STIBITZ: So, my view of pre-clinical data prior to Phase I trials is -- I mean, we would only be talking about safety studies. I think we are not surprised, that we kind of expect sponsors to do proof of concept, to do studies that convince them that proceeding with this makes sense, but in terms of what's actually required to go into the first human study we're really looking at safety, and I don't know if it's been clearly --
july10-850	DR. GRUBER: No, I mean, I just wanted to add to what Scott was saying but I think this is sucha new area that we are actually, and this is part of the reason why we are having this workshop, because we would like to hear from you, too, you know, what makes sense, you know; what should be, you know, recommended. We are not having any requirements right now. I think this was already clearly stated by Doran to say that we're not asking for the typical GLP repeat dose toxicity studies that we have been asking for other products the Office of Vaccine regulates; and that we are also right now, we don't borrow from the small molecule drug development paradigm, but -- and it was also mentioned if there are some directed safety studies that we feel would be needed, you know, when we have the discussions with you when you come and propose a clinical trial that is something that we can then further elaborate on, but at this point this is a fairly new area and field for us, and we're really, you know, trying to map out a non-clinical/clinical development program that makes sense and that is feasible and scientifically defensible, and that's actually one reason why we're having this workshop because we also would like to hear from you, you know, what does make scientific sense.
july10-851	DR. GRUBER: I think you've heard, you know,proof-of-concept studies, you've heard, you know, characterization data that were outlined in Scott's talk, you know, in vitro studies as applicable, and that's where we are right now. But, again, I mean, we invite comments from the audience on these -- on these questions.
july10-852	DR. CARLSON: And I should have started the panel off by saying what we're looking for is really a discussion between the regulators and interested parties to try and figure these things out in some instances.
july10-853	DR. FIORE: So, I just want to add that the pre-clinical/non-clinical studies are often very important for you to inform your development plan, and if you have those studies, you know, we'd love to see them, but they could be more important for you in some cases than they are for us.
july10-854	AUDIENCE MEMBER: Fair enough. Thank you.
july10-855	AUDIENCE MEMBER: I have a question for colleagues from FDA, and this topic is about the phage substitution or phage addition in the approved cocktail, for example, and it has different, of course, subtopics like CMC and clinical efficacy.
july10-856	AUDIENCE MEMBER: The question is how do you think the industry and regulators would initiate thediscussions? What would it take to replace the phage in the approved cocktail from the CMC standpoint and from the clinical efficacy standpoint?
july10-857	AUDIENCE MEMBER: For example, stability data, we cannot generate let's say two years real-time real condition stability or if you're talking about clinical efficacy if the requirement would be go for Phase II, Phase III again this would make this impossible. Would you please elaborate on these a little bit? Where do we start to discuss this?
july10-858	DR. FIORE: For myself and my colleagues here do you mind defining when -- are you talking about coming in with a defined cocktail and then switching out or are you talking about a panel of phages?
july10-859	AUDIENCE MEMBER: The defined one.
july10-860	DR. FIORE: Defined one, okay.
july10-861	DR. STIBITZ: Well, again I'll ask a question back to you. Are we talking about a licensed product?
july10-862	AUDIENCE MEMBER: Yes.
july10-863	DR. STIBITZ: And then you want to change the phage makeup.
july10-864	AUDIENCE MEMBER: Right, yes, to replace or to add an additional one, for example.
july10-865	DR. STIBITZ: Right. So, my understanding, and my colleagues can correct me if I'm wrong, is that you could submit a BLA supplement to make those changes to the product.
july10-866	DR. STIBITZ: Now, exactly how at some point it could be different enough that we consider it to be a new product, but I think the devil as always is in the detail. So, are we talking about a similar phage from a genetic perspective and it's a variant? Is it a brand new phage that, you know, you just isolated?
july10-867	DR. STIBITZ: I think -- I mean, we can talk about, you know, exactly how we want to pursue that in the structure of our regulatory process. In other words, is it a new BLA? Is it an amendment and so forth? But I think in general it will be possible with, you know, the same CMC information and enough information about the applicability of that phage to have it included.
july10-868	DR. STIBITZ: I know that's not terribly precise but I think it's the best --
july10-869	AUDIENCE MEMBER: This is going to be treated in a case by case depending on the data available, right?
july10-870	DR. FIORE: I just want to clarify because what I heard you say is Phase II and Phase III, butScott's referring to a marketed product. When you use the word 'license' we mean already approved and marketed, and out there for clinicians to use.
july10-871	DR. FIORE: So, during your IND development you would submit that to your IND, or if you had a master file, which I am a huge proponent of, which would include all your CMC information, you would submit it to your master file or IND with the same type of information that you would submit with your other phage products.
july10-872	DR. FIORE: After licensing it's a little bit different. It may be slightly more complicated and more expensive, but nonetheless it would go through the process that Scott was talking about.
july10-873	DR. GRUBER: Yeah, I just wanted to add that it may be a little bit premature to discuss the type of product characterization data that's required after you have licensed a defined, you know, phage cocktail, because we have to actually see first what really makes sense, what product characterization data would be required, you know, all the way -- and if clinical data even would be required. I'm not saying that this would be the case but I think we are a little bit ahead of ourselves. I think the criteria or the type of information requested to support a supplement to a license for a defined phage cocktail is something thatwe need to discuss, but once out there I think we would have a set, you know, of required information. I would not think that this would be case by case at that point in time, but I think right now we need to get some clarity first if you -- you know, let's say you have a defined cocktail that is not licensed or you were to swap phages, you know, what type of studies, what type of data would be needed. I think this is something that we would need to start in order to really define what is requested once these products are licensed.
july10-874	AUDIENCE MEMBER: Okay, thank you.
july10-875	AUDIENCE MEMBER: I understand there is a general assumption of safety for the most part, but I was curious if there are specific safety concerns. For example, I saw some recent findings about impact of phage therapies on the microbiome. So, I'm just curious your thoughts on that or if there are specific safety concerns that you may have going forward because I -- not that it was sort of brushed aside but I do understand in the field there is a general assumption of safety.
july10-876	DR. FINK: So, I presented a couple of, you know, safety considerations that one might think about in my talk that are related either to, you know,direct effect of the phage material on human tissues or indirect effects such as microbiome changes. I don't know if anyone else has any more specific --
july10-877	DR. GILL: Do you want to go first? You go ahead.
july10-878	DR. STIBITZ: All right. This will be short. I mean, I think the problem -- we have the tools to look at changes in microbiome that might be associated. We don't have the knowledge to interpret what those changes mean. So, in many ways we're in the same region that we're in with FMT and live biotherapeutic products to some extent.
july10-879	DR. STIBITZ: And what was the other -- oh, and the other thing is just, I mean, certainly there will be some changes to the microbiome, but I think we all think that those will be more acceptable than the massive changes you get with wide spectrum antibiotics.
july10-880	AUDIENCE MEMBER: A couple of small observations. Any decisions --
july10-881	DR. CARLSON: We had a little more feedback on the last question here. Just a second.
july10-882	DR. GILL: The other thing that we've talked about is, you know, the possibility of horizontal gene transfer made by phages and that can be screened for. We're looking for transducing phages. And so as wasbrought up earlier some phages they degrade the host chromosome early in the infection cycle, and so if you use only phages that do that they're unlikely to transduce.
july10-883	DR. GILL: And another thing is that it depends on how the phage is packaged, their DNA into the head. So, some phages are quite permissive and others are very site-specific, and so if you have a phage that doesn't necessarily degrade the host chromosome, but if its DNA packaging is very, very specific to its own DNA then I think we were looking -- it's not that it will never ever transduce, but as long as the transduction is lower than what you normally get, you know, just from the normal traffic of DNA in that ecosystem, then I think it should be okay.
july10-884	DR. CARLSON: Just to follow up briefly on the question of microbiome damage. Joe, is this something you guys consider in terms of antibiotics, even single-species antibiotics? Is that something that's looked at as a safety signal?
july10-885	DR. TOERNER: That's a good question and we have not specifically looked at that issue. There are a number of concerns with it. How are -- you know, how are the cultures ascertained; what's the -- you know, how do you go about knowing what the microbiome-- it's such a dynamic -- yeah, how would you begin to characterize what's considered to be normal and what's considered to be not normal.
july10-886	AUDIENCE MEMBER: Okay. I was just going to say there are quite a few steady state late stage infections, chronic area infection, possibly Randy Fish's work on toe ulcers where there is no standard of care, and those can provide a way in. I'll say if you want to know about that I'll tell you afterwards, happily.
july10-887	AUDIENCE MEMBER: The second thing is, of course, in regard to the last question doses in phage therapy can be very tiny indeed, microgram, nanogram, even down in one study to picogram doses. So, input toxicity is an issue that needs to take that into account.
july10-888	AUDIENCE MEMBER: But my actual question was for Scott, and it was -- you made a very interesting comment that a GM product, it will be about what was added and how you're changing the GM agent which is then introduced. So, how would then would be regarded a zero residue removal? For example, taking out a lysogeny cassette from a phage where there are no lytic phages as with Clostridium difficile. If you did a zero residue removal of the lysogeny cassette, how would that be regarded?DR. STIBITZ: Well, that's the only kind of removal we do in my lab, but you're talking about a completely clean deletion, for example, in-frame in a repressor gene, correct?
july10-889	AUDIENCE MEMBER: Yes.
july10-890	DR. STIBITZ: So, I mean, I think the answer is really the same. I think phages that have been genetically modified or genetically engineered are not viewed really any differently than wild type phage with the exception that we know a change has been introduced and therefore we will want to understand the results of that change.
july10-891	DR. STIBITZ: So, I think when you're adding something there are perhaps more questions than when you're simply removing the repressor.
july10-892	AUDIENCE MEMBER: I have one question and second one is like a suggestion. The first question is, is there any chance to use historic safety data for phage therapy for this type of, you know, approval process?
july10-893	DR. STIBITZ: You're talking about historic controls for a clinical trial?
july10-894	AUDIENCE MEMBER: Right, historic safety data. Historic phage --
july10-895	DR. STIBITZ: What the occurrence would havebeen without the intervention, is that correct?
july10-896	AUDIENCE MEMBER: Right.
july10-897	DR. FINK: Or are you talking about historical safety data?
july10-898	AUDIENCE MEMBER: Historical safety data, mainly historical safety data.
july10-899	DR. FINK: Yeah, I don't know that we would really consider that. I guess it would depend on exactly what the nature of the data is.
july10-900	AUDIENCE MEMBER: Okay.
july10-901	DR. FINK: Is it with the same product? Is it with a closely related product? How closely related? How long ago? How similar were the, you know, monitoring procedures to the procedures that, you know, we would typically require to determine safety? All of these are questions that, I think, you know, kind of stack the deck against, you know, relying on historical safety data.
july10-902	DR. FINK: So, I don't want to come out and say under no circumstances absolutely, but it does seem a little bit unlikely for, you know, any particular given phage product what historical safety data might contribute to supporting licensure.
july10-903	AUDIENCE MEMBER: Okay.
july10-904	DR. STIBITZ: Do you have a particularexample in mind?
july10-905	AUDIENCE MEMBER: Yes. One thing was like 1931 or sometimes they did a Staph or a -- I think it was a Staphylococcus, the clinical trial in USA. Yeah, so they did a trial and they showed that 31 percent cases they are successful and there is not much adverse effect, something like that it was -- so, this type of data, can you mine this type of data, mining, and can, you know, produce to FDA to find out that what they think about it, you know.
july10-906	DR. GRUBER: I don't have a lot add to what Doran just stated. I think it would really, really depend. So, let's say if a sponsor would come and propose that safety information to us as supportive or pivotal demonstration of safety for a product in a given target population against a specific condition in 2017, I think we would, of course, look at that data, but I don't think, you know, we can give you an answer here on the podium to say yes, that would be acceptable or no, it would not be acceptable. I mean, it really would depend.
july10-907	DR. GRUBER: But I have to agree with Doran. I think it's rather unlikely.
july10-908	AUDIENCE MEMBER: So my second point is this. I am hearing a lot of -- about the problem withthe transductions. With this transduction things is happening in the environment, you know, millions and millions of time, and not only that, the plant biologists use phage randomly to hose down the trees and other things and they don't, you know, check all of their phage or phage composition, you know, for transduction ability.
july10-909	AUDIENCE MEMBER: So, why are you worried so much about that, you know, little transduction? What is going to happen when they inject some phage, you know, in human body?
july10-910	DR. STIBITZ: So, this is the way that I think of it and I think I've convinced my colleagues to think about it. It's sort of a belt and suspenders approach.
july10-911	DR. STIBITZ: If you're using strains to propagate your phage for therapy that are completely free of any troublesome genetic material, it's probably not as important. But it seems more and more likely as we're talking about isolating phage from nature for a particular patient isolate, that maybe -- and then perhaps adapting that phage to that isolate, it seems more and more likely that we will be growing the phage on virulent strains, and in that case I think it becomes essential to make sure that you're notdelivering to the infection site, you know, additional little care packages with weapons in them because this is not a theoretical concern at this point. You can measure the degree, the number of transducing particles in a lysate.
july10-912	AUDIENCE MEMBER: So then why EPA doesn't control it? Because in environment if you release this type of transducable phage it can cause problem to transfer the antibiotic genes and other things.
july10-913	AUDIENCE MEMBER: So, my point is that why they don't control it and why when we come to this type of, you know, clinical treatment at that time we consider it so much. Environmental biologists are using phage, lots of phage. They don't do all those type of study and they release this phage for farm and also for poultry and industry, and they are using it, you know, to clean the poultry housings.
july10-914	DR. STIBITZ: I'm not positive I understand your point, but I think what we're getting at is perhaps adding 10 to the 9th, 10 to the 10th phage particles into an existing infection with what 10 to the 5th, 10 to the 6th, 10 to the 7th, transducing particles if it's a transducing phage.
july10-915	DR. STIBITZ: So, I mean, I think -- I believe you're making the argument, and correct me if Imisunderstood, that this is happening in nature all the time.
july10-916	AUDIENCE MEMBER: Yes.
july10-917	DR. STIBITZ: And so I think it's largely a numbers game to some extent.
july10-918	DR. LEHMAN: I'd also posit that the risk assessment for that is a little bit different when you have a human patient in front of you than in an environmental setting.
july10-919	AUDIENCE MEMBER: (Away from microphone.)
july10-920	DR. LEHMAN: It could easily be asked in the other direction as well. If the human therapy field finds it important, should the animal side of things and the environmental side of things also find it important. There are two directions in which to ask that question, and I know that at least -- my knowledge of the food animal portion of this is somewhat limited, but I know that in at least some cases the phages are intentionally applied after the animals have basically been removed. They've been removed from interaction with the rest of the herd or the flock.
july10-921	DR. LEHMAN: I'm not saying that that's happening in all cases but I know some of the people who are working on that do care about that because they are concernedabout confining the effect to just the treated population so as not to just have broad environmental exposure.
july10-922	DR. LEHMAN: The comment was that EPA is asking these questions now.
july10-923	DR. CARLSON: Go ahead.
july10-924	AUDIENCE MEMBER: I have two easy questions and one comment. I know that a number of guidance you have new antibiotic development for a range of indications. I understand from your talk that we can reference those guidance for phage development, right? That's one.
july10-925	AUDIENCE MEMBER: The second one is a comment. The comment is that when we choose the standard treatment and we use the data actually for any drug where it's actually developed, when it's new and at that time it's no resistant, itâ€™s actually sensitive bacteria, therefore the data usually it's generated when it's -- everything, it's sensitive, no resistance. But when the time you compare with it actually it's very high resistance, so the data in the literature usually not reflect the situation. So, that's my comment.
july10-926	AUDIENCE MEMBER: Actually whether it's non-inferiority or superiority, we would choose the marginal use at that time, sometime can be difficult.DR. TOERNER: Thanks for the comment. Also our guidance documents clarify that when you are doing the non-inferiority analysis you have to ensure that the control drug has activity and is shown in in vitro susceptibility to be susceptible to the control drug in order to establish non-inferiority to the investigational drug. And so that is part of the population that's used for the efficacy analysis in a non-inferiority trial.
july10-927	AUDIENCE MEMBER: The last one I ask that you opinion on the definition of the standard of care treatment. So, what is it? Is it the most commonly used drug or it's a drug you find in the society guideline?
july10-928	DR. TOERNER: We define standard of care therapy and there's a definition we provide in the guidance documents, and it's a drug that's approved for the treatment, but we recognize in some cases it may not have that specific approval yet standard of care guidelines provide the recommendation for its use, and so we say if there's enough data you can provide to us a rationale for why you want to use a particular comparison drug, and there may be very good reasons for doing that.
july10-929	AUDIENCE MEMBER: Yeah.DR.
july10-930	TOERNER: You may want to have a blinded trial, and the only comparison drug that's administered twice daily, maybe one that doesn't have that specific indication. Yet it's recommended in treatment guidelines, or that the twice daily dosing isn't in the FDA labeling, but there are other data that support uses of twice-a-day administration. So, we are willing to be flexible and you just -- you know, sponsors can just provide a strong rationale in the use of the comparator drug and why it's felt to be effective.
july10-931	AUDIENCE MEMBER: That's good because I do come across all these situations. I do see society guidelines recommend a drug which is not approved in the country. I do see recommended drug in the guidance it's not the most prescribed drug either.
july10-932	DR. TOERNER: And it's important to recognize too, clinical trials are global, and so there are some drugs that are available in other countries that -- and they're available here but they may not have that specific indication that they have in the other countries, and we recognize that and look to professional societies for their guidelines as well.
july10-933	AUDIENCE MEMBER: Thank you.
july10-934	DR. FIORE: I would like to add to what Joe said to address your first comment about the guidances.
july10-935	DR. FIORE: So, Center for Drugs and Center for Biologics we do have some shared guidances and we also have separate guidances, so just to keep that in mind, and a draft guidance is a draft because we're still accepting comments on them whereas the final guidance is final.
july10-936	DR. FINK: And just to add on to that in case it isn't already clear. We don't have a guidance that is, you know, specific for -- that specifically covers phage therapy at this time. So, while Joe went over a number of CEDR guidances for antimicrobial products that, you know, portions of which may be relevant to development of phage therapy products those guidances were not written with phage therapy in mind, so just a caveat.
july10-937	AUDIENCE MEMBER: Thank you. That was the best possible lead-in to my question.
july10-938	AUDIENCE MEMBER: So, we've talked about sort of two different arenas today. One is phage therapy and development of phage therapies and one is development of products, antimicrobials that meet an unmet need. And to your point exactly, there's a little bit of a disconnect inmy mind between what is most important to FDA on the unmet need side versus on the phage side, and I think that taking a step back we can sort of anticipate that phage are going to be, at least initially, in the clinic used in areas of unmet need, maybe areas of MDR or maybe areas where there is a second or third line defense rather than adopted out of the gate as a first line use. Therefore, this kind of puts them, we can predict, into an unmet need use kind of situation.
july10-939	AUDIENCE MEMBER: What I'm hearing is that for the antimicrobials that address an unmet need there's a lot of emphasis on PK and pre-clinical data whereas it's kind of the opposite for phage, where the PK situation is very hard to nail down because of the self-replicating nature of phage, not as concerned about the pre-clinical animal models, not even looking at the toxicology necessarily, and sort of moving straight into the later phases.
july10-940	AUDIENCE MEMBER: So, how do you synthesize the conversation that you all have had around phage development versus the conversation around products that address an unmet need when truly what we're talking about is a product in the Venn diagram of both of those things that overlaps both?
july10-941	DR. FINK: So, can I take this first stab?Okay. I think the point that I took away from Joe's talk is that for these products that are intended to address an unmet need, and phage therapy, as you've said, certainly, you know, would fall into that category for certain uses, it's going to be challenging to accumulate clinical data, clinical trial data of the type that would usually support licensure for antimicrobial products.
july10-942	DR. FINK: And so what CDER has decided and what their advisory committee has agreed with them on is that some, you know, less robust package of clinical data could conceivably be supported with animal model data as well as PK data because those PK data are very useful. Now, for phage therapy products PK data may or may not be useful.
july10-943	DR. FINK: And so if we were to, you know, go along a similar path, you know, we might say that licensure or demonstration of effectiveness could be supported by some, you know, package of clinical data that's feasible to achieve, plus some animal model data where the animal models are reasonably relevant, plus whatever other non-clinical or in vitro data might help to inform the effectiveness of the product. And so, you know, it may not turn out to be PK data, itmay turn out to be something else. I would, you know, love to hear from the audience out there what -- what do you think those data should be.
july10-944	DR. GRUBER: I just wanted to make one additional comment before we let you answer, and that is, you know, I think we're not really looking at this point to reconcile what, you know, is asked in the world of, you know, anti-infectives and, you know, phage therapy.
july10-945	DR. GRUBER: I think what was interesting is, you know, the paradigm that Joe's division worked through to see, you know, how can clinical trials for these type of products be conducted to support development and licensure, and we, you know, invited Joe today to really, you know, explain this to us to see how they approach this very complex field and to see can we borrow, are there some common, you know, themes or elements, but I don't think we are at the point yet that we can say okay, you know, this is sort of the paradigm that we would follow for phage therapy clinical trials, yet there are some interesting approaches and we would love to really discuss those further, and again hear your perspective on that.
july10-946	DR. FIORE: I'm sorry, if I could just addto that. One of the elements that could also possibly be added to in a package is something that I haven't heard mentioned although I did see, I think, maybe on Joe's slide, is post-marketing, and then also some element of our expedited programs which is a guidance document. Thank you.
july10-947	AUDIENCE MEMBER: And I had one more comment to follow up as well, and I apologize I neglected to introduce myself at the beginning. I'm Lucia Mokres. I'm the chief medical officer of EpiBiome, which is a small company working on bacteriophage therapies.
july10-948	AUDIENCE MEMBER: And I want to disagree with the comment that it's too soon to think about post-market manufacturing and changes to manufacturing. Early stage companies are really at the forefront at a lot of this development. A lot of big pharma companies are not willing to take on the enormous risk that it would take to get a phage therapy progressed through a clinical program.
july10-949	AUDIENCE MEMBER: As such, we're really contingent or dependent on the investment of venture capitalists. There is no grant -- amount of grant funding and non-diluted funding in the world that will bring a product all the way through the market, and one of thequestions that we always get from potential investors is what happens when resistance develops or a new strain emerges or, you know, like is this going to be like the flu vaccine that gets updated. And if we can't answer that they won't invest.
july10-950	AUDIENCE MEMBER: So, I'd like to encourage everybody in the room to kind of not be afraid to have those conversations early because they do matter and early stage companies do need to grapple with them, at least have an idea of what that might look like earlier than one might think if one had a continuous revenue stream and could just kind of cross that bridge later.
july10-951	DR. GRUBER: Yeah, the point is well taken and perhaps I was misunderstood. What I was trying to convey here is that we're right now trying to really get our arms around, together with interested product developments, to see what are the criteria about which we can characterize, you know, a new phage to be introduced in a defined cocktail. And as long as we don't really have that clear and mapped out under the IND, you know, how can we provide guidance here, you know, for something that may be approved in the future?
july10-952	DR. GRUBER: But you're point is well taken. I mean, this is -- you know, clinical development strategies,what would be required, you know, overall thinking about this, you know, how -- how the tests and methodologies apply then to a licensed product, I mean, is something that's, of course, part of the discussions to be having with the product developer during the IND stages. Thank you.
july10-953	DR. FIORE: And just to add what Dr. Gruber said, it's going to be possible. We just can't give you a concrete answer. It's not like it's unfathomable. It's going to be possible. We just can't give you an exact concrete answer exactly how you're going to do it at this point in time.
july10-954	DR. CARLSON: We can do one or maybe two more questions. We're pretty much out of time for the day.
july10-955	AUDIENCE MEMBER: I'm JeShaune Jackson from EpiBiome as well. Promise we didn't plan or practice the synergies there but, you know, but great presentations individually and collectively a ton of knowledge so far on this panel.
july10-956	AUDIENCE MEMBER: My question goes to another kind of question that we get asked sometimes, too, and that's if you're treating sometimes like non-life-threatening diseases, where we talked about a single bacteria and, you know, Pseudomonas and all these other ones, but if it's non-life-threatening, what is the potential option for doing like over-the-counter or off-the-shelf or, you know, or even like a nutraceuticals route as a -- like at what point does the FDA have to step in and regulate that for phage therapy?
july10-957	DR. FIORE: If you're planning to use a product to cure, treat, mitigate or prevent a disease you need an IND. It doesn't have to be life-threatening. In fact, we have many products that luckily aren't used for life-threatening situations, but you need an IND and you go through the IND process, and we certainly can help you with that.
july10-958	DR. FINK: The requirement -- one of the requirements for expanded access use, and I'm thinking about particularly emergency use for single patients, is that the product has to be intended to treat a serious or life-threatening disease or condition. So, it can be serious or life-threatening.
july10-959	DR. FINK: What does serious mean? Well, there's a -- we typically draw on our guidance for expedited development of drugs to treat serious diseases or conditions, and under that guidance serious is defined as it causes a substantial impact on day-to-day function.
july10-960	DR. FINK: So, if the patient is suffering substantialimpact on day-to-day function from their disease or condition then that would be considered serious and would qualify for expanded access use.
july10-961	DR. FIORE: I apologize. I thought you said non-serious.
july10-962	AUDIENCE MEMBER: I'm saying non-serious, like, you know, acne, uncomplicated UTIs or like skin care, women's health.
july10-963	DR. FIORE: So, if you're trying to treat you would need an IND and we would help you through that. So, the IND process is for any drug development. So, we don't -- it wouldn't be -- if it came to us it wouldn't be a nutraceutical or anything like that.
july10-964	DR. GABARD: Maybe a couple of ideas to fuel the discussion. From our own experience with three different regulatory agencies I can provide some information to you.
july10-965	DR. GABARD: Regarding the kinetics of the phages, what we have been agreeing with the three agencies is that we would test the concentration of phages at a thousand-fold -- one hundred to a thousand-fold above what was expected to be administered to the patients in healthy mice and in healthy pigs.
july10-966	DR. GABARD: So, the mice got one hundred the times ofphage that we provided to the patients, and the pigs got one thousand-fold times the amount of phages that would be provided to the patients. Those animals were healthy without any bacterial infections, and we followed the course of the disappearance of the phages in organs and in fluids, and that was agreed by the agencies.
july10-967	DR. GABARD: Concerning the effect of the phages in infected organisms, during the course of the Phagoburn studies we also have been following the concentration of the phages day after day each day of the treatment during 14 days to see what was happening to the amount of phages as the bacterial infection was disappearing, and that was agreed also by the authorities.
july10-968	AUDIENCE MEMBER: Thank you.
july10-969	DR. CARLSON: I think at this point unless anyone on the panel has anything else to say we're going to have to end the discussion for now, but I'm sure everyone is willing to stick around for a little while if you have more questions for them, and we'll continue again tomorrow starting at 8:30.
july10-970	DR. CARLSON: Roger, do we have any announcements or anything for tomorrow? No. Okay. Don't forget to bring your I.D. badges back tomorrow or it will be difficult to get into the building.Thanks, everybody. We'll see you tomorrow. (Applause.)
july10-971	DR. CARLSON: (Whereupon, at 5:00 p.m., the workshop in the above-entitled matter was adjourned, to reconvene at 8:30 a.m. the following day, Tuesday, July 11, 2017.)
july11-1	DR. RANALLO: Good morning, everybody. Sorry about that. Good morning. I hope everybody's up. Okay. So we're going to start on time today. My name is Ryan Ranallo, I'm a program officer here, at NIAID, and I'm going to be your moderator for the entire day today, something the organizing committee didn't tell me before they signed me up for this. Nevertheless, hopefully we'll get through it all day today.
july11-2	DR. RANALLO: So one thing that I wanted to just note is how in two years, how things have changed significantly since the last time we've held a phage therapy meeting, and so, with that, I think we have a couple of large buckets of topics today, phage engineering being one of them, and essentially looking at phage for different uses, including, you know, transmission and decolonization.
july11-3	DR. RANALLO: So, with that -- oh, the only other thing I would say is if you have any questions about whether or not your slides have been loaded for speakers, please check in the back. Marcus has been great all day yesterday, and certainly today as well. So for the first talk, it's a tag team talk of Col. Zapor and Lt. Col. Tyner. Col. Zapor is the deputy commander of operations at the Walter Reed Army Institute of Research, and Lt. Col. Tyner, who I first met actually when I was a post-doc at NCI and -- in Building 37, is the director of bacterial diseases branch, which just actually happens to be my old department where I spent 10 years at Walter Reed working on enteric vaccines. So, without further ado, I'm going to introduce Col. Zapor and Lt. Col. Tyner for our first talk.
july11-4	DR. ZAPOR: Okay. Good morning, everybody. Thanks to the organizers for inviting me to speak at this conference. Unfortunately, I'm only here for the morning session because of conflicting obligations, as well as secondary to car problems, but -- so I'll be here until lunch and then depart after that.
july11-5	DR. ZAPOR: As you heard, I'm splitting my 30-minute block with a colleague, Lt. Col. Tyner, so I'll be cognizant of the fact that I have 15 minutes to speak to ensure that he has 15 minutes as well.
july11-6	DR. ZAPOR: So the purpose of this talk, I was asked to speak about potential therapeutic indications for bacteriophages and first thought we would kind of address some of the limitations of the current -- antibiotics and the current problems. So antibiotics of course have been the mainstay of therapy in the -- for the treatment of infections for decades, but there have been some unintended consequences. Everybody of course is familiar with the issue of the emergence of multidrug-resistant organisms, in some cases extremely drug-resistant organisms, or even pan drug resistance.
july11-7	DR. ZAPOR: Moreover, antibiotics, as effective as they are, are not 100 percent specific. In the parlance of my profession, we unfortunately see considerable friendly fire, especially with the broad-spectrum antibiotics such as the carbapenems, and so oftentimes the antibiotics are effective in eradicating the intended target, but have the unintended consequence of killing benign, or even beneficial, bacteria as well.
july11-8	DR. ZAPOR: This is evidenced, for example, by the emergence of C. diff colitis in patients who are on broad spectrum antibiotics.
july11-9	DR. ZAPOR: Other limitations with antibiotic use of course include the emergence of drug resistance. I've already spoken to that. Some types of infections are less amenable to treatment than other types. So infections which involve abscesses or other sequestra, antibiotics generally don't penetrate abscess fluid very well, some less well than others. Rifampin works fairly well, but there are many other antibiotics that are inactivated in abscess fluid. Aminoglycosides come to mind.
july11-10	DR. ZAPOR: Additionally, the presence of a foreign body can make infections difficult to treat. Foreign body -- we've seen a considerable number, a very large number, of war wounded coming back from Iraq and Afghanistan status post blast injuries with retained foreign bodies. Some of these can be removed surgically, some cannot. Some are intentionally left in place.
july11-11	DR. ZAPOR: Each of these FBs becomes a potential nidus for infection. They get colonized with bacteria, oftentimes bacteria that elaborate glycocalyces or make a biofilm, and there are very few antibiotics that can reliably sterilize biofilms.
july11-12	DR. ZAPOR: Other considerations include patient anatomy. So I gave the analogy or offered the example of war wounded. Patients who have had blast injuries oftentimes have interruptions in their blood supply, they have interrupted vasculature, and all the tissue prior, distal to the injury becomes ischemic, starved for oxygen, starved for blood, and antibiotics can only work where they're delivered, and if antibiotics are not delivered to vascularized, oxygenated tissue, then they don't work very well. It's very common for us to see patients who have ischemic limbs, necrotic tissue, retained foreign bodies, and antibiotics just don't work very well. More often than not the intervention of choice for those patients is cold steel, for example, amputation, rather than medical therapy alone.
july11-13	DR. ZAPOR: And then there are other considerations such as the rapid metabolizers. We know that there are some patients who just inherently metabolize and inactivate antibiotics and other drugs more rapidly than other patients.
july11-14	DR. ZAPOR: And then we always have to be cognizant of patients who have drug allergies or some other contraindication to antibiotics. So, examples that come to mind, beta lactam allergies, which are fairly common, nephrotoxicity associated with aminoglycosides, associated with vancomycin and so forth.
