 So we have now the third and last lecture by Joshua Bytes. Before he starts, just to remind you a a little bit of how to ask questions. If you have a question, please post it in the chat or use the raise hand feature on the screen of Zoom. I'll post in the chat the instructions to use that feature once again. So please, Joshua, if you want to share the screen, you can start the presentation. Thank you very much. Okay. Perfect. Thank you, Jacoble. Can you hear me as the sound louder? It's perfect. Good. And then share. Can I see my slides and hear me? Yes. Okay, well, welcome back folks to this third lecture. I'm trying to make each connected but also stand alone in case people pop up and just see this one. So this will be the third lecture on virus micro dynamics spanning principles ecology and therapeutics and because therapeutics was the last in that sequence after the Oxford comma. I will be focusing on therapeutics today. I want to again remind folks I started earlier in the week with some principles of credit prey like interactions between virus and microbes and consequences from population to evolutionary dynamics to even co evolutionary dynamics. And then in the Tuesday lecture began to go in a slightly different direction confronting these paradigms by looking specifically at outcomes of infection at the cellular scale that don't necessarily lead to lysis and the production of more virus particles, but instead, potentially long term associations in which the fates of the virus and the host become entangled. And today I will revisit again the lytic paradigm in a specific context and applied context will try to leverage what we know about bacteriophage bacteria interactions with the purpose in mind that is to try and improve treatment of multi drug resistant infections and obviously that's clearly motivated by a real world application. And to focus on this real world application I would remind folks many of you probably don't need reminding that, in addition to SARS CoV two there are a lot of other hazards around and one of the biggest ones that continues to be a problem globally is the emergence and spread a multi drug resistant bacterial infections and the CDC categorize these in different levels and the levels have to do a with the extent of harm of the pathogen, but also the extent to which we are running short of therapeutics, including last line therapeutics or therapeutics that themselves cause harm when given. And these here are some of these listed scrap groups and pseudomonas originosa multi drug resistant staph aureus and I say are going to read a CDF and so on. And the issue here spans, obviously one not only impacts now but looking forward to the extent to which there are those who are concerned one of the most prominent. I would say statements on this topic is this O'Neill report, looking at deaths attributed to antimicrobial resistance MR every year now compared to other majors of causes of death. And the challenge of course, looking forward 30 plus years is the danger that would be associated with the spread of these multi drug resistant infections that would then be untreatable and estimates that rival that even that of cancer in terms of mortality. And in terms of some of the research spending, you can see at present there's just has not been the same prioritization within the NIH obviously that is changing, but more needs to be done to really to move this field forward. And part of this is that for a very long time may no major new types or class of antibiotics were developed there were some recent discoveries that provide some optimism with respect to discoveries based on environmental microbes including texabactin. Again, the crisis here is a serious one already, and is bound to get worse unless we really start to confront and take new approaches to address how to how to treat antimicrobial resistant infections, particularly those that are multi drug resistant. And one of the ways that people have begun to reconsider is to use phage. And I reminded you earlier in the week, or explained to you if you hadn't heard before that phage are highly abundant in natural systems where there can be 10s of if not more virus particles per milliter natural systems, whether in water, aquatic system soil, and other microbiomes and these can become the basis for candidates by a natural reservoir as it were, of potential treatments. This study and kind of broad scope description of virus host impacts is described in this book by Carl Zimmer, but my point here on the right is that there's this diversity of potential treatments that if we can harness maybe use productively to try to treat these infections. And in fact this is increasingly what has happened. This is five years ago, this is now five years ago. There was a first multi center clinical study of phage therapy in serious burn victims. And the idea was to try not just as a compassionate use case but do an industry standard clinical trial to look at both tolerance and effectiveness, not just does it not do harm, or is it tolerated but it actually is effective in treating antibiotic resistant infections. And this was meant specifically on burn wound patients so that the presumption here at the outset was that these could be colonized by either Coli or soon most original setup, and there will be a control and would be compared against the usual treatment and to see if the phage treatment showed a benefit. The problem about a year later was there was a series of delays size and scope and in fact there just weren't enough participants included in the trial to get the kind of power they needed to determine efficacy. And part of that was because the design of the trial presumed that they were going to use a cocktail of phage against a single type of pathogen. E. coli or pseudomonas and of course what is unfolding in many of these wounds is that these are complex communities. And so they weren't able to actually do have the right inclusion criteria and only a dozen plus of the intended hundreds plus patients were included you can see that, even if that were to work in those 15 you just wouldn't have the power to make those calls. Nonetheless, that even though this was a bit of a setback, there have been a number of promising developments. And those are often through compassionate use cases. These are two well known examples on the left. This is Dr. Patterson who was in a coma in near death and was treated with a phage cocktail and this is was a collaborative effort that was using essentially information on phage of acidibacter boughmownie as a way to treat this infection. And this is again been well documented you can read about this particular story. His wife is described quite a lot of the story as well. And a similar story this is again by Carl Zimmer. And you can see here on the bottom right, this is Paul Turner from Yale, and you can hear more about his work, also online in slides where he, again, literally these viruses fished out of a lake may have saved a man's life and advanced science these phage found in natural systems which were able to infect and lyse pseudomonas originosa and I'll just talk a little bit more about this particular system and the rationale