Scientist Stories: Nathalie Garçon, The Future of Adjuvants are mRNA Vaccines

So I think we'll get started. It's just after 10 o'clock. Hello, and thank you to everyone around the world for joining us for this week's lab meeting. My name is Kristin Aboud. I am the science editor at the Human Vaccines Project, and I will be your moderator today. The latest Omicron subvariants, BA.4 and BA.5, are now responsible for the majority of new COVID-19 infections globally. These highly infectious subvariants are able to sidestep antibody responses induced by either vaccination or prior Omicron infection, and in some places have led to an increase in hospitalizations, though there isn't yet any definitive, definitive evidence showing that they cause more serious disease. BA.5 is particularly worrisome to some experts who have classified it as the worst COVID variant yet. Given its ability to evade prior immunity. As SARS-CoV-2 continues to evolve and spread globally, new and improved COVID-19 vaccines are a top priority. Today we will hear about how adjuvants may play a role in future vaccines for emerging or existing infectious diseases. With that, it is my great pleasure to now introduce today's speaker, Dr. Natalie Garçon. Dr. Garçon is a biological pharmacist by training, earning two PhDs, one in pharmaceutical science and the other in immunotoxicology and immunopharmacology. After working as a postdoctoral research fellow and a research assistant at Baylor College of Medicine, Dr. Garçon set up and led the vaccine adjuvant and formulation group at GSK Vaccines. Eventually becoming Vice President of the Adjuvants and Technologies Innovation Center there, where she provided leadership on new vaccine technologies. In 2014, she joined the French technology research institute Bioaster as Chief Science Officer, later becoming CEO. Her expertise in immunology and vaccine adjuvant and formulation technologies is widely recognized, and she holds more than 100 patents in these areas. During today's presentation, please send me your questions using the Q&A function in Zoom. Feel free to use the chat for general comments, but please don't post questions there. I will ask our speaker a broad selection of your questions after the presentation, and we will have about 20 minutes for discussion. With that, it is my pleasure to introduce Dr. Nathalie Garçon.

Thank you very much for the introduction. Good morning, good afternoon, maybe good evening everybody, and although I think we get used to the webinar session for presentation, it's always nice to see the people you're talking to, but I'll do my best to be understandable and not go too fast. So as Christian told you, the I have to do a maneuver, but hopefully I'm not gonna mess up.

We've already had our first year of COVID and I still can't do it.

Yes, perfect. So I'm going to spend about half an hour talking to you about adjuvants, their future, and an obvious question of will, could they, should they be part of mRNA vaccine as a standalone or as adjuvant we know today. So I think it's always good to look at the past when you want to have an idea of the future and remember how adjuvants actually occurred. And clearly at the beginning it was a question of chance. And that there was chance, through chance, that Cody used DNA as a vaccine in the context of cancer, solid tumor. He realized that injecting some bacteria into these individuals, some of them through a resection of their tumor. Then later on in the early 20th century, in 1925, Gaston Ramon, who later became one of the directors of the Pasteur Institute, realized that in order to increase the production in horses of diphtheria toxoid, adding particles to the toxoid tremendously increased the antibody response but also decreased the necrosis that you could observe at the site of injection. Not long after that, Kenny introduced the notion of aluminum salt as an adjuvant for vaccine. And finally, during that period, Freud Freund, sorry, introduced what's known as a Freund adjuvant, complete or incomplete, which is based on water in oil emulsion. So if we look there, we already have the basis of pretty much what defines the adjuvant that we see today. We have immunomodulator, Coly, which is a CpG, it's a TLR9 agonist. Gaston Ramon was LPS, or another immunomodulator. And glenial fluid that introduced what, what in the classification that Adjuvant now considers as carrier. And then when you look at the evolution of the discovery and understanding of microbiology and immunology, they pretty much have that in parallel. But clearly it is the discovery in immunology that made the vaccine evolve the fastest in the 20th and 21st century. And then we went from chance to necessity when, when HIV occurred. There was a very intense renewal of work on adjuvants, hoping that it would be possible to design a vaccine that would be efficacious. At that time, it was observed that none of the adjuvants as a single or combined entity were sufficient to induce a protective immune response, and that retriggered a lot of work done in that area. Combined with the fact that recombinant antigen emerged and was giving also the opportunity to develop vaccines in much more disease and much more pathogens, although it was observed that those recombinant antigen alone were not sufficient, so adjuvants were necessary. As you can see, there's quite a number of