july11-15	DR. ZAPOR: So, for all these reasons, antibiotics, as effective as they are, as reliable as they have been, they do have their limitations, and, as a consequence, we're forced to explore alternatives.
july11-16	DR. ZAPOR: So what are some of the pros and cons of using phages as therapy? This is a table I put together with which you may or may not agree. In the pro column for phages there's long history of use. Everybody knows that phage has been used in Eastern Europe for many years, and at one point in time early in the 20th Century, phages of course were available by prescription in this country.
july11-17	DR. ZAPOR: Phages are ubiquitous, they're fairly easy to isolate, they're much more specific than our antibiotics. We don't see that friendly fire, so to speak. Phages potentially are active against MDROs. Probably benign, as far as the patient is -- patient goes.
july11-18	DR. ZAPOR: Phages are bactericidal. At least the lytic phages are. Phages, I think, are gaining acceptance. Certainly in Europe, both East and Western Europe, phages are getting more use and have a wider acceptance. And then phages also provide an opportunity to present an opportunity for us to pave the way in publishing evidence-based, peer-reviewed articles supporting their use.
july11-19	DR. ZAPOR: On the con side, although phages have been used for many years in Europe, there is a paucity of high quality literature. Much of this literature has not been translated. We've got some folks over at the WRAIR, at the Walter Reed Army Institute of Research, we've asked to translate some of this literature.
july11-20	DR. ZAPOR: Phages need to be propagated under controlled environmental conditions. Phages are highly specific, and so just as that may be an advantage, that could be disadvantageous as well if we're looking at patients with polymicrobial infections, or if we have a phage that's only specific against a particular species or strain, then we may be forced to look at cocktails in order to sufficiently treat a patient with an infection.
july11-21	DR. ZAPOR: Bacteria can acquire resistance to phages. We don't yet know what the host response will be. You know, the role of antibodies formed against phages, for example. I know over at the WRAIR there is a lot of concern about phages being lysogenic rather than lytic, and I know that's a concern from a regulatory standpoint as well.
july11-22	DR. ZAPOR: Phages are viewed skeptically in the United States. I will tell you, as an infectious diseases physician, that a lot of my colleagues are very critical about phages. You know, they see this group as a little eclectic, and phages are a little bit like voodoo.
july11-23	DR. ZAPOR: I don't mean that to sound pejorative or facetious, but, you know, I'm here trying to tell you from a clinician's perspective how I think we can get a wider acceptance of phages therapeutically. But I know that I've engaged some of my colleagues over at the hospital where I spent 12 years, engaged some of my colleagues over at the hospital about clinical trials, and I get this kind of raised eyebrow response. So that poses a challenge.
july11-24	DR. ZAPOR: So my opinion, for whatever it's worth, probably worth about two cents, we don't know if in vitro activity yet portends in vivo activity. In other words, if phages will behave or will perform for us in the laboratory as they do -- in the clinic rather, or in the operating room as they do in the laboratory.
july11-25	DR. ZAPOR: Moreover, we don't yet know what the clinical indications might be. I don't imagine there are many people in this audience who are arguing that phages will be effective against every infection conceivable. Rather, what we need to do is identify those in particular clinical indications for which there is a use for phages.
july11-26	DR. ZAPOR: But that said, my perspective at least is the issue of emerging drug resistance forces us to consider modalities and therapeutics that maybe we wouldn't have considered years ago. So I think our backs are up against the wall, figuratively speaking.
july11-27	DR. ZAPOR: I think that phages amongst the clinical community are most likely to be accepted and considered useful if we offer them as adjunctive therapy: to be used with antibiotics, perhaps with surgery, to be used in situations in which medical management alone is problematic or antibiotics might be ineffective or contraindicated, or -- and I think this is a big selling point, and I know, I believe there's at least one surgeon in the room -- if we can tell the surgeons we have a therapy that may potentially obviate the need to remove infected hardware.
july11-28	DR. ZAPOR: I can tell you, as an infectious diseases physician, I spend a lot of time consulting, or providing consultation with orthopedic surgeon colleagues, and the last thing they want to hear from the ID doc is the hardware has to come out.
july11-29	DR. ZAPOR: That poses technical challenges, both with the removal and subsequent replacement of hardware. And so if we can tell the surgeons we have a modality which may enable the patient to retain the hardware, I think then you're going to get some buy-in from the surgeons. And lastly, look, whether or not phages live up to their expectations, at least we'll be able to do -- you know, with the experiments we're doing, at least we'll be able to say, you know what, we studied these rigorously, we subjected them to the rigorous scientific method, unfortunately, phages don't work, but we know we did the experiments right, controlled studies, and these were our conclusions.
july11-30	DR. ZAPOR: So what are some of the potential indications? Abscesses and other infections in which antibiotics have limited activity. So one that comes to mind, for example, is osteomyelitis, right? Bone infections.
july11-31	DR. ZAPOR: Mainstay of therapy for osteomyelitis is place a PIC line, give the patient six weeks of intravenous antibiotics, take the patient to the OR, debride the infected bone, all right? And if there's hardware involved, the hardware may have to be removed. More often than not, it has to be removed.
july11-32	DR. ZAPOR: So, boy, it would be great if we could offer phages for the treatment of osteomyelitis. Now there's some intrinsic limitations to that. Ischemic bone is not vascularized and, you know, it may have an issue getting phages there in the first place, but that remains to be seen. Pocket device infection -- the one that comes to mind would be something like a pacemaker infection. Pacemakers are very common in this country. They're placed in a small pocket over the pectoralis muscle over the chest. When they become infected they generally have to be removed because untreated pocket device infections are potentially very dangerous, as you can imagine.
july11-33	DR. ZAPOR: With pacemaker leads, these go into the myocardium, the heart muscle, and the last thing you want to do is have infected pacemaker leads leading into the myocardium, and so they have to be removed.
july11-34	DR. ZAPOR: I think, as far as this goes, we may end up really looking more for a prophylactic role for phages than a therapeutic role because I think we would be hard-pressed -- we'd have a difficult time selling the cardiologist on retaining an infected pacemaker, you know, while we inject phages into the pocket.
july11-35	DR. ZAPOR: I think it may be an easier sell to say at the time you place the pacemaker in the pocket, why don't we add some phages that are active against the common culprits at gratis: Staph aureus, right, or coag-negative Staph.
july11-36	DR. ZAPOR: Orthopedic hardware-associated infections such as patients with intramedullary rods, external fixators, plates and screws -- very common. Since the wars in Iraq and Afghanistan in 2003 and 2001, respectively, the commonest reason for consultation at Walter Reed for ID has been 20 something year-old male, status post blast injury, traumatic amputation, placement of hardware, now with a hardware-associated wound infection.
july11-37	DR. ZAPOR: As I mentioned earlier, telling the surgeons that the hardware has to come out is usually not met, you know, with a good reception. Boy, it would be great if we could introduce a therapeutic that would enable us to salvage hardware.
july11-38	DR. ZAPOR: Burn infections. These are typically associated with very drug-resistant, slimy, gram-negatives such as Pseudomonas aeruginosa and some other related GNRs. Maybe there's a role there. Essentially, anything with biofilms. Catheter-associated urinary tract infections. We know that every patient with a catheter in his or her bladder eventually will acquire bacteriuria. That is bacteria in the urine. Many of those patients, most of those patients over time will go on to have catheter-associated urinary tract infections.
july11-39	DR. ZAPOR: How do we treat those? We remove the catheter, we give them antibiotics, and we put another catheter in, and so it's only a matter of time until they become re-colonized and re-infected. Maybe there's a role for phages there, obviating the need.
july11-40	DR. ZAPOR: The other one I want to address quickly is mesh infection. Surgical mesh, right? You go and you have your herniorrhaphy, you have your hernia repair, surgeon puts in nylon mesh or Gore-Tex mesh, that becomes infected.
july11-41	DR. ZAPOR: Removal is very difficult. It's not as simple as just snipping some sutures and just plucking it out because it gets epithelialized, the tissue grows over that mesh, and now you're looking at an en bloc resection. Maybe there's a role for phages there.
july11-42	DR. ZAPOR: And then other potential indications include patients with cystic fibrosis, right? These patients have lung infections, chronic recurring pulmonary infections characterized by very drug-resistant, gram-negative bacteria such as Burkholderia cepacia, Pseudomonas aeruginosa, and so forth. Extremely drug-resistant -- multidrug-resistant organisms. Some other indications I'm not going to address may be the treatment of patients with bacillary dysentery.
july11-43	DR. ZAPOR: Our priorities at the WRAIR. Right now we're interested in looking at orthopedic hardware- associated infections. I'm going to hand off to my colleague in a second to talk about some of the experiments we're doing there, and also perhaps using phages to treat patients with Shigella, shigellosis, bacillary dysentery.
july11-44	DR. ZAPOR: So, look, this is a 39-slide presentation. Yesterday I narrowed it down to 29 slides. I'm only on five. I think I'm out of time. I told you I'd be cognizant of my time, so I'm going to stop here. I will be here until the lunch break. If anybody would like to discuss this further, I'll happily stick around for a bit. Otherwise, I'm going to hand it off to my colleague, Lt. Col. Tyner.
july11-45	DR. TYNER: Good morning. Hi. I'm Steve Tyner. Those of you that were here for Schooley's talk yesterday probably saw my name in one of his slides, and I think my phone number, too. Joke's on him, though. I didn't turn -- I haven't activated my voice mail.
july11-46	DR. TYNER: So I'm going to try to run through this quickly. I think Col. Zapor did a great job, and other speakers have done a good job of highlighting where the problems are. This is just to emphasize that my group works on primarily two areas: militarily-relevant wound infections, and we'll talk a little bit later about bacillary dysentery, or shigellosis.
july11-47	DR. TYNER: You guys know that. So we have two different approaches. One of these approaches, which is this library-to-cocktail approach that you're going to hear from Dr. Biswas and Dr. Regeimbal later, is really a collaboration with the Navy. We interact with the Navy with this to help evaluate the therapeutics that they develop. We do not develop precision cocktails on the Army side.
july11-48	DR. TYNER: What we do work on de novo in-house, Dr. Mikeljon Nikolich who is participating in this workshop, is -- fixed cocktails. So these cocktails are what we call sort of a broad host range, which is really kind of a misnomer, but essentially it's an expanded host range phage, so it targets more strains within a bacterial species than some of the other phages.
july11-49	DR. TYNER: I'm going to talk about fixed cocktails first. These are a number of the different studies that Dr. Nikolich has been working on, he and his team. We partnered with Eliava to look at Sb-1, which is a Staph aureus phage.
july11-50	DR. TYNER: We've expanded that host range in our lab. We've been isolating phages for ESKAPE pathogens, and then beginning to try to select phages for biofilm degrading properties, as well as engaging with one of the other departments that I lead, which is the wound infection department, to look at phages and antibiotics and whether or not there's synergy or not.
july11-51	DR. TYNER: We recently were recipient of an award with JCVI, and I think Dr. Fouts is here, in the back. We're going to be a partnering institute with them.
july11-52	DR. TYNER: Pre-clinical studies. We've been looking at aeruginosa, so, phage against aeruginosa in a wound model. And then, more importantly, clinical studies. We were a partnering organization with AmpliPhi in a phase one safety skin trial study that was held at the Walter Reed Army Institute of Research, or the WRAIR. That was done last year.
july11-53	DR. TYNER: This is an example of some of the phages that we've found against Shigella. Actually found 50 lytic phages. They're active against a bunch of different species of Shigella.
july11-54	DR. TYNER: In fact, the best three phage cocktail was active against 90 percent of the strains from the panel of Shigella isolates from the Armed Forces Research Institute of Medical Sciences. That's that acronym that says AFRIMS. They're located in Bangkok, Thailand. That's an Army lab in Bangkok. So we're beginning to look at assessing our best cocktails for shigellosis in our pre-clinical models which are mouse, guinea pig, and non-human primates, which we all have internal at Walter Reed.
july11-55	DR. TYNER: So for fixed cocktail I'm going to shift now to some of the work which is a little bit more in-depth with precision cocktails. Again, this is a collaborative effort with the Navy.
july11-56	DR. TYNER: Not to belabor the point, I'm sure Dr. Biswas is going to go into much more, and better, detail for the system that he's created than I can, but essentially what they are doing is developing synergistic phage cocktails so that when you lose activity with one phage in this cocktail, another one is still active against the particular bacteria that you are targeting.
july11-57	DR. TYNER: This work that was published in AAC is a collaborative effort between Dr. Regeimbal, who is sitting in the second row over here, and one of my scientists, Dr. Anna Jacobs, who's the second author on this, in which we looked at a five-member phage cocktail and assessed it in a skin and soft tissue infection model. This was against a MDR Acinetobacter baumannii that we isolated from a war wounded subject from Walter Reed in 2010. What this graph shows is that the -- there was a phage they call AB Army 1 which was very active against capsule positive acinetobacter.
july11-58	DR. TYNER: It basically removed all the capsule-positive organisms, and resultant organisms that were left that were resistant were capsule-negative, and so we went back and the Navy found four more phages that were active against the capsule negatives. So, in combination, this eradicated the baumannii phage infection.
july11-59	DR. TYNER: Just briefly, this is the model. These are CP-treated, or cyclophosphamide-treated, animals. These are mice. Then they follow up with three treatments. After the dorsal wound punch, there's a treatment about four hours after, and then for a couple days. Then we measure the wound and we do in vivo imaging.
july11-60	DR. TYNER: On the left you can see the phage cocktail by day five by IVIS has basically removed the wound pathogen, and on the right you can see the biofilm on the occlusive dressing is much less robust in the phage cocktail-treated animal than the animal that was with PBS.
july11-61	DR. TYNER: So the cocktail resulted in a reduced bio-burden, prevention of wound expansion, and a decrease in biofilm formation. So we were very excited about this because this is a great proof of concept for the process that Dr. Biswas and team have developed.
july11-62	DR. TYNER: So we wanted to move further with this, and so we started thinking, where can we innovate? Where we need to innovate is in areas, because we're the military, that are militarily-relevant. I think Col. Zapor did an excellent job of identifying some areas that have cross-over civilian military potential.
july11-63	DR. TYNER: The top on the list for us is orthopedic hardware-associated infections. These are mainly biofilm-mediated. The principal organism that's causing this is Staph aureus. Then we also have an effort looking at enteric infections. So we believe that phages in this setting are going to be an adjunct to antibiotics, and we want to understand how they work in pre-clinical models.
july11-64	DR. TYNER: So I'm going to walk you through the orthopedic hardware-associated infections. We did this in collaboration with the U.S. Army Institute of Surgical Research which is located down in San Antonio, Texas. That's where the Army Burn Center is located. They do a lot of trauma research there, and so they have a very well-developed rat femur pin infection model where they look at therapeutic adjuncts to prevent orthopedic hardware-associated infections.
july11-65	DR. TYNER: So in this animal, day zero, the animal has a cut down, and then there's a non-union segmentation done in the femur, and it's spanned with a wire. Then Staph is added into the wound at that time. The wound is closed. Six hours later they open it back up, they wash it with nine liters of isotonic saline, and then they debride it, much like we would any other service member that's in a -- that's been injured. When they first arrive to the first surgical facility, that's how they're treated.
july11-66	DR. TYNER: We treat then at six hours, and then 24, 48, 72 hours. At that point we stop treating, and then we wait for 14 days, and then the animals are euthanized and we evaluate whether or not there's been a reduction in CFU.
july11-67	DR. TYNER: So off the top this is -- for those of you that are phage guys you're looking at this and saying why aren't you treating all the way through, and there's a good reason for that. The reason is there's a boatload of information, published information, that this organization has done with this model. We need to have a baseline of where we need to begin before we can start modifying the pre-clinical model and modifying how we add therapeutic adjuncts into their system.
july11-68	DR. TYNER: So this is a very challenging model, and you're getting ready to see some data that's not overwhelming, but I don't want to take the wind out of the room. All right. So this is the data. The inoculum was one times ten to the five CFU.
july11-69	DR. TYNER: Phage treatment. We did local, as well as systemic. You can see the different doses that we did there. We did local only, systemic only, and local and systemic, and what we had is we had a slight reduction at day 14. Remember, that's 11 days after the last treatment with phage of aureus in the bone, as well as on the hardware.
july11-70	DR. TYNER: So it's slightly encouraging. It's encouraging particularly because this is a very challenging model. It's also an extremely challenging organism to treat, and it's in a biofilm.
july11-71	DR. TYNER: So there's a number of different things that encouraged us, and we're moving forward and trying to come up with our next steps, one of which is to modify this model so that we shorten the time and we're able to take earlier time points and begin to look at the effectiveness of phage much earlier in the system.
july11-72	DR. TYNER: But I like to focus on the positive. What this did for me, if you're going to look at orthopedic hardware-associated infections, then you need to evaluate your phage activity against biofilms. They are evaluated against biofilms, but the process by which the precision cocktails, and I think the fixed cocktails for the most part, are derived are phage are isolated against organisms that are pretty fat and happy.
july11-73	DR. TYNER: They're planktonic organisms. Staph itself changes its extracellular receptors quite substantially when it's in a biofilm as opposed to planktonic state.
july11-74	DR. TYNER: So if we're actually interested in clinical problems where biofilm is the problem, and that's the reason why it's challenging to treat, then we need to think about how we're isolating phage or how we're assessing phage activity against biofilm.
july11-75	DR. TYNER: In this model there was concomitant antimicrobial use, and we need to assess phage activity with concomitant antimicrobials. I think some papers have recently come out. There was one in January that looked at in vitro phage plus antimicrobials.
july11-76	DR. TYNER: I don't think phage is going to work with every single antibiotic, and we need to assess and understand how well they work both in vitro and in vivo as we're moving forward because, unlike a basic science lab, I'm not interested in studying the phage. What I'm interested in doing is building a therapeutic.
july11-77	DR. TYNER: So I'm looking at making different efforts that we can plug and play and add into a therapeutic development pipeline. The precision cocktail is in collaboration with our Navy partners.
july11-78	DR. TYNER: And then, of course, you know, how phages are administered is an important point, but I think it's less important early than the phage activity against biofilms and with concomitant antibiotics.
july11-79	DR. TYNER: I have been charged to get us back on time. So I've got one more -- I think one more slide that I'm going to show you.
july11-80	DR. TYNER: This is a biofilm assay that Dr. Jacobs has been working on where we're looking at a phage cocktail, I think this is a precision cocktail, against a biofilm. So it's the Staph biofilm. You can see there there's a nice dose response against phage. The biofilm was grown in TSB, plus one percent sodium, plus one percent glucose.
july11-81	DR. TYNER: The literature suggested that this was one of the more accepted ways to grow a Staph biofilm. Literature's a little all over the place, I think, in terms of how people grow these.
july11-82	DR. TYNER: TSB is in no way a non-nutritive media, it is a nutritive media, so that's one caveat, but there is a dose response to phage. So the biofilm was grown for 24 hours, remove all planktonic cells, so it's a fairly -- it's a mature biofilm, from an in vitro perspective. We add phage for 24 hours, then we do CFU and look at absorbance.
july11-83	DR. TYNER: You see a nice dose response, and then you see about a log, log and a half reduction, almost two log reduction, in CFU after treatment. So the phage work in in vitro setting against biofilm.
july11-84	DR. TYNER: So what's going on in vivo? Why is it so difficult to treat in vivo? I think it's a whole â€˜nother hurdle that we're trying to come up with a technical solution for.
july11-85	DR. TYNER: So, with this, I'd really like to thank my colleagues. I really have to thank my colleagues not just at the Walter Reed Army Institute of Research, but at the Naval Medical Research Center, in particular, BDRD. And some of those colleagues are sitting here, in the second row, and then Cmdr. Stockelman's over here, three rows back.
july11-86	DR. TYNER: Without their engagement, their input, their energy and intelligence, it would have been very hard to get to this point. Thank you.
july11-87	DR. RANALLO: Okay. Thanks, Stu, and thanks, Col. Zapor. I appreciate it very much. So we're going to transition a little bit to the next talk. It's by Dr. Breck Duerkop who just recently started his lab in 2016. He post-doc'ed with Lora Hooper at UT Southwestern. His talk is going to focus on Enterococcus and receptors and resistance mechanisms.
july11-88	DR. DUERKOP: All right. Good morning. So I'd like to first start out by thanking the organizers for giving me an opportunity to spend a little time talking about my fledgling laboratory that I just started at the University of Colorado, where we're interested in a number of different aspects of phage biology, one of them focusing on receptors that phage utilize to infect and kill gram-positive pathogens like Enterococcus. All right.
july11-89	DR. DUERKOP: So just a little bit of background on phage receptors in gram-positive bacteria. So there's a number of different moieties on the surface of gram-positive cells that can be targeted by phage, and these include standard polysaccharides that coat the surface of the cells, peptidoglycan which, you know, obviously forms a thick layer around the body of the gram-positive bacterial cell, and then other more interspersed polysaccharides like wall teichoic acid, lipoteichoic acid.
july11-90	DR. DUERKOP: Our interest has primarily been in membrane proteins that are, you know, embedded in the cell wall of gram-positive bacteria, and how phage target these receptors.
july11-91	DR. DUERKOP: So I would argue that gram-positive receptors are kind of understudied in comparison to receptors in gram-negative bacteria, especially in classic organisms like E. coli, but, due to the fact that we're interested in the potential for therapeutics for phage, I think there's a need to better understand the gram-positive cell surface in terms of how phage interact with that molecular body.
july11-92	DR. DUERKOP: Interestingly, I kind of didn't realize this, but there's a lot of interest in phages in the dairy industry for industrial applications due to the fact that large dairy fermentations can usually be destroyed by organisms that are utilized during fermentation by phage such as Lactobacillus and Lactococcus. All right.
july11-93	DR. DUERKOP: So the focus of my lab is really looking at Enterococci, and so these are facultative anaerobic gram-positive bacteria, and they're natural commensals that are found both in the intestine and in the oral mucosa. E. faecalis and E. faecium represent the most common drug-resistant versions of this genre, and they can, under certain environmental perturbations, like antibiotic treatment, go on to form intestinal dysbiosis that can lead to sepsis.
july11-94	DR. DUERKOP: So over the last several years we've been collecting phage from wastewater. This is just an image showing the Dallas/Ft. Worth water reclamation facility where we've sampled a lot of different areas. What we found is that wastewater, as many of you know, is a very abundant source of phage, and specifically for Enterococcal phage.
july11-95	DR. DUERKOP: So we can find these phage in fecal-contaminated water sources, whether this is primary effluent coming directly out of the flow at the facility, or even some of the, you know, more processed water further down the line.
july11-96	DR. DUERKOP: These sewage phage are actually quite effective at killing E. faecalis, and so we've been isolating these over time from these samples and purifying them to high purity to then study their interactions with E. faecalis.
july11-97	DR. DUERKOP: So I'm going to talk to you today primarily about one phage, but what we found was that we have two more or less identical phage at the genetic level. They have some polymorphisms that, you know, make them a little bit different at the nucleic acid level, but primarily these phage are about 97 percent identical. We call them VPE25 and VFW.
july11-98	DR. DUERKOP: I'm just showing you here the genetic organization of these phage. They're modular, as many phage organize their genomes in terms of organizing different regions of the genome in terms of their gene content.
july11-99	DR. DUERKOP: So we've been interested in kind of exploring these Sipho phages as potential targets that can be used to manipulate Enterococcal communities, but the first question we really wanted to answer is how do they actually interact with the cell surface of E. faecalis?
july11-100	DR. DUERKOP: So what we did is we grew E. faecalis in the presence of these phage over time, and we just isolated resistant colonies that came out of these growth cultures. What we found after doing some genomics to basically map resistant genome reads to our reference strain, we found that phage resistance mapped to a membrane protein that was encoded by a gene called EF0858. EF0858 is a homologue of two different proteins that have been described in the literature, one called UEB and Bacillus subtilis, which is known to be involved in phage absorption for a particular phage called SSP1, and then in Lactococcus lactis it has been termed PIP for phage infection protein, and so we kind of went with that nomenclature for our E. faecalis homologue.
july11-101	DR. DUERKOP: So what I'm showing you here is a cross-streak, and you're going to see several of these throughout the talk. Really what this is is we just take the bacteria of interest, we streak it in one direction on a plate, we take our phage of interest and counter-streak that, you know, vertically, and we can look for the presence, or absence, of killing.
july11-102	DR. DUERKOP: What we see is that with VPE25, it can effectively kill wild type E. faecalis. If we knock out PIP by making a clean deletion, you can see that you're no longer susceptible to infection. And we can complement this. So this shows that PIP is sufficient for infection of E. faecalis.
july11-103	DR. DUERKOP: So we wanted to learn a little bit more about PIP. So not much had been really, you know, studied in the literature, other than the fact that it was involved in phage infection. So, due to the fact that we have many genomes now available, we kind of compiled a number of these PIP homologues across the Enterococci, specifically in E. faecalis, and we just aligned these proteins.
july11-104	DR. DUERKOP: What we found was that the N- and C-termini of these proteins are actually quite conserved; however, there's a large extracellular -- there's a large variable region in the center of this open reading frame. What we found was that this variable region, or this region of high diversity, actually maps to a predicted extracellular domain that would, in theory, be on the outside of the cell.
july11-105	DR. DUERKOP: So we were curious if this diverse region actually played any role in the biology of E. faecalis during phage infection. So what we did is we took our two phages and we did cross-streaks -- I'm just showing you this here on a very crude heat map -- where we looked for the sensitivity, or the resistance, of these different phage based on whether thy could be infected by one phage or the other.
july11-106	DR. DUERKOP: What we found was that a number of strains could be infected by both phages, and some phages could actually only infect one strain or another. When we actually did alignments of this variable region in PIP, what we found was that they clustered identically to their susceptibility pattern. So what you can see here is all the strains that cluster in black are susceptible to both phage, whereas the ones in blue are only susceptible to VPE25, and then, vice versa, the ones in red are only susceptible to VFW. So what this told us is that the diverse region in PIP likely drives phage tropism for the surface of the E. faecalis cell.
july11-107	DR. DUERKOP: So we wanted to test this genetically, so what we did is we took a strain called E1Sol E. faecalis, and if you just, you know, reference the map on the right, E1Sol is actually resistant to VPE25, but susceptible to VFW.
july11-108	DR. DUERKOP: So if we actually express the V583 version, which is our standard wild type strain that we work with in the lab, in E1Sol on a plasmid, you can change tropism. So that's what we're showing on the second from the top cross-streak.
july11-109	DR. DUERKOP: And then if we cross that V583 version of PIP into the chromosome and make a clean insertion onto the genome, we get a similar phenotype.
july11-110	DR. DUERKOP: But I think the most important thing is if we actually engineer a plasmid that only has the variable region from V583 that's different from E1Sol -- so this is the last, the very bottom cross-streak you're looking at -- that's sufficient to drive tropism change.
july11-111	DR. DUERKOP: So what this tells us is that the variable region in the surface protein is likely driving the specificity of VPE25 for the surface of the E. faecalis cell and, most likely, the infectivity of those phage.
july11-112	DR. DUERKOP: So then we asked another question. Can we actually, you know, go outside of E. faecalis, and can we expand this to related organism such as E. faecium? This became a little bit more I guess muddy in the sense that when we over-expressed wild type V583 PIP in E. faecium we saw a somewhat mild killing effect on our cross-streak assay, as you can see there.
july11-113	DR. DUERKOP: If we actually spike this phage into growing culture, what we found was that it could inhibit growth, but it didn't actually collapse the culture in terms of, you know, real robust killing like we see with wild type E. faecalis.
july11-114	DR. DUERKOP: So we wanted to learn a little bit more about this. So what we actually did is we actually looked at phage transcription, and we looked at a number of genes -- and I'm just showing you one open reading frame here -- in the presence, and absence, of phage in the different strains. What we saw is that there's, you know -- after 30 minutes there's a large transcriptional up regulation of this ORF123 in our wild type E. faecalis. You can see in our delta PIP mutant that there's virtually no transcription below baseline, or above baseline.
july11-115	DR. DUERKOP: However, in E. faecium what we saw is we saw kind of an intermediate transcriptional phenotype in the wild type version, and then when we expressed PIP in E. faecium we saw that this was elevated by several logs.
july11-116	DR. DUERKOP: But we were never able to actually recover phage from these cultures. So you'd add phage to these cultures, it would slow their growth, but when you titered those cultures you were never able to get more phage out than what you put in.
july11-117	DR. DUERKOP: So what we determined was that these phage are actually infecting, they're replicating inside of E. faecium, but they can't actually get out of the cells. So that's what I'm showing you here, in this bottom graph on the right.
july11-118	DR. DUERKOP: So we basically took these cells and we lysed them by sonication, and then we were able to liberate a number of different -- a number of phage from these bacteria. So what this tells us is that E. faecium actually has a receptor that is sufficient to promote infection, but that once the phage get inside the cell and replicate, they can't actually get out. So that means there's something defective about the holin, or the lysin that doesn't allow the cell to actually be lysed from within.
july11-119	DR. DUERKOP: So I think this is something that should be considered in terms of when we're thinking about engineering phage. If we don't see infection, it doesn't necessarily mean that -- or killing, it doesn't necessarily mean infection is not happening, it may just be that the -- a downstream mechanism has been blocked.
july11-120	DR. DUERKOP: So then of course we wanted to try and apply these phage to an animal model to see if we could decolonize E. faecalis from an environment where it's a native organism, and so we've set up some experiments using germ-free mice. So I come from Laura Hooper's lab. We study -- most people study epithelial cell interactions in the microbiota, so we have many germ-free mice that are accessible to us.
july11-121	DR. DUERKOP: So what we did is we took germ-free -- male germ-free mice, we colonized them initially with E. faecalis, and then six hours later we gave them a single phage treatment. Then we monitored colonization levels at 24, 48, 144, and 216 hours. So six days, and nine days.
july11-122	DR. DUERKOP: We observed a number of interesting things. So initially, at 24 hours we see a modest reduction in the colonization levels of E. faecalis, about roughly a log decrease; however, over time we saw that these levels came right back to levels similar to untreated animals.
july11-123	DR. DUERKOP: And when we actually monitored the phage abundance in these animals over time, we saw that the PFU recoverable from the feces actually decreased considerably.
july11-124	DR. DUERKOP: So we were interested to know whether or not this was due to the fact that maybe the phage were not getting access to the bacteria or if we had the outgrowth of resistant bacteria.
july11-125	DR. DUERKOP: So we looked at bacteria that were coming out of these feces and we sequenced a number of these PIP alleles in E. faecalis -- in these strains coming out of the mouse feces, and what we found was that by 48 hours we were upwards of 75 percent non-susceptible strains coming out of the mice, and by six days we were virtually at 100 percent of the isolates were receptor-deficient E. faecalis.
july11-126	DR. DUERKOP: These were receptors that had not evolved changes in the variable region, but they were mostly truncations, or insertion mutants, or polymorphisms that led to the generation of stop codons.
july11-127	DR. DUERKOP: So the next question, and this is kind of really where I think we're starting to take some of this work, is I kind of talked to you about this protein called PIP, but, you know, what does it do? I mean it probably did not evolve as a protein that's meant for phage to infect.
july11-128	DR. DUERKOP: So one of the things that we're interested in is identifying novel surface receptors using phage to better understand proteins in gram-positive bacteria that might be utilized for lifestyle.
july11-129	DR. DUERKOP: So if you look at the domain organization of PIP, it has several interesting domains. So obviously it has this variable region in the center, but at the N-terminus it has this YhgE PIP domain which is actually conserved in some type 7 secretion proteins that are considered to be part of the potential apparatus of the type 7 secretion system in Staph aureus, and then at the C-terminus, interestingly enough, there's a major facilitator super family domain.
july11-130	DR. DUERKOP: These domains are largely involved in transport of small molecules either inside or outside of the cell. So the fact that PIP is highly conserved across the Enterococci, not just in E. faecalis, but E. faecium, and the fact that phage use this to actually infect the bacteria probably suggests to me that, or it suggests to me that this is likely an important protein for some component of its lifestyle.
july11-131	DR. DUERKOP: So we set up an experiment where we took wild type E. faecalis and our PIP mutant that was marked with a tetracycline cassette and we just did a co-colonization in antibiotic-treated mice.