were used to treat this fistula. So there was a lung associated infection. And again that was another example of a compassionate use case and Paul and his team, including Ben Chan have done this multiple times now successfully treating individuals using pseudomonas phage. So it's said to initiatives that go beyond compassionate use cases, but to the, to the scale of institutes, clinical trials, and even really institutes dedicated to phage therapy, including treatments not just pseudomonas and not just money but here's an example for my go back to him from Graham Hatfield schoolie Helen Spencer and colleagues. So, if you can look in the past few years you'll see an increase in interest a significant increase in interest with respect to using phage as the basis for treating these multi drug resistant infections in a diversity of pathogens. So let me go backwards in time to the origins, and just coming past really almost the centennial anniversary of the discovery of phage from the very outset, Felix Darrell and colleagues were thinking about therapeutics as one of the potential uses of this new discovery right bacteria phage phage means to devour this organism this virus that was able to devour bacteria. And here's an example from his book after being assured that no harmful effects attended the ingestion of the sugar bacteria phage this treatment was applied for therapeutic purposes to treat dysentery. The very outset Felix Darrell and colleagues thought about phage of course that over time this was supplanted by the widespread availability of antibiotics, but we now face new challenges. And the critique has been to some extent with respect to phage therapy that there's not been some transformative development or technology that means it's open season. Of course the need is greater, because of the emergence of multi drug resistant infection the question is, is there been a development that could say that the chances of using this effectively have increased. And I would argue that the answer to that is actually yes there has been developments, not only on the genomics or engineering side to improve the, let's say the production and and characterization of phage that are available for treatment that even the principles really ecosystem aware principles and I think this is why it really fits in to this workshop because these are really ecosystem aware principles that are now being used to try to improve treatment and one of them has been developed by Paul Turner and colleagues and I've given you three references here in the bottom left in case you're interested in following. One of the things that's really there is that a virus and I'll get back to this at the end of my talk, a virus and an antibiotic can be used together. There's a particular class of efflux punch in which essentially bacteria pump out antibiotics so that they are not affected by them or continue to to divide and and proliferate. So phage can absorb to these efflux pumps and uses as a means to gain access and entry to the cell. So if you apply both at the same time you can see how this could provide an evolutionary trap. If on the one hand the bacteria, some bacteria were to mutate and block this efflux pump so phage can't get in but then the antibiotic can't get out. So don't mutate that particular efflux pump. Well then sure the antibiotics can get out but the phage can get in and this is precisely the rationale by using antibiotics and these particular efflux pump targeting phage together that Paul Turner Benchand and others have managed to push this forward. Another idea I'd like to talk about with respect to synergy again an ecosystem or surgery is our work collaboratively with Laurent de Barbue and colleagues to think about phage not as the only actor in the system, but rather as part of an ecosystem in which there critically are immune cells. So I'd like to explain today how phage may not necessarily be the sole sterilizing agent as it were a veterinary system, but rather are working synergistically with the immune system where we can think of them as working synergistically with the immune system to eliminate bacteria. And if we think about that that transforms the scope in which bacteria phage therapy may be more effective and also gives us some indication in which it may not be effective. So what I'll try to do today is really span this scale from models to mice and talk about roots towards a modern immunophage therapy. And I will do this in three parts again with the interest of trying to be have a self contained lecture and I know for those of you who were here at the prior to the first part each time I make this first part a little shorter but I will just keep this in mind as the motivation for two principles underlying immunophage synergy going beyond just phage bacteria interactions but including immune components and then talk about work on curative treatments of otherwise fatal respiratory diseases using bacteriophage and immunomodulated mice as well as return to some of the issues involved in antibiotic phage synergy. Okay, so in this first part, as I've described earlier in the week, the paradigm for these lidic phage bacteria interactions originated in many ways with Campbell 1961 but also Bruce Levin in the late 70s. The idea that there's a prey the bacteria in the predator, the virus, and this means that we can think about the dynamics arising in terms of some predator prey cycles in the absence of that temperate mode which I talked about on Tuesday. In these models to remind folks we have some carbon source of nutrient some bacteria some virus, there's going to be new resource coming in resources going out. These recent will be taken up leading to division of the cells infection and lysis, and you see these oscillations and again the key point here for phage therapy is if we want to get rid of the bacteria. It's important to keep in mind that in these kinds of models we don't necessarily expect although there could be large excursions we don't necessarily expect generically that the virus that can kill one cell will eliminate the population but rather will drop down the population density but coexist with it. And that's the point of the slide that there will be the the cycles but it doesn't lead to the joint extinction it leads to coexist and I'll be at a lower density. We have to consider the fact that there's more microscopic mechanisms, and there can be delays between adsorption and lysis but even in those cases, again, we don't find that the addition of phage and the absence of other factors. And that need lead to the elimination of the host but in contrast could lead to coexistence and oscillations. So we have this virus that we'd like to add in a phage therapeutic sense, but from an ecological perspective thinking about these as prairie dynamics if we've eliminated this temperate phage route. We expect there to be coexistence and likely to be oscillations. It's also shown these oscillations can be problematic. Not only because it means the host hasn't been eliminated, but also because evolution can happen and resistant host can emerge. And so this initial phage that you might have wanted to use as a therapeutic, sure it'll be