adjuvants that were developed, and today there's more than 11 adjuvants, I believe, that are in licensed vaccines. The last one between Matrix M for COVID, and that is in Novavax vaccine. So there are quite a few adjuvants that have been developed, and they are in very different vaccines when you look at that. So now that we've seen the past, it's also good to understand the present. And during all those years, I think we are— there's a body of data that has emerged that can pretty much qualify what should be an ideal adjuvant. Clearly, the first thing is it should not have and cannot have any bystander effect. What it means is the adjuvant needs to act on the antigen, induce an immune response against the antigen which is administered with it, but nothing else, no bystander effect. It needs to have a clear mechanism of action, which is something possible today, less so 10 or 15 years ago, but the comprehension of the mechanism of action can educate and direct which, for which disease or which antigen you can use it, but also can generate information that are of great value for the safety evaluation of the adjuvanted vaccine. It may, it should effectively activate humoral and cell immunity with no adverse reaction across population, and it's, if not, should, and if not, it should be tailored to the population that will best benefit from it. What that means is that you may have an adjuvant that is great for adults, less so for pediatrics, or not sufficient for elderly. There is a way to use the same adjuvant by modifying something to make it amenable to all populations, but by design, an adjuvant should be adapted to the population that it is going to be used in. And finally, it should be easy to produce, store, and administer.. And this should be actually at the beginning. If you cannot develop an adjuvant that will not be easy to produce or store or administer, it will never be used otherwise, and it doesn't help. So what have we learned today? We know that current adjuvants can be natural, so they are part of a plant or a microbe. Part of a plant like saponins, a microbe like CPG or NPL and their other TLR4 agonist molecules. Sorry, it can be emisynthetic, which means that it is built on a natural product and the final molecule is synthesized, or fully synthetic. And for each of those categories, that has impact on the CMC and the regulation aspect of the adjuvant. The revolution in the understanding of how innate immune system senses microbes has brought a huge opportunity for their design and their development, and even beyond, with the use of some of those molecules in areas that so far have not been successful for vaccines, such as mucosal immunity or intradermal one. So there's still a lot of potential that can be tapped on. One size will not fit all. There's a One antigen— sorry, one adjuvant will not be necessarily the best for any situation or any disease you're looking at. It has to be adapted to the antigen, the target population, and what is the immune response you need to induce. And that's very important. You cannot force an adjuvant to be the best for a vaccine if it's not adapted to the criteria. And finally, the combination of two adjuvants together can be synergistic, but this is through a different mechanism of action. And that will— I will show you that with ASO1, and that's the only one so far where you combine two known adjuvants, in that case QS21 and MPL. The final mode of action, mechanism of action of the combination is not the same than you see for MPL and QS21. So what have we seen? What are the benefits of adjuvants? Well, they can You hope that they can overcome poor immunogenicity, and that has been shown for recombinant antigen, that they can increase the duration of the immune response. That has been clearly seen with HPV vaccine, and that was in particular in the context of aluminum or aluminum MPL, that it can reduce the dose of antigen that you can use in the context of high demand, like in the context of pandemic emergency, and this is called antigen sparing, and that has been seen in the context of the 2009 flu pandemic for both, both emulsions that were used. And finally, you hope that adjuvant could be able to improve the immune response in specific and special population, and this is the case for Zoster, where the adjuvant used in a recombinant vaccine has shown a remarkable protection in the population, well above 80 years old. So they do pretty much tick the box for quite a lot of things you want for a vaccine, considering the diversity of the antigen, the diversity of the population, and the diversity of the needs. So to go a little bit further, you need, for in the context of those adjuvants, you, you need to have— sorry, I have a problem here. So the effect of combined adjuvants, I told you earlier, the effect of a combined adjuvant molecule can be superior to the sum of these parts, and that's what you can see here. In the case of ASO1, you take MPL alone, looking at specific antigen-specific T cells. The MPL alone is here, T21 alone is here, and when we when you combine both, the effect is higher than the sum of its parts. So that's what's called a synergistic effect. More importantly, the effect actually of those combined molecules is different from their single parts. MPL is the TLR4 agonist. QS21 acts through the caspase-1 pathway. And when you combine MPL and QS21, it's a