july11-132	DR. DUERKOP: What we found was that by comparing the competitive indices, so the ratio of the wild type to the delta PIP, over time, during colonization we found that the wild type outcompetes the PIP mutant by about -- after about two weeks. We see about, you know, roughly, on average, about a log out competition.
july11-133	DR. DUERKOP: So what this tells us is that PIP may be involved in niche adaptation, it may be involved in some aspect of colonization, and so we're going to spend some time now in the future to really kind of run down whether or not this plays any specific role in colonization.
july11-134	DR. DUERKOP: Okay. So I've talked to you so far about phages that infect through a PIP mechanism. So what about phages that infect in a PIP-independent manner. So there's a phage that some of you may be familiar with. It's a very old phage. It's called NPV-1. It was originally isolated by Gary Dunny's lab from wastewater back in 1990. It's a Sipho phage that has a non-contractile tail and a prolate head, and it has -- compared to our VPE25 and VFW phages, it has a very limited host range.
july11-135	DR. DUERKOP: So you can see here that it only infects, out of at least the collection -- the strains that we tested from our collection, it only infects two: OG1RF and JH1. It infects in a PIP-independent mechanism because it can kill OG1RF delta PIP mutant and it can also kill the wild type, but it can't kill V583.
july11-136	DR. DUERKOP: So, again, we wanted to, you know, use genomics to figure out what the receptor is for NPV-1, and so we did -- we used a similar strategy to what I described to you earlier in the talk. We came up with one isolate that we call OG1RF-C. It's an NPV-1-resistant strain, and it was generated from a confluently lysed agar plate of OG1RF delta PIP.
july11-137	DR. DUERKOP: We did whole genome sequencing on this strain, and we found three polymorphisms. We found a polymorphism in epaR, which is a sugar transferase, bgsB, which is a glycosyl transfer, or glycosyltransferase, and then iolA, which is a malonic semialdehyde dehydrogenase that's involved in inositol metabolism.
july11-138	DR. DUERKOP: So we were interested in the first two because these are actually enzymes that would be involved in changing, potentially, the surface of the bacterial cell. So the epa cluster in Enterococcus has been well-characterized by Barbara Murray's group in Houston over the last decade or so, and it's a polysaccharide that's composed of numerous carbohydrates, including rhamnose, glucose, and others.
july11-139	DR. DUERKOP: So we went in and we made an in-frame deletion of epaR, and what we found was that if you delete epaR, similar to the OG1RF-C strain, you get resistance to NPV-1.
july11-140	DR. DUERKOP: We also made an in-frame, a single in-frame deletion of bgsB, and this did not result in resistance to NPV-1, but it doesn't necessarily mean it's not involved in resistance because, if you can see, OG1RF-C tends to be a little bit more resistant than the delta epaR mutant, so these may actually work together in some way dur -- to promote a fully-efficient infection. So, in conclusion, what I've told you is that some lytic Enterococcal phages use a conserved membrane protein that we call PIP-EF, or the exopolysaccharide Epa, in E. faecalis, an extracellular variable region actually determines phage specificity for E. faecalis hosts, and that phages can temporarily reduce E. faecalis abundance in the mouse intestine, yet resistance is rapidly re -- acquired, suggesting that, you know, cocktail methodologies might be more applicable in this situation.
july11-141	DR. DUERKOP: And then PIP-EF conservation among the Enterococci may be linked to efficient intestinal colonization.
july11-142	DR. DUERKOP: So kind of some of the future directions where I kind of see some of this work going and how, you know, this will contribute to the phage -- to the field of phage biology, and also phage therapy, is we're in a good position now to expand the repertoire of virulent phages that infect E. faecalis. I know there's a number of them out there, and I'm learning more and more every day.
july11-143	DR. DUERKOP: So we're returning to wastewater to -- for new virus discovery. We've actually received 20 Enterococcal phages from the Navy from Biswajit Biswas who generously provided those for us, and we're going to spend a significant amount of time looking for putative receptors for a number of those phages, and then we've started to establish methods for the genetic modification of existing phages to alter receptor specificities and CRISPR technology.
july11-144	DR. DUERKOP: So I guess, from a broader perspective, can we actually use phages to identify conserved proteins that might be indispensable for Enterococcal lifestyle? So phages target surface proteins that are conserved, and sometimes these are important for, you know, the viability of the cell.
july11-145	DR. DUERKOP: So, for instance, PIP-EF looks like it is involved in colonization, but also, the epa cluster of polysaccharide genes has been shown to also be a colonization determinant, so this may be a useful method that we can use to identify novel proteins that could be targeted for other types of medical applications or drug applications.
july11-146	DR. DUERKOP: And then I think a broader, more kind of hand waving direction is what are the physiological effects of phage predation in the intestine? That's something we're very interested in. You know, does phage predation have an effect on the global community of commensals that are in that environment, and how does that impact the host? Does phage predation select, you know, on select bacteria actually influence the biology of the mammalian host, such as impacting innate immunity, adaptive immunity, things of that nature?
july11-147	DR. DUERKOP: So, with that, I need to acknowledge a number of people. I need to acknowledge, of course, my lab, which has just started at the University of Colorado.
july11-148	DR. DUERKOP: I really need to acknowledge Dr. Kelli Palmer at UT Dallas who's been an active collaborator throughout the course of all of these studies, my former mentor, Laura Hooper, for allowing me to take a phage project in a direction that was very different from what the lab traditionally works on, and then some of my new colleagues that I've started collaborations with here.
july11-149	DR. DUERKOP: So I thank you for your time, and I can take questions if there's any time left. Thank you.
july11-150	DR. RANALLO: Yeah. So we do have time for questions if anybody has any questions for either of the speakers this morning. I apologize. I didn't give you guys time for questions.
july11-151	AUDIENCE MEMBER: I'll ask a question. So in your resistant mutants, I mean, so you look at just kind of killing in liquid and that kind of sensitivity. Do you do like adsorption rate experiments or anything like that to see if it's gone down or absent?
july11-152	DR. DUERKOP: Yeah. So we've done some adsorption experiments, especially with the PIP mutant, and there's no adsorption difference. So I just didn't have time to show that data, but that data's published. The phage adsorb fine, but what we think is happening is that the -- is that PIP actually promotes DNA entry into the cell.
july11-153	MR. DIXON: Morning. I'm Dennis Dixon from NIAID. I'm an interloper from the other room. I wanted to come in and commend Col. Zapor for his point on reluctance of the infectious diseases community when actually confronted with phage as an experimental possibility.
july11-154	MR. DIXON: I do see the same disconnect between give us something new, we have to have some alternative and something innovative, and then when you present the community with this as an option, would you be interested in moving forward with this, well we don't know, it looks so different and we don't know if it'll work, even though you have a DSMB in place that's monitoring safety, and you have all the steps you need to determine evidence to guide your decision so you will know.
july11-155	MR. DIXON: So I liked your idea about specialty populations, where maybe the mainstream ID doc doesn't seem them on a recurring basis, moving to things like spinal cord injury, where you know the consequences of repeated catheter insertion, or the plates and implants from surgery, because surgeons generally have no reluctance to give something such as antibiotic, even if it's not exactly an antibiotic.
july11-156	MR. DIXON: So that might be worthy of further discussion, on how to start to have discussions to engage the community that's going to be necessary to buy in to any clinical evaluation. Thanks.
july11-157	MR. CHEN: Yeah. Good morning. Rong Chen from Phagelux. I have question to Dr. Tyner. I am very interested in your wound model. I found it very interesting to see on the slide, it looks like the topical application is better than systematic, right? Its look like at least similar, or even better. That's my understanding.
july11-158	MR. CHEN: So, and another question is that did you found any difference between -- in the systemic use between IP, IV, and SC?
july11-159	DR. TYNER: Okay. Thanks. You're right. It looks as if perhaps putting the phage into the -- you're talking about the rat model with the orthopedic heart? Yeah.
july11-160	MR. CHEN: Yeah.
july11-161	DR. TYNER: So it looks like that might be a little bit more effective, but the N is so small and the effect right now is not large enough to really draw a definitive conclusion.
july11-162	DR. TYNER: The other delivery was IP. We did not -- we have not yet tried IV or SC, but we have to solve the -- part of the issue with the effect of the therapeutic on reducing the biofilm, before we start looking at the delivery method, although delivery method is important. You're right.
july11-163	MR. CHEN: I notice there's a difference on the dose between your topical and the systemic. It's 2.5 and 1.75. They're different because of dose, or difference is because of route?
july11-164	DR. TYNER: That's a good question. I'm happy to discuss that with you after. We probably should rope Dr. Jacobs in for that discussion. Thanks.
july11-165	AUDIENCE MEMBER: Hi. Nancy from Phagelux. I just have a couple of questions here for your prosthetic joint infection models. I was wondering if you had looked at the activity of phages if you pre-treat your nail or your implant versus if you do the post-treatment after the infection has started.
july11-166	DR. TYNER: That's an excellent question. We have not done that yet. Might also be interesting to look at whether or not if we deliver antimicrobials first, then add phage, if there's a difference than if we add phage first and then do antimicrobials.
july11-167	AUDIENCE MEMBER: And maybe a follow up question on that. Well, maybe more a follow up question on what Rong was discussing. Have you tried to do the phages intramedullarly? So you would just make a hole inside of your tibial cavity, put the phages in it, and see how the infections would result. Think it might be very different from --
july11-168	DR. TYNER: That's an excellent point. No, we have not tried that yet.
july11-169	AUDIENCE MEMBER: Okay. Thank you. Thanks.
july11-170	DR. RANALLO: Okay. Thank you. Let's move on to our next speaker, Dr. Paul Turner from Yale University. We heard a little bit about Paul's work yesterday, but we're going to hear much more in-depth detailed information about how selective pressures can reduce virulence and sensitize against antibiotics. So the -- Paul's talk is using phage to select for evolution or reduce virulence in pathogenic bacteria. Thanks, Paul.
july11-171	DR. TURNER: All right. Good morning, everybody. Pleasure to be here. I'd like to thank the organizers for inviting me. So what I'm going to do today, first talk will be a little bit about my background and the mission that we have in my laboratory.
july11-172	DR. TURNER: I have a very broad interest in the evolution of microbes, and we focus a lot on viruses, so on the left are very familiar pictures for this audience of phages and bacteria, but we also look at other types of viruses, especially mosquito-borne viruses. So we do evolution experiments on dengue virus, and chikungunya virus, and some other human pathogens.
july11-173	DR. TURNER: So what I want to do today is demonstrate for this one project how there was a nice move from basic research, longstanding interest of mine in evolutionary biology, that in a very short period of time led to, you know, we're on the cusp now, we hope, of investigational new drug status and continuing to pursue that for phages, especially a phage that we found in a lake in Connecticut that -- you heard a little bit about that yesterday from my colleague Deepak. Okay.
july11-174	DR. TURNER: We like to address big questions, and here's kind of a big question. Why are there so many species on Earth? As an evolutionary biologist, it's very obvious to me that evolution involves compromises.
july11-175	DR. TURNER: So one of the most misunderstood concepts in biology, unfortunately by the lay -- public, is how evolution occurs.
july11-176	DR. TURNER: So what is not at all controversial and what Darwin first, and best, articulated is that organisms interact with their environment, and the variants that leave more progeny, are the ones that end up being enriched in those populations, and the traits that they have end up dominating populations through time. So the only controversy is how much people want to believe that that happens in humans.
july11-177	DR. TURNER: But the main point is that natural selection often leads to trade-offs, and I'm finding that trade-offs in my career are a very prevalent thing that we observe in our research.
july11-178	DR. TURNER: Essentially, it works this way. If you improve in one trait, it doesn't necessarily mean that you're going to improve in other traits simultaneously, and often you sort of give up the ability to perform another trait well. This opens up niche space for organisms that do the opposite. So, in this way, you have, through eons of time, species diversity evolving on the planet.
july11-179	DR. TURNER: The gentleman on the right is one of my colleagues at Yale, Steve Stearns, and he is very famous for life history theory, which is this general idea that traits cannot be simultaneously maximized. An interesting general trade-off, this is a talk for a different day but you see this also in viruses, is that survival versus reproduction is something that is a difficult thing to maximize on both sides.
july11-180	DR. TURNER: This, I would say, is one of the cornerstones of evolution by natural selection, and you can demonstrate it in Drosophila populations, but also in viruses. That if they evolve greater reproduction, it might take away from their stability, and vice versa.
july11-181	DR. TURNER: So I want to step back a little bit to a system that is not a phage of humans, but it is one of the first phage systems, virus systems, that I started working on in the 1990s.
july11-182	DR. TURNER: So this is a phage called phi-6 that infects Pseudomonads, especially Pseudomonas syringae pathovars, and it's a well-characterized system with a segmented genome. I started working on it because of its segmentation and RNA genome because it mimics genetics of human pathogens like influenza and hanta viruses.
july11-183	DR. TURNER: So you have a cartoon of the familiar lytic infection cycle, and in the middle here there's a picture of these phage particles, visible as little, white spheres, that are lined up along the type 4 pilus of these bacteria.
july11-184	DR. TURNER: So this is the initial receptor site for this phage in nature, and the type 4 pili are also what these bacteria use to twitch across a leaf surface and enter into the stomata. So this is absolutely essential as a structure for these bacteria to get inside of a plant and to be pathogens. And, not surprisingly, you see this a lot in phage biology and other virus systems. These viruses have evolved to use as a receptor something that is absolutely essential to their hosts.
july11-185	DR. TURNER: What we have seen in the laboratory is that the resistance to the phage in vitro easily occurs if the bacteria simply shed these pili. They get rid of the type 4 pili.
july11-186	DR. TURNER: Now, this is a bacterial pathogen of some interest in agriculture. It causes halo blight disease, which is a big deal in crop production of beans. So if they had this option in nature they would be out of luck in terms of bacteria surviving in their natural environmental.
july11-187	DR. TURNER: If the pilus loss occurs, they cannot get inside of the leaf, as I mentioned. So I would call that a conditional virulence factor, meaning that if you simply took the bacteria and you put them in a plant, they will happily function as pathogens.
july11-188	DR. TURNER: So what I would assert here is that the interaction of the phages with these bacteria demonstrates that the bacteria can easily be forced into an evolutionary trade off. If they evolve resistance to the phage, then this lowers their pathogenicity.
july11-189	DR. TURNER: You know, I'd seen this for a very long time, since the mid-'90s, and it was of interest to me simply because I was using this phage in experiments. Maybe about four or five years ago, really in earnest, my group started looking at this property in phages of humans in human -- phages of human-associated bacteria, of course.
july11-190	DR. TURNER: So could you use the same principle to drive our thinking in developing, or at least finding, better candidates for phage therapy. So here, the general question is can phage therapy also exploit evolutionary trade-offs?
july11-191	DR. TURNER: By now, at this point in the conference, this is a little familiar to people, but firstly, antibiotics are becoming less useful, MDR bacteria are on the increase, Pseudomonas aeruginosa is particularly worrisome for CF patients, severe burn and immune-compromised patients.
july11-192	DR. TURNER: So what we've focused on are efflux pumps, which I think are these fascinating complexes of proteins that span the inner and the outer portion of the cell of bacteria like Pseudomonas aeruginosa. These efflux pumps are transport proteins that help the bacteria efficiently remove a wide variety of drugs from the cell.
july11-193	DR. TURNER: They have a lot other properties as well. They function in host colonization, evasion of host immunity, and biofilm formation, but obviously this is a big problem in Pseudomonas aeruginosa. That if you throw an antibiotic at it and it manages to get in, it can be very effectively pumped out.
july11-194	DR. TURNER: So efflux pumps are typically chromosome encoded, they're genetically conserved -- that turned out to be important in the study that I'm going to focus on, and I'll try to remember to get back to that later -- they are generally found in gram-negatives, and for many antibiotic classes, but not all, these are the major determinants of how the resistance would occur for the antibiotics.
july11-195	DR. TURNER: So kind of a useless slide at this point. Phage therapy is amazingly interesting, and we should invest in it further.
july11-196	DR. TURNER: So here is another cartoon to help illustrate a point that really is the core of this project. So this is a lytic infection cycle, very obviously. If you use a phage to target a bacterium, then, in essence, I would expect, as an evolutionary biologist, you're going to get the same problem that often occurs any time an organism faces a selective challenge. It's going to be selected to change.
july11-197	DR. TURNER: So now I'm showing the bacteria in this cartoon. It is now presenting different-colored -- blue-colored proteins now that is not able to be used by this phage to enter and initiate the infection cycle. So if I throw a phage at a bacterium, the natural consequence is it's going to select for increased phage resistance.
july11-198	DR. TURNER: So wouldn't it be cool if that came along with increased antibiotic sensitivity? That's not only cool, but that's also the take home of my talk as well otherwise I wouldn't be suggesting it. So this genetic trade-off between phage resistance and antibiotic sensitivity would of course improve antibiotical -- antimicrobial therapy options and would extend the lifetime of our current antibiotic arsenal.
july11-199	DR. TURNER: And I want to really emphasize that. So if you have drugs that are approved currently and they're in use, if you can use phages to interact with pathogenic bacteria and convert them into genotypes that are susceptible to something that's already approved by the FDA, then you have a faster track to being able to use phages, I would say, in therapy.
july11-200	DR. TURNER: So we found such a phage. It's abbreviated as OMKO1 for outer membrane knockout one. It's in the family of Myoviridae. It's a lytic phage that binds to that outermost protein in many of the very commonly found efflux pumps in P. aeruginosa, these Mex system efflux pumps.
july11-201	DR. TURNER: We confirmed that using a mutant knockout library that we got from University of Washington. So we know that when the genotype that has the oprM gene knocked out, that is the only strain that this phage cannot infect.
july11-202	DR. TURNER: So we discovered in sequence this phage which has a pretty whoppingly large genome, but we found that in 2016, and it does force this genetic trade-off that I mentioned. The phage-sensitive bacteria can efflux antibiotics, but they're killed by the phage, and the phage-resistant mutants have an impaired ability to efflux antibiotics. So that demonstrates the interaction. Again, that was found in a contaminated lake in Connecticut called Dodge Pond.
july11-203	DR. TURNER: So probably obvious to many people in the room, but I want to make sure you understand the core thing that we're measuring in the table that I'll show in a moment. So what you should keep in mind is that the evolution of P. aeruginosa resistance to this phage causes sensitivity to certain drugs.
july11-204	DR. TURNER: So how you easily measure sensitivity to drugs for bacteria is through a MIC assay, minimum inhibitory concentration. So this agar plate has a lawn of bacteria growing on it, and imagine you've got a strain that is in that lawn that grows up right next to a Kirby-Bauer disc that you had placed on the lawn, and that has antibiotic leaching out from it. If it doesn't care about the antibiotic, it grows up right to the edge of the disc.
july11-205	DR. TURNER: Well, what I'm emphasizing is that strains of these bacteria that become resistant to the phage no longer have that property. So they are one mutational step away from having a much larger killing zone and a much greater sensitivity to antibiotic.
july11-206	DR. TURNER: So I'll show you that in the following table that was sort of a compilation of the data that we presented in the 2016 paper.
july11-207	DR. TURNER: So let's begin first with -- efflux pump literature does implicate certain antibiotics and antibiotic classes for which efflux pumps function, and it's pretty rock-solid evidence.
july11-208	DR. TURNER: So if we begin with tetracycline and erythromycin, you can see that the isolate MIC has the number shown in the third column, and when these bacteria -- and basically what I should emphasize, that this table is kind of a compilation of data from multiple bacteria, but I'll get into that more in a moment.
july11-209	DR. TURNER: So the phage-resistant isolate MIC changes dramatically. You'll see in the final column there's a fold increase drug sensitivity that's a very impressive number.
july11-210	DR. TURNER: Now we move on to -- efflux pump is associated with these other four antibiotic classes, but the evidence isn't as rock-solid. Nevertheless, you get a change in the isolate MIC versus the phage- resistant isolate MIC. It's not as dramatic of an increased drug sensitivity, but the asterisks are showing you how these agree with break points for clinical importance. So it has now changed the bacterium to a clinically relevant resistance to susceptibility instead.
july11-211	DR. TURNER: And finally, efflux pumps are not involved in penicillin class antibiotics. Moving them out of the cell. This is due to other types of mutations that happen in the chromosome. You can think of this last example here as a control, and, not surprisingly, we saw no change in the fold increase drug sensitivity.
july11-212	DR. TURNER: So everything agrees with my assertion that the interaction of the phage with the efflux pump protein is placing selection pressure on these bacteria to change, and they change in a way that makes them a better outcome for humans in terms of our ability to treat them with existing drugs.
july11-213	DR. TURNER: I'll now show you a bit of the unpublished data in my talk. I think I have time for this. Not very many slides of it.
july11-214	DR. TURNER: So this is a cartoon that probably you can figure out this is a bacteria biofilm. The problem with these little, red, I guess they're circles, trying to get through that biofilm at the bacteria is that a biofilm is very resilient to antibiotics getting in.
july11-215	DR. TURNER: If you have the phages that are interacting with the biofilm and they can disrupt it and allow those cells to become exposed to the antibiotic, then you can get a synergistic activity of killing for the phages and the antibiotic.
july11-216	DR. TURNER: So what we thought is really the promise of this phage and, frankly, why it worked in a patient -- and I'll talk about that more in a moment -- is that there's a synergistic interaction that is expected.
july11-217	DR. TURNER: So here are some unpublished data where -- focus on the taller bars in each one of these examples. I'm kind of in shock and awe that there's very little in the literature on commonly-used substrates that you place in the human body and the ability of bacterial biofilms to form.
july11-218	DR. TURNER: We know this, surgeons know this very well, and yet you don't see very much in the literature of the ability of, say phages versus antibiotics to tackle that problem. So these data illustrate that point.
july11-219	DR. TURNER: The three bars on the right in each case show you that in a control versus these two antibiotics, there's really no action of the antibiotic in disrupting the biofilm and reducing cell density, whereas the phage alone, which is the bar on the left-most in each one of the categories, this is this phage and its ability to break apart the biofilm.
july11-220	DR. TURNER: The asterisks show you the cases of where the combination of the phage and the bacteria -- I'm sorry -- and the antibiotic are doing a better job at killing the bacteria than the phage alone, and in the majority of the cases, that's what we observe. So that's a very promising result.
july11-221	DR. TURNER: So I said that the data that I showed you quickly from the '16 paper were for a variety of strains. Indeed, this worked for laboratory model strains PA01, PA14. It worked on clinical isolates from multiple sources.
july11-222	DR. TURNER: It also worked on environmental isolates, bacteria that we pulled directly from an estuary, and also from human homes in the Louisville, Kentucky area. Everybody, if you don't know this, you generally have Pseudomonas aeruginosa growing at least in your kitchen sink, if not in your bathroom sink as well.
july11-223	DR. TURNER: So the objective is to examine the impact of this phage on a much larger set of isolates, and that's what we have as submitted grants to NIH, as well as to the Cystic Fibrosis Foundation.
july11-224	DR. TURNER: The objective is, with FDA approval, we would use this phage to treat chronically-infected human volunteers. So yesterday you did hear about this one case presented by Deepak where we did successfully treat an MDR P. aeruginosa biofilm infection that was associated with aortic arch replacement. That case study is still in review, but we are optimistic that it will come out soon.
july11-225	DR. TURNER: Nevertheless, we were able to talk about this publicly, so we mentioned it in media presentations, on public radio international, People's Pharmacy, and Carl Zimmer, the science writer, had a very nice piece on this late last year, so you can go look for it on the web, if you choose.
july11-226	DR. TURNER: The objective for the future work is to test the safety and efficacy of this in animal models. So I think this is a very interesting project, where it went to discovering something that was found through a natural product sort of pipeline, to bring something interesting that might be useful for translational medicine, and quickly we found a patient and we helped the patient, and now we're doing, I would say, a lot of backfill. So we were awarded an NIH pre-clinical services award, where there's a contract to a team at University of Louisville who are testing the safety and efficacy in a mouse model for lung pneumonia in immunocompromised patients. So that study is still underway. I can't tell you very much about it.
july11-227	DR. TURNER: Some of the controls in that study had to be repeated, so the entire thing is being repeated next month, but I found this data set to be pretty interesting. What that laboratory at Louisville did was, even though the experiment has to be repeated, they sent us tissue samples from the mice in this three day experiment that -- we were able to retrieve phage from the animal tissues that were subjected to phage trying to control the infection.
july11-228	DR. TURNER: So UNC-D is this pathogenic strain that they use in their pneumonia model, and focus on the data set in gray there, the left-most one. It's showing the efficiency of our phage that we sent them and its ability to grow on that pathogenic strain relative to our typical lab strain that we would use to enrich it, PA01. And they don't grow as well on the pathogenic strain, but they grow on it.
july11-229	DR. TURNER: So after only three days, in the vast majority of these cases, the phages we isolated from those tissue samples are remarkably better by orders of magnitude in growing on the target bacterium.
july11-230	DR. TURNER: As an evolutionary biologist, I will tell you that impresses the heck out of me because this is a DNA phage, and I think it is demonstrating if you put it in this very novel environment of a mouse -- animal -- an animal with -- that is used in the experiment, there is strong selection pressure on it to do its job very well in targeting the bacteria that are there and present for it to grow on.
july11-231	DR. TURNER: So my point is that strong selection can happen in vitro, and even stronger selection can happen in vivo in some circumstances.
july11-232	DR. TURNER: So I'll finish up by saying that we want to continue with our clinical application of OMK01, and we did acquire the IND in 2016 for compassionate use. We have a teleconference, I found out only yesterday so I didn't put it on this slide, with FDA next month to talk about the possibility of this phage going into clinical trials.
july11-233	DR. TURNER: The targeted diseases are ambitiously, hospital-acquired pneumonia, CF-associated pulmonary infections, catheter-associated UTIs, and burns.
july11-234	DR. TURNER: We thought we would make faster strides in agriculture. I'll have you -- I'll just be completely transparent and honest about that. So we know that a lot of agricultural systems we rely on to feed an ever-hungry world are having just as big a problems with antibiotic-resistant bacteria: the shrimp industry and many leafy plants, so the development of phages for bio-control and agricultural systems, I think, has amazing promise as well, and that's something we would like to get into eventually.
july11-235	DR. TURNER: So I'd like to acknowledge the folks who actually did the work because all I do is look over people's shoulders and make them nervous. I really have to credit my lab group for being very bold about taking on risky projects, and also bold about me showing embarrassing pictures of them from the murder mystery party that we have annually.
july11-236	DR. TURNER: The individual in the middle, I don't know if you can see him, this is the patient who was treated who is now back to work, and this is Ben Chan -- he was the primary person on this project -- to the right. He's a research scientist at my lab group.
july11-237	DR. TURNER: We're in that picture showing, or we're giving a thank you card to the patient, as well as a phage plush toy. I don't know if you can see that, but that's what he's holding. So I'd like to thank Deepak, as well as John Wertz, another one of my longstanding collaborators at Yale, and the funders for the project. Thank you for listening.
july11-238	DR. RANALLO: We have plenty of time to take a few questions.
july11-239	AUDIENCE MEMBER: Hi. Nancy from Phage Lux. I have a question. We find in our lab that the presence of Pseudomonas usually inhibits the way that Staph aureus bacteriophages are able to infect Staph aureus, and I was wondering if you would expect the same results on polymicrobial biofilms, or if you would expect the same kind of selection pressures. Or would it be different in polymicrobial models?
july11-240	DR. TURNER: So I don't know your data because I haven't seen them, but maybe one possibility is if you have a phage that you're using against a target bacterium but it has maybe an ability to passively bind to something else, especially another bacterium, it's probably going to weaken the ability of the phage to do its job. So you could have in a polymicrobial setting sort of a weakened ability for the phage therapy to work.
july11-241	DR. TURNER: We haven't seen that with this particular phage, but I would agree that that's just one of the very many interactions of the phage therapy candidate with a diverse community of bacteria that we need to address and study further. I guess that's my only answer to that.
july11-242	AUDIENCE MEMBER: Two quick questions, Paul. First, have you tried selecting for resistance changes in the pump that would give you resistance? Because they should be in the external loops of the --
july11-243	DR. TURNER: Right.
july11-244	AUDIENCE MEMBER: Have you tried that yet?
july11-245	DR. TURNER: No, we have not tried that yet. Yeah. It's all been kind of just what is phage doing to interact with the bacterium, and what's the mutational spectrum of the bacterium response.
july11-246	AUDIENCE MEMBER: Right. And you also said the phage didn't grow as well on the pathogenic strain.
july11-247	DR. TURNER: Right.
july11-248	AUDIENCE MEMBER: So when you say that, is that just reduced EOP or what --
july11-249	DR. TURNER: Correct. Correct. Just reduced EOP.
july11-250	AUDIENCE MEMBER: So it's likely to be a restriction escape?
july11-251	DR. TURNER: I'm not sure what's at the root of it, but it's kind of remarkable that this phage grows very well on a wide variety of genotypes of Pseudomonas aeruginosa, so --
july11-252	AUDIENCE MEMBER: Yeah, but if it's got a restrictions problem with that strain, then it'll just take one escapee.
july11-253	DR. TURNER: Exactly. So we have to examine that. You know, it's kind of reminding me, ambitiously, of if you had a phage that transcends all genotypes of a species and it doesn't infect other species, then you do have a species-specific drug in phage therapy. So I'm not claiming that that's what this is, but maybe a modified version of this phage would be closer to that. But I hear what you're saying. Yeah.
july11-254	AUDIENCE MEMBER: My question's kind of similar. I was wondering whether you had tried selection with an antibiotic that you're trying to re-sensitize to and phage at the same time.
july11-255	DR. TURNER: Right.
july11-256	AUDIENCE MEMBER: Try to generate those mis-sense mutations and understand the resistance frequency. Whether you really are going to reduce the barrier to resistance by maybe co-dosing using it as an adjunctive therapy.
july11-257	DR. TURNER: Yeah. I guess maybe the way I could have answered the prior question is we are trying some of those experiments, and, you know, I'm not sure why, but there's kind of a remarkable inability of the bacterium to regain antibiotic resistance when it sees this phage.
july11-258	DR. TURNER: I think what is going on is it's placing selection pressure. We're looking for mutations in oprM, and we're actually not finding them. I think that there's something else epistatically happening to make them more resistant to the phage, and then when you remove the phage -- we've cultured them for up to 10 days afterwards in the absence of phage and they don't go back to being antibiotic-resistant, so that suggests there's something going on.
july11-259	DR. TURNER: That they're happily growing, but they're sort of -- they lost the ability to have a toggle switch that moves back. It's not like efflux pump repression and --
july11-260	AUDIENCE MEMBER: Right. So you're not co-administering, you're first selecting for resistance to the phage and then later looking for --
july11-261	DR. TURNER: Oh, I see what you're saying. Correct. Yes.
july11-262	AUDIENCE MEMBER: Because the eas -- it's a un -- it's a non-essential protein. The easiest way to get resistance is to knock it out. And it's not going to revert back on its own without selective pressure.
july11-263	DR. TURNER: I agree.
july11-264	AUDIENCE MEMBER: It's similar to what we saw with the PIP protein with the Enterococci where the strains that were not susceptible had point mutations, but when you select for resistance, all you get is knock out after knock out out of it.
july11-265	DR. TURNER: Right. Right. So I have to admit we have to look at that further, but, anecdotally, I would have predicted we would have seen a lot more of that by now, and we're not. So I think there's something interesting going on there that maybe has not been shown biologically in phages. I just don't know.
july11-266	AUDIENCE MEMBER: Okay. It's important because if you're going to go into the clinic and do the co-administration adjunctive therapy to antibiotics, you want to know what that resistance --
july11-267	DR. TURNER: Completely agree. Let me emphasize, though, when we did treat the patient, we put a useless antibiotic in at the same time, okay? So that worked.
july11-268	AUDIENCE MEMBER: Okay.
july11-269	DR. TURNER: All right. Yeah.
july11-270	AUDIENCE MEMBER: So, nice presentation.
july11-271	DR. TURNER: Thank you.
july11-272	AUDIENCE MEMBER: I have a specific question for you. You mentioned that you like to expand it for environmental uses, the phage.
july11-273	DR. TURNER: Uh-huh.