effective on some, but maybe not all of the host that one is targeting. And I bring this up today as part of this talk is because if we think about trying to use phage as a therapeutic, it does provide a counterpoint to some of the standard assumptions of phage therapy. Yes, viruses can kill individual cells. But that doesn't mean they eliminate entire host population in fact we should expect they may coexist with host populations and may even leave the evolution of resistance to the loss of top down controls instead of controlling at some low densities. We may be back exactly where we started where now we have a phage resistant bacteria and we have lost that the efficacy of this candidate therapeutic. And you can read more about this as I said in my book for some of the context that goes into why these principles hold. The field is aware to some extent of these issues and there's been a approach to respond to them. The approach or cocktails, not the kind of things you might have at 6pm in Milan, in normal times, when you're having a spritz or whatever it is one has in in the aperitivo, I believe it's called, which would be a delight to have and share. There are two kinds of cocktails. These cocktails involve a combination of bacteria phage, each with a different sort of feature and you can see a schematic here on the upper left. The idea might be that if there are target bacteria, maybe some of them get stopped on the outside. Some of them get stopped on the inside, but there may be enough coverage here that the bacteria are going to be laced by this collection of diverse phage. If you go to Georgia, the other Georgia in the former Soviet Republic, these are actually available as OTC phage over the counter phage you can buy them at a pharmacy. There's been some question marks vis-a-vis what is exactly in some of these OTC phages are just phage or maybe also antibiotics and there's been effort certainly to engineer phage to have different characteristics, for example, different targeting receptors and even different contents, in other words gene content so that they can deliver different, whether it's toxins or other other features that may lead to the lysis of target cells. This is good news in some respect, but we have to keep in mind that some of the concerns of standard phage therapy still remain even with cocktails. Yes, cocktails may kill more but not all and there may be trade-offs with coverage because you're making a choice about which particular phage to use and if you have in some sense a constraint of the total density of phage that you're able to combine. We see natural systems that are diverse, that are coexisting with host populations and it's true theoretically as I described in my Monday lecture that there can be coexistence, albeit just amongst more diverse communities. So you may be using this complex cocktail, we just may have a situation where now there's complex coexistence rather than a single oscillatory dynamic. And yet there still can be this problem of evolution, now it may be slowed, but there's now in some sense just a more complex Luria Delbrook experiment where there can still be that kind of escape. Okay, so that in some sense points us in the direction of new principles and I'll begin to explain those in a moment. Are there any questions at this stage? I don't imagine there'll be so many given I'm covering things but with a particular purpose in mind. So there is no question in the chat of Zoom or in the YouTube chat but if anyone wants to raise hand and ask a question I think it's a good moment to do that. I imagine there'll be more questions after part two, once we've said that. Okay, so let me just keep going here. So, this kind of gives us a direction to be somewhat skeptical about bacteriophage therapy but I'll try to explain why other things are happening that may give us more confidence. So let me give you a contrast everything I just said basically implies like why is this working? Why should this even work yet? So, certainly with a mirroring model within mice and obviously there are these compassionate use cases that seem quite promising. There have been controlled studies, and this is one by Laurent de Barbieu from about a decade ago, in which mice are infected with an acute respiratory model of Pseudomonas originosa. As you can see here, notably, these have a fluorescent mode so they should actually visualize the spread of Pseudomonas originosa in the control versus in the phage treated mice and you can see the difference here in terms of intensity. And on the right, you can see the outcomes in terms of survival between what happens when you don't use phage, one to 10 ratio, and here as many phages there are bacteria, or 10 times as many. And there's a contrast here between no mice surviving and all of the mice surviving. So clearly this is working at some level this actually works despite everything I just told you. So I think this should raise questions and raise questions about principles by which what may hold for a phage bacteria system the absence of other players may fundamentally be altered in the context of an immune system. In fact, Bruce Levin Jim bull proposed an early model to try to explain some of the differences and their ideas are shown here where you have bacteria which can be infected and laced by phage linked to more phage. These bacteria may stimulate an immune response and the stimulator immune response may therefore inhibit the growth of bacteria through immune killing. And now you're comfortable with these kinds of equations here we have bacteria viruses, the immune response. There's an implicit resource model here which I've left out. So there's cell division infection and immune killing. Note the virus particles are leaving from the outside but then being regenerated cow later, right. When a burst beta are produced, and there's also viral decay, and we see here we also have immune stimulation so there's more bacteria leads and more immune response, and this immune response directly leads to kill. So this was the model they proposed, and they made these claims that when there were was an active immune response, then there was a market difference from the case in which there was not and I'll try to explain the difference here. Here we have in the absence of phage, a case in which you have susceptible and resistant populations. And here we have only the immune response. And what you can see is that here there's a threshold they imagine that would be a critical bound leading to the mortality of this particular organism. And their claim is when you add phage the system goes up, but it never crosses this critical bound. And the phage are in some sense responsible for the elimination of these sensitive bacteria. Although this is a good idea, and it is very promising there's also somewhat of a problem here first of all you'll notice that in this case, without phage, the bacteria were eliminated after about 12 hours but when you add the phage it actually seems to take longer to eliminate the bacteria. And the other issue is that if I were to move this threshold just a little bit. Then in fact, even in this case we might still have a crossing that generically, you