different pathway through SIK activation. And this is important. There's still quite some work to do on that combination to fully understand the extent of the ability of the adjuvant to act on different types of vaccines. The adjuvant can bring pan-protection and increase the breadth of the immune response. That's what it means is that putting an antigen with an adjuvant, you can protect through variants of that pathogen. That has been seen in the context of LMPN and HPV. Two genotypes in the vaccine combined with that adjuvant can protect against more than 12 different genotypes closely related. And in the context of emulsion and influenza, clearly emulsion can protect across close variants of the genotype of influenza. And that finally, adjuvant can protect better than the original pathogen, and especially in frail population. And this is what has been seen with MPLQS21 when comparing in comparing the, the ability to protect against zoster, the attenuated viruses, which is about 10 logs PFU of the virus, then the pediatric vaccine gives a protection which is below 30% over 80 years of age. However, a recombinant antigen with that adjuvant can protect over 90% against zoster over 80 years of age, which Somehow it is doing better than nature. So I think there's still a lot of things we can learn from nature and improve upon to target a new area of interest. So basically, that's the status of what we know. And in all of that, being a pharmacist, I have to talk about that. I think one has to recognize the power of the formulation. If you look at the formulation, it is critical to know exactly what is the type of molecule you use and to understand how you can prepare them. The current constituents of adjuvants are mostly all based on knowledge of the '90s, and those are the ones that are on this slide. But the emergence of the understanding of the innate immunity, and in particular the TLR pathway, has brought new molecules that can be used for those. Purpose. If you look today in the licensed adjuvants, you have mainly as immunomodulator the MPL, which comes from a bacteria membrane, CPG, which is part of a microbial DNA, QS21, or saponins as in Matrix-M, which are— sorry, Quillase, a mix of saponins, which are extracted from the bark of a tree. Those are really what come from the '90s. However, when— and we have seen that in the context of the pandemic— natural availability of natural products may be difficult at large scale, especially when you have to secure them in a very large quantity. So there has been quite some work done to produce either any synthetic molecule, and in particular, if you look at the case of QS21, so on the left, this is a natural molecule. This is a— this one is a natural molecule, and this one is an emisynthetic molecule which allows to produce exactly the same chemical entity and can be produced at very large scale. The debate there becomes the, the ease of manufacturing and the cost of the manufacturing, but yet it is possible to have an emisynthetic molecule from that fairly complex biological molecule. There's a question also that is of interest, and especially if you look at in the context of the teloreceptor for agonists. And today there is only one TLR4 agonist which is used, which is known as MPL. Actually, MPL is not one molecule. MPL is a combination of quite a few molecules that are called congeners. A lot of work is done to have synthetic molecules that can be produced. And the fair question is to ask if a mix of molecules is, or a single molecule is as efficient as a mix of molecules in a diverse population. And that's something that still remains to be seen for those molecules, as the diversity of the molecule should be more amenable to the diversity of the Toll-like receptor agonists. The other question is, uh, the, the— it is possible with this synthetic molecule to link the antigen and the adjuvant together, and theoretically that should tremendously improve the immune response because you target one cell. Both the antigen and the adjuvant are in the presenting cell. So, and there, there are preclinical work that have shown that indeed this, this works very well, and that can reduce tremendously the amount of antigen adjuvant you give. And that remains to be seen in humans. In mice, it's clearly efficient. Finally, when you look at the adjuvant design, you really have to keep in mind the innovation versus the discovery. Another way to say that is data generation versus striving to an objective that you have set. It is very easy to develop a lot of adjuvants on the bench. However, when you need to have one that is efficient, safe, easy to produce, and be able to use in a vaccine, this is what you have to strive for. And that's really what has to be done. One example, actually, is for the ASO3 adjuvant. There was quite a few prototypes that were produced, yet the one that was selected One of the criteria was the stability of the emulsion over time in stringent conditions and the ease of production. And the reason why the vitamin E was included in that emulsion versus MF59 was because it was seen that in the '90s there was a full respiratory virus vaccine that was developed and that was based on vitamin E and was, was very efficient. That's demonstrating the ability of vitamin E to increase innate immune response, and that was the purpose of introducing it. Less is more. When you design