july11-274	AUDIENCE MEMBER: So how do you isolate it in the environmental application? Because selected pressure on used phage, it will, you know, generate resistance population. So how you overcome those resistance bacteria in the environmental situation?
july11-275	DR. TURNER: Right. So what I should have said, I didn't want to confuse, is we have other phages that do the same thing for different target bacteria, and I would say they're actually not that hard to find. So we found them for cholera, Klebsiella, Shigella, et cetera. I think it's more a matter of looking for them in the right way.
july11-276	DR. TURNER: So your question is if you deploy it in a large scale in an agricultural field, what will happen?
july11-277	AUDIENCE MEMBER: Yes.
july11-278	DR. TURNER: I would think you're going to get resistance to it, and it may fail ultimately. An intriguing basic research question is whether you can run through the co-evolution in the laboratory and, in a sense, get a cocktail that is, you know, the ghost of evolution future or something like that, right, and then you use that.
july11-279	DR. TURNER: I think that that's an intriguing idea. I have no idea if it will work because evolution can take many paths, right? But --
july11-280	AUDIENCE MEMBER: But --
july11-281	DR. TURNER: Yeah? Go ahead.
july11-282	AUDIENCE MEMBER: I agree with you, but my problem is that if phage is that effective, and if we can make a broad spectrum cocktail to prevent all these things, all of these phages are present in the environmental situation --
july11-283	DR. TURNER: Yes.
july11-284	AUDIENCE MEMBER: -- but we don't see the phage has eliminated all the bacteria on the surface of the Earth right now. So I think, my -- this is my personal opinion, that phage can be used as like antibiotic, but it cannot be used as disinfectant.
july11-285	DR. TURNER: I agree. Yeah. Yeah. I'm a big believer in spatial models, and you have local sort of, you know, pros and cons to things in biology. So, yeah, I see exactly what you're saying, but I am not worried that we would change the landscape of bacteria on this planet with selection pressure due to phages because they've existed together for billions of years on the planet.
july11-286	AUDIENCE MEMBER: No, no, I'm not worried about that, I'm worried about the effectiveness of that phage application, because within a couple of hours, the resistance population will start over dominate the system --
july11-287	DR. TURNER: Yeah. We should talk more further because I -- yeah -- I have lots of ideas about ways to test it in the field, and I know exactly where you're coming from.
july11-288	AUDIENCE MEMBER: I have, first, one question, and then one comment.
july11-289	DR. TURNER: Sure.
july11-290	AUDIENCE MEMBER: The question, have you tried much working with small cell variants like you tend to find in the cystic fibrosis lung? I've been particularly curious, also, about small cell variants of Staph.
july11-291	DR. TURNER: Right. Not yet. So that is in the realm of these large repositories of strains that we're trying to acquire to test the generality of this phenomenon for clinical isolates coming directly from CF patients, okay? So we can kind of get at that variation through those experiments.
july11-292	AUDIENCE MEMBER: By the way, we did once work with 200 CF strains from Univers -- from Children's Hospital in Seattle, and we were able to find phage against all but about eight of them, and of those, four actually turned out not to be aeruginosa. We checked them using the 16S ribosomal marker.
july11-293	DR. TURNER: Yeah.
july11-294	AUDIENCE MEMBER: You do find them working in other parts of the world as well. When I first got started with phage back in '97, then -- or started with Pseudomonas phage, I should say -- I'd always worked with E. coli -- we got a bunch of strains of phage from Tbilisi that had been isolated against wounds and burns, and they worked against all of -- all but one of the 18 strains of cystic fibrosis we got at that point.
july11-295	AUDIENCE MEMBER: So from a completely different use and comdip -- completely different part of the world, they worked.
july11-296	AUDIENCE MEMBER: And, again, the one that they didn't work on turned out later -- not actually to be aeruginosa when we did -- DR. TURNER: Yeah, yeah. AUDIENCE MEMBER. So that's something to think about tied in with it.
july11-297	AUDIENCE MEMBER: In terms of how low you can get them, in the oceans they're -- it's completely controlled by phage in terms of what the high levels are. What they do is you do -- it's like the red tide situation. They are at such low levels, about 10 to the fourth per ml, and so are the bacteria below that, and it's only when they get higher than that that the phage can find them enough.
july11-298	AUDIENCE MEMBER: So if you get a sudden bloom of e. coli O157, as we saw in sheep models, then you can activate the phage that are naturally there --
july11-299	DR. TURNER: Oh, I see. Because they're in the system already is what you're saying.
july11-300	AUDIENCE MEMBER: They're in the system already --
july11-301	DR. TURNER: Yeah. Yeah.
july11-302	AUDIENCE MEMBER: And they work, actually, better. That seems to be what's going on in livestock to keep them in balance.
july11-303	DR. TURNER: Right. So a radical idea would be whether you can decrease antibiotic administration to CF patients by at least giving them a lower dose of antibiotic and a phage which helps their quality of life, and the phage is sitting around in case a variant emerges. That kind of a thing.
july11-304	AUDIENCE MEMBER: And to keep them lower in that kind of way.
july11-305	DR. TURNER: Yeah. Yeah. I agree.
july11-306	AUDIENCE MEMBER: So nice work. Keep it up.
july11-307	DR. TURNER: Thank you. Thanks, Betty.
july11-308	AUDIENCE MEMBER: Thanks.
july11-309	DR. RANALLO: Okay. So I just want to thank the morning speakers. I am going to take programmatic liberty and give us a 25 minute break, so we'll be back here at 10:30 for the next set of speakers. Again, thank you.
july11-310	DR. RANALLO: (Whereupon, a short recess was taken.)
july11-311	DR. RANALLO: So we have a little bit of a change in our agenda. Frank Ramig had a personal emergency and is unable to make our conference, our workshop today, so we're going to start off with Dr. Roy Stevens from Temple University.
july11-312	DR. RANALLO: DR. Stevens is a professor of endodontology at Temple University's Kornberg School of Dentistry, as well as a professor of microbiology at Temple University's Katz School of Medicine.
july11-313	DR. RANALLO: Roy is going to talk to us a bit about engineering phage and phage products to disrupt Enterococcus faecalis biofilms.
july11-314	DR. STEVENS: Okay. Well thanks --
july11-315	DR. STEVENS: (Away from microphone.)
july11-316	DR. STEVENS: (Pause.)
july11-317	DR. STEVENS: Okay. That's better. Well I'm still delighted to participate in this wonderfully informative workshop, so thank you for organizing this.
july11-318	DR. STEVENS: So this morning I'd like to speak to you about a phage genetic engineering strategy that we've been exploring in my laboratory. What you see on the screen here are a couple of phages that we've isolated in our laboratory.
july11-319	DR. STEVENS: Since my laboratory is located in a dental school, as Ryan alluded to -- endodontology, by the way, for those uninformed in that area, is root canal treatment. I don't hear any moans, so that's good.
july11-320	DR. STEVENS: So my laboratory is located in the dental school so it shouldn't come to anybody's surprise that the phage that we've isolated infect oral bacteria.
july11-321	DR. STEVENS: So, for example, the Siphoviridae phage on the left infects strains of E. faecalis and was originally isolated from a root canal of an infected tooth -- an infected root canal of a tooth. The Myoviridae phage on the right was -- infects strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans, and this was originally isolated from dental plaque of a periodontally-diseased tooth.
july11-322	DR. STEVENS: Most of my discussion this morning is -- about genetic engineering is going to be directed towards the E. faecalis phage.
july11-323	DR. STEVENS: So to start out I think I should say a little bit about a rationale for genetically engineering phage for phage therapy. So what I have on the screen here is a simplistic schematic view of the conventional paradigm for isolating phage that are used in phage therapy, and this is going to be very familiar to everybody in the audience here.
july11-324	DR. STEVENS: Typically, phage are isolated from the environment, whether it's sewage, or water sources, or animal effluents and so forth. The isolated phage are typically tested for host range.
july11-325	DR. STEVENS: In the last 60 years or so phage are also characterized morphologically by EM to describe the morphotype, and then in the last 20 years or so phage that have been isolated and planned for use in phage therapy often are sequenced, and then, typically, there may be some clinical trials or animal studies prior for use in phage therapy. This approach has been -- the overall success of this approach is largely due to the rate abundance of phage in the natural environment. However, there are limitations to the -- to this approach, and some of them I have listed on this slide.
july11-326	DR. STEVENS: So using this approach, basically there's a random isolation of phages. It's a relatively hit or miss approach. The saving grace again is the fact that phage are so abundant, plentiful, so that it makes it possible for, in most cases, the process to succeed in any event.
july11-327	DR. STEVENS: Using randomly isolated phages for phage therapy run the risk of employing a virus with an unpredictable, or even undesirable, property, so, obviously, we wouldn't want to do that. Randomly isolated wild type phages may, in fact, lack qualities that would improve their therapeutic performance, so just using a wild type phage, we may be missing some advantages.
july11-328	DR. STEVENS: Genetic manipulations of virulent phage may be problematic. Of course there's no convenient way for selecting for recombinant mutants, or positive selection of desired recombinant mutants with the desired characteristics. And finally, as we see over and over, what's necessary to be used in phage therapy are basically phage cocktails because of -- the host range limitations of any one specific phage may necessitate using cocktails, and this may complicate safety evaluations needed for clinical development.
july11-329	DR. STEVENS: That's not to say that there are no genetic strategies for modifying virulent phages, and I have several of these strategies listed on this slide, but even in these cases the same issue applies, or the same issues apply. There isn't really any good, positive selection system available, recombination rates are relatively low, and in vitro manipulation of a large, synthetically-assembled DNA molecule is tep
july11-330	DR. STEVENS: -- technically difficult.
july11-331	DR. STEVENS: So we are looking for an alternative way of modifying a phage to make it perhaps more useful in phage therapy, and our strategy essentially involves starting out with a prophage of a temperate virus and winding up with a recombinant phage of a virulent virus.
july11-332	DR. STEVENS: Basically what we do is it allows us to use conventional bacterial genetic strategies to make modifications in the genome, in the prophage genome, and ultimately change the region of the genome that controls lysogeny such that the resulting virus is no longer capable of lysogeny. So we convert it into a virulent phage after we do whatever other recombination work we want to do in the prophage.
july11-333	DR. STEVENS: So by doing this we actually have sort of an oxymoron. We have a prophage of a virulent virus, which to most phage people probably wouldn't make sense, but this is basically what we are able to achieve.
july11-334	DR. STEVENS: So it's a three step process, in which we initially make -- we replace, or delete genes in the prophage that we wish to change. In the second step we use a second allelic exchange mutagenesis to delete lysogeny-related genes and replace the wild type promoter that drives lytic cycle functions with an exogenous inducible promoter.
july11-335	DR. STEVENS: And so what we're -- in doing these manipulations we can easily select for lysogens that contain the recombinant prophage by simply plating the reaction mixtures on antibiotic-resistant plates and recovering the recombinant lysogens.
july11-336	DR. STEVENS: In the final step we can induce the phage using appropriate inducing agents to produce the virulent version of the original temperate virus. So let me give you an example of how this works using one of the phages that I showed you earlier in the talk. This is the E. faecalis phage that we isolated in our laboratory from an infected root canal.
july11-337	DR. STEVENS: This clearly is not a phage that anybody in their right mind would consider as a candidate for phage therapy in its wild type state. Upon isolation it was identified as a temperate virus. It's weakly lytic, and it has a narrow host range.
july11-338	DR. STEVENS: So the isolation procedure for this phage was nothing very unusual. We isolated it, again, from an infected root canal; that is, we isolated Enterococcal strains from an infected root canal, we plated these out on selective media for Enterococci, we got -- we recovered E. coli clones, we picked clones and we induced with mitomycin c, and then we test the resulting cell-free culture medium for plaques against the panel of E. coli strains, and this is what you see. Small, somewhat turbid plaques.
july11-339	DR. STEVENS: If you grow them up and purify the phage and -- you can do EM analysis, and this is what the phage looks like.
july11-340	DR. STEVENS: So when we purified the virus we further analyzed the genome. After sequencing the genome we found that this virus has a genome consisting of 42,822 base pairs, distributed among 65 open reading frames. And thatâ€™s many -- as has been mentioned by other speakers here today, typical of many, many other phages. The genes are arranged in functional modules, as you see illustrated in this diagram.
july11-341	DR. STEVENS: We focused on one region of the genome, which you see here, and it appears that the apparatus that determines lysogeny, or lytic functions, are found within this region of the genome. That is, the establishment and maintenance of lysogeny is basically determined here.
july11-342	DR. STEVENS: Now if we look at this in a little bit more detail we can see that there's open reading frame 31 which is predicted to code for an integrase, open reading frame 36, which is predicted to code for a cI-type repressor, and open reading frame 37, which is predicted to type for a cro type repressor. In between 36 and 37 there is a regulatory region, which we'll look at in a little bit more detail.
july11-343	DR. STEVENS: And, as we'll see shortly, transcription in the right direction results in lytic infection, transcription in the left direction results in lysogeny.
july11-344	DR. STEVENS: This is that region between 36 and 37. You see that there is a stem loop structure, and to the right there's a promoter that controls transcription of cro and the remainder of the lytic functions, and to the left is a promoter that controls transcription for the cI repressor and the lysogeny functions.
july11-345	DR. STEVENS: So how do we go about doing this? We design a vector in which there are homologous regions upstream and downstream of the lysogeny genes, and between these two homology regions we have an antibiotic resistance marker and we have a inducible exogenous promoter. In this case it's the nisin promoter.
july11-346	DR. STEVENS: So upon homologous complementation, this will permit complementation between the vector and the prophage, and ultimately, in a small fraction of the cases, there will be an allelic exchange, and the result of that will be a pro phage that now has the antibiotic resistance marker and the nisin-inducible promoter in place of the lysogeny genes and the wild type promoter that was in the original prophage.
july11-347	DR. STEVENS: So this actually represents, as I mentioned before, a -- now a prophage of what is now a virulent pha -- virus. The lysogens that now contain this construct can easily be selected on antibiotic-resistant plates, in this case with erythromycin, and those clones can then be induced using the appropriate inducer, in this case nisin, and you can get the phage out, and that phage will have the properties of the virulent virus.
july11-348	DR. STEVENS: So what we've done by doing this is to cause the deletion of all the lysogeny-specific genes of the prophage and replacement of the wild type promoter with an exogenous inducible promoter, in this case the nisin promoter, and this will yield a virulent variant that is incapable of lysogeny since it has none of the genes needed for lysogeny, and, furthermore, it's not sensitive to repressor repression since it has an exogenous promoter that's not sensitive to repressor.
july11-349	DR. STEVENS: So we've changed this genome on the left from the wild type to the recombinant genome you see on the right.
july11-350	DR. STEVENS: If we compare the wild type to the genetically-modified as you see in this slide, you can see that there is a noticeable change in the host range. We have -- the wild type had a very limited host range. As you can imagine, the wild type temperate virus is subject to repressor repression, whereas the genetically-engineered version is not sensitive to repressor, and so it can, in fact, infect other lysoge -- lysogenic strains. If we take that genetically-engineered phage and we inf -- and use it to infect biofilms, we can see a very dramatic result. On the left you see controlled biofilms of two strains of E. faecalis. JH2 is a vancomycin-sensitive strain, V583 is a vancomycin-resistant strain.
july11-351	DR. STEVENS: This is a live dead stain, and you can see a very rich biofilm that was formed in this system. In the phage-treated biofilms you see almost complete elimination of the biofilm in the JH2 strain, and an almost as complete elimination wi -- in the vancomycin-resistant strain.
july11-352	DR. STEVENS: In fact, what's -- where I found interesting in this is that if you do a cut through the biofilm, you can even see the death of the cells throughout the depth of the biofilm.
july11-353	DR. STEVENS: This is actually a concern of -- in terms of being able to deal with biofilms. It's been, you know, postulated that cells at the depth of biofilms are protected in certain ways from agents that are going to be used for treating them, and yet here we see bio -- in a biofilm all the way to the bottom of the depth of the biofilm predominantly dead cells.
july11-354	DR. STEVENS: If we want to take the recovery, we can see that in both the cases of the JH2 strain and the V583 strain there is a substantial diminution of the recovery, there's basically a two log drop, at least, in the recovery, and the amount of detectable residual cells recovered is quite small.
july11-355	DR. STEVENS: In addition to testing a biofilm formed on a glass slide, it turns out, for all the non-dentists in the audience, that infected root canals also produce biofilms inside the tooth, the -- depending on what kind of infection it is, and so we fabricated a dentin infection model in order to test the effects of phage on infections of the dentin.
july11-356	DR. STEVENS: So here, in this model, we fabricated a cylinder made out of the de -- the root of a tooth, which is basically all dentin. This dentin cylinder is then sealed inside the encasement of a disposable needle cap, and then that is put -- assembled inside a -- the cap of a needle and buffer can be placed in the lower portion of the cap, and either this -- the E. faecalis can then be injected into the root canal, which you see in the center of the dentin cylinder.
july11-357	DR. STEVENS: After incubation for a period of time, the phage can also be introduced into the root canal. The result of that is -- to the remaining bacteria is shown on the next slide.
july11-358	DR. STEVENS: You can see with the vancomycin-resistant strain there's a dramatic drop in recovered E. faecalis from these infected root canals. For a reason I -- we not quite clear about yet, the decrease in the J -- in the vancomycin-sensitive strain is not very impressive. We're curious about that, and we'll probably be looking at that further.
july11-359	DR. STEVENS: So it appears that you can genetically alter phage to change its properties and make the phage more usable and useful in -- as an antimicrobial agent.
july11-360	DR. STEVENS: Now, in addition to looking at the phage itself, we also looked at products of the phage. In this portion of the phage genome you see a series of genes that appear to be related to the lysis of the cell. There are -- there is a lysin, a holin -- I'm sorry -- a endolysin, and the ORF28 gene product appears to be an amidase-type lysin.
july11-361	DR. STEVENS: So what we did was we PCR-amplified the ORF28 gene using the phage DNA as a template, we cloned the gene into an expression vector in tandem to a GST tag, and so we got this vector as you see on the right.
july11-362	DR. STEVENS: If we transform that into an E. coli cell and express the gene and then make a sonic extract of the E. coli where the gene is being expressed, we get this mixture of, basically a gemisch of all of the components of the E. coli cell, including the produced ORF28-GST fusion product.
july11-363	DR. STEVENS: We can put that through a glutathione affinity column which will bind to the GST, the glutathione, as transferase protein, which is associated, or attached, or fused to the ORF28 lysin. Then, by adding excess of glutathione, we can elute off that protein.
july11-364	DR. STEVENS: In the gels that you see in the lower left of this slide, you can see that after putting it through a column several times, we get basically a homogenous preparation of a protein of 72 kilodaltons. And you'll notice that the ORF28 gene product is predicted, or the sum of the ORF28 gene product and the GST fusion tag is 72.5 kilodaltons, so this appears to be a purification to just about homogeneity.
july11-365	DR. STEVENS: What's interesting is if you take -- if you spot some of this material onto a lawn of any of the
july11-366	DR. STEVENS: -- or many of the strains of E. faecalis that we have in our collection, you can see that it produces a very noticeable and distinctive lytic zone in these different E. faecalis strains, including vancomycin-resistant strains.
july11-367	DR. STEVENS: So out of 99 strains that we've tested so far, just a little over half of them are quite sensitive to this lysin. Of the 99 strains, two of them, two of the E. faecalis strains are vancomycin-resistant, and both of those are sensitive to the lysin. So vancomycin resistance, as in VRE strains, does not pose a problem to the lysin.
july11-368	DR. STEVENS: Adding this substance to a suspension of the E. faecalis strains causes a very rapid clearing of the suspension. In about 15 minutes you can start to see a precipitous drop of the turbidity of E. faecalis suspension, so the reaction occurs quite rapidly.
july11-369	DR. STEVENS: Again, you can use that purified lysin that we got from the phage on a E. faecalis biofilm. On the right you can see what the biofilm looks like after it's been treated with this lysin. The left is, of course, a control. You can see an obvious difference.
july11-370	DR. STEVENS: And you can see -- if you quantitate the recovered -- the recov -- the cov -- recovery of the residual cells from the treated versus the controlled biofilm, you see that there's about a two log drop, and to very low levels. So the lysin, as well as the phage is very active in disrupting E. faecalis biofilms. Okay.
july11-371	DR. STEVENS: So as we've seen in other presentations today, there are many, many other E. faecalis phages that have been isolated and characterized, and out of many of these, the lysins have also been identified, sequenced.
july11-372	DR. STEVENS: We've compared the sequence of the ly -- the ORF28 lysin that we're -- that we got from our phage to each of these other lysins, and, surprisingly enough, when we look -- when we do a BLAST analysis, we see only very moderate homology between the EF11 ORF28 lysin, which is what we've been working with, and each of the lysins of the other E. faecalis phages.
july11-373	DR. STEVENS: So I'm no -- I won't go through each one individually, but you can see easily that there's only a modest percentage of identity between these two lysins.
july11-374	DR. STEVENS: In another phage, phage 1 -- this one was I think the one used by Fischetti's group in isolating the lysin that they published on -- again, you can see only a moderate degree of homology between our lysin and the lysin of the phage 1, and so on and so forth for each of the other E. faecalis phages that we analyzed, and so this is sort of a summary of that.
july11-375	DR. STEVENS: If you, you know, go down the list, there appears to be only 10 to 20 percent identity between the lysin that we obtained from this phage and any of the other E. faecalis phages, which is curious to me because these are all E. faecalis phages and, presumably, they all have to lyse the same, or very similar, cell walls in order to go through a lytic cycle, and yet they are obviously different.
july11-376	DR. STEVENS: So one thing that we would like to do is actually compare the host range, if you will, of the lysin that we have to the host range of many of these other phage lysins and see if there's an overlap or not.
july11-377	DR. STEVENS: So, with that, I'll conclude my talk. We're trying to produce a super phage that will be super useful in phage therapy.
july11-378	DR. STEVENS: Before we close up shop I have to thank the -- all the contributors to this work. Hongming Zhang is a research scientist working in my lab. Tina Buttaro is a professor at the medical school who set up all of the biofilm assays. She's done a lot of work in E. faecalis biofilm analysis.
july11-379	DR. STEVENS: Derrick Fouts who is here, in the audience, somewhere in the back I think, helped. He is a staff scientist at JCVI, and he was -- played a major role in the sequencing and annotation of our phage genome. Lastly, but not leastly, Justine Tinoco was a graduate student who did many of the assays that you saw in this presentation. So, with that, I thank you. DR. RANALLO: Okay. So we have some time for questions if anybody has, anybody has any questions. AUDIENCE MEMBER: It's an interesting observation that you don't see a lot of homology between your lysin and those of other phages. I'm just curious what the identity -- if you exclude yours and look at how similar those other lysins are to each other, is there also dispar -- is there disparate relationships between those as well? DR. STEVENS: I haven't really done that. That would be interesting to do as well. I mean you can do each permutation of each of them against all of the others and see. But, again, I know that you're very interested in cell wall structure, and, you know, I'd be very interesting -- interested in learning more about the potential binding sites for the lysin. That may also be something you're interested in as well, whether each of these lysins have a different target on the cell surface or not. We just don't know that. DR. RANALLO: Actually, I had a question.
july11-380	DR. STEVENS: Is it -- did I understand correctly that the antibiotic resistance marker, once you're done, is still present? You know what --
july11-381	DR. STEVENS: Right. It's a great point. I did not mean to imply that the genetic engineering is completed. This is mark two or three of the manipulations that we've been doing. Before this could be used in a patient certainly, you're absolutely right, we would have to use a different strategy for just eliminating the antibiotic-resistant marker, and there are markerless methods of doing that as well. Yeah.
july11-382	AUDIENCE MEMBER: So, yeah, just one quick question. Have you looked at using Hidden Markov Modeled -- Modeling -- predictive modeling for the structures of the various lysins that you're looking at to see if there's an overall structural fold that's held in common?
july11-383	DR. STEVENS: No. Haven't done that.
july11-384	DR. RANALLO: Okay. So we're going to continue on with the engineering theme with Dr. Timothy Lu from MIT. Tim is a rising star at MIT. He's an associate professor and leader of the synthetic biology group in the department of electrical engineering and computer science and department of biological engineering at MIT. Tim's going to talk to us about engineered phages for the dia -- for diagnostics and therapeutics. Take it away, Tim.
july11-385	DR. LU: All right. Thanks a lot for the opportunity to be here. I think it's a really exciting forum to be able to talk at. I also want to thank Dr. Stevens earlier for basically introducing why we want to engineer bacteriophages. So I'm going to walk through some of the work that we've been doing in our own group to try to engineer bacteriophages for a variety of applications. I think they're pretty interesting, you know, chassis to play with.
july11-386	DR. LU: Before I start, you know, I'm involved with several companies involved in sort of commercializing bacteriophages -- I wanted to list them here -- including BiomX, Eligo, and AmpliPhi, as well as Sample6.
july11-387	DR. LU: So my lab is really focused on synthetic biology. Really what we're excited about is really this exponential increase in our ability to genetically engineer stuff. That might include viruses, it might include cells. Today I'm going to focus primarily on viruses.
july11-388	DR. LU: So we're excited, really, by this exponential improvement in our ability to read and write DNA, and how can we leverage that to modify organisms or viruses for useful applications.
july11-389	DR. LU: So this has been a challenge for us since I was doing my Ph.D., and one of the questions that I started off with was could we try to engineer bacteriophages for therapeutic, as well as diagnostic applications.
july11-390	DR. LU: Initially we were inspired by this challenge which I think we've heard about already, which is that can we get away from this idea of using broad spectrum antimicrobials and move to a paradigm where we use narrow spectrum antimicrobials to either treat infections or, I think, actually, a potentially even more exciting opportunity, or at least equally exciting opportunity, is to modulate the microbiome.
july11-391	DR. LU: If we're going to do this we need strategies that allow us to do diagnostics and therapeutics. So if you have a narrow spectrum antimicrobial but you can't quickly tell whether an infection is going to be susceptible to it, from a clinical perspective, it's going to be really hard to deploy.
july11-392	DR. LU: So I think we've been focused on trying to develop tools to allow you to build rapid diagnostics. So can you engineer phages as a diagnostic tool? So I'll tell you briefly about that effort, and then for the remaining time I'll tell you about some of the effort to now engineer bacteriophages in a variety of different ways, primarily using them as gene therapies for bacteria, and how we can then use that to modulate bacterial populations in targeted fashions.
july11-393	DR. LU: So I'll start off with the diagnostic application. Sort of alluded to this earlier. Really, what we want to ultimately do is enable precision therapy, right? So we do precision therapy increasingly for cancer. Why don't we do that for infectious diseases?
july11-394	DR. LU: Well one of the things we need to enable that is a rapid diagnostic platform, and, ideally, something that's relatively easy to use, point of care, and can give us information about what bacteria we're actually going after.
july11-395	DR. LU: If we can do that, then we can potentially couple that with narrow spectrum antimicrobials. In some cases that might be phage therapy on its own. I think we've heard a lot of great examples here about combining phages with other antimicrobials. I think that's a very potentially powerful way to go about it, especially if you start coupling some of the strategies we heard earlier from Dr. Turner and others. So we're excited about coupling the two together, and so a -- you know, after my Ph.D. we decided to try to see if we can actually try to solve the first part of this problem. Can we develop diagnostic tools that allow us to rapidly diagnose the presence of microbes?
july11-396	DR. LU: So here's the basic idea. And this is an idea that the field has worked on for the last 20, 30 years in terms of building reporter phages, but I think we're quite excited that we've been able to now commercialize this and actually do the genetic engineering of these phages at a point where it's actually applicable at industrial scale.
july11-397	DR. LU: So the idea is really simple. We know that phages can be narrow spectrum, and so we can identify phages that are selective for certain bacterial populations, and then we can genetically engineer those bacteriophages to basically force the cells that they infect to produce some sort of reporter.
july11-398	DR. LU: So in this particular case we're engineering the bacteriophages to deliver some sort of reporter gene, like a very strong luciferase, and basically what happens is the bacteria get infected by the phage, they start generating luciferase, and now, with a reader, you can basically detect whether there's light coming from your bacterial population.
july11-399	DR. LU: This has allowed us in -- to build diagnostic tools that give us readouts of presence of bacteria in a population in a few hours.
july11-400	DR. LU: So the initial application for this technology we started off with was actually for the food industry. It was a little bit lower hanging fruit for us initially when we started the company. So we started off going after Listeria -- so Listeria is one of the major food pathogens -- and subsequently we have tests for Salmonella and E. coli sort of in the pipeline.
july11-401	DR. LU: Longer term I think this technology, especially as we get better and better at engineering these bacteriophages, has a broad range of applications in the clinical space, being able to do, potentially, rapid diagnostics for other clinically-relevant systems.
july11-402	DR. LU: So here's just a comparison for the sort of phage technology we've developed to compare it to sort of conventional assays that are used in the food industry. Like PCR or immunoassays, those can be quite slow, primarily because they require a primary enrichment step. So both of those methodologies require you to grow the bacteria for a period of time so that it's -- the test is either sensitive or specific enough. Number one, most food companies don't want to be growing large amounts of pathogens on site so they often ship that out, that adds additional time, and then the enrichment time itself adds time to the actual assay.
july11-403	DR. LU: So our goal was to try to see, can you develop a test that you can run on site that's easy enough to use, that you can basically take non-trained biologists, basically, you know, potentially high school or college-trained technicians, teach them to run this assay on the factory, and be able to get a result on the same day. So you can come in the morning, do an assay, see if the food has, for example, bacteria or not, and then you'll make a decision on what you do with that.
july11-404	DR. LU: So I'm happy to tell you that we spent a few years and developed actually a test that's, frankly, pretty simple and easy to use. So basically one version of this test looks as follows:
july11-405	DR. LU: We have basically a sponge they use to swab some sort of surface. You then put a bit into a bag, you add the bacteriophages, you let it sit in the incubator for about six hours. Then you take a little bit, an aliquot of the liquid there, stick it into a very simple luminometer, and then you basically read is there light or not? Based on that information, you can make a diagnosis of whether there was a particular bacteria, in this case Listeria, in the sample that you were taking.
july11-406	DR. LU: The system is quite easy to use, and so we've been able to deploy this in a variety of sort of large food processing plants where people basically -- we don't need to train technicians, as you might need if you're performing PCR-based assays to carry this out.
july11-407	DR. LU: In addition to this, we don't really have time, but the cool thing about sort of having a cheap and easy to use diagnostic is then you can couple that with analytical tools. So we've now developed methods where you can sort of geo-locate where assays are being taken on the factory floor and really build sort of analytical cloud-based tools to see where contaminations are happening on your factory floor and how you stop that from happening.
july11-408	DR. LU: So one of the reasons we started off with the food application is because you can go through and get a test that's industrially used in a relatively short order. So we got the certification from this AOAC institute for the detect test.
july11-409	DR. LU: But now we've also been working on a variety of therapeutic, sort of clinically-relevant sensors. Here's just an example. I don't have sort of like the more finalized data to show you, but just thought I would just point out some, you know, that this can work to detect bacteria in other formats.
july11-410	DR. LU: So in terms of saliva, urine, and blood, we've done this sort of testing. In this case we have Salmonella that we can detect pretty -- relatively quickly. In about a few hours you can detect down to about one or 10 CFUs/ml. So I think there's app -- sort of potential applications for this technology that you can envision beyond just the food industry.
july11-411	DR. LU: So I think I told you about some more efforts to try to develop rapid diagnostic tools with phages. I think they're very useful and already sort of making an impact in the industrial space. I'll spend the rest of the time talking about some of the therapeutic applications that we've been envisioning and what we've been focused on over the last, I would say decade.
july11-412	DR. LU: So I think we've heard about this previously. I think one of the areas that we're excited about is this idea that, potentially, we can engineer, or evolve, antimicrobial agents in a -- to keep pace with the evolution of resistance, bacterial resistance.
july11-413	DR. LU: I think the challenge with going after bacteria is they're, you know, probably going to outsmart anything we throw at them eventually, but if we can keep -- at least keep pace with them in the development of novel antimicrobial agents, maybe we can at least keep pace with their development of resistance. So if they take a step forward, can we take a step forward ourselves with a counter-measure.