don't need phage to eliminate the bacteria in this first model so why do we even need this in the first place this model seems to imply that the immune system always works, which clearly is not the case. We tried to modify this level model and extend it in two key ways. First of all, by implying that the immune stimulation has a capacity can't just keep growing without bounds first of all that can itself cause damage, but also there's a limit to the extent to which the immune response can be stimulated. The other part is that bacteria can initiate density dependent defenses, whether the quorum sensing or biofilms or virulence factors that can evade the immune response. So even a stimulated immune system may not be able to eliminate bacteria. Once the bacterial density gets high enough so you see this term new term in the denominator so these two red terms here. These negative feedback loops this first one is carrying capacity, and here's a density dependent response. What happens in this model. So it's the same model more or less as 11 bone model except with these two additional biological features. First of all, if we were to get rid of the immune system just ask what happens when we have bacteria phage together we see coexistence as we expect these kind of predator prey models. And if we eliminate the phage that unlike the lemon and bull model which bacteria are eliminated here in fact bacteria increase and get to a point where they reach a steady state, the immune response is on but can't eliminate the bacteria know there's there's an infection. And when all are combined what you can see is that we have these dynamics between phage and bacteria the immune response is turning on, and eventually actually the bacteria does get eliminated only when both are together so neither alone can do it, but together they can. What we also see here notably is that the back phage disappeared some way our local extent before that of the bacteria so you can even understand here that in the end it is the immune system in this model that fundamentally eliminates the bacteria, rather than the phase and what the bacteria are doing in some sense are dropping the densities of bacteria to a level that can be controlled by an immune response. So this is what we call immunophage synergy, it's the elimination of bacteria through this tripartite dynamics. And just to point out here that this doesn't always work. Notably it can work in a larger regime that we initially expected here results for the final state of bacterial density and phage densities as a function of the decay rate meaning fast decay of viruses inside the host and slow rates and the absorption of phage to bacteria so on this upper left we have long lasting phage that are very good at absorbing and lysing cells and down here on the lower right. These are rapidly decaying phage that don't do a good job and as you can see that in this bottom section, we expect and find both theoretically and then the simulations that we have bacteria and the phage can't get a foothold and are eliminated. When you see both colors that means there's a coexistence regime and initially we expected this only to work in this upper left hand wedge but we actually found a larger regime where the bacteria were eliminated and therefore also the phage but that's okay. And this is through a dynamic mechanism that these oscillations in abundance can actually lead to opportunities in the troughs where the immune system can eliminate the bacteria and then the phage as well. So we have a large regime and parameter space where we expect that immunophage synergy is possible. And just to give a synopsis here. In other words, there's a bacterial introduction which proliferates the immune system responds but is unable to clear it. So immunophage whether because it breaks up biofilms or just directly reduces densities means there's a decrease in density and with a decrease in density the immune system can overcome the bacteria also leading to elimination of phage. Okay. So those are the key ideas that make this different that really thinks of bacteriophage therapy as part of an ecosystem and thinking about feedbacks in that system. So maybe now there may be questions before I move on to the later parts. One question from the chat from Sylvia, who asks, with respects to antibiotic therapy, does phage therapy have the potential to be more bacteria specific and cause less side effects on the good microbial community? That's one of the key benefits of using phage. It also is the one of the key negative parts of using phage because of the specific nature of phage rather than broad spectrum antibiotics, it means that you have to make sure that the phage can target the specific bacteria that one has with another reason why people use cocktails, but it should not have the same side effects as targeting other cells. So it is both good and bad news with respect to use. Great. Any other question? Yes, there is one question from Ankit, please. Hi. So I was just wondering, like, since you're talking about coexistence and elimination, like, do you also sometimes consider systems where you might have a very large number of bacteria species and, yeah, and it's maybe different, yeah, pathogen. Let me try to answer that in two ways. First of all, we are looking at acute infections and part of the reason my group has been focusing on acute infections is I think, frankly, the challenge of using bacteriophage to eliminate a complex multi-species community is going to be harder. So in terms of these steering complex networks, nonetheless, in this next part, I will address what happens when there are resistant mutants and how do you deal with the fact that this is not just one bacteriotype, but actually there can be susceptible and resistant types. So in that sense, I will address it, but in the broader sense, that remains a big challenge of how does one in some sense use phage either to steer or influence or control complex bacteria communities, and that would be relevant, for example, to gut communities, or even some of these surface communities. So we're focusing on these acute infections purposefully. And I would say that even in the treatment, for example, of bacteriophage, I'll note though, Paul Turner is working in CF and treating CF patients, and then absolutely there are complex communities, but there tends to be a disproportionate impact of Pseudomonas originosa, so there can still be significant benefits. So, you know, those compassion use cases are in situations where the community is more complex, even if they're targeting a subset. Thanks. Great. Any other questions? I'll keep going. Yeah. Oh, there is one. With the concern that the elimination of bacteria. With the concern that the elimination of bacteria will be more effective with the synergistic involvement of phage and of the immune system, how will the bacteria elimination work in immunodeficient immunocompromised? That's a great question, because if you look at the title of the slide, that's exactly what I'm going to talk about. So that person, whoever that was, we can let's get to it. Okay. So this third part we're going to address precisely the question that this individual