an adjuvant, once you go for licensure, you have to demonstrate that every part is needed for the adjuvant, that there's nothing you have added there that doesn't have any added value to the purpose of, uh, to the intended purpose. And that's again, you see the same slides That's exactly what's demonstrated here. Here you need both MPL and QS to have the optimum response, and you need the liposome to stabilize the QS21 and avoid this reactogenicity, this necrotic effect. So you need to have an understanding of what's important and be able to define and justify it. And finally, again, think simple, because when you develop adjuvants, they only get more complex along the way. So the the simpler it is at the beginning, the less difficulty you're going to have later. So, oops, uh, sorry, sorry, there's a problem here, but this is important. The— for the— so when I was saying each part needs to be justified, here you see that both immunomodulators are justified, and for the immune response which is induced, you could argue that liposomes are not necessary. However, if you look at those slice of muscle that have been stained. Here, this, this one received QS21 alone, and I don't know if you see it well, but that has moved. But you see here, what you see bluish and light pink, actually this is where the QS21 alone was injected, and that induced necrosis at the site of injection. Basically, cell died, and the recovery was through fibrosis. So this is not something you want to see. Now, if you put QS21 in liposome, you have absolutely no necrosis that can be observed, yet it is still an immunomodulator. So here, that's another example that the immunomodulator is important, the formulation is critical also for the use of that molecule. So if we look at the way vaccine development is done today, it's really based on the understanding of the host and the pathogen.. And if we want to summarize those, the pathogen-related challenges are based on their nature. If you have multiple serotypes, genotypes, the antigen drift, are there— do you have moderate efficacy through nature, the duration of the protection, life complicated, life cycle or not. And on the other side, which is as important, is the population you are targeting, whether it's elderly, chronic disease patient, infant, or immunosuppressed. They all have different reasons to have an immune system which is different, whether it's fitted or it's, uh, it's reduced for because of immunosuppression, or reduced because of immunosenescence, or through chronic disease. All those will impact what is the best formulation you can use. But also, when you look at it, the formulation will be key for the adjuvant, but also the optimum adjuvant you will use. Considering the antigen, the disease targeted, and the population. So beyond all of that, what could be next? Clearly, beyond the first line of defense, although adjuvants are critical and central for the induction of innate immunity, there is an interplay with a different cell type that is involved, and the sensing of adjuvant that should be better and more delineated, understood, and capitalized upon to be able to define and use them in other circumstances. The optimal immunoallogeneic immune response, the— and that's going to be my next question— can adjuvant mRNA approach be combined so that you can have a the best of both worlds when and where you need it. And by best of both worlds, I'm talking about a strong optimal immune response and CD8 T cell response. Beyond TLR, there are numerous other patent receptors that are being evaluated as alternative immune potentiators, and they are targeting an alternative immune response, whether it's STING for Th17 and mucosal immunity, whether it's or that different type of molecule, they are opening the way for other arm of the immune response to be tapped on, alone or in combination, which open the door to different type of vaccines as well, through different routes of administration. I can't see my slide, but I think it's persistence. So one of the key questions for vaccine is how can we induce strong persistence, and what would be the key factor to do that? And also today, we do have some knowledge of what is expected, but we should build more on the knowledge we have today on adjuvants to educate us on the key factors that will induce an immune persistence and allow again to improve on the existing adjuvant or develop new ones. Persistence of the response is essential to not only the efficacy of the vaccine, but the willingness to be vaccinated. Above and beyond that, and going further than what is currently used today, you can think about metabolic adjuvants. There is a new concept of metabolic state of myeloid cells that can impact on their innate response, but also on the capacity to stimulate T cells. Those metabolic adjuvants really opened a new avenue of immune stimulation through the optimum regulation to get induced and should be considered as an alternative candidate molecule, not only in the context of combination with antigen but also as immunomodulator in therapeutic approach. This is— immunometabolism is something that is really exploding today. The microbiome, there is a recognition that the microbiome can initiate, modulate the host defense function, and it's particularly level of innate immune response, and that can open the door to new approaches to adjuvantation. Although