july11-414	DR. LU: So we've developed a variety of phage engineering-based technologies to do this. We heard a lot yesterday and today about engineered bio -- about bacterial biofilms and how they can be a problem.
july11-415	DR. LU: From my simplistic engineer's perspective, I sort of think of biofilms like fruit Jello, where the fruit is sort of like the bacteria and they make this gelatinous matrix that makes it very hard to clear out the bacterial contamination, so it poses a challenge for any antimicrobial agent youâ€™re trying to develop.
july11-416	DR. LU: Similarly, biofilms can be associated with antimicrobial resistance. I think we've heard a lot about the challenge of going after antimicrobial resistance so I'll skip over this.
july11-417	DR. LU: In our lab we're primarily focused on going after the gram-negative pathogens. I think we're particularly focused on this because of the great need for novel antimicrobial agents, especially going after these specific pathogens.
july11-418	DR. LU: So early on -- actually, this is some of the Ph.D. work I was doing together with Jim Collins. We started to think about how do we develop target therapies to go after biofilms, right? We know that biofilms are involved in a lot of medical-related issues, and as well as in the food or industrial space, biofilms are part of a major sort of burden on industry. Current methods for going after this including mechanical disruption or chemical-based methods are not necessarily the most effective.
july11-419	DR. LU: So one of the strategies we came up with, this was back in 2007 when we published this, was this idea that we could engineer bacteriophages to express biofilm-degrading enzymes. If you actually look at natural phages, some phages actually carry these enzymes with them to allow them to access biofilms or to degrade polysaccharides.
july11-420	DR. LU: What we tried to do is to demonstrate that you could actually synthetically encode the expression of these enzymes into an artificial phage. So in this particular case we took a model phage T7, showed that you could genetically modify it to express a biofilm-degrading enzyme, and the idea is if you could then sneak just even a little bit of that bacteriophage into the biofilm, you could generate this loop where you generate more enzyme, it breaks up the biofilm, and hopefully help propagation of the engineered bacteriophages.
july11-421	DR. LU: So we showed in this particular experiment that if you compare the engineered bacteriophage, which has dispersin B, an enzyme that is known to disrupt certain types of biofilms, with a control or untreated, we could get, in general, two to four orders of magnitude increases in our ability to eradicate these biofilms, even with a very small dose of bacteriophages to start.
july11-422	DR. LU: In addition to that, so we -- you know, that was like the initial work that we did, and we quickly realized that perhaps the bacteriophages could then be extended to other sorts of applications. So could we use the bacteriophages not just to degrade bacterial biofilms, but to potentially synergize with other treatments that are already in use. So one of the strategies that we started to look at was whether we could actually engineer phages sort of like gene therapy vectors for bacteria to deliver payloads into bacterial populations that allow them to have an effect on antimicrobial resistance, for example.
july11-423	DR. LU: We heard a lot earlier about sort of diverse mechanisms by which bacteria can become resistant to antibiotics, including sort of discrete mechanisms like exporting the antibiotic or degrading the antibiotic. In addition, there are sort of bacterial defense mechanisms, for example, the generation of reactive oxygen species and sort of the triggering of certain response pathways inside the cell, that could be potentially targeted with an engineered phage if you think about it really -- not the phage as a sort of direct killer, but as a gene therapy device.
july11-424	DR. LU: So here's a very simple schematic of some of the work that we did. If you envision antibiotics inducing DNA damage that induces some sort of let's say a DNA repair response that allows a cell to survive, what if we could try to potentiate that sort of strategy by engineering a phage. In this case, this is a phage that's not lytic, it's lysogenic, that potentially can deliver a gene inside of the cell. Here we used a particular protein, LexA3, which suppresses the SOS response. The idea was we wanted to test whether we put these two strategies together, can you get a potentiation of killing.
july11-425	DR. LU: So we actually looked at this engineered bacteriophage, phage lexA3 which is shown here in blue, in comparison -- sorry -- in combination with three different classes of antibiotics: quinolone antibiotics, which in this case is ofloxacin, aminoglycoside, gentamicin, as well as a penicillin class drug, and showed that in all cases, if we combined the bacteria -- engineered bacteriophage together with the antibiotic we -- you got a potentiation of killing by several orders of magnitude. This was simultaneous treatment.
july11-426	DR. LU: We also looked at what happens if you can take sort of bacteria that are already resistant to drugs. So here's an example where these bacteria were already resistant to ofloxacin.
july11-427	DR. LU: We showed that, you know, normally if you just apply ofloxacin on its own, really, these bacteria don't really get affected by very much, maybe an order of magnitude of killing. Combine this together with the engineered bacteriophage, we again get a very significant potentiation of the killing effect. So we then went on to test this in an animal model of infection. We basically took E. coli, infected the bac -- infected mice, and then tried to treat with either antibiotic alone, which is shown in black, the solid black line, or the combination therapy, the engineered phage, plus the ofloxacin antibiotic, and showed here in blue we can -- found sort of increased survival with the combination type approach.
july11-428	DR. LU: So I think, moving forward, it would be quite interesting to explore how, you know, engineered -- phages can be engineered in this fashion to try to synergize with antibiotic -- particular antibiotics, or, as I'll show you a little bit later, where we might be able to engineer phages to try to directly re-sensitize bacteria to antibiotics or kill selectively antibiotic-resistant pathogens.
july11-429	DR. LU: So that sort of leads us to this next story. So when I first started the lab I had two very talented students, Rob Citorik and Mark Mimee, who wanted to take this to the next level and think about, can we build even more targeted strategies as antimicrobial agents.
july11-430	DR. LU: So we started off thinking about, again, this problem of broad spectrum antimicrobials, which generally address either protein-based targets or other sort of, you know, cell wall synthesis type mechanisms. What if we could actually develop antimicrobials that act at a very different level, right?
july11-431	DR. LU: So if we want to really realize this dream where we can make a new antimicrobial base really quickly, then one of the best ways, potentially, to do that is if we can just make sort of sequence-specific antimicrobials. Because we can make -- we can sequence DNA really easily, and then we can make -- we can print DNA really easily, right? It's a lot easier for us to do that rather than develop a new drug with a target-specific protein.
july11-432	DR. LU: So what if we could actually enact specific targeted pressure against undesirable genes at the level of DNA? So in order to do this we actually started off using zinc finger and TALE factors, but quickly realized that the CRISPR system was a more powerful way to do this.
july11-433	DR. LU: I'm sure everyone here knows how CRISPR works, but just briefly to mention that we sort of think about the Cas9 enzyme, which is shown here, this Mickey Mouse structure, as a molecular scissor, it's directed by what's known as guide RNA, to target a specific location of DNA and cause cutting.
july11-434	DR. LU: In human cells people use this for genome editing because the cutting event leads to repair pathways that repair the DNA in a specific way. In bacterial systems that lack, you know, very robust repair systems, this can induce cell death.
july11-435	DR. LU: So the idea here would be very simple. What if you could actually engineer a bacteriophage -- we also did this with bacterial conjugation in sort of like a -- we could talk about this later, if you're interested -- we -- sort of like in a gene drive type methodology. You can imagine spreading sort of self-transmissible plasmids everywhere that contain this. But here, because this is a bacteriophage meeting, I'll just focus on the phage-based data.
july11-436	DR. LU: So what if you could make a phagemid, right? So this is not a propagating phage, it's just a virus structure that contains a piece of DNA that just delivers the DNA. So, again, this is really thinking about phages as a gene therapy vector.
july11-437	DR. LU: So what if we could package the Cas9 system into a phagemid, and then use that to deliver this Cas9 system into targeted bacteria? The idea would be that in bacteria that contain a specific gene that you don't like, like a resistance gene, antibiotic resistance gene, you could cause DNA cleavage, cause those cells to die, but in a related bacteria that doesn't have that sequence, they would be fine.
july11-438	DR. LU: So here's the experiment we did. We took a bacterial cell. This is, in this case, E. coli that contains a genomic target. Here we tried two different settings. So we had wild type E. coli, as well as E. coli with the gyrase A mutation that confers quinolone resistance, and then we developed two different RNA-guided nucleus type phagemids, one that targets the ndm-1 beta lactamase gene -- this is -- as well as one that targets specifically this mutation, gyrA.
july11-439	DR. LU: So in the base case, in the wild type cells, you basically transduce them with this phagemid, and it basically showed there's no really toxicity that you can see with this sort of approach, but if you then follow up with using this engineered, what we call the sort of RNA-guided nuclease phage, or the CRISPR phage, to deliver into these cells, we can get a very selective killing of the bacterial population with the gyrase A mutant, but not the ndm-1 targeting mutant.
july11-440	DR. LU: So this, in effect, shows us that we can actually -- if we can achieve efficient delivery of payloads into a specific bacteria either using phages, or conjugative methods, or other methods of these Cas9 type elements or other CRISPR systems, could be useful for causing site-specific cleavage, as well as then cell death.
july11-441	DR. LU: Because of time I won't show you some of the other data we generated in this paper. You could take a look. We also show that you could actually target plasmid-borne targets. Depending on the context of that plasmid, you could either just cure the cells of the plasmid without affecting, really, toxicity against the cells, or cause cells to die.
july11-442	DR. LU: Other applications of this technology potentially using the CRISPR system as a diagnostic tool. So here, this idea is, again, very simple. If we can engineer these phages to cause cleavage, the cleavage event, at least in some bacteria, triggers the SOS system, and then if you have a reporter that turns on some sort of GFP or luciferase, you could then use this for very sequence-specific diagnostic tools.
july11-443	DR. LU: We did two examples of this. Here, one where we have E. coli with the ndm-1 plasmid from a clinical isolate. We show that with the cognate RNA- guided nuclease you see an increase in GFP fluorescence in that case, and similarly in the E. coli that contains the gyrase A mutation, you get a very selective increase in GFP fluorescence.
july11-444	DR. LU: So, again, I think if you're going to build very specific killing tools, you need very good diagnostics that go with them. There's a lot of improvements we can do upon this, but this is a proof of concept that you might be able to use this methodology for sequence-specific diagnostics of bacteria based on their genomic sequence.
july11-445	DR. LU: So I think one of the things I want to follow up on is, you know, we've talked a lot in this conference, I think almost every talk in this conference has really been about infectious disease applications of bacteriophages, and, indeed, that's the traditional way of thinking about phage therapy, and then -- we are quite also excited about that potential approach.
july11-446	DR. LU: I do think that actually one way for phages to maybe have a broader usage is to sort of think about phages as a way of modulating the microbiomes, right? So when we're using bacteriophages to target a population, oftentimes the bacteria that we're going after are not just like sort of the dominant player there, they might be one small member of a sort of consortia of bacteria that have a wide range of effects on our immune system or health, and so if we're going to go and try to modulate the microbiome, we need tools that are very narrow spectrum and targeted to be able to modulate specific members of the microbiome.
july11-447	DR. LU: Right now the tools that we have are relatively crude. So we have fecal transplants, which is like taking an entire ecosystem and trying to slam it onto another ecosystem, we have antibiotics that sort of act as like sort of a nuclear bomb on your microbiome.
july11-448	DR. LU: So I think if we're thinking more precisely about replacing or delivering things into a microbiome we need tools like phages, potentially, or other sort of narrow spectrum antimicrobials that can be useful for this type of approach.
july11-449	DR. LU: So I would advocate that thinking -- going forward, as a field, we should think not only about the infectious disease applications, but let's think about the microbiome-type applications. There's a couple advantages here that we can talk about maybe later in the forum, one of which is that potentially you can avoid sort of only talking to the ID docs. No offense to ID docs, but really expanding upon the indications that you can go after with engineered phages or, you know, natural phages for microbiome-associated diseases which are now being implicated in a variety of different areas, including GI health, neurodevelopment, et cetera. Really opens up the scope of what this powerful tool is that we all have a lot of interest in.
july11-450	DR. LU: So we've been thinking about how do you start modulating microbiomes, and can you test the specificity of this. I'm just only going to show you in vitro stuff because it's the stuff that's been published so far, but we're certainly doing a lot of in vivo work in this now.
july11-451	DR. LU: Here's an example using the CRISPR phage. It's to target a three-member consortia, right? So all through these bacteria here, blue, purple, and orange are all susceptible to the phagemid, they can all be infected by the phagemid, but only -- they all have different genomic signatures.
july11-452	DR. LU: So, for example, if we apply phage B, which targets the B gene in this population, we only want to kill the bacteria and we want to leave the other guys happy, right? So imagine if you had a bunch of E. coli in your body. One of them was bad, you want to get rid of that, but affecting everyone else. Similarly, we do that with this phage A gene.
july11-453	DR. LU: So here's an example where we have the three member community. If we apply the very specific ndm-1 bacteriophage, we can basically knock down the ndm-1 tool -- sorry -- the ndm-1-containing bacteria, and similarly with the gyrase A mutation.
july11-454	DR. LU: So we're starting to think about these phages not just as like antimicrobials, but, really, can we do what RNAi did for genetics. But think about this at a population level. Can we build phage-based tools or other conjugative-based tools that allow us to do specific knock downs of a bacteria in a population. All right.
july11-455	DR. LU: So this is a summary of this particular approach. I sort of mentioned sort of genomic targeting, but the paper actually describes other stuff. Targeting plasmids, for example. And we're sort of thinking about this like gene drives, where people try and eliminate mosquitoes in a population. What if you could eliminate bacteria also, similarly in a way. This is kind of radical, we haven't figured out the regulatory path, but I think, technically, it's actually very doable. All right.
july11-456	DR. LU: So in the remaining time, I know we have lunch afterwards, I thought I'd tell you some -- just finally, the other things we're working on in the lab. I think we've quickly realized after all this work that phages are a really useful tool. We can engineer them to deliver all sorts of cool payloads into the bacteria. The challenge ultimately, and this is similar to the gene therapy field, is that like delivery is still the challenge.
july11-457	DR. LU: So how do we achieve delivery? How do we get the bacteria that we want, and be able to engineer the phages to do what we want, and deliver the right thing to the right place? So we heard about this earlier from the previous speaker, about sort of classic phage hunting. I think there's a lot of benefit for that side of -- type of approach. The primary benefit is that you come out with natural phages, and, potentially, the regulatory hurdle I think is lower with those sort of things.
july11-458	DR. LU: But I think there is a great opportunity for thinking about phages as an engineerable biotechnology. So one of the things we've been interested in doing is to try to adapt this idea of the antibody, but conceptualize this with the phage scaffold.
july11-459	DR. LU: So what if you could take a phage and keep most of the phage the same and simply switch out some parts of that phage to redirect its activity against other bacteria? If you could do this, there might be some advantages.
july11-460	DR. LU: First, most of the phage scaffold is the same. It just makes genetic engineering of that phage scaffold really easy, right? We can develop one set of tools and just use it over and over again.
july11-461	DR. LU: Secondly, manufacturing potentially could be easier, right? So if we don't have to worry about sort of manufacturing 20 different phages that are completely different from each other, if we have one phage that's quite uniform and simply tweak, for example, the tail fiber to change a spectrum, that might be beneficial.
july11-462	DR. LU: So this idea of the phagebody we did some work on a few years ago, and we continue to do stuff now. I have a couple papers in review on this idea. Basically, we wanted to show that you could actually take phages and swap tail fibers, right? So it's previously been shown in -- from the literature that phages can make hybrids, for example, and you can change specificity based on that strategy.
july11-463	DR. LU: Could we develop an engineering pipeline to enable that tail fiber swapping more efficiently? So here's a very simple concept. Can we take the red phage, which normally goes after the red bacteria, and instead make it go after the blue bacteria. The way we do that is by giving the red bacter -- phage the blue bacteria phage's tail, right? So we swap them. All right.
july11-464	DR. LU: So we heard earlier about some of the challenges of engineering bacteriophages. One of the challenges with engineering bacteriophages, especially lytic ones, is they kill bacteria, and most of the tools that we have for engineering anything is reliant on sort of bacteria staying alive.
july11-465	DR. LU: So we had to come up with an alternative strategy. Fortunately, you know, the folks at JCVI, and others, have developed tools based on yeast that allow you to do very efficient genome engineering.
july11-466	DR. LU: So what we realized is if you could capture the phage genome into a yeast artificial chromosome, you know, the phages are pretty happy in terms of living there because they don't really kill the yeast, and now you can propagate the yeast, do whatever you want with it.
july11-467	DR. LU: The other cool thing about yeast is it's really good at DNA assembly. So you can make different fragments genomically and assemble them together. You can then extract the phage genome out and then transform your bacteria, and then you can then, in many cases, actually boot up viable phages. It's pretty cool.
july11-468	DR. LU: So here's an example where we did this. We basically wanted to just recapitulate some of the experiments that had been known previously on T7, T3 hybrids. So here's an example where we basically took T7, and we grafted the GP17 tail fiber protein from T3 onto the T7. We built two different constructs, one where we swapped the whole gene 17, we also swapped just a portion of gene 17 between the two phages, and we showed that this basically was sufficient to cover host range switching activities.
july11-469	DR. LU: So basically, the T7 with the T3 tail fiber basically looks like T3 in terms of its host range, the T3 with the T7 tail fiber looks like T7 in terms of its host range.
july11-470	DR. LU: We've also now done this with other bacteria -- sort of targeting other types of bacteria. Here, this is a very simple example where we simply mutated several point mutations in the T3 genome to make T3 phage now go after Yersinia.
july11-471	DR. LU: We've also done other experiments where we actually start swapping tail fiber components between bacteria that target different species. So here we took Klebsiella K11 and we swapped several components onto the T7 scaffold.
july11-472	DR. LU: So one of the things we're trying to explore is really how modular and flexible this strategy is going to be. Initially, we were hoping that the tail fiber itself was going to be sufficient for conferring this host range switch. With K11 we realized that this was not actually going to be possible, and so we ended up having to swap the GP11-12 structure which really composes this sort of tubular structure, as well as the tail fiber, in order to get sufficient tail fiber swapping.
july11-473	DR. LU: Here's just a demonstration that the T7 with the Klebsiella tail components infects Klebsiella, the K11 phage with the T7 components infects E. coli. Here's both on plaque assay, as well as based on killing. The T7, K11 shown here in the green does have about three or four orders of magnitude of killing, although it's not as good as the native bacteriophage, and we're trying to figure out why that might be the case.
july11-474	DR. LU: Similar as what I showed you earlier. The ultimate goal here is can you produce a population, a cocktail of these bacteriophages, whether directly having lytic efficacy or delivering some sort of payload, but go after a population of bacteria.
july11-475	DR. LU: And so here, again we just show that you could actually make cocktails of these bacteriophages, for example, T7 plus T7 containing the K11 tail fiber -- so basically, these phages are majority similar -- except of the tail fiber component -- and show that when you put these things together, you can then eliminate one or more species in a sort of rational way from a microbial population, as we show here.
july11-476	DR. LU: So, with that, sorry to run a little bit over time. I think I just want to reiterate this idea that, you know, infectious diseases is great, we should continue going after that, but I think, personally at least, we're very excited about sort of the opportunities here for modulating microbial communities and thinking about microbiome type applications.
july11-477	DR. LU: This wouldn't be possible without a very good lab and talented group of people. So Rob Citorik and Mark Mimee did the work on the CRISPR phages, Sebastian Lemire, as well as Hiroki Ando, did the work on the engineered phage bodies. With that, I'd like to thank you for your time and take any questions.
july11-478	DR. RANALLO: So that was a lot to unpack, Tim, but I'm sure we have -- we certainly have time for questions if anybody has any questions. Oh, and just for my FDA regulatory colleagues, in terms of engineering, that pain in your head, that's called a new headache.
july11-479	DR. RANALLO: (Laughter.)
july11-480	AUDIENCE MEMBER: Hi. That was a super, super cool presentation.
july11-481	DR. LU: Thanks.
july11-482	AUDIENCE MEMBER: I have a couple of questions for you. Have you tried using nanoswitches for your bio detection assays? Sort of a similar approach, but with a nanoswitch.
july11-483	DR. LU: What do you mean by a nanoswitch? I'm not familiar with the term.
july11-484	AUDIENCE MEMBER: You could use your bacteriophage, couple it with your whatever, luminescent release thing, and when the bacteria binds, it will make a conformational change that will release that light.
july11-485	DR. LU: I see. So you're saying that instead of like forcing the cell to express some sort of payload, to have some sort of reporter that, based on its conformational change, when the phage binds, it sort of switches on activity?
july11-486	AUDIENCE MEMBER: Yeah.
july11-487	DR. LU: No, we haven't done that. That's an interesting concept. Potentially could be a faster way of doing the detection.
july11-488	AUDIENCE MEMBER: It would be. And did you think about coupling your bacteriophages with nanodots or nanoparticles? And so you would amplify your signal and get a real time detection.
july11-489	DR. LU: Yeah. No, I think that's a great idea. Think it's worth thinking about. Thanks.
july11-490	AUDIENCE MEMBER: Okay. That's my comment.
july11-491	DR. LU: Okay. Thanks.
july11-492	DR. TURNER: That was really nice. I have a question about -- the evolution of modularity does help you with the swapping of tail fibers, and then you found out that you had to kind of go deeper into the structure to make it work.
july11-493	DR. TURNER: What about the mode of replication inside of the cell, where there is kind of a phage that used to a stamping machine, order of replication has to do something else. Do you have any evidence yet that you can take the approach and move into a very distant cousin, you know, a very distant relative, and what are the challenges?
july11-494	DR. LU: Yeah. So I think the challenge is, as you mentioned, if you want the phage to replicate, it sort of goes -- you not only need to be able to bind and deliver DNA, but you need that sort of DNA to be functional.
july11-495	DR. LU: So in this particular experiment we actually started off with phages that we knew could replicate in the target host. So, for example, we just took Klebsiella phage DNA, electroporated it into E. coli, it could boot up.
july11-496	DR. LU: I think that's going to be a challenge with some more distantly related applications, so I think there we're going to need to be able to identify chassis that potentially either have broad host range capabilities in terms of replication capacity, or you might have to build sort of cocktails that are based on sort of nearly related bacteriophages.
july11-497	DR. LU: The one area that we are quite excited about is actually not thinking about the phages as totally a lytic tool, but, really, just delivery. So if you could just deliver stuff and you could have some other mechanisms of action, you know, based on the DNA that you've encoded, then I don't have to necessarily worry about the phage having to replicate in order for us to have the activity.
july11-498	DR. LU: So sort of more thinking about them as sort of purely just biocapsids that you can swap around is another -- is actually one of the big areas that we're trying to move this technology into. It's a little bit simpler.
july11-499	DR. TURNER: Nice.
july11-500	DR. LU: Thanks.
july11-501	AUDIENCE MEMBER: Excellent presentation. So this engineering things is very attractive, but my question is if the receptor starts changing when you use this engineered phage, what is your remedy? Because you spend too much time and money to develop this engineered phage.
july11-502	DR. LU: Yeah, yeah, that's a great ques -- so I didn't show you here because the paper is currently in review right now, but we have some strategies to engineer phages and -- sort of at a high level, build very dense libraries of bacteriophages that you can then easily find sort of new vectors that overcome that. So I think, at a vague level, that's what we've been able to do.
july11-503	DR. LU: I think certainly phage evolution, I mean bacterial evolution is always going to be a challenge for I think phage-based approaches, whether you're using it for lytic applications or non. So I think -- I'm not going to say that you're going to ever be able to deliver -- sort of have a methodology that's going to always work universally, but I think we need high throughput strategies to keep up with the pace of evolution. I think that's the only way we're going to keep up with bacteria.
july11-504	DR. LU: So, but they're always going to try to outrun us in terms of their ability to evolve resistance to whatever we're throwing at them.
july11-505	AUDIENCE MEMBER: So your concept is developing an engineered phage library to tackle all the problems.
july11-506	DR. LU: Yeah. I think there's a lot of regulatory questions about how that might be applied, but like if we can get these phage-bodies sort of to work and we have a common scaffold, but we're simply changing certain components to get around the bacteria -- keep pace with as the bacteria resist, then, from a technological level, we can definitely do it. From a regulatory perspective, I'm not sure how they would view that.
july11-507	DR. LU: But from -- if we can do it technically, I'd rather show that first, and then maybe figure out the details of the regulatory afterwards.
july11-508	AUDIENCE MEMBER: Right. It look ready for the venture capital aspect because you can take the patent on those, you know, modified phages, but nature already prepared these phages into the 10 to the 31 titer. You need to just harvest them and use it.
july11-509	DR. LU: Well I think that's certainly an interesting -- yeah. So I think there's sort of like the natural phage groups, and then there's a engineered phage group.
july11-510	DR. LU: I think the challenge, at least from my perspective, if you want to enhance the natural capacity of some of the phages through genetic engineering at least, if you're going to make a cocktail of like 50 different, well even just like five really diverse phages, it just becomes really hard to genetically engineer those in any industrially-relevant way, and so we're trying to set up methodologies where you sort of have well-defined things that you can manipulate over and over again. It just makes it a lot easier to commercialize. Yeah. Yeah. Thanks.
july11-511	DR. RANALLO: Okay. So we have ample time for lunch. We're going to be back here -- oh. Thank you, morning speakers, very much. It was excellent. We're going to have some more discussion on engineered phage and natural phage during the panel discussion. I hope you guys can all join us. We're going to come back here a little over an hour, at 12:40. So we're off for lunch until 12:40. Thanks.
july11-512	DR. RANALLO: (Whereupon, at 11:35 a.m., the meeting in the above-entitled matter was recessed, to reconvene at 12:40 p.m. this same day, Tuesday, July 11, 2017.)
july11-513	DR. RANALLO: A F T E R N O O N S E S S I O N (12:40 p.m.)
july11-514	DR. RANALLO: So we're going to get started again. In interest of our speakers, for the last three speakers, I did give everybody a little bit more time for lunch. So -- not that much time. So we're going to get started here again with this last session on future directions.
july11-515	DR. RANALLO: Andrew, or Andy Camilli is going to start us off about -- talking about prophylactic use of bacteriophages against cholera. Andy Camilli is a Professor in the department of molecular biology and microbiology at Tufts University School of Medicine, and is also an investigator with the Howard Hughes Medical Institute.
july11-516	DR. RANALLO: I'm really excited to hear his talk on prophylactic use of bacteriophage against cholera, so, without further ado.
july11-517	DR. CAMILLI: All right. Good afternoon. I guess this is the dangers of being the first talk after lunch. Hopefully I'll also keep you awake. So, yeah, I'm going to talk -- so I guess I'm one of the rare talk about using phages for disease prevention.
july11-518	DR. CAMILLI: I think, you know, this is a -- kind of a unique example, cholera, as I'll tell you about, but I think it's interesting to keep in mind that there could be some other diseases and some other situations where phages could potentially be used prophylactically.
july11-519	DR. CAMILLI: Some other, I think, unique parts of my talk compared to what you've heard so far is, you know, we've heard a lot about the traditional paradigm of finding phages from the environment, from sewage, and finding ones that are active against the bacteria you want, and so one of the important parts of my talk is that we get our phages from the same environment where you want them to work, and I'll try to point out why I think that's important.
july11-520	DR. CAMILLI: So just conflict of interest statements. So, along with two of my post-docs, we founded a company called PhagePro, and I'm currently a scientific advisor.
july11-521	DR. CAMILLI: Okay, so the science. So cholera, as you probably all know, is this profuse watery diarrhea and vomiting disease. Virtually all cholera in the world and in the previous pandemics that we're able to have data on the strain have been caused by this O1 sera group. That's an LPS type.
july11-522	DR. CAMILLI: There's about 150 different serogroups known for this species, so it's interesting that virtually all cholera is caused by this O1 type. That's important because I'm going to show you later that this is a receptor for many phages, this O1 LPS.
july11-523	DR. CAMILLI: The secretory diarrhea and vomit are filled with Vibrio cholerae, and this is a highly transmissible bacterial pathogen, as I'll show you in the next couple of slides. It's got a high death rate, so prompt treatment with rehydration therapy is very important.
july11-524	DR. CAMILLI: There is an oral whole cell killed vaccine for cholera, but it only gives partial, short term immunity. There's a lot of research on trying to come up with better vaccines.
july11-525	DR. CAMILLI: So we've talked -- we learned a lot about d'Herelle yesterday. He was the one who first discovered cholera phages, and he noted that often he would find these virulent phages coming out in cholera patients' stool samples, so it's fun to a hundred years later still be working on, you know, I wouldn't say rediscovering what he's done, but making use of it in modern times.
july11-526	DR. CAMILLI: So this slide kind of shows the classic view of the life cycle of cholera. So a susceptible person drinks contaminated water, they get cholera. The bacteria colonize the small intestine, make cholera toxin, and they get this profuse watery diarrhea that results in what are called rice water stools. This contaminates the water further and this -- you get this vicious cycle.
july11-527	DR. CAMILLI: But it's been appreciated for a long time, but there's been some recent studies that have really pointed out this tremendous problem of rapid household transmission.
july11-528	DR. CAMILLI: So some recent papers have shown that the infection rate jumps two orders of magnitude, from about 2.5 per thousand via water-borne to about 230 per thousand if you're in a household where somebody comes down with cholera, so this means that about 23 percent of the households are exposed.
july11-529	DR. CAMILLI: The peak incidence of these secondary cases in the household is two to three days after the index case. that's a huge problem during cholera outbreaks. There's not enough time to go in and vaccinate the household contacts.
july11-530	DR. CAMILLI: So this is one idea we had, is perhaps we could use phages in a prophylactic manner to protect these, you know, the household contacts. The idea is that maybe by doing this very efficiently, we could perhaps blunt outbreaks.
july11-531	DR. CAMILLI: So in thinking then about this idea of prophylaxis using phages, we had a number of questions that we have come up I'm sure in other people's minds as well.
july11-532	DR. CAMILLI: So the first is are there Vibrio cholerae-specific lytic phages that are virulent in the human small intestine where the cholera is happening? I think this is an important point.
july11-533	DR. CAMILLI: We all screen for phages in the laboratory but it's been known for many years from many pathogens that they alter their surface properties during infection as opposed to growth in a flask in the laboratory, and so perhaps the receptors change. I'm going to talk a bit about that today for some of the phages we're going to talk about.
july11-534	DR. CAMILLI: So if there are such phages, what's the biology of these phages? What receptors do they use? What insights can you get from looking at the arms race between the bacteria and the phages? What are the mechanisms of Vibrio cholerae escape from these phages, because of course they will escape. And, importantly, do escape mutants remain infectious? Then finally I'll address this last question, can phages protect from cholera in an animal model.
july11-535	DR. CAMILLI: So this work on this started off a number of years ago with a former post-doc, Kimberley Seed. She's now an assistant professor at UC Berkeley. In collaboration with my collaborators in Dhaka, Firdausi Qadri, and in Boston, Stephen Calderwood, we took advantage of this great collection of glyceroled and frozen rice water stools that Firdausi Qadri's been keeping in her freezers for years.
july11-536	DR. CAMILLI: So we did this respective study just going back and getting a little bit of a frozen stool sample and then screening for phages in the stool sample.
july11-537	DR. CAMILLI: So these are three different stool samples per year, and what you can see is we found plaques in a number of these stool samples, and we were able to isolate the phages, sequence them, and put them into families and learn a lot about what these phages were.
july11-538	DR. CAMILLI: But for this slide, what's important is once we had the sequence and we saw how highly conserved these phages were, we were able to design PCR primers to go back and screen these stool samples in a more sensitive manner. When we did that we found something surprising.
july11-539	DR. CAMILLI: So there was a much more prevalence of these phages in these stool samples, and this one, IC -- we call ICP1 was omnipresent. It was in every patient's stool sample, which is really -- to us, was shocking. I'll say that these three phages are still around to this day. Even last year and this year's sampling shows that theyâ€™re still the phages we find in cholera patients' stool samples.
july11-540	DR. CAMILLI: So this kind of goes against that idea that there's a huge diversity of phages. This is a bacteria that lives out in the environment, that infects people. You'd expect a lot -- a huge diversity of phages. But it's not true. Apparently there's selection for phages that are really fit during this -- these epidemics and going into humans.