raised was just what happens in immunodeficient or immunomodulated systems. So let's go and try to deal with that. So this goes back now a few years. Everyone here recognizes Liverpool. If you've ever been to Liverpool, you'll notice that they've modified the central church to have this giant phage capsid on top. You might not have noticed that this was just for the display. And this theme here is really I get by with a little help from my friends, because this became not just a theory project, we're really a collaborative theory project spanning both the US and France. One of the first presenters on this session was Dwayne Roach, formerly a pastor at the time but now recently professor, assistant professor at San Diego State University. And they were doing in parallel, unbeknownst to us, an experimental study of phage therapy efficacy and immunomodulated mice precisely the point we've just raised in other words taking a multi drug resistant to Pneumonous originosa which is that same system that I showed you from Laurent in 2010 which causes acute pneumonia, which is fatal in mice, and treating with pack P one, which was shown as I showed in that 2010 study to prevent fatal acute pneumonia when given at least that one to one or 10 to one levels in vivo. And again, I've shown you this before here we have these readouts hours past post treatment of the control versus the phage therapy treated mice showing the effectiveness of phage therapy. Here we have complete difference in terms of survivorship note survivorship in the control group 100% in the phage group here we have radiance measurements, which get to be at levels of detection. After one, two or three days and remain there, whereas the saline treated mice then via the compassionate treatment then are not extended because they show distress and all die after 24 hours. However, using the same phage in a different mouse model and here I'm only denoting this word anti gr one which I will get back to later. What you can see is that we have the original treatment, and then we have a treatment and the fate treatment in this modulated mouse, and in all cases, even though one is using this phage that was effective in the immunocompetent mice, it is no longer viable in this modified system here with radiance that look no different post infection again the two hours notes the fact that phage are added two hours after infection just to contrast these are days and this is hours post infection. And although this is not good news obviously for these particular mice, this is good news with respect to the idea and advancing principles of trying to be more effective of how and when to use bacteria phage. And the challenge of course, here is that we have to then bridge the gap between these in vitro models and in vivo outcomes so we initiated a collaboration with Dwayne with Laurent and their team. And we had these discussions, and we figured well you know we are models are basically predicting this we I had a poster without any experimental data at the time. They had experiments without any theory we got together and chatted. And we began to adapt it to the particular absorption rates and birth sizes and other details of the system. And this is what happened at first. Here is in our model, two hours after the sense of bacteria increasing. And the reason for this is that the post immunity is responding we add phage. And if you can see there's like a cliff, and clearly something has gone wrong, it's a miracle cure. So in our models initially it looked like a miracle cure, even with phage and bacteria didn't even need the immune system and the reason is that at the absorption rates that they expected and the birth size and lane periods and so on. But this many bacteria would immediately lead to clearance and clearly that's not the case. So there was a gap here in terms of time scales. And the models that we developed in a well mixed chemostat that is not exactly how things work in the lungs of a mouse, that if a bacteria is killed on one lobe by a phage that doesn't mean the phage immediately gains access to bacteria in the other load. And so of course is that we have to think more carefully about the immune side can we diagnose the basis of this, which are the effector cells that might be involved in this synergistic clearance. So the first thing that we revisited was this notion of a linear attack rate. So today is inspired by work in epidemiology where it's well known that heterogeneous mixing can lead to nonlinear interaction rates. We began to think about modifying this in some sense force of infection this absorption rate not based on linear contact rates, but nonlinear interaction rates due to heterogeneous mixing. And also another problem here is has to a phage saturation that locally there may be elevated levels of phage, but if they are infecting the same bacteria they don't get to kill it more than once. And in a linear model in some sense they do. And so here we also took into account another wage in which there may be a nonlinear response between phage density and absorption and killing due to phage saturation. So phage m and PS for these different functional forms. We then took this model which we have phage and bacteria and an immune system, but instead of having this linear term here, we use this heterogeneous mixing model or a fade saturation model and found instead of having time scales of basically instantaneous elimination, it was a day using the same parameter related parameter sets for bacteria to be cleared here you can see it in these two models. We also did the same thing with resistance and here I think there becomes another insight, which is throughout this I have phage only targeting these sensitive vector phage sensitive vector. On the other hand, there can be resistant bacteria and the question is, how does phage, how do phage and affect ourselves together lead to the elimination, when the phage cannot eliminate these infect and lyse these resistance cells. And the same as you can see is elimination of both, and the rationales as follows, which is that there can be the proliferation of resistant bacteria, but because phage are targeting eliminating sensitive bacteria that the stimulated immune response, can then in the absence of these large numbers of sensitive bacteria can eliminate the much smaller numbers of resistant bacteria and someone drew a red line on my slide which is really cool. I don't know how that happened. You can see then that this then is controlled, not by the phase but rather by immunity. And so this also gives an insight as why this is very different than the version I explained before that by making these models explicit you can see that there can be despite the mutations, as long as there is this notion of an ecosystem and immune cells a chance for phage therapy to work, even if we don't target every particular resistant mutant there's still a chance that this can. However, we don't quite know which of the immune effector cells are going to be responsible for this particular synergy. And one of the ways in which they probe this was to take different types of immuno modulated mice and use the same system. And one of these is called my 88 minus