this is certainly something that is a long path forward, there is no doubt that there is a connection between both, and that could be an additional avenue for modulation of the immune response in an individual with or without adjuvant. And finally, CDL T-cell response, and this is a big gap. Today, I think we have seen that there is no adjuvant that is capable of, with a recombinant antigen, to induce a CD8 immune response in naive individuals. And the question really is, could the combination of live or mRNA vaccine, which is able to induce CD8 immune response with proper adjuvant, could you have that arm of the immune response with a strong and persistent humoral response, which is not seen today. So that brings us to the second part, which is exactly what, what I was saying, and is that possible? Well, I think again we should do a step back and look at the evolution of the type of vaccine. The original one was the whole microbe approach, sorry, inactivated vaccine. We went to live attenuated because there is certainly an improvement of immunogenicity and hence efficacy. We went to viral vector vaccine, and the idea that you have one-size-fits-all, you just change within your vector, viral vector, the antigen you're going to use, and that's one process. There's a lot of advantage to that: one process, one master file. And then we went to, about the same time, we went to the subunit approach where why not use part of the pathogen which is the one which is protective, which means that it will require adjuvant. Two benefits to that: it was the reactogenicity was much less, and that will depend on the adjuvant, but also that allowed to produce huge amounts of vaccines and open the door to a broader mass vaccination. And finally, the genetic approach, which is the nucleic acid vaccine, whether it's DNA or mRNA. But here, clearly, the idea why, why producing, having antigen produced in in expression system and purifying and characterizing when you could use DNA or RNA to produce directly the antigen in the, the own body of the individual, of the animal that will be vaccinated. And clearly that was reducing tremendously the production of the cost and the time of production. And that's certainly what was a huge and clear added value of mRNA and what allowed for their such a fast use in the context of the pandemic, and this is certainly one of the great aspects. So the question now is, again, understanding the formulation is key. If you look at DLNP, which is a classical formulation for mRNA, they are made of different structures. You have the classical part of what's called the liposome, so an artificial vesicle, cholesterol and lipid, that do the structure. You have what is called pegylated lipid, and this is, this, those here that are present at the surface, and they do improve the stability, especially in structure where you have only a lipid layer. This is not always stable, it's like a micelle, so that does improve the, the stability of the structure. However, they do render macrophage invisible— sorry, those structures invisible to the macrophage, and which may sound counterintuitive for vaccine. And those have actually coined stealth liposome because they have been designed in particular about 20 years ago for gene therapy, where when you give them IV, they have the ability to not be seen by the immune system and be able to do several passages in the bloodstream, allowing them to reach different sites to be able to transfect and have their effect. They are positively charged, and because they help balance the negative charge of the nucleic acid, which is here, so it's good. However, and that the first thing you learn when you work on liposomes, they are very toxic, and their toxicity comes from their ability to destabilize a cell membrane. In that context, however, because you do have the PEG they do not reach the cell membrane, and in that sense, they are much less toxic than positively charged liposomes. One additional thing that has been changed is to use what's called ionizable cationic lipid. Those lipids are positively charged when they are in an acidified environment, and they're neutral in the blood, so they're their curves originally has been used as a positive thing when depending in which environment they are, they can be either positive and bind to the mRNA in that case, or be neutral and be able to be in, in the blood or interstitial fluid. Finally, the phospholipid and cholesterol are the classical component of lipid or lipid nanoparticle, and they do contribute to the particle structure its size. Cholesterol can induce fusion of the membrane, and it promotes transfection efficacy. So that's what LNPs are today. Now, there's one thing which is important when you look at the structure and any combination of a recombinant antigen, and basically this is what the mRNA produces. It does produce a recombinant piece of the antigen within the cell that it transfects. You still need to have the induction of the immune response. And actually, this is known as the three critical steps of the induction of the response in the context, in particular, of humoral immune response. When you have your LNP that delivers the mRNA, it is in, in the cytoplasm, the protein is produced and proteasomes intervene, it is degraded, picked up by the reticulum endothelial, connect to the MHC class II, which is presented at the surface