july11-541	DR. CAMILLI: Now you might ask why do we get plaques in some cases and no plaques in others, and I'll say that a lot of these stool samples have a high titer of phage in them. And the reason is we look for plaques
july11-542	DR. CAMILLI: -- we isolate a single colony from that stool sample and use that to screen for plaques, and so the reason is that because often the Vibrio cholerae in that stool sample is an escape mutant that's resistant to the phage.
july11-543	DR. CAMILLI: So starting with that first phage, ICP1, that phage that's omnipresent, we then asked basic questions. What's the receptor? It turns out if you mix this phage with Vibrio cholerae in the lab, you very quickly get escape mutants.
july11-544	DR. CAMILLI: The escape mutants are truncations, or alterations, to the LPS O1 antigen. That's the receptor for the phage. So this is what the O1 antigen looks like.
july11-545	DR. CAMILLI: Vibrio, as I said, high frequency escape, and the reason -- the way it does this is two of the genes within the biosynthetic locus for this O1 antigen have this run of As, and so at a very frequency you get slip strand mispairing during replication and you get a frameshift mutation.
july11-546	DR. CAMILLI: When you get a frameshift mutation there's stop codons downstream of these poly A tracts that now become in frame and you make a truncated product. The result, and this is work that Kimberley Seed did, is for the manA frameshift mutants you have a less dense O antigen on the surface, the phage don't like that, and the wbeL frameshift mutants are missing this tetronate modification and the phage can't plaque on them either.
july11-547	DR. CAMILLI: So this high frequency escape is apparently evolved as a built-in mechanism within the bacteria, but we don't see these escape mutants, these frameshift mutants coming out of cholera patients. The reason for that, and this I think is an important principle that's been mentioned a couple of times in other talks, is that the receptor in this case is an important virulence factor. The O1 antigen, this was shown by Matt Waldor and John Mekalanos years ago, is critical for infectivity, and so these frameshift mutants are anywhere from 10 to a thousand-fold attenuated for virulence in, in this case, an infant mouse model of colonization. You can revert these frameshift mutants back and they regain virulence.
july11-548	DR. CAMILLI: So this is why we don't see these high frequency frameshift mutants coming out of cholera patients, is they're -- they lose virulence. Yet some of those stool samples that I showed you where there was a circle, you can detect the phage by PCR, but not by plaque, the Vibrio strain in that stool sample is resistant.
july11-549	DR. CAMILLI: So we then asked, well what's the mechanism of resistance of those Vibrio cholerae clinical isolates, and we used whole genome sequencing to show that these contained this unique island. It's an 18 kilobase island called the PLE, for phage-inducible chromosomal island-like element.
july11-550	DR. CAMILLI: You can see, here's a strain with the PLE. It's resistant, and it's called phage-inducible because it's been shown in analogous phage-inducible islands in gram-positives that upon phage infection, these things pop out of the genome as a circle, replicate, steal packaging material of the helper phage, and that's how they're transmitted.
july11-551	DR. CAMILLI: So we designed these outward-facing PCR primers to be able to detect this excision and circularization of this element and, lo and behold, within five minutes of adding ICP1 phage, we can detect this circle.
july11-552	DR. CAMILLI: This excision and replication is not induced by other phages, and it gives immunity to this phage only. So it's kind of a phage-specific immunity system that the bacteria have evolved. And it turns out there's four different versions of this PLE in circulating clinical isolates. And Kimberley in her own lab is trying to figure out how these PLEs give resistance to ICP1.
july11-553	DR. CAMILLI: But what we do know is that it works very well as a defense mechanism against this phage. So it reduces -- in a culture can reduce production of phages by five orders of magnitude.
july11-554	DR. CAMILLI: Now we did occasionally come upon a stool sample from a patient where the Vibrio cholerae in that stool sample had a PLE, and yet ICP1 could still form plaques on it, so that there was more going on there. To figure that out, we just sequenced the phage isolates, and what we discovered is that this -- these phages that could plaque on a PLE plus host had their own CRISPR/Cas system. We published that a few years ago.
july11-555	DR. CAMILLI: This shows the Cas genes. So this is, as far as we know, the only phage-encoded, naturally encoded CRISPR/Cas system. So here's the Cas genes. There's two CRISPR arrays, and here's four different isolates with CRISPR arrays. Upon sequencing and looking at these spacers we immediately learned the mechanism, because all these spacers in this green color are perfect matches to proto-spacers in the PLEs, either PLE1, 2, 3, or 4.
july11-556	DR. CAMILLI: In fact, this phage from 2011 has spacers that target all four of the known PLEs, and so this phage is the first component of a phage cocktail I'm going to tell you about in a minute.
july11-557	DR. CAMILLI: So the next question was is this CRISPR/Cas system fully functional? Can it acquire new spacers, which would be an amazing property for a phage. And, indeed, it can. If we delete spacers so it can't target, and we infect Vibrio cholerae, we'll get rare plaques where the phage has acquired new spacers against the PLE. This just shows some of those newly-acquired spacers. So this is a phage that has an adaptive immune system that it can use against Vibrio cholerae, which I think is going to be a unique aspect of a phage cocktail.
july11-558	DR. CAMILLI: So I have time to talk about one other phage real quickly. So ICP2 was not -- wasn't omnipresent, it was more scattered. We still find it. We found it last year, and in this year as well. It's also in Haiti, and we're working on a manuscript right now for that.
july11-559	DR. CAMILLI: This is a completely different phage, and what we found is its receptor is not the LPS, but a surface protein, a porin called OmpU. So this is just a predicted structure of this porin that sits in the outer membrane. There's these loops sticking out to the surface of the cell.
july11-560	DR. CAMILLI: What we found is that in some patients that were shedding Vibrio cholerae and ICP2, we found isogenic escape mutants of Vibrio cholerae. And sequencing them we saw that they had mutations, precise -- it's not deletions or stop codons, it's amino acid changes in these two outer loops, and so we hypothesized that the phage tail fiber probably interacts with this. And we have some unpublished data that confirms that. That this is what the phage tail fibers engage with. So these types of point mutations are kind of hard to get, if you think about it numerically. It's much easier to delete a gene or mutate it in other ways that just knock out the function.
july11-561	DR. CAMILLI: But it turns out that OmpU is critical for virulence of Vibrio cholerae. During infection it switches the major porin from a porin called OmpT to this one called OmpU.
july11-562	DR. CAMILLI: So, again, during infection in the presence of this phage, Vibrio cholerae is between a rock and a hard place. It needs to express OmpU, it needs to express its O antigen, and yet these phages are using those as receptors. So I think that, or we think that that's part of the reason for the success of these three phages.
july11-563	DR. CAMILLI: Now some patients will be shedding these point mutants, but what's interesting is when we look at the publicly-available database of cholera strains that have been sequenced from many patients from many parts of the world, we don't see these point mutants.
july11-564	DR. CAMILLI: They don't become fixed in the population. We just see the wild type and variant ompU sequence. And we have some data to show that these point mutants do have a subtle fitness cost, and we think that that's why they -- there's probably evolutionary pressure to revert these mutants back.
july11-565	DR. CAMILLI: Now some patients were shedding escape mutants against ICP2 that made a normal OmpU, at least by gene sequence, and so we went in and figured out how these are escape mutants. It turns out they have null mutations in a gene called toxR, and this just shows a few examples: stop codons, mutations, and critical residues.
july11-566	DR. CAMILLI: Now why would mutations in toxR give escape? Well it turns out that toxR is a positive regulator of ompU during infection. Again, Vibrio has this switch from OmpT to OmpU during infection.
july11-567	DR. CAMILLI: What's interesting is toxR is also a major virulence regulator. It regulates the cholera toxin genes, it regulates pilus that's needed for colonization, and so these escape mutants are rendered avirulent. We wanted to show that taking some of these point mutants that we got out of human patients, and showing that they're highly attenuated in animal models. So this is now two examples of this where escape mutants can escape the phage, but they're attenuated, or have fitness costs.
july11-568	DR. CAMILLI: So we've put together this cocktail of these three phages that we find -- year in and year out we're finding in cholera patients in Bangladesh and tested them out for prophylaxis. Again, keep in mind this idea of preventing household transmission.
july11-569	DR. CAMILLI: So this is not a novel idea. Of course, phage therapy was -- back in the 1920s and '30s was tried for cholera, but a lot of those studies weren't well-controlled. It's not clear if it worked or not. I'd like to point out that this well-controlled study, clinical study that was done in 1971 unfortunately showed that a phage cocktail did not have efficacy in a -- again, in a well-controlled study.
july11-570	DR. CAMILLI: And I would think nowadays cholera is such an acute disease where, really, it's rehydrating the patient and giving them antibiotics. That's the treatment. I don't foresee therapy being used, at least not in and of itself, in treating cholera patients.
july11-571	DR. CAMILLI: The idea of prophylaxis is an old one as well for cholera, but recent studies haven't shown that it works. So this study from the Sarkar lab used an adult rabbit model of cholera, and they basically showed it didn't work. The phages lost orders of magnitude titer within a few hours, and it didn't bode well, but we forged ahead, thinking that maybe our phages, which I'd like to think have evolved to be virulent in the context of the cholera small intestine, maybe they will work.
july11-572	DR. CAMILLI: So I'm going to show you some data from Mimmin Yen who's here with us and another post-doc, Lynne Cairns, where we've tried out this idea. And we've recently published this this year.
july11-573	DR. CAMILLI: So we have two animal models for cholera: the infant mouse, the infant rabbit. Cholera will not infect adults, except for adult humans, which we have no data for.
july11-574	DR. CAMILLI: So first the infant mouse model. So a typical experiment is we'll give them 10 to the seventh pfu of single phages, or the cocktail, we wait three hours, that's the transit time of liquid through the small intestine, then we challenge them, and then we'll determine the load of Vibrio cholerae 24 hours later.
july11-575	DR. CAMILLI: So here we see that the load in the no phage group of mice is very high, and with single phages we get different levels of reduction of the load of the bacteria. ICP1 not so good. Not surprising. I showed -- told you that's this high frequency frameshift mechanism. The cocktail worked the best, and ICP3 worked the best.
july11-576	DR. CAMILLI: Now when we look at the Vibrio cholerae that are still in these animals at 24 hours, we see escape mutants. That's no surprise. We see escape mutants for ICP1, 2, and 3. The concentration of phage in these animals generally reflects the load of the bacteria. The more bacteria there are, the higher the load of phage, and that's -- of course you'd expect that.
july11-577	DR. CAMILLI: So based on this first experiment showing the cocktail seemed to work pretty well, we asked, well how long do the phages last in the intestinal tract? So we gave them about 10 to the seventh, 10 to the eighth of these individual phages and looked at retention. What you can see is they were retained pretty well out to 24 hours, although ICP3 really starts to go away by 24 hours.
july11-578	DR. CAMILLI: So I'm going to show you some prophylaxis experiments where we give the cocktail and we test longer times, up to 24 hours, the idea being, for humans, they could drink the phage cocktail once or twice a day.
july11-579	DR. CAMILLI: So when we look at longer times of prophylaxis we see a different story. The bacteria do colonize. So we see this bimodal protection at six and 12 hours between giving the phage cocktail and challenging them. Within 24 hours, all of the animals are colonized. I'll note that the load is about eighteen-fold lower.
july11-580	DR. CAMILLI: When we go in and look at the bacteria that are colonizing these animals, many of them are escape mutants. They're escape mutants that have lost the receptor, and so theyâ€™re -- they have lost virulence.
july11-581	DR. CAMILLI: So now I'm going to switch to this infant rabbit model. The infant mouse model's a model of colonization, they don't really get profuse diarrhea like humans do, but infant rabbits do get profuse diarrhea like humans.
july11-582	DR. CAMILLI: So we give the infant rabbits 10 to the tenth pfu -- that's the combination of the three -- we wait three or 24 hours, and then we challenge them. So what you can see is the rabbits that don't get phage are -- have a high titer, they're very sick, they lose a lot of body weight. We have to euthanize them once they lose 10 percent of their body weight.
july11-583	DR. CAMILLI: The three and the 24-hour prophylaxis times were protected to varying degrees. Again we see a bimodal protection for the three-hour prophylaxis, and in the 24-hour, just like in the infant mice, they're all colonized, but here, the load is about 300-fold lower. And, again, if we go -- and we've done exhaustive studies on what Vibrios are still there. Many of them are escape mutants to one, more rarely two, of the phages, but we don't see escape mutants to all three of the phages in these populations, and a lot of these escape mutants we see are avirulent.
july11-584	DR. CAMILLI: So, again, I think it's part of the reason for the success of these phages in nature. And so -- that these animals are colonized, but with mostly avirulent strains, the hope would be, well they don't have disease. Indeed, if we look, we don't see any symptoms of cholera in these animals.
july11-585	DR. CAMILLI: If you go and look at the percent body weight, there's no significant loss of body weight. So no phage, no Vibrio cholerae challenge. They lose a little bit of body weight because they're away from their mothers for this duration of this experiment.
july11-586	DR. CAMILLI: The no phage prophylaxis group, I mentioned they're all very sick. These had to be euthanized much earlier than any of these other animals. But, again, no body weight loss, and that's consistent with the lack of seeing any symptoms.
july11-587	DR. CAMILLI: So we're hoping that this can work in a similar manner in humans by reducing the load of the bacteria, or perhaps preventing the bacteria from colonizing. I point out that here we administer a huge dose of the bacteria. During household transmission I would -- we don't really know the dose that people are exposed to, but hopefully it's not tremendous numbers.
july11-588	DR. CAMILLI: So the last thing I'll tell you is these three phages, we've look at other gram-negatives, they appear to be very specific for Vibrio cholerae, but we wanted to show that they don't alter the gut microbiome, and so we did this experiment that I'll mention quickly where we have a heat-killed phage group, a group of adult mice that got the live phage cocktail, and then as a positive control for a change, vancomycin.
july11-589	DR. CAMILLI: Now we looked at phage coming out in the stool pellets and it kind of declines, but even at 60 hours we still see phages. So we looked at the microbiome at zero, one, and two days, and the bottom line is the antibiotic-treated group going from T zero to one, to two days has this tremendous change in the fecal microbiome, as you'd expect, but our heat-killed and our phage-treated all cluster together. There is no substantial change. If you blow this up, there's no pattern of change in the microbiome.
july11-590	DR. CAMILLI: So we expected this, but it's nice to show this, that the phage cocktail does not alter the gut microbiome. So just to summarize, we have these three virulent phages that we find repeatedly coming out in cholera patients naturally, and all three phages use receptors that are essential virulence factors so this limits escape within humans. When there is escape you have these avirulent mutants, and so probably this is reducing the pathogenesis in some humans that are asymptomatic or have mild symptoms.
july11-591	DR. CAMILLI: One of our phages has its own CRISPR/Cas system, an adaptive immune system that can keep pace with Vibrio cholerae's PLE defense system. This phage continues to be prevalent today with the CRISPR/Cas system, so it's part of its success.
july11-592	DR. CAMILLI: Then I showed you that a cocktail of these three phages can be used to prevent infection and reduce infection in a high-dose challenge in animal models. And then finally, that the cocktail, as would be predicted, doesn't substantially alter the intestinal microbiome.
july11-593	DR. CAMILLI: So I mentioned the people that did the work during the talk. I should also mention Andrea Wong's working on ICP2 receptor work, and Dave Lazinski's the senior researcher in my lab who has a hand in a lot of this stuff. And I thank my international collaborator here, Firdausi Qadri. Thanks. Happy to answer any questions. AUDIENCE MEMBER: Hi. So do you see any changes in expression of cholera toxin in your escape mutants?
july11-594	DR. CAMILLI: Yeah. So the toxR escape mutants that come out of some humans with this phage in their stool are avirulent in animal models, and they don't express the cholera toxin in the entire toxR regulon, which inc -- has many virulence factors.
july11-595	DR. CAMILLI: Other alterations to cholera toxin, we haven't seen that yet. The other escape mutants, like the LPS rough mutants, they still have the virulence regulon intact, but they're avirulent for another reason. They need their LPS for colonization.
july11-596	AUDIENCE MEMBER: Two questions. One is is the PLE induction purely a matter of gene dosage or are there also genes actually turned on? Secondly, what -- can phages -- escape PLE, and, if so, how do they do that?
july11-597	DR. CAMILLI: So we don't yet know what induces the PLE to excise and replicate. We know it's specific for ICP1. And Kim, I can't -- I mean she -- I saw her recently. She has some data where she kind of is figuring out what causes excision, and she hasn't told me the details so I can't tell you, but it's some --
july11-598	AUDIENCE MEMBER: So it's gene dosage clearly goes up, and as the --
july11-599	DR. CAMILLI: Oh, you mean their excised element?
july11-600	AUDIENCE MEMBER: Yeah --
july11-601	DR. CAMILLI: Oh, that thing replicates to a copy number of about a thousand, which is tremendous.
july11-602	AUDIENCE MEMBER: Oh. So it's going to be a huge dose of whatever it is it's delivering.
july11-603	DR. CAMILLI: Yeah. But once the signal has been given to excise, then it just takes off replicating.
july11-604	AUDIENCE MEMBER: And so if -- phages can escape PLE, can they not? I mean I saw it's 10 to minus six or something like that when you --
july11-605	DR. CAMILLI: But it's only through the CRISPR mechanism.
july11-606	AUDIENCE MEMBER: So you can't get point mutations in the phage that doesn't have a CRISPR.
july11-607	DR. CAMILLI: We have no mutants, other than the CRISPR/Cas system, that can overcome the PLE defense system. That being said, have we looked very hard?
july11-608	AUDIENCE MEMBER: I was going to say, you can't repeal the law of phage genetics, right? It'll find a way, I would think.
july11-609	DR. CAMILLI: Yeah. I mean there -- maybe there's isolates in our, but the dominating is the CRISPR/Cas systems. CRISPR/Cas.
july11-610	AUDIENCE MEMBER: I was just wondering, of the 77 percent of the household contacts that don't get it, have you or your collaborator looked to see if they have signs of having some of your phages?
july11-611	DR. CAMILLI: There are now some NIH-funded projects to start to look at that, look at the -- look more at this household transmission, and why do some people get cholera and others don't.
july11-612	DR. CAMILLI: I would hypothesize, it's speculation, that sometimes they're the lucky ones that got a dose of phage at the same time they encountered the bacteria, but that's pure speculation. D'Herrelle was on to this stuff, you know, a hundred years ago, hypothesizing similar things.
july11-613	AUDIENCE MEMBER: There are obviously several parallels between the PLE and SaPIs. Do you have any evidence that they might be packaged by the ICP1 machinery?
july11-614	DR. CAMILLI: Yeah. So the SaPIs in Staph aureus are these chromosomal islands that pop out. They steal packaging material. They're transmitted at a high frequency that way. They don't -- they interfere with the helper phage a little bit, but not much. Vibrio cholerae PLE, there's no homology with the SaPIs, other than an integrase gene.
july11-615	DR. CAMILLI: So we don't know if there's a common ancestor, but they knock down phage infection almost completely, so we think they're different in that sense.
july11-616	DR. CAMILLI: Vibrio cholerae -- so the bacteria lyse and release those thousands of circles, and Vibrio is naturally competent. That's probably the major mode of transmission of this element. But Kim Seed, my former post-doc, does have some evidence that there is some packaging, very low level packaging, and she's trying to work out the details of that.
july11-617	DR. RANALLO: Quick question. So do you ever see ICP1, 2, and 3 in the same stool? Have you been able to detect that?
july11-618	DR. CAMILLI: Yeah. So we see -- rarely, we'll see two of the phages in a stool sample, but we've never seen all three in a stool sample. Because it's a good question. Why -- well it would be -- it would not be in the phage's best interest to prevent cholera. They need it for dissemination. So it could just be a predator/prey. Like the household contacts that don't get cholera, maybe they have all three.
july11-619	DR. RANALLO: Okay. So we heard a little bit about Tom Patterson's story yesterday a few times. We're going to continue that with the next talk.
july11-620	DR. RANALLO: DR. Biswajit Biswas from the Naval Medical Research Center is a phage team leader at the Biological Defense Research Directorate at NMRC in Fort Detrick and his title is rapid emergence of phage-resistant bacteria during phage therapy of a terminally-ill patient who was infected with a multidrug-resistant Acineto baumannii."
july11-621	DR. BISWAS: Hello. Good afternoon, everybody, and thanks the organizers to allow me to present my data of the recent phage therapeutic applications in human.
july11-622	DR. BISWAS: So my topic today is rapid emergence of phage-resistant bacteria during intravenous application of phage therapy of a terminally-ill patient who was infected with the multidrug-resistant A. baumannii.
july11-623	DR. BISWAS: You know, you hear the -- you heard all the story yesterday from Dr. Schooley. Today I'm going to mainly discuss about the bacterial mutation leading to the phage resistance during this therapy. So this is the disclaimer. I have to show it. I have no conflict of interest to declare.
july11-624	DR. BISWAS: So I work for U.S. Navy at Biological Defense Research Directorate at Fort Detrick. Currently, our phage-based programmatic efforts are the -- can be, you know, explained in three different part. There are therapeutic applications of phages, prophylactic applications, and diagnostic applications.
july11-625	DR. BISWAS: Our therapeutic applications, we are generally working with natural phages. Prophylactic applications, we try to use some lambda phage to modify to make vaccines. In this aspect, a long time back when I used to work for a company, I prepared a vaccine, cancer-based vaccine for using phage display technology which is in phase 1 clinical trial currently at BDRD. We are making some vaccine specific for targeting for malaria and prevention.
july11-626	DR. BISWAS: So for diagnostic applications, we are currently developing some rapid diagnostic process for using phage. So for therapeutic applications, we are currently working with MRSA, VRE, and Klebsiella, Pseudomonas, and baumannii. So these are all based on natural phage applications. For engineered phage side we are developing some sorts of, you know, delivery systems to deliver some lethal genes to neutralize the bacteria which are mainly in stationary phase, because stationary phase bacteria is very difficult to treat with, you know, phages.
july11-627	DR. BISWAS: So, lastly, the phage components which we are trying to clone is like some source of some endolysins and lysozyme genes. This is ongoing projects.
july11-628	DR. BISWAS: So 2013 -- you know, I joined the BDRD at 2010. During that time I was working to develop natural phage therapy for Bacillus anthracis. That was very interesting work.
july11-629	DR. BISWAS: But in 2013 we got some seed money to develop some therapy, natural phage therapy, for Acinetobacter baumannii and Staph aureus, so we joined with Navy Wound Department and Army Wound Department to develop some animal model to use to develop phage therapy for Acinetobacter baumannii infections. Mainly wounds infections.
july11-630	DR. BISWAS: So why we are interested for this? Because during the Iraq War we saw the type of -- last Iraq War we saw 30 percent of -- 35 percent of clinical infection was caused due to A. baumannii infections. Currently, WHO prioritized A. baumannii as their priority number one organisms for antibiotic resistance problem.
july11-631	DR. BISWAS: You see that there are near about 60,000 to 100,000 infections reported at USA and 13,000 in all five European, you know, countries. This data is part ER reported cases. So there are a lot of A. baumannii problems.
july11-632	DR. BISWAS: So when I thought about these projects, how to develop these, we were thinking about different approach. So I talked about using a very broad spectrum monophage because previously we develop such type of treatment for VRE bacteria at NIH. So we thought that probably it is possible to find a monophage.
july11-633	DR. BISWAS: Then next one was to -- what about a cocktail, fixed cocktail with phage therapy? Then we thought about to make some engineered phage also. Soon we realized that none of these things will probably work for A. baumannii treatment because A. baumannii is very, very diverse. The clinical isolates are very diverse.
july11-634	DR. BISWAS: So monophage -- find a monophage is very -- prospect of monophage is very difficult. Also the -- if we try to use cocktail, the -- probably resistance will pop out. Engineered phage is a lucrative idea, but it take long time and lots of manpower.
july11-635	DR. BISWAS: So we lastly thought about to use natural phages and to direct -- this was towards more than personalized and precision approach. So we start harvesting phages, lot number of natural -- large supplies of natural phages from environmental samples.
july11-636	DR. BISWAS: So the process is very simple. I think yesterday somebody asked what is the process? How you isolate the phages from the nature? It's very, very easy. We get sewage water, and then near about 300 ml of sewage water we put tryptic soy agar powder, just the raw powder, and then inoculate them with a little bit, 200 microliters of actively growing culture. In this case it's A. baumannii against which we are looking for phages.
july11-637	DR. BISWAS: So in this primordial soup everything start growing, and the smell is not pleasant. You know, the whole lab starts smelling horrible. But anyway, so after that, within the -- within six to -- six hours toward 18 hours later, we harvest samples from there, we filter-sterilize those samples or chloroform treat to deactivate all other bacteria, and then we plate them against the bacteria against which we are looking for these phages -- this way we can find many phages simultaneously, sometimes for many different bacterial isolates -- and we make our phage collection libraries.
july11-638	DR. BISWAS: So right now we are near about 208 A. baumannii phages in our collections.
july11-639	DR. BISWAS: So recently we have opportunity to test the strength of -- about this natural phage library. This is specifically that case which associated with UCSD. You know, the UCSD -- one of the USCD case. This case actually, the case history was reported yesterday by Dr. Schooley, but for the newcomer, I'm just presenting it again.
july11-640	DR. BISWAS: The patient was a 68 years professor psychiatrist from UCSD, and he was traveling to Egypt during Thanksgiving time. He developed pancreatitis in Luxor, and he was hospitalized. During
july11-641	DR. BISWAS: time -- that time, probably he was infected with this multidrug-resistant A. baumannii.
july11-642	DR. BISWAS: They transfer him in Frankfort where they found this multidrug-resistant baumannii from his pancreatic pseudocyst, and he was evacuated, ultimately, to UCSD, his home station. Home hospitals.
july11-643	DR. BISWAS: So here you can see the -- these pictures were provided by Dr. Schooley. You can see the growth of the abscess in the biliary duct. So I'm avoiding these slides because we don't need to put it there.
july11-644	DR. BISWAS: Previously, also, we developed our unique system to evaluate all the natural phages simultaneously to find out their therapeutic efficacy. In this process we actually use microwell plates, 96-well microwell plates. We diluted the phage serially, and then we used some control, bacterial control and media control, and then we infected all of these wells with the same number of bacteria.
july11-645	DR. BISWAS: During this time we also -- in the media we add a dye called tetrazolium dye. So during active bacterial respiration, tetrazolium dye start to reduce, and during this process the dye start changing color. So the color change from light yellow to a very dark purple.
july11-646	DR. BISWAS: So we scan these plates in a machine called OmniLog, TM system. In this machine a camera every 15 minutes take a live picture of these plates. So this is actually a graph which produce from every 15 minutes monitoring the bacterial growth.
july11-647	DR. BISWAS: So here you see that when we collect the data from the machine and plot it, you see the growth rate of different bacter -- same bacteria in presence of different phage. So this is the bacteria control. You can see it. So it is actually you are monitoring the phage-bacterial interaction in real time.
july11-648	DR. BISWAS: So when we receive this, you know, request from UCSD to provide some phage for treatment, we immediately pull out 98 A. baumannii phage from our collections, we very rapidly use our robots to distribute all the phages, and then we inoculate it with the patient's isolates, whatever we receive from patient.
july11-649	DR. BISWAS: So within 16 hours -- 16 to 18 hours, we found 10 of the phages which are active against this patient's bacteria. So that particular isolate we call TP isolate because the person who was -- from whom we gave this, you know, isolates, is -- his name was Dr. Tom Patterson.
july11-650	DR. BISWAS: So now the question is how we select this personalized phage. You know, phage for this personalized phage therapy. We found four phages, I mentioned, and then we monitor their activity in the BioLog system. We see all these phages are very virulent.
july11-651	DR. BISWAS: So we didn't have a chance to monitor their receptors activities or anything like that because the time was short, so we selected these four phages, and then we studied and we found that they can combinely reduce the bacterial growth completely. This is the control bacteria.
july11-652	DR. BISWAS: So we pull out all these four phages from our collections, and then we make a small-scale lysate, then we grow a large-scale lysate. From there we -- this is near about a 3.8-liter culture. We purify it through tangential flow filtration systems, and this is actually a diafiltrations where we exchange the media against buffer, and that also helps to reduce the LPS, some extent.
july11-653	DR. BISWAS: So then it goes through the continuous cesium density gradient purification process, and then we isolate the phage bands. So here you can see the phage bands. These phage -- after we collect these phage bands, generally the titer is 10 to 11 per ml during this time, and we dialyzed it very rapidly, filter-sterilize, and then, you know -- this was done separately.
july11-654	DR. BISWAS: Then we combined all those phages together and did a sterility test and produce investigational drugs for personalized cocktail, for use.
july11-655	DR. BISWAS: So I like to mention for this therapy the source of the therapeutic phages came from two different places. So phages provide by the Center of Phage Technology in Texas A&M Universities are AC4, CP12, CP21, CP24. AC4 actually came from AmpliPhi. Here, in Biological Defense Research Directorate, we produce four phages, which are Ab phage 1, 4, 71, 97. Later also, we provide another phage that is AbTP3 phage 1. I will talk about it little later.
july11-656	DR. BISWAS: So you can see that -- here is the phage therapy dose per day. This is actually our cocktail phage, what was used intravenously. The phage administration start two days before, but that was for the inter-cavitary wash.
july11-657	DR. BISWAS: Seventeenth March, Dr. Schooley start giving this phage intravenously, and this is the number of time he injected it. So you can see that -- how many times he give this -- use this phage.
july11-658	DR. BISWAS: So I like to mention, also, that our phage was never used directly for inter-cavity wash, so always this phage was used for intravenous administration.
july11-659	DR. BISWAS: So during this process Dr. Schooley also harvested the bacteria from the patients. So those bacteria call -- we -- those isolate we named as TP1, TP2, TP3. TP1 was isolate before giving our phage, and TP2, TP3, and TP4, TP4.1, all these things was isolated after giving phage therapy. So the source of all these bacteria is mainly from pancreatic drain. You see their date when they're isolated, 21, 23rd, 9th May. Like that.
july11-660	DR. BISWAS: So we were very interested to see what is going on into the -- into bacterial side, you know, so we use BioLog system to monitor this -- our phage activities on these different TP1 isolate -- TP isolates. So we see that before the phage was given, the isolate which we call TP1 isolate, the -- all phages are very, very active.
july11-661	DR. BISWAS: So after the phage therapy, which was done at 17 March, and this isolate TP2 was harvested
july11-662	DR. BISWAS: 21st March, we see the phage is not that much active on this isolate anymore. We see the -- all these four phages are not that active like, you know, what was -- they were very active before, or very virulent.
july11-663	DR. BISWAS: So we took the TP3 isolate and we ran it in our BioLog machine, and we see they are completely resistant. So we did the same thing with the A&M phages. We see that they have also, in initial stage of TP1, before given phage, they were partially active.
july11-664	DR. BISWAS: Because all these phage can make plaques, but they are not that very virulent like what they were -- our phages were. But later on we study this. In the TP2 isolate, you see they are still little bit active, and then TP3, they're completely inactive. So this is the composite profile. So this is actually phage came from the Texas A&M, and this is a phage came from -- used by Navy. U.S. Navy.
july11-665	DR. BISWAS: So the resistance pop out. So what is the solution? So we thought about to find another phage immediately, and this time we went to environment directly, environmental samples, and we used this resistant bacteria to find out another phage, and here you see this phage. This phage is a unique halo former, so you can see the halo around this phage, clear phage spot.
july11-666	DR. BISWAS: We tested that new phage on original isolate, parental isolate, and also the resistant bacteria. So we see that, you know, these particular phage, which we call AbTP3 phage 1 is very active TP1, and also TP2 and TP3. TP2 figures are not given here. So that means this phage is very active, its parental isolates and the resistance population.
july11-667	DR. BISWAS: So we need to produce another cocktail so we run the BioLog assay using this new phage. You can see these phages can active up to seven hours, but then after that, resistance start popping out. So we thought, what about to pick up another phage from our previous cocktail, which is AB71, and combine these two. When we combine, we see there is a complete remission of bacterial growth.
july11-668	DR. BISWAS: So we prepared a new phage cocktail, which call -- which we call Navy phage cocktail 2, using AbTP31 and Ab phage 71. So these are the phages which we used from our side. These are phage -- electronic photograph of those phages.