which is immunity activation deficient mouse. In other words the signaling is deficient so you can see here an example of survival and radiance. Here we have a wild type saline case, but here are these immuno deficient mice in which when you add phage, the survivorship is no longer 100% but drops down. And what you can see our radiance is that they seem to be the same. The phage looks like it may be improving things but then there's a reversal. And that reversal in our models we also expected to happen because the phage are eliminating the sense of the bacteria, but resistant bacteria are increasing. And because the immune spawn system is present, but it's not responding correctly, then there's the uncontrolled proliferation of resistant bacteria. And in fact that's precisely what they found when they actually looked and isolated these bacteria at the end they were all resistant to the phage. And that's in the model as in the system, it's the proliferation of resistant bacteria in the absence of immune control that leads to the failure of therapy, which then points to innate effect ourselves. And I'll just point out that one of the interesting stories that they explored was examining the potential for phage therapy to work in the absence of innate and adaptive limits of sites and turns out they could. So the synergy is not with innate lymphoid B cells or T cells and obviously the adaptive part may be less surprising but the fact that it wasn't with the innate lymphoid cells is promising and also suggested that the synergy was likely to be with neutrophils. And that is in fact what I showed you in that first system when you have these neutral panic or depleted neutrophil depleted mice, then yes in fact the addition of phage does not lead to survival and you see the uncontrolled proliferation of bacteria. So, again, as I said pointing to the synergy between neutrophils and phage as being an essential alliance required for effective therapy. And I'll make one more point here before wrapping up the section, which is that you can also use this as a prophylactic in the sense that in a few experiments they added phages, four days in advance and then added the bacteria and still it was effective at preventing infection fatalities of the mice 100% of these pre treated mice survived and none of those treated with saline and we also expected this to happen. The reason why we examine this case is because in certain circumstances there was concern that the turnover or decay of phage would be so fast. Blood treatments that shown to be very fast but they had done the work just looking at the decay which seemed to be on the order of nine to 10 hours which meant that a large dose given four days before there was still going to be enough around in terms of the decay of phage for this to be effective as it was. And the other interesting point here is that it doesn't seem to have at least the addition of these phage to lead to differential production of cytokines in other words there wasn't an immune response that might directly target the phage on its own. Which would also be problematic, and that would maybe limit the efficiency of phage therapy at the moment as you can see, I'm thinking about phage and bacteria phage and immune system synergizing with respect to elimination of bacteria and adverse impacts on phage by the immune system could obviously limit that but there's no evidence of significant priming of host immunity. Maybe that's a good place to stop as well just in case I have a few last slides I think I'm mostly on time. Yes, so there are a couple of questions so one is from the Zoom chat from Lehan who is asking the following so biologists have been using vectors to do genetic manipulations. Is this one of the ways to design phages to target specific bacteria? Right, so there's all sorts of different ways if you want to modify bacteria crisper system phage delivery systems and so on. In this particular case we're really thinking about phage not as a means to modify as a way to deliver something but rather to kill and proliferate. So this nothing I'm saying today would prevent interesting work in those directions but really thinking of phage as agents of mortality here. Of course you could also think of using a phage derived product like a license as an agent of mortality and people are using license also as whether pre treatments or therapeutics, but I'm not investigating a particular using phage as delivery vectors in this step. That would be interesting but it's not the scope of this particular work. Yes, I think he has a very related question, which is to get the new sets in action, can one use phages to make bacteria express some peptide that are already known as pathogens to a new sense. So just say that we're exploring some of those things now I don't have any results I now understand when the person's going at that has been of interest to us to do something more than just killing in light of this synergy. And we don't have any results to share yet but yes that is certainly of interest to us. So if there is a question from the YouTube chat chat so Julia is asking, would it be possible to apply ideas from control theory, optimal control stochastic control to the system, such as in the immunophage synergy model. I have a paper out this year in the bulletin of mathematical biology which does that I didn't include that because I had, I was going to run out of time and I included a different study that came out in M systems but if that individual is interested. You can look at one Lin Lee at all we've collaborated with your I warty who's a control theorist here Georgia Tech to do precisely that and I'll just make a comment that the challenge always an optimal control is that you have a sense of timing. But we all can worry about misbefuscation of the model. And so in that paper what we did is try to take the lessons from our optimal control results and use them to guide something that could be done in practice, which is the delivery at the outset of what we think is the right dose and combination. And we're also looking to try to convert that more into a feedback control case by taking data not just at the outset and projecting forward in time but also taking new measurements and responding appropriately. We're actively working on that as part of an NIH grant that funds a study, and you can see our first work in that direction again in Lee at all with myself and your I warty, and join them in the bulletin mathematical biology 2000. If there is not any other question I think you can go to the next. Oh, okay, there is one question in the zoom chat by Sria Rama is competition and or cooperation play any role in controllability. I assume that person is referring to maybe competition cooperation between bacteria. Maybe if so, I certainly would think that this goes back to my point about using page to treat multi component infections right multi species infections and then absolutely we need to be thinking about those kinds of interactions here with in a species. If we have systems, particularly cases like CF, or that have a longer time to develop biofilms, then if I think of that as a cooperation mechanism within bacteria of the