july11-669	DR. BISWAS: All these phages are Myoviridae. This is phage cocktail 1. Probably they had the same phage and they are using same receptors. And this is the Podoviridae, which is AbTP3 phage 1, which can kill the parental and the resistance isolate.
july11-670	DR. BISWAS: So what is going on in the bacterial side? Is it -- we thought -- first we thought that it may be the capsular difference between the parental and the phage-resistant bacteria because previously we developed another model for A. baumannii AB 5075 for wound infection. This one for wound infection in mouse model.
july11-671	DR. BISWAS: So we have five phages that time we used: AB phage A, B, C, D, E. We observe the AB phage A can produce plaque on the AB 5075 bacteria, but other phages, AB phage B, C, D, E has no effect. So you see here the AB phage A can, you know, prevent the infection up to six to seven hours. Then the resistance pop out.
july11-672	DR. BISWAS: But when we mixed any of these -- any of the other phages with AB phage A, we see complete remission of bacterial growth. So, but this phage alone, this phage cannot make any plaques on this AB 5075. Surprisingly, when you mix AbB, C, D, and E, they cannot prevent the bacterial growth. Here is the curve.
july11-673	DR. BISWAS: So to understand what is going on, we collect the bacteria after phage exposure, and then we monitor their surface, using the Raman spectroscopy, and we see that there is a specific peak appear if the bacteria has capsule. Is the peak appear in 979. But if bacteria lose capsule, then it become plain.
july11-674	DR. BISWAS: So we realize that after exposure to the AB phage A, the bacteria, you know, the selection pressure move the bacteria from live variant, to capsular variant, to a smooth variant, and that smooth variant then can be infected with the other phages, which are AbC, D, and E.
july11-675	DR. BISWAS: So we realize that AB 5075 is cap-positive. When we expose them in AB phage 1, they become AB 5075 cap-negative, and they can -- then they can be killed by other phages. So we thought the same thing is probably happening here. So we monitor the TP1, TP2, TP3, you know, with Raman spect and we found that there is not much difference. They are all same in 900 peak.
july11-676	DR. BISWAS: So then we though that let's stain the capsule itself. We stained the capsule and we found some difference in the thickness of the capsule. Here is the three pictures. So this is actually TP1, this is TP2, and TP3. You see that TP3, the capsules is less thick.
july11-677	DR. BISWAS: So, to understand better, we sequenced the whole genome of all these TP isolates, TP1, TP2, TP3, and you can see here this, you know, comparison of all these three different bacteria. This is compared to TP1, TP2, and TP3. The outermost ring is TP2, the innermost ring is TP3, and these are the reference bacteria.
july11-678	DR. BISWAS: You can see that these TP1, TP2, TP3, as compared to each other, they are very similar, whereas in reference bacteria they are very different. This indicate the heat map. The blue means, you know, they match properly.
july11-679	DR. BISWAS: So we look deep and we found that insertion of two mobile elements in TP3 disrupt the gene for a cell surface protein. Excision of mobile elements that is present in TP1 joins two hypothetical protein sequence into one TP2 -- in one, TP2 and TP3.
july11-680	DR. BISWAS: Genes for the outer membrane protein CarO is truncated in TP3 and missing one amino acid -- missing several amino acids that would form a surface-exposed loop. Maybe that loop is contributing in the receptor. Within capsular biosynthesis region TP1 and TP3, glycosyl transferase genes also differ.
july11-681	DR. BISWAS: So all these findings, this one for CarO was very interesting to us, and also the glycosyl transferase gene, because it can change the thickness of the capsule.
july11-682	DR. BISWAS: So we further analyze that one, and here you see the CarO proteins in TP3 is missing, this part, and this cause a loop formation. CarO protein was also responsible for carbapenem resistance. So here you see the glycosyl transferase protein involved in capsular biosynthesis. There you see the gap of the two SNPs.
july11-683	DR. BISWAS: So we are investigating this more, and we don't know exactly what is causing this phage resistance yet, but we will going to dig it more.
july11-684	DR. BISWAS: So just to inform you that when we produce this Navy phage cocktail 1 and 2, we also estimate the LPS, and our LPS for cocktail 2 was 10 to the three EU per ml, and this titer was near about 10 to 11 to 10 to 12, so when we diluted it we maintained the FDA-recommended guideline 5 EU per kg per hour recommended per dose. So it is possible to make, you know, LPS-reduced phage prep using the cesium density gradient.
july11-685	DR. BISWAS: So we also estimate the plasma phage concentration. Here you see after just giving the phage, phage titer goes 1.8 times 10 to the four per ml of blood, but is rapidly reduced. It's mainly probably the liver and spleen entrapment of the phage in human body.
july11-686	DR. BISWAS: So phage stability. We also study the phage stability in Ringerâ€™s solution because they diluted the phage in Ringerâ€™s solution. So you see phage is very stable in the Ringerâ€™s solution, and there is no difference between this in the buffer and the Ringerâ€™s solution titer.
july11-687	DR. BISWAS: So we also monitor -- because Dr. Schooley reported that the phage be -- I'm sorry -- the bacteria become minocycline-sensitive, so we monitor their activity against minocycline and phage combined. So you see the minocycline, one microgram per ml, you know, is not -- cannot prevent the bacterial growth completely, but when it -- and the bacteria -- and the phage alone cannot prevent the bacterial growth, but when we mix phage and minocycline together, you see that its diminish the bacterial growth. So there is some synergistic effect.
july11-688	DR. BISWAS: So we study that effect before also with some other bacteria, and we can see very eas -- very clearly that for Staph aureus, gentamicin, nafcillin, and cefoxitin work very well with phage and antibiotic. So here you see the bacteria and antibiotic, here you see the bacteria and phage, but when we mix bacteria and antibiotics, and phage, you see the complete, almost, inhibition of bacterial growth. All these study was done simultaneously in a BioLog system.
july11-689	DR. BISWAS: So recently we do -- did a -- you know, investigate the effect of meropenem in antibiotic-resistant K. pneumoniae, and we see that very little amount of phage and antibiotic can prevent the bacterial growth.
july11-690	DR. BISWAS: So we exposed near about four microgram per ml of meropenem and carbapenem-resistance K. pneumoniae, and you see that bacteria completely growing in presence of antibiotics, but in presence of very little phage, it's even in 0.0 -- 0.01 MOI, the phage and antibiotic can prevent the bacterial growth. So the phage and antibiotic, some antibiotic, can produce a very strong synergistic effort. So outcome of the phage therapy. Phage therapy was started -- actually, Dr. Schooley described all those things, but I'm just reading the slide here again. Phage therapy started as inter-cavitary installation at day 109, which were continued at six, 12 -- six to 12 hourly intervals. During this time, patient was unresponsive and -- to commands and had developed renal failure.
july11-691	DR. BISWAS: So over the next 36 hours clinical condition was stable, but he remained comatose. He needs pressors, and his renal hepatic functions was declining. After 36 hours of infection of inter-cavitary installations of the phage cocktail, phage therapy was introduced through intravenous route and five times 10 to the nine phage was given intravenously. That's our Navy cocktail.
july11-692	DR. BISWAS: After intravenous administration -- the patient tolerated that intravenous administration very well. After that, he came out from his coma. After intravenous application he came out from his coma, and then he start talking with his family, and for the first time in several weeks, that things happen. He was sick for last three months, almost.
july11-693	DR. BISWAS: So Dr. Schooley describe all those phenomenon yesterday. I'm not going to go very details of that. So finally what happened, over the ensuing three weeks patient's mental status continued to improve and he was fully conversant and lucid. He was weaned off the ventilators, and his pressors were
july11-694	DR. BISWAS: gradually weaned and were discontinued.
july11-695	DR. BISWAS: So the conclusion from my side is -- from our study, that modified OmniLog system is an ideal platform for studying phage bacterial interaction because you can monitor many phage-bacterial interactions simultaneously in real time with using this system.
july11-696	DR. BISWAS: Precision phage cocktail suppress emergence of phage resistance. Phage therapy can resensitize bacteria to antibiotic against which it has previously acquired resistance. Different phage-resistant phenotypes are observed depending on the phage-host combination studies. Antibiotic phage therapy synergy is possible.
july11-697	DR. BISWAS: So here you see the patient before given phage. Post-phage treatment and he's reading cards. Here you see he's watching and telling that science saves lives.
july11-698	DR. BISWAS: Just a couple of slides. This is the acknowledgment. This whole things was possible because our support from our captain, Dr. Mateczun, and LCDR Theron Hamilton. He's actually my boss, and he is a very fine Navy officer. So -- no, he is really brave. He actually activate me to do these things.
july11-699	DR. BISWAS: Our lieutenant commander, Luis Estrella, Mr. Matthew Henry, and Mr. Javier Quinones worked day and night to make this preparation, phage preparation to send it to UCSD. I also like to mention that we are currently working with Adaptive Phage Therapeutics to develop this system further to provide it for general public.
july11-700	DR. BISWAS: DR. Carl Merril who is actually -- is my mentor also, I worked previously at NIH with him for a long time. So from the Food and Drug Administration I like to give thinks to Cara Fiore who actually approved the eIND process. This is the end of the story.
july11-701	DR. RANALLO: Any questions?
july11-702	DR. RANALLO: (No response.)
july11-703	DR. RANALLO: Okay. SO, with that, we have our last speaker. Jimmy Regeimbal is going to talk to us about phage and personalized medicine. The essence of his talk is to look at a well-characterized library to build personalized cocktails. So Lt. Regeimbal is currently stationed at the Navy Medical Research Unit in Lima, Peru, where he's expanding the isolation of natural phages from remote and unique environmental samples. The title of his talk is phage therapy against MDR strains - Overcoming the double-edged sword of phage specificity.
july11-704	DR. RANALLO: Jimmy, it's all yours.
july11-705	DR. REGEIMBAL: Okay. Good afternoon. Once again, my name is Lt. Jimmmy Regeimbal. I'm stationed at the Naval Medical Research Unit No. 6 in Lima, Peru, but prior to that I was at the Naval Medical Research Center in Silver Spring, Maryland, which is where a lot of the work I'm going to be talking about was actually done.
july11-706	DR. REGEIMBAL: I'm also very aware that I am the last presenter of the last session on the last day of what is a very packed meeting, and it is tempting my natural ability to be reckless a little bit, so I might actually be a little bit more provocative than I was originally planning on being. Sorry about that.
july11-707	DR. REGEIMBAL: So I tend to beat a fairly specific drum, which is this idea that -- well first let me get through my disclaimer because I have to do that. Although I am a uniformed service member, I'm speaking on behalf of only Jimmy at this moment. These are my opinions. I am not speaking on behalf of the Navy or the DoD.
july11-708	DR. REGEIMBAL: So within the Naval Medical Research and Development enterprise, it's really a collection of labs all over the planet. The Army has one as well, and so we work in very close partnership with them.
july11-709	DR. REGEIMBAL: So, actually, I should say just from the very beginning that everything we have been doing, and everything that we are doing, has been in very close collaboration with the Army, specifically the Army Wound Infections Department, but also the Bacterial Diseases Branch, in general, at the WRAIR, and all of our animal model data, for example, will be -- was worked out in collaboration, in very close collaboration with that group.
july11-710	DR. REGEIMBAL: But, generally, on the Navy side, we have groups that are interested in population level cocktails and engineered phages, phage diagnostics, phage vaccines. Obviously the project that I was most associated with was the natural phage therapy developing, using a library-to-cocktail approach.
july11-711	DR. REGEIMBAL: I sort of think this is one of the most durable and robust ways of generating a phage-based therapeutic, and, really, to wrap your head around what I really think this product actually is is I think you need to view it through the paradigm that the product is actually the library, and the application of that product to any individual case is actually the cocktail.
july11-712	DR. REGEIMBAL: So our product is a little different. It's a little bigger. I think it's important to view it through that paradigm to really understand what it is I'm trying to do, or what we are trying to do.
july11-713	DR. REGEIMBAL: So I'm not a very sophisticated person so I wanted to start back from the very bottom and ask the question of what are we actually trying to do when we try to use phages as therapeutics? Really, you're exploiting a predator/prey interaction. It's a horrible, but extremely helpful, analogy.
july11-714	DR. REGEIMBAL: What you're doing is you're actually trying to generate an artificial situation. And I use that word purposefully. It's an artificial situation in which a collection of phages can drive a contained and local bacterial population to near extinction. That's what you're asking it to do, and that's actually a fairly big ask. It's kind of hard to get phages to do that. To ask a phage cocktail to do that not only in one person, but in every person at a population level is an enormous ask, in my opinion. So if you do this sort of reductio ad absurdum thought experiment and then you imagine you have a phage on the planet, or a cocktail, or let's just say it's one, and it can kill every single strain of Pseudomonas aeruginosa, imagine a world where that existed.
july11-715	DR. REGEIMBAL: What would happen over a period of time? That broad spectrum phage would eventually kill all the Pseudomonas aeruginosa, and then you would not find that phage anymore because it ran out of its host and it hit a biological dead end. But that's what people are actually trying to do when they're looking for truly broad spectrum stuff.
july11-716	DR. REGEIMBAL: So that situation's almost selected against in nature because it would result in a biological dead end. So I think it's much more advantageous to just realize that exploiting that predator/prey interaction involves asking the phage to do something that, anthropomorphically, they don't want to do, and so you have to engineer that situation in which that phage cocktail can drive a local bacterial population to near extinction.
july11-717	DR. REGEIMBAL: So a lot of my talk is how we arrived at that. It's going to seem comically simplistic, but I'm doing that on purpose. So when you -- when -- the first way you try to engineer that artificial situation is you use a ridiculously large population of bacteriophage, at like 10 to the seventh, or 10 to the tenth, 10 to the eleventh.
july11-718	DR. REGEIMBAL: These are numbers we use all the time, but that's actually an enormous number of individuals at a population level. With that enormous number of individuals comes a whole lot of sequence diversity and a host range, and those are related to each other, but they're not exactly the same.
july11-719	DR. REGEIMBAL: So I have here a sequence diversity. In any bacterial population you're actually going to have a consensus sequence and some distribution around that consensus. This is grossly oversimplified, but it helps me illustrate my point.
july11-720	DR. REGEIMBAL: This is actually going to vary in at least four dimensions. Not smooth distribution around the consensus, but you have four nucleotides, you have indels, you have rearrangements, and so what you actually have is a cloud of closely-related bacteriophage.
july11-721	DR. REGEIMBAL: Then you're taking that cloud of closely-related organisms, the N-dimensional cloud, and smashing it into a bacterial population which itself is an N-dimensional cloud of closely-related bacteria, and the collision between those two populations is actually your therapeutic. So population dynamics really matter.
july11-722	DR. REGEIMBAL: If you talk to microbial ecologists, a lot of them don't even consider -- I'm a biochemist by training. It's just -- to give that disclaimer, I'm not a phage biologist. It gets me into trouble with phage biologists.
july11-723	DR. REGEIMBAL: But population microbial ecologists don't even sometimes view phages as being predominantly bactericidal, they view them as agents that can introduce bacterial diversity with antibacterial populations, and one of the major mechanisms for doing that is by killing off huge swaths of local bacterial populations and allowing those resistant mutants to outgrow, and so that's already happening in nature all the time, and we're actually trying to fight against that. We're trying to get them to drive the population all the way to extinction.
july11-724	DR. REGEIMBAL: So what happens when you infect a phage into a bacteria, right? We've gone through this over and over again, where you basically have a phage that infects. Over a certain amount of time you're eventually going to get resistance. So it starts off where your sequence diversity is enough to cover your strain of interest and that strain resides into the host range of that particular phage.
july11-725	DR. REGEIMBAL: Eventually, resistance is going to pop out, it's going to pop out outside of the host range, and the sequence diversity is no longer enough to cover it.
july11-726	DR. REGEIMBAL: So how do people get around that? Well, they go let's build a cocktail. That gives you a lot more sequence diversity to play with, you have a larger aggregate host range to deal with, and so when you treat the bacterial infection with those -- with that phage cocktail, it might take a longer period of time, but eventually, you're still going to get resistance.
july11-727	DR. REGEIMBAL: This will happen every single time a phage interacts with a bacterial population, even a cocktail of phages, and so eventually you're going to get a host, or a bacterial strain that pops out and is now resistant.
july11-728	DR. REGEIMBAL: But if your product was the cocktail, what do you do now? What do you do if you started with a cocktail and you have a whole bunch of strains that are -- just lie outside of coverage from that fixed cocktail in time?
july11-729	DR. REGEIMBAL: If you started with a library you have far more sequence diversity to play with, if you build your library correctly you can have far more aggregate host range to play with, and now it's a question of finding the correct phages in your library that could cover any clinical-relevant -- clinically-relevant strain that comes in to the lab.
july11-730	DR. REGEIMBAL: So what you do is you have an arrayed library that's characterized -- I'll get into that in a second -- you screen using robotics and an algorithm for screening, which we have developed on the Navy side of the house, and you have to feed that through an assay that Dr. Biswas just recently talked about, but I'll come back to it in a second.
july11-731	DR. REGEIMBAL: What this assay does is it actually helps you find what we are terming as synergistic cocktails, cocktails that show internal synergy between the phages.
july11-732	DR. REGEIMBAL: So a more traditional cocktail is all the phages interact with, and infect, the parent strain of an infection, you get a several log reduction, sometimes up to four logs and so it could be really huge, then -- but eventually you're going to get resistance, and that will happen every single time at some frequency. Some low frequency.
july11-733	DR. REGEIMBAL: When we generate our synergistic cocktails through our iterative screening process, what we can actually do is find a collection of phages that work together, whereas one phage in the cocktail will infect the parent strain of the infection, you'll get several log reduction, that strain will become resistant so that phage will no longer work -- that's what you see here, in the middle -- eventually another phage in the cocktail which now didn't infect before now can infect that emergent strain, and so you have these phages working in series to drive a bacterial population to near extinction, even if the phages cannot go in reverse and infect the previous iterations of the phage.
july11-734	DR. REGEIMBAL: Sorry. The phage cannot infect the previous iterations of the strain.
july11-735	DR. REGEIMBAL: What we're also seeing, just like everyone else is noticing, is that when you get phage resistance, which finally will emerge even against our synergistic cocktails, those bacteria are usually way lower, they have reduced virulence, and they're often more sensitive to antibiotics. So that's also a mechanism that these cocktails are using to drive bacterial populations to extinction.
july11-736	DR. REGEIMBAL: So when we go back in these synergistic cocktails and we ferret through our workflow, another controversial aspect of this that I think is actually important whenever possible is to actually manufacture the phages you're trying to use therapeutically to the degree possible on the target strain. Everyone hates that idea because you'll be using an MDR clinical isolate to manufacture, at some level, phages.
july11-737	DR. REGEIMBAL: The reason why I think that's actually kind of important to think about is because any time a phage interacts with a bacterial culture you're going to get some level of host adaption. That host adaptation will happen every single time.
july11-738	DR. REGEIMBAL: Again, what you can imagine is imagine you have a consensus sequence of your phage and it's perfect for infecting your target, but then you manufacture that strain, or that phage on a manufacturing strain.
july11-739	DR. REGEIMBAL: What if the sequence is optimized here for infecting the manufacturing strain? What will happen is the -- when you grow that phage the new consensus sequence will shift. The sequence that was optimized for the manufacturing strain will become the new consensus sequence of that new emergent population of phages.
july11-740	DR. REGEIMBAL: That in vitro might be completely undetectable. In a diffusion-controlled environment you might not even notice that ever happened, but in vivo, when you put it back into an animal, for example, what we've noticed is that you have three-dimensional architectures. You have an immune system that's constantly trying to remove those phages.
july11-741	DR. REGEIMBAL: That might be massively consequential, and you didn't really know it at the time. You could have shot yourself in the foot and shifted your population away from being optimized to your target, even though in vitro you can't even detect that shift. So if this is really possible, I think you should manufacture in the target strain if you can.
july11-742	DR. REGEIMBAL: So that's sort of the way we envisioned how this would -- could work, and then we actually went and did it. So the way we build our libraries is we go to some of the worst places you would ever want to go.
july11-743	DR. REGEIMBAL: We go to wastewater treatment facilities, we go to standing cesspools. This is the training population in Fort Benning Georgia where guys are swimming in a pond. There's phage to Staph there.
july11-744	DR. REGEIMBAL: It turns out that ships are probably a pretty good way to look for phages because of the way they deal with what's called brown water -- you can use your imagination for what that is -- and it's in a really big tank on the ship. So that is probably a good place to go.
july11-745	DR. REGEIMBAL: In Peru, this is one of our favorite spots. We have five spots that look just like this, and they are filled with household refuse, diapers and fecal matter, food waste, trash. Animals water here. We've actually found a dead dog in it several times. It's very unpleasant. It's actually downstream of a local hospital, so you get hospital runoff.
july11-746	DR. REGEIMBAL: Actually, the best place to find phage, or the best time to find phages is right -- is about a day or two after a rain storm because this would become, essentially, a static culture. Couple days after it rained you get this churning event, you get new stuff introduced in the environment. We find a burst of phages about two days after a good rain storm.
july11-747	DR. REGEIMBAL: We have about five sites like this. I actually wrote a grant to try and do global phage harvesting at every place that DoD has a lab. I don't know if it's going to be funded yet, but what we want to build is one of the most robust libraries against all the clinically-relevant ESKAPE pathogens that the world has ever seen. That's what we're trying to do with the infrastructure of the United States Military, but I don't know if it's going to be funded. Once you build your library, again, Dr. Biswas talked about how you would isolate phages. What we're currently doing is we'll use clinically relevant strains of the ESKAPE pathogens, for example, that are local to the site of phage isolation because we want to get the best -- that would be the best soil to sort of grow your phages in from that region.
july11-748	DR. REGEIMBAL: What we're trying to do is build a diverse phage library against clinically relevant ESKAPE strains. So once you get the -- a culture supernatant that's rich in phages that you care about, this red arrow is extremely important because that's going to be the arrow that is the characterization that is required to transition your phages from just environmental isolates to what is needed to be an arrayed library, for inclusion in that library.
july11-749	DR. REGEIMBAL: So that arrow is probably going to be very expensive, it's probably going to involve sequencing, but a lot of the characterization requirements aren't even worked out yet. Eventually, what we want to build is an arrayed phage library in that way.
july11-750	DR. REGEIMBAL: What we're going to be doing is iteratively screening it on a per person basis to come up with a personalized therapeutic cocktail. The way you do that is you have a phage library, you're not sure which phages are going to be used, but a strain comes in from the clinic -- so this was the example of how we demonstrated this in an animal model.
july11-751	DR. REGEIMBAL: Our target organism is A. baumannii I5075, which is a clinical isolate from an osteomyelitis patient. We have a version of it that expresses luciferase. It's got the lux cassette.
july11-752	DR. REGEIMBAL: So the idea would be you would screen this phage library using -- against your target pathogen using our iterative process in the assay -- the BioLog assay that Dr. Biswas just presented, and what it helps you do is find phage that work synergistically, but you don't have to know the underlying mechanism of that synergy.
july11-753	DR. REGEIMBAL: We've figured it out in this case, and it has to do with capsule production. So the Army actually had a great phage which could infect 5075 and it causes a lag in growth at about six hours, and then you get a resistant population that pops up. That resistant population is uncapsulated.
july11-754	DR. REGEIMBAL: Then the Navy had four phages that infected that version of A. baumannii 5075, the uncapsulated version, very well. You blend them all together and you get a complete killing event that lasts way past 20 hours. It goes well out to over 36, even maybe 72. When you do see resistance, which will pop up eventually, it's stochastic. It doesn't happen in every version of the culture.
july11-755	DR. REGEIMBAL: So essentially what you have is four phages that basically do nothing. They have no detectable activity against this isolate, you have one phage that sort of just delays its growth for about six-ish hours, but when you blend them together you have a possible therapeutic that gives you a complete killing, or at least as near as we can come to complete killing.
july11-756	DR. REGEIMBAL: You've engineered the artificial situation in which you're almost driving a bacterial population to extinction.
july11-757	DR. REGEIMBAL: Although we know it here, you don't necessarily need to know the underlying mechanism for that, which would allow you to screen through potentially dozens of these kinds of events without having to know the underlying mechanism for how that synergy's working as long as you know the phages you're starting with are safe.
july11-758	DR. REGEIMBAL: And so we tried this in a mouse animal model. I think Col. Tyner presented this this morning, so I'll just go through it as quickly as I can. It was a 60-animal study. The only reason I show this busy aggregate picture is because if you look in the PBS groups, what you see is that we had some adverse events.
july11-759	DR. REGEIMBAL: We don't want to use death as an endpoint in this model, but sometimes it happened accidentally. There were also two cases in which we had to euthanize PBS-treated animals because they developed paralysis. The location of the wound is on the back of the mouse. We got tissue invasion that led to hind limb paralysis, and so we had to euthanize those animals. But we never saw those adverse events in any of the phage-treated mice.
july11-760	DR. REGEIMBAL: So to give you a cleaner picture to look at, essentially, this is an aggregate picture, or a representative picture. You have a PBS-treated group on days one, three, and five. The group treated with just the Army's phage that -- it's the capsulated version of the baumannii, and then the full five membered cocktail.
july11-761	DR. REGEIMBAL: In the PBS-treated group, again we saw about five fatalities. All those animals lost way more weight, and they all developed eye infections. So it's frequent that they start to groom each other again, and they all had massive eye infections. These animals were very sick. We never saw any of those events in any of the phage-treated mice, and in -- basically, in the full five membered cocktail we were able to lower bioburden by IVIS signal, and we were also able to lower bioburden not only by intensity, but by area.
july11-762	DR. REGEIMBAL: So you can't really get better than the surgical wound, but you can get far worse if the bacteria invade neighboring tissue, which happened in the PBS control cases and didn't happen in the full five-member cocktail. The cocktail actually can -- restrained the bacteria to only being in the original surgical wound.
july11-763	DR. REGEIMBAL: We also had no detectable necrosis in the phage-treated mice. Again, in the PBS-treated group it advanced outward and you got necrosis in the surrounding tissue, and that didn't happen. The wound never got larger.
july11-764	DR. REGEIMBAL: So then, as a result of that, the phage-treated wounds got -- remained smaller and closed faster, which allowed them -- basically, we concluded that the -- this proof-of-concept cocktail was able to treat a multidrug-resistant infection in mice. This technology development was actually the foundation for the work that was then used to compound a cocktail in the eIND case in California. What we also noticed, which is also the same thing that everyone is noticing, is that phage can push around bacterial populations. One of the ways the -- our phage cocktail could push the bacterial population was to become less virulent.
july11-765	DR. REGEIMBAL: So 5075, when you -- so we have a very simple Galleria mellonella model, that was worked out again by the WRAIR, the Army side of the house, from the wound infections department, and basically, you have a wax worm, you inject it with a bacteria.
july11-766	DR. REGEIMBAL: If the wax worm shrivels up and dies, the bacteria was virulent. It's a very easy assay to do. So if you inject wax worms with wild type 5075, the capsulated version of the bacteria, all the worms shrivel up and die by four days.
july11-767	DR. REGEIMBAL: You can use any number of controls that don't make a capsule, and any of the mutants that popped up from our synergistic cocktail also had -- were uncapsulated. If you inject those into the wax worm, they essentially survive.
july11-768	DR. REGEIMBAL: So you've basically taken a phage therapeutic and was able to render a bacterial infection, or render a bacterial isolate less virulent. This is happening in lots of different cases. There's lots of ways we've even seen that today, where bacteriophage can alter bacterial virulence in the emergent resistant populations. We can do that as well with just phage you might find in the sewer outside of this building, as long as you compound the cocktails correctly.
july11-769	DR. REGEIMBAL: Again, we also see, which Dr. Biswas went over just a second ago, is that our phages -- the phages -- the kind of phages that we're finding can also synergize with antibiotics. So not only can we develop cocktails that have an internal synergy amongst the phages, but the phage, like everyone else is noticing, can synergize with antibiotics.
july11-770	DR. REGEIMBAL: This is an example of Kleb. I think he just actually went over it so I'll just briefly go over this. We can see, even with low concentrations of phage in the presence of meropenem, you can reactivate the activity of meropenem in some way in the presence of antibiotic, or in the presence of phage.
july11-771	DR. REGEIMBAL: So it could be that a strategy for phage therapeutics maybe to start is that you're never going to convince a clinician to stop using an antibiotic, so maybe we should just embrace that and say the first application for a phage could actually be, and the way to augment antibiotic therapy, and possibly even reactivate an antibiotic that hasn't even been used in 20 years. That could be a potential strategy, assuming that we can get it to work.
july11-772	DR. REGEIMBAL: So just generally speaking, the Navy phage therapeutic program, in my opinion, I think a phage therapeutic that's based on a library-to-cocktail approach is actually the most robust and the most durable way of generating phage cocktails that will actually be efficacious in the clinic.
july11-773	DR. REGEIMBAL: I think it makes -- and we've actually demonstrated this. We've showed it in animal models, we've shown it in a human compassionate use case. We can show that we can alter virulence, we can show that we can alter antibiotic sensitivity. Essentially, it's all based on phages that can be found all over the planet in the wild.
july11-774	DR. REGEIMBAL: So what we're limited now by is just the availability of wild phages that we can then characterize, do the correct husbandry, and build the -- a library the world has never seen. I think we're poised to be able to do that.
july11-775	DR. REGEIMBAL: So, in thinking about some of those issues, I think there's probably some -- numerous regulatory concerns, because that would mean lots of things that -- would be different about this kind of technology. The first is we have to really figure out what is required to move a environmental isolate of a phage into a phage library and have that be called safe.
july11-776	DR. REGEIMBAL: What does that mean? Do you require full genome sequencing? Does that genome have to be closed? Are draft genomes okay? Can we use PCR in certain cases? I also think the library will probably have to be iteratively updated.
july11-777	DR. REGEIMBAL: So I heard yesterday people were talking about, well what if I have a fixed cocktail and I want to swap out a phage over time? And if you start to think about that, and if your product was the cocktail and you want to already start swapping out phage, that starts to sound a lot like a library-to-cocktail approach, just with a very small library.
july11-778	DR. REGEIMBAL: So I would invite you to come over to the dark side and just embrace the library-to-cocktail approach. It would mean you have to change a lot of things, potentially, but it's a very robust idea, I think.
july11-779	DR. REGEIMBAL: So, as clinically-relevant strains drift, we'll constantly have to be updating our library. There will no such thing as even a fixed library. Maybe you have to do it every year, every six months, every two years. It's hard to say. In terms of manufacturing, I understood -- you know, I think it's a good idea whenever possible to grow the bacteria on the MDR strain, the target strain of interest, so that you host adapt to the correct and most appropriate strain.
july11-780	DR. REGEIMBAL: If that is your strategy, then your scale up isn't a 300 or 1,000-liter fermenter making a lot of GMP phages. What you're doing is it's a question of scale-up according to bandwidth. How often can you compound a personalized phage for individuals per unit time? So that scale-up is a little different than the way you would currently think about normal CMC for drug manufacture.
july11-781	DR. REGEIMBAL: That would also mean that every time I compound a cocktail and I grow it on the target pathogen of interest, I would never give those phages to anybody else. They would be one-offs. It's just how many times can you do those one-offs per unit time.
july11-782	DR. REGEIMBAL: And, again, this would also affect clinical trials. I think that the clinical trial caveats for phages have been beaten to death, so I can just sort of skip over that.
july11-783	DR. REGEIMBAL: Finally, I'd just like to say that when I first joined the Navy four years ago I had no idea I'd be working on phage. I'm actually a biochemist by training. The people that I've had a chance to work with have been fantastic, both in the Navy side, the Army side.
july11-784	DR. REGEIMBAL: And now, down in Peru, we actually have a very eager team because in Peru, for example, and, actually, all over South America, MDR Pseudomonas, MDR baumannii is an extremely massive problem. You hear cases in the newspaper all the time of a young girl, for example, who goes in for appendicitis, she gets an IV line placed two days before for some reason, she got a Pseudomonas infection, and then three days later they had to cut off her arms and her legs because nothing would work.