same species, then also yes we have to think about ways to address issues of emerging properties of populations that may then lead to recalcitrance or making it different, more difficult to treat with phage, if phage can penetrate those biofilms or somehow overcome those collective defenses. I only have a few minutes left so maybe I'll go to this fourth part is that okay. I can now go in one direction and again, I didn't add the optimal control work but wanted to get back to this example from Paul Turner, and just elaborate a little bit more on it, which is, again to recall that there are. There's an original so that use these antibiotic efflux pumps. So in other words, if an antibiotic is present, they can be pumped out, increasing survival but yet this phage on K one uses these efflux pumps in some sense isn't as a surface receptor to inject genetic material into the cell. Of course, there could be a mutation, which means that some pseudomonas may have lost this efflux pump or at least do not have the surface receptor so that now they can no longer pump out antibiotics but of course, phage can't get in. So in these two examples, and there may be a continuum of expression here, there may be at least on the archetype side, antibiotic sensitive types which are also phage resistant and antibiotic resistant types which are phage sensitive. And this is precisely the rationale behind the dual use of antibiotics and phage together. And this is what it was reported in really a pioneering study by Benjamin Chan at all with Paul Turner as the senior author leading the study and describing it here. There's a caveat though that concerned us that there may be issues about targeting the wrong strain. So for example, if there is a fence set phage sensitive an oculom. Then phage may work, whereas if the initial community is largely phage resistant, right by antibiotic here in this particular case I haven't had antibiotics, then even all the things I said before about immunophage synergy may no longer work because if it's the bulk of resistant bacteria, they may rise to such high densities that we don't get the benefits of them being driven down. So this is just to point out that once we have sensitive and resistant cells, then targeting really matters and we think this also matters with respect to the this antibiotic phage joint treatment. So what we did is take our original model, and instead of just having phage and immune cells and a sensitive and resistant type, we also are going to have including antibiotic and I'll label these two with A and P meaning a is sensitive to antibiotics and P is sensitive to phage. And so now we have a specific kind of mutation which just doesn't make it resistant, but now becomes sensitive to the use of antibiotic. And we wanted to explore what might happen in these kinds of models. One of the things that we realized initially is that a combination therapy might restore efficacy to mis-targeted phage therapy. So if the inoculum was initially phage sensitive, and one adds phage and that's going to work. But if it's an antibiotic sensitive inoculum and use phage, then of course you're just going to get the emergence of these BA types. And therefore, the combination therapy here in both cases there's a background antibiotic applied can lead to elimination in both. So our first point here is that we think that in this class of systems, clearly this phage antibiotic combination is going to be better, irrespective of what the initial breakdown of that population is. Whether it tends to be more on the sensitive to bacterias, phage side, or to antibiotics. The other thing that I think is important is it the inclusion of immune responses we think again is an implicit and hidden part of what makes this effective. So we went and explored the outcomes of combination therapy with immunity, and you can see in both cases we get an elimination. But in the absence of an immune response, we end up getting again this persistence of phage and bacteria with some background lower levels of the antibiotic sensitive bacteria, they're being reduced by the use of antibiotics. But the phage and antibiotics, even though they're working synergistically, can't clear things because phage that intrinsic relation between phage and bacteria means that we end up getting generically coexistence rather than elimination. So we tried to put this all together, envisioning that we have variation of the inoculum type here, all antibiotic sensitive, all phage sensitive, the level of antibiotic and here is the MIC concentration, the minimum inhibitory concentration. And what I'm showing you here are bacterial densities, only with antibiotics which you end up getting is except in this very corner case, essentially the elimination of the antibiotic sensitive type, but the persistence of bacteria that are sensitive to phage. When you add immune responses so if there's an active immune response there's some regime in which there's clearance but there's still a large regime in which there's not. When antibiotics and phage are added together but not having the immune system, you can see that in all these cases we don't expect elimination despite the synergy. Although densities are driven lower, there still are persistent levels and when you use a lot of antibiotics, then instead of having the antibiotic sensitive bacteria you get largely phage sensitive bacteria when you don't use much antibiotics below the MIC, you get antibiotic sensitive bacteria. So in other words, you're selecting for the type but not eliminating them. But notably when all of these players come together we expect a large regime including in the sub inhibitory concentration levels where there's robust elimination of the bacteria. And only when you basically don't use much antibiotics at all, you end up selecting for these antibiotic sensitive types. So the point here is that the combination of antibiotics and phage is good and it's particularly robust when there is an active immune system. So the conclusions here I hope I've explained this tripartite model of phage immune bacteria dynamics, it really is an ecosystem aware approach. And thinking about phage as part of an ecosystem may also redirect our attention to how to think about the development of therapeutics that in vivo now shows that the curative success is not just dependent on the phage, but also on the immune response that a fade to a neutrophil alliance in terms of is maybe necessary for therapy that was revealed with his immunomodulated mice studies, and also synergy can help resolve the resistance problems, because the immune response can eliminate both susceptible resistant pathogen. And we're working now on generalized symmetry to include commensals I didn't talk about the today, as well as antibiotics, and also keeping in mind that once we are aware of these kinds of feedbacks it may be that trying to get candidate phage, we should be thinking about ways in which phage can make it easier for the immune response to work rather than thinking of the efficacy of killing on its own. And with that, again just want to thank collaborators here pointing out I talked about this M systems paper here at the end but also there is