july11-785	DR. REGEIMBAL: I mean there -- this problem is everywhere. It might not be as visible in the U.S., but it's everywhere. It's a particular problem for the military because our wounded service members were coming back with some of the most severe injuries that you could think a human could survive, and they did, and then they got an infection which required even more surgery and more removal of tissue.
july11-786	DR. REGEIMBAL: That just sort of can't happen, so we have to come up with a solution for this problem. Personally, I think a library-to-cocktail approach using natural phages is one of the most robust I've seen as a potential solution for this.
july11-787	DR. REGEIMBAL: So thanks again for everybody on the list. They're awesome. Doing science with them is a lot of fun. If you have any questions, I'd be happy to answer them.
july11-788	DR. REGEIMBAL: Yes, sir?
july11-789	AUDIENCE MEMBER: If I could ask a question with respect to someone who's run a successful phase 2 trial with a fixed cocktail. Well, yeah, the only one. Agreed. But the only one. Two things.
july11-790	AUDIENCE MEMBER: First, antagonistic co-evolution. Your fixed X will drop outside the circle, but the circle will then spread to find it again. I've got a really good chapter written for a book I'm editing right now by Brockhurst on that. It is a fact, and it does happen. Phages aren't fixed the way a chemical is.
july11-791	DR. REGEIMBAL: No. No.
july11-792	AUDIENCE MEMBER: You know, that is an argument that I've used many times in the past. Second, you don't, I agree, expect a phage to eliminate its dinner. That's not what it does. That's not what it does ecologically.
july11-793	DR. REGEIMBAL: Right.
july11-794	AUDIENCE MEMBER: And, again, I've said that many times. But if you can get the number of phage down below quorum sensing, down below pathogenic effect, down below -- sorry -- bacteria, down below pathogenic effect tissue damage, then you have got responses in the body which will help to clear it. Not only the adaptive, but the non-adaptive immune response. Even physical clearance, cilia in the ear, cilia in the lungs.
july11-795	AUDIENCE MEMBER: So is elimination actually required? I mean most antibiotics won't eliminate but they'll drop it down below the pathogenic threshold and the body can then cope, to quote me. Isn't that the possibility with a cocktail, regardless of the outlying Xs?
july11-796	DR. REGEIMBAL: So I would answer my ques -- your question this way. I am not ready to down-select any modality. I was meaning to be sort of tongue-in-cheek provocative, but I don't think anyone who's in the room ready to down-select what modality we should use.
july11-797	DR. REGEIMBAL: I do think fixed cocktails would have lots of clinical applications. But when you use a fixed cocktail you're making, you know, several hopes, or maybe assumptions is an easier way to say that. Your assumption is that you can -- your cocktail will cover enough clinically-relevant strains to give you some sort of efficacy. You're hoping that your cocktail will knock down the infectious target in all people to a degree that can show clinical efficacy, you're hoping that your emergence of resistance is infrequent enough to give you clinical efficacy, and you're hoping that it can do all of those things for a long enough period of time to make it economically viable to sink the $50 to $120 million in your product you just sank.
july11-798	DR. REGEIMBAL: So while that is possible, I do think we should all desire a better alternative, and I think that's not a mysterious alternative. It already exists, it's just a lot more complicated to bring to the market, which is to start with a library and just personalize as best we can.
july11-799	DR. REGEIMBAL: So I fully admit, for example, retrospectively, after people compound personalized cocktails for a while you might empirically discover that a fixed cocktail in that dataset is great, and so every time you're making a baumannii library you have the same handful of phages in all those cocktails, so just start with those.
july11-800	DR. REGEIMBAL: But I think that should be decided empirically downstream, not today when there is no commercialized product in the U.S., for example. AUDIENCE MEMBER: Or you could take it the other way and go with the cocktail to start with, and people who come through that get the personalized approach.
july11-801	DR. REGEIMBAL: Fair enough.
july11-802	AUDIENCE MEMBER: Because the people in Peru living in that alley you showed won't have the resources to do the personalized approach, I don't think.
july11-803	DR. REGEIMBAL: Well, fair enough. I understand the argument. And, like I said, I meant to be a little bit provocative. I obviously am not ready to down-select anything. I just wanted to be a champion -- or not a champion, that's the wrong word, an advocate for this kind of idea because I think it's seen as the sort of weird fringe in a world of weird fringe.
july11-804	AUDIENCE MEMBER: They're all weird, but everywhere you get a Sith, you get a Jedi.
july11-805	DR. REGEIMBAL: Fair enough.
july11-806	AUDIENCE MEMBER: I have two -- one question and one point to make. So it sounds like the Navy may take care of Acinetobacter baumannii, and we won't -- I mean, look, this is a governmental intervention at that point. So if the Army would do one, and the Marine Corps would do one, Air Force would do one, and the Coast Guard would do one, we'd have six of the ESKAPE pathogens and there would be no commercialization, it would be provided by the military, and I think that's a great idea.
july11-807	AUDIENCE MEMBER: Secondly, there is a problem with the -- I mean I -- and we did it under the time pressure and so did -- and that is growing your therapeutic cocktail on the pathogen itself. We have seen in multiple cases if you infect a bacterial strain, you will induce prophages.
july11-808	DR. REGEIMBAL: Yeah.
july11-809	AUDIENCE MEMBER: So that's one of the problems there's going to be. Of course, in an eIND situation, that's a risk you just take, right? But trying to put it, when -- to non-eIND situations, I think you would have to make sure that the pathogen is not going to induce prophages carrying the very toxins that made the patient sick already.
july11-810	DR. REGEIMBAL: Well I would ask this question. If you're going to use phages, in general, in a person, whether you grow that phage in the target strain of interest --
july11-811	AUDIENCE MEMBER: A numbers game. I mean you're --
july11-812	DR. REGEIMBAL: I understand that. But you're going to get burst events downstream within the human, and so whether that happens five minutes before or five minutes after you push it into the IV -- I'm not sure I understand the --
july11-813	AUDIENCE MEMBER: In the liter culture or three-liter culture you're growing, you're going to have a lot -- an awful lot of those phages, and they can -- phages can lysogenize way beyond the domains where they can make plaques or grow virulently. Just something to be concerned about. Because we've seen phages become -- one percent of the total phage population is induced prophages when you're super-infecting with a virulent --
july11-814	DR. REGEIMBAL: Yeah. And that's why I would also just add the additional caveat that if I -- if you were to do that, you could never use those phages in anybody else. It would be a one-off. Those phages that grown on that target pathogen would only go back into that person in an attempt to limit those kinds of outlying events, or side events.
july11-815	DR. TURNER: That was an intriguing talk. I guess the comment, in defense of evolutionary biology, is that I don't think any species wants to go extinct, but the vast majority of them have in the history of the planet. A phage doesn't want to eliminate its dinner, it just doesn't mean it won't happen.
july11-816	DR. TURNER: So I guess I just want to make sure the audience understands that, you know, humans drove smallpox virus into extinction, and it certainly wasn't in the interest of that virus, variola virus, to have that outcome, okay? But let's just put that comment aside.
july11-817	DR. TURNER: It was intriguing what you said about, you know, if you do groom the phage on the patient strain you may get adaptation that is specific to it. I agree with that. But another core principle in evolutionary biology is correlated response to selection.
july11-818	DR. TURNER: You could just as easily groom it on that, and it's actually very good on other strains as well. Because that explains how this gets into humans very readily, you know. It was not groomed on humans. So I think it's an open question --
july11-819	DR. REGEIMBAL: Absolutely.
july11-820	DR. TURNER: And that bears more research.
july11-821	DR. REGEIMBAL: Absolutely. So, in my opinion -- well, again, this is Jimmy talking, this isn't Lt. Reigembal. There's a lot of work that has to be done to bring this kind of product to the next step. I mean we would have to show that -- whether manufacturing on the host versus a manufacturing strain would actually make a difference. It might be that you get clinical efficacy without the need to doing that.
july11-822	DR. REGEIMBAL: But what I'm saying is that what everyone needs to realize is that regardless of your modality, though, you are smashing two populations together, and those population dynamics really matter. Most people just talk about -- well I don't want to -- it's a gross characterization.
july11-823	DR. REGEIMBAL: But frequently what you hear about is lytic spectrum, and host range, and that kind of stuff, but, really, you're going to -- all of molecular biology is selecting for the rare event. That's like all you ever do. That rare event can happen weirdly at any time if you're mixing any kind of 10 to the eleventh population with a local population that's in equal numbers.
july11-824	DR. REGEIMBAL: So my goal was to bring some of those kinds of ar, or those kinds of issues to the table. But, no, I don't think that tomorrow I necessar -- well it depends on how sick I was because I saw it work. But I think there's a lot of work that still needs to be done in this space of personalized therapeutics. DR. TURNER: Yeah. I didn't want to sound hypercritical because I think you're raising a lot of interesting questions that need to be studied.
july11-825	DR. REGEIMBAL: Yep.
july11-826	AUDIENCE MEMBER: I like your idea to be able to bank for the whole world. I think it sounded like a very big task.
july11-827	DR. REGEIMBAL: Yeah. Yes, it is. That's why I might not get funded. But I try.
july11-828	AUDIENCE MEMBER: The clinically relevant bacteria are changing, so, from your experience, how often you have to monitor to ensure you're current?
july11-829	DR. REGEIMBAL: I mean that's an open question because what does it mean to monitor? Are you monitoring only in vivo? Sorry. Are you monitoring only in vitro, or you're monitoring in vivo using some sort of animal model?
july11-830	AUDIENCE MEMBER: If you built a bank that either covered the whole world, you have to ensure for each country all the clinical-relevant bacteria is covered --
july11-831	DR. REGEIMBAL: Yeah, but -- yeah, and that's -- obviously it's a -- but that's a problem of scale, it's not a problem of techno -- if you have engineering solutions, if you have other kinds of solutions -- I mean that's a -- it seems to me like even though it's big, it's not difficult. It's not easy, but it's not difficult. It's just you have to get larger in scale.
july11-832	AUDIENCE MEMBER: And I'm interested to know how far you been on that road now.
july11-833	DR. REGEIMBAL: In terms of trying to build a large library? So we have -- right now I have med students from Penn State in Peru harvesting phages for me in some of the worst places you would ever want to go. We have gone into Honduras, we've gone into a lot of -- basically in Central and South America we have lots of sites that we're now going to.
july11-834	DR. REGEIMBAL: I'm trying to go international over into southeast Asia, as well as Africa. We have -- the military has infrastructure there, both Navy and Army labs. But the problem now is funding. It's not even willing partners. There's people with those labs ready to go. They want to be involved in this effort.
july11-835	DR. REGEIMBAL: I think it's an effort that if we build a diverse enough library, it will -- it might be great source material for people who think that a therapeutic phage cocktail that's fixed could be the best modality to go with. I might have a whole bunch of interesting phages you might want to try. But we're just -- really, it's limited now by funding. I'm just waiting to see if that happens.
july11-836	AUDIENCE MEMBER: Okay. Last one is can your bacteria or your -- or the information about these bacteria be shared?
july11-837	DR. REGEIMBAL: I don't know the answer -- which bacteria? The phage or the --
july11-838	AUDIENCE MEMBER: What you got in your bank. Can that --
july11-839	DR. REGEIMBAL: I'm not sure about that. I can't speak to that because I'm not sure if it can be shared outside the DoD or with our partners. I don't want to say the wrong answer. I have to ask nine layers of people before I can almost make any decision, so, but I can figure --
july11-840	AUDIENCE MEMBER: I shouldn't ask here. I'll ask --
july11-841	DR. REGEIMBAL: Yeah. Yes, sir.
july11-842	AUDIENCE MEMBER: First of all, it's incredibly exciting to see the world having gone from basically two phages being looked at in some detail to people all over the world doing this kind of enormous amount, and I want to say that's incredible.
july11-843	AUDIENCE MEMBER: How can one get, for example, students various places involved in doing things that could be helpful and other people involved? What suggestions do you have in ways like the Phage Hunters program, but going to ones that are perhaps more broadly useful?
july11-844	DR. REGEIMBAL: I mean I don't know that we have -- like, so the military, to my knowledge, does not have a common repository in that way, and I'm not even sure that you would want the military to be that kind of repository.
july11-845	AUDIENCE MEMBER: I'm not saying necessarily the military, but for guidance, just encouragement.
july11-846	DR. REGEIMBAL: Oh, I mean I guess word of mouth at this point is the only place I know to go. I mean the students that came down to work with us, they were planning on working on something else, a clinical study, and I just said, well you could do this idea, and all of them wanted to do it because they all saw, hey, this is unique, it's getting out into the lab, but it's also getting out into the -- to doing some of the more grimy field stuff. I mean really grimy field stuff.
july11-847	DR. REGEIMBAL: So it appealed to them on that level. It's a way of doing tropical medicine and mixing it with sort of a laboratory setting. So advertising it, I guess. I have no other answer for that. AUDIENCE MEMBER: Have you published anything about how you're doing that that one could get their hands on?
july11-848	DR. REGEIMBAL: No, ma'am. No. We're still in our stages of -- like, so all my phages are sitting in a freezer in a fridge in Peru, waiting to figure out the correct export for that.
july11-849	DR. RANALLO: Okay. So we're at the end of our presentations. I have a couple of announcements. One, the organizers would like me to at least investigate the possibility of making the presentations that we've heard over the past two days publicly available, so I'd ask speakers who are still present to reach out to one of the organizers. I'll just mention them by name -- Roger Plaut, Scott Stibitz, Paul Carlson, and Randy Kincaid. Those are the only, at least off the top of my head.
july11-850	DR. RANALLO: So, as I said, I'd like to ask the organizers, or the speakers to consider that with the, you know, with the possibility of perhaps doing some small redaction.
july11-851	DR. RANALLO: And then the last thing is, again, we're ready for our panel here. We have until 3:00, and I don't have on my agenda that there's a break, so we're going to just transition really quickly into a panel. This panel is with our speakers today, as well as with Scott Stibitz from CBER. So we're going to get that going so stick around, please. We'll only be a few minutes getting speakers up here.
july11-852	DR. RANALLO: (Pause.)
july11-853	DR. RANALLO: Just also to round the bases, I've been up here all day, I'm fairly exhausted, but I can tell you that there are a few areas that we heard today that I'd like the panel to opine on and perhaps address specific questions from the audience.
july11-854	DR. RANALLO: So we heard, you know, we heard talks on novel uses and future uses, so specifically looking at prophylactic or preventative use of phage. I think that, to me, is very intriguing, and an area that we have not discussed in terms of -- we haven't covered that.
july11-855	DR. RANALLO: Another is in terms of phage engineering and looking at how we can serve to, you know, genetically modify phage to make them more useful or to have them as tools to study bacterial populations.
july11-856	DR. RANALLO: So, with that, I don't have any specific questions for the panel. I certainly think, like I said, I would like -- maybe I could start off and, Andy, maybe press upon you a little bit, again, thinking about cholera phages and just trying to understand how in a high event situation such as household transmission of cholera one could, I won't say run a clinical trial, but at least, you know, conceptualize how that might be done.
july11-857	DR. CAMILLI: Yeah. I mean, you know, in Bangladesh and in India there's two outbreaks per year. Pretty reproducible. During those outbreaks there's certain places, like the icddr,b hospital in Dhaka gets thousands of patients a day.
july11-858	DR. CAMILLI: So they've run a number of household studies over the years, various investigators, various groups, and so the mechanism's there to do this, where they would -- they could incorporate into a household study where they go and teach them about transmission, and cleanliness, and chlorination of water, et cetera.
july11-859	DR. CAMILLI: They could do a trial with a small number of households where they -- the household contacts take the phage cocktail. That's the ideal field trial. We need to get the money to do that. We have the product ready. And then you would look. This high rate of transmission, 23 percent, is a easy target. Do you lower that or do you not?
july11-860	DR. RANALLO: Is there a role in a -- and I don't know the status or -- in a human challenge model or something like that, or -- and I think I mentioned this perhaps earlier, just the idea of an attenuated strain just to look at the dynamics of how this would occur and what the rate of excretion would be?
july11-861	DR. RANALLO: Because I think you mentioned that there was a tenfold -- two log increase in transmission rates. Can that be predicted by the excretion of a rice water stool in a clinical setting? Is that a first step or is that --
july11-862	DR. CAMILLI: Well with animals you can mimic this transmission. You know, with the infant rabbit model, they will transmit it either naturally, just have the baby rabbits together, or you can take some of the stool and transmit it so that you can easily model that in the laboratory.
july11-863	DR. CAMILLI: In households it's not clear how the cholera is being transmitted within the household. I mean you can imagine somebody comes down with cholera and it gets all over the place, maybe it gets in the water that they're drinking, but nobody really knows. Nobody's looked at that yet.
july11-864	AUDIENCE MEMBER: Maybe just an idea for these -- the people in this room. Based on the things we have had to do all with the individual case treatment, we have been discussing that with AmpliPhi Bio Science already, and I wonder if would not be possible to set up some kind of a database with a standard format, simple format, of all the patient cases that we are starting to treat to try to harmonize the information on these patients treating with phages in a way to provide more information and to get that through the -- information available to the regulatory authorities in USA and in Europe.
july11-865	DR. STIBITZ: I think that's very hard to do. You have to ask the question of who's going to fund it. I think it's -- you're talking about, for example, a database entry for each case where phage therapy has been attempted --
july11-866	AUDIENCE MEMBER: Yeah.
july11-867	DR. STIBITZ: -- with certain minimal details. I mean I think that the FDA lacks the regulatory authority to do that unless it were under an IND. I think it's something that a group of concerned scientists and/or citizens could organize, and I think there would be great value in the sense that it would capture the denominator.
july11-868	DR. STIBITZ: Currently, we hear about the successes, and I think Dr. Gorski's presentation I think was certainly an eye-opener for me for somebody who's really, you know, kept the records so that we are getting a -- estimates for the efficacy of phage therapy at least in one modality. He's standing right behind you, so he might want to respond.
july11-869	DR. GORSKI: -- would be to reduce the dose patient within clinicaltrials.gov, even though clinicaltrails.gov is restricted for clinical trials. For example, we did so. We did not update the information -- I'm sorry about it -- but our, not trial, but our experimental therapy is registered within clinicaltrials.gov.
july11-870	DR. GORSKI: I don't know if it's legally possible. That's another question. But if it is, why not?
july11-871	DR. STIBITZ: Right. I think I'm on pretty safe ground saying, and I can look for nods from my colleagues, but I don't think that's something that the FDA could mandate or be that instrumental in doing. I'm not seeing nods, so maybe they'd like to respond.
july11-872	DR. TYNER: So I have something just briefly to add perhaps for consideration is I like the idea of having a database. I think the important part of a database is some level of harmonization of the data you collect.
july11-873	DR. TYNER: Previous life I was a malaria researcher, and the malaria community realized when it did meta analysis that they couldn't compare one study to the next, and so maybe it's incumbent upon some folks, perhaps in this room, to sit down and talk about what it would be -- what are the things we would want to collect and at what time points, et cetera.
july11-874	DR. TYNER: I realize it's not a clinical study, but if there was some level of agreement, consensus on the information that you collect, it sure does make it a lot easier to compare as you're beginning to put all these different eIND cases which are disparate enough and different enough that they're hard to compare in the first place.
july11-875	DR. STIBITZ: Before I let Jay ask the next question I just wanted to add there's -- it seems to me in this scenario there would be a very strong incentive to report positive data and a very weak incentive to report negative data.
july11-876	AUDIENCE MEMBER: Hi. So I want to ask you to focus for a minute on a different area of anxiety. Not so much efficacy, but safety, perhaps in a more global perspective. A number of you touched on it, but I guess I didn't feel like I really got a fully developed response, especially from the phage engineering folks.
july11-877	AUDIENCE MEMBER: Certainly in Dr. Duerkop's discussion he mentioned the great anxiety that the dairy industry has about phage gone wild, and that's a major concern in that universe of the dairy industry and fermentations that you don't want to get extinguished.
july11-878	AUDIENCE MEMBER: I don't think anybody's talking about a phage that gets out in the world and destroys every gram-negative on Earth and unleashes other horrible problems, but certainly in our microcosms that we work with, not so much patients, but maybe hospitals and other little microorganism universes, can you imagine any adverse effects in those universes that you should worry about, especially with an engineered product, not necessarily a product that's been co-evolving with these organisms for hundreds of millions of years.
july11-879	AUDIENCE MEMBER: Just fantasize about your worst nightmare and then we can -- then we could just -- after you verbalize it, we could just sleep better. How about that?
july11-880	DR. LU: Right. I mean I could write a Hollywood script on it. I mean I guess it's the same question as asking, you know, any genetically-modified organism ... can you accurately assess every possibility that could happen, right?
july11-881	DR. LU: So I think we have the same debate over GM foods or, you know, oncolytic viruses that are engineered in a variety of different ways. So I'm not sure that I can a priori predict to you all the bad things that can happen with engineering. I think we can probably take off things that we might expect to look for, right?
july11-882	DR. LU: So we don't want to be able to transduce genes between organisms at a greater rate than we might naturally be able to do that. We might want to test that our engineered phage, as a well-defined spectrum, it doesn't hit -- won't go commensal, or good bacteria or bacteria that we're worried about in the dairy industry, for example.
july11-883	DR. LU: I think if the requirement's going to be that we have to take an engineered phage to a standard where I can prove to you with a hundred percent certainty that there is zero risk, I think that it's sort of an impossible bar for an engineered construct across.
july11-884	AUDIENCE MEMBER: No, no, no, no. But somebody's going to ask you to do an environmental impact assessment for sure of some sort, and I think
july11-885	AUDIENCE MEMBER: -- I just think the conversation is worth having. I think it's worth having among scientists rather than in the --
july11-886	DR. LU: Yeah. So I think doing an impact assessment of spectrum and transducing capability of an engineered phage versus its natural counterpart could be worthwhile to do. I'm not personally concerned that engineered phage would be any worse in that particular context, but I think -- I mean I -- certainly we can define assays that we can all agree make sense, but I don't want that to necessarily turn into like GM is necessarily bad.
july11-887	DR. LU: I think it's always a risk/benefit trade-off of, you know, sometimes it makes sense to do it, in other cases the natural phages make a lot of sense. If you can get great efficacy with the, 'natural phages,' that have been, frankly, evolving for a very, very long time, then why not go ahead with that?
july11-888	AUDIENCE MEMBER: Just to add a little bit to that, so our center is collaborating with a large pharmaceutical company to generate phages to treat Pierce's disease which is destroying the wine industry in California, and there's no way we'll ever be able to use engineered phages.
july11-889	AUDIENCE MEMBER: The California EPA has already said never, ever, ever, and then the -- and our EP -- the U.S. EPA doesn't seem to be quite as negative, but the California EPA says no way.
july11-890	DR. LU: Yeah. So I think certainly environmental applications is probably a very, very difficult way of using engineered phages. I think if we're talking about serious human disease, then I think the bar is probably different.
july11-891	DR. RANALLO: So, yeah. One of the things that I've been thinking about over the last -- this -- today here is this idea of phage cocktail and the utility of phage cocktails for dealing with resistance generation. Do you think that thereâ€™s a specific engineered solution to that in terms of engineering phage to not â€“ I guess my question is a phage cocktail almost always going to be the solution, or is â€“ do you feel that thereâ€™s an approach that â€“
july11-892	DR. LU: Yeah, I think the engineer â€“ I think the phage cocktails make a lot of sense currently, given the data. I think we have some stuff in the works that shows that you can integrate certain properties into single phages that are quite interesting.
july11-893	DR. LU: So I don't want to say too much about that right now, but I think there is the possibility of integrating multiple properties into a single phage, and -- but I think, you know, the cocktail approach seems to do pretty well in most cases, so I'm not saying that that should be thrown out the window.
july11-894	DR. RANALLO: And -- sorry. Yes?
july11-895	DR. BISWAS: So I just like to mention one thing. Also I prefer natural phages, as engineered phages are great to attack stationary phase bacteria. Because stationary phase bacteria, to kill them with natural phage is very difficult sometimes. So something to deliver lethal gene or something in those bacteria using engineered phage is a very good idea.
july11-896	DR. RANALLO: Yeah. And I guess that's my question for developers, in general. You know, we heard Scott say yesterday basically on this topic is that engineering is not bad, it's just you have to explain why and prov -- and likely incumbent upon the developer to develop assays, or at least to have some way to address the issues at least that are brought up, or that, you know, that -- I'm sorry.
july11-897	DR. LU: Yeah. No, I agree. I mean I think we have to justify why we're doing it and -- in some sense certainly to the regulators, but also to our own time. Like why am I spending all this time doing it? So I think there are certain properties that we would need to be able to demonstrate why the engineered phages make sense.
july11-898	DR. LU: I think the other thing, to address sort of some of the comments brought up earlier, I think one of the reasons to go to an engineered construct is if you can perhaps remove the replicative ability of these phages and sort of simply use them as delivery. I think that's an alternative way we've been thinking about sort of hopefully addressing some of these concerns about sort of freely replicating genetically-modified mutant viruses.
july11-899	DR. RANALLO: Okay.
july11-900	AUDIENCE MEMBER: Sorry. Real quick. I don't think it's ironic that you two are sitting right next to each other. I think when you talk about diagnostics and you talk about personalized medicine, can you just kind of touch quickly on like the time-sensitivity of having to deliver these therapeutics pretty rapidly and how important like the complement of each other are.
july11-901	DR. BISWAS: So your question is how we can mark this diagnostic approach with personalized phage cocktail, right?
july11-902	AUDIENCE MEMBER: Yeah. So like say you need to identify what the pathogen is pretty rapidly, and then you need to come up with a cocktail just as rapidly. I guess what stage of -- you know, is it in hours, or is it in days, or is it in weeks right now, and at what point do you think that --
july11-903	DR. BISWAS: So currently, the systems which we developed, we can figure out within six hours, almost, that bacteria is going to be infected with those particular group of phage. If you don't know the bacteria, what is the bacteria if you have a group of phage, you can use them to find out that -- what is that bacteria is -- actually.
july11-904	DR. BISWAS: Not only that, you can also study the antibiotic-resistance pattern of the bacteria in presence of phage because you can use the antibiotic, you know, in the bacteria, and then let them grow, and then use phage to see that they are affected or not.
july11-905	DR. BISWAS: The bacteria inhibit the -- if the antibiotic inhibit the bacterial growth, then you will not see the signal. The phage will not multiply, and they will not produce any signal. So you can do all these things in one single run, actually.
july11-906	AUDIENCE MEMBER: And on the personalized medicine side, how quick do you think that we can kind of get cocktails together that can be effective to treat --
july11-907	DR. BISWAS: Oh, okay. So last time when we prepared these we need three days. Three to four days. Three and a half days, almost. People took much more time to transfer the phages. But in real-time scenario, if we have previously prepared phage already, and cesium chloride and LPS, you know, removed phage, then we can use it right away.
july11-908	DR. BISWAS: But if we are looking at, like Colonel Regeimbal mentioned, that we need to grow it into the patient's bacteria, then we -- it takes little bit longer time. So that -- in that case, probably three and a half days will be fine.
july11-909	DR. BISWAS: But its depends. If we are -- and the people working the night shift, we can do it within two days.
july11-910	AUDIENCE MEMBER: Since we're being controversial this afternoon, I'll just pick up on one of Tim's comments. Removing the ability to replicate. When you use a therapeutic dose of penicillin you use about 10 to the 19 molecules. Now, penicillin isn't a single hit kill, and phage can be. I appreciate that.
july11-911	AUDIENCE MEMBER: But equally, penicillin, 350 daltons, gets through things easily. Phage, pick a dalton range, doesn't. So maybe those two balance out. So you -- maybe you need to use the same number.
july11-912	AUDIENCE MEMBER: If they can replicate, they can produce what they need, but if they can't replicate, okay, great, if you're putting it onto a situation where you can see the infection. You can dump on the amount of non-replicating phage you need. But if you're relying on it passing through wax, or body surfaces, or mucus, or whatever, I've done a few mathematical calculations on this, and I know Steve Abedon disagrees with me, but I come up with a dosing level, depending on the size of the phage, of somewhere between 400 and 1,000 kilograms.
july11-913	AUDIENCE MEMBER: So is not removing the ability to replicate going to be slightly problematic in some situations?
july11-914	DR. BISWAS: Definitely.
july11-915	DR. STIBITZ: Well I think I can get partly there. I mean ampicillin doesn't kill with a single molecule per cell.
july11-916	DR. RANALLO: Okay. So in -- with regard to finishing on time, what I wanted to do is maybe -- unless we have any other questions for the panel?
july11-917	DR. RANALLO: (No response.)
july11-918	DR. RANALLO: Seems like we got them all out during the day. So I'd like to, you know, thank the
july11-919	DR. RANALLO: -- well I'd like to thank everybody who came up for the panel, and I'd like to thank all our speakers today. I think it was a wonderful day. Then we're going to conclude with Dr. Mike Kurilla who opened the meeting yesterday with some concluding remarks. So thank you, panel members, thank you, speakers.
july11-920	DR. RANALLO: Mike, you're up for concluding remarks.
july11-921	DR. KURILLA: Well we've come to the end and -- of the workshop. I hope that the comments I've received over the last two days from staff and from individual participants are representative.
july11-922	DR. KURILLA: I'm always encouraged by the sort of crude marker I use that at the end of the meeting, if the density of occupancy of seats is similar to what it opened with, that obviously a lot of people have found a lot of useful things to stick around.
july11-923	DR. KURILLA: I have to say, personally, you know, this is now our second phage workshop, and I can tell you that, having been an undergraduate student back in the late '70s and actually had the honor of meeting Max Delbruck who introduced, I think, molecular biology to the world by studying phages, I never anticipated in a medical career that this would be something that would be realistically considered, and the amount of interest and focus that is being applied is very heartening.
july11-924	DR. KURILLA: So Dr. Marks from the FDA opened the workshop, and he noticed -- he remarked on the importance of history, phages being a little over a hundred years old, but that, with a little bit of effort, you know, they could be an example of the old becoming the new new, and I think that's been clearly evident in what's gone on. For that new new it's going to require a continuous input, both in terms of guiding developers on the current and evolving regulatory perspectives, which was a major focus on what we discussed here for the last two days, as well as encouraging continued investment in the scientific foundations that are needed to fill the gaps of knowledge, as well as to identify new, and potentially exciting opportunities that phages can offer us.
july11-925	DR. KURILLA: So the first and second sessions of the workshop focused on the clinical use of phages and regulatory perspective and, really, in terms of their applications to what we're seeing as probably the -- one of the most critical in developing unmet medical needs, that is, antibiotic resistance.
july11-926	DR. KURILLA: While antibiotics may be considered one of the real gems in terms of 20th-century medicine, the 21st century may see a much more limited utilization because we know that they are so easily overused.
july11-927	DR. KURILLA: In addressing these unmet needs we can't underestimate that phages clearly offer us, potentially, a new solution set, but it's still going to require quite a bit of effort in order to establish that regulatory guidance that defines product expectations, as well as the types of clinical studies and trials that are going to be needed in order to achieve regulatory approval.
july11-928	DR. KURILLA: The third session gave us a forecast of future possibilities and emphasized the value of incorporating lots of different perspectives in terms of looking at phages, and we're beginning to appreciate the understanding of co-evolutionary relationships and trade-offs that impact bacterial resistance and are likely to inform strategies for future development of phages.
july11-929	DR. KURILLA: We can also appreciate the newly-realized powers of genomics, bioinformatics, synthetic biology that will shed new light on phage evolution and prospects for useful and clinically-relevant modifications that will be desirable.
july11-930	DR. KURILLA: We saw some of the perspectives drawn from military medicine and ongoing challenges seen due to injury and exposure to initially unique, but becoming more commonplace infectious agents.
july11-931	DR. KURILLA: So these are just examples of the kind of cross-fertilization that these types of conferences afford us going forward, as well as just the overall value of communication with different communities, all with the goal of treating and preventing infectious disease. //
july11-932	DR. KURILLA: So, on my part, we and my FDA colleagues would like to thank all of you for making this a successful conference. We really want to encourage everyone to continue the communication, momentum, and collaboration that is building towards developing phage-based solutions for the future. Thank you very much.
july11-933	DR. KURILLA: (Whereupon, at 2:55 p.m., the meeting in the above-entitled matter was concluded.)

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