that other bolder math bio paper, which focus on control theory, and additional thanks to our experimental researchers at Pasteur as well as to the Turner Group at Yale for their collaboration on this theory analysis of their fascinating antibiotic and phage evolutionary trade off mechanism. And with that, I'll be happy to take a few last questions. Okay, thanks a lot, Joshua for this very nice lecture so that we have time for questions. I don't see any question in the chat, either zoom or if you have any please either write it or raise your hand. Yes, there is a question by Martina, please. It is always nice to hear you to hear your lectures. I, it's a bit of a bit of a leap. I think, because there is all this idea that let's say the right quantity of phages might promote bacterial diversity, and in this sense, we might can. I mean, if, you know, probably far away future. Do you think we could use back to phages to as probiotics. Phages is probiotics. Yeah. Okay, and instead of bacteria is probably for using, say, phage in some sense, I think, I guess it depends on how we're going to think about the term probiotics. Meaning do you want them to be residents, because maybe if we add phage that are hanging out and eliminating the wrong bacteria that could be good, but maybe you mean something else like you'd actually like them to become residents, which Yeah, like a for the gut microbiome in the sense that you need a diverse community. Yeah. So, so there's an interesting paper a few years ago, and it's a shame that I'm dropping the name of the first author but the last author is Mark Young was published in PNAS on a healthy gut viral, which looked at the relationship between in some sense the diversity of phage and outcomes with respect to health, finding that they were related. And I think there are many questions left to be answered about mechanism is that essentially a reflection of healthy gut microbiome or we see phage of those microbes, or is it actually the inclusion of phase that is leading directly to health benefits. I don't think we know the answer to that. I think the fact that you're raising it in some talks I've made a schematic I wish I had it here of a yogurt with extra phage, because you know that when people will go to the store, and pay lots more money for bacteria and other back, let's say called a bacteria infused probiotic yogurt, right. But right now the market for. If you told people, I'm put extra viruses in your yogurt, I don't think I'd have a good market share. Maybe that will change with time. I don't have that slide here today where I've created my fake campaign, but you're raising an important point. And that's in fact what we'll be doing but I think we still have a ways to go. Thank you. Great. Any other questions. Hi, I have a question. Yes, please. Sorry, I'm going to start the video. Hi. Thank you very much for the nice lecture. The question was about always diversity. I think in part you replied before, like, of course you were talking about like an acute face of the illness, so not a big specific diversity of the of the macro organism to be killed by phages or by the immune system. But what about like the, the, the introspecific diversity that might be caused by the, like the evolution of the micro organism of its own different and differential like sorry, defense against phages, like in the model than. Yeah, so in some of my earliest work in this space and thank you for the question. One of the first problems I worked on also with Simon Levin is co evolutionary dynamics arising from precisely the mechanism that you describe in which in some sense, catalyzed diversity, which then leads to catalysts of more phage and so on. And we've worked in other cases showing that you can have co evolutionary dynamics and diversification stimulated by these interactions. In the context of therapy. That's I mean that's fascinating from a fundamental sense but they may not be the right outcome you want from a therapeutic sense. We see here already, even if there was a pre existing phase resistant mutant. Right. In the event that there was this immune response that got eliminated so in some sense selection operated because of your addition to phage and led to a proliferation but it could be controlled. In other sense, there's also something that we've been working on and I'll see if I can, while speaking find it for you but there's a new paper on bio archive by Justin Myers group, talking about co evolutionary phage training. And I will. It's something that we've been thinking about from a theoretical perspective. I don't know if I can write to everyone in the chat. Maybe once maybe I'll send it to jacobo he can write to everyone in the chat with AC the idea is that it may be possible to look forward in time. Finding what kinds of phage might arise because of the emergence of new mutants, but then go back and apply those at the beginning to stop the emergence in a therapeutic sense. We always have to keep in mind I mean these co evolutionary systems are so cool and fascinating we want to understand them but in a therapeutic sense they may not be our friend, per se. So we have to think about maybe ways to leverage and that's one that we've been very interested in exploring. Great. Yes, this is a question that is there is a question in the chat is related to a question that was already asked, and Edgar is wondering whether we can engineer phages so that it to facilitate a better recognition of the bacteria by the new system. The answer to that is we've been working on that for quite some time and in principle, probably yes in practice which we're working on, maybe harder and maybe others are also working in this space but that's clearly one that is, as you can see implied by my work here, where there are maybe reasons to think about phage not just as agents of mortality but also as synergistic elements, potentially even adjuvants and in that case it does reshape the way that we might want to engineer phage, as was pointed out, in order to have better ecosystem effects, not just killing or lytic activity in the absence of that ecosystem. These are all and I would just say since we're almost at time. Just to thank you for everyone in the audience for these questions. Obviously, if people want to follow up, I'd be delighted. And also to point out that this field I've tried to introduce topics in which the story isn't totally written yet that story needs to be written by all of us so I hope that maybe some of you might be interested in what's happening here and work in your own universities to try to advance this ecosystem, the eco-evolutionary approaches to understanding viruses and their microbial host so thank you. Thank you very much, Joshua, for these very great lectures and it was really great, thank you very much for the involvement in this and thanks again to everyone for attending the school. We'll meet each other again tomorrow at the usual time, I guess. Yes, no, a little bit later than usually at 2.30 Italian time with the first lecture by Andrea right now. So thank you again, Joshua and thank you everybody for being here, thanks. Thank you, Joshua. Thanks all thanks again. Take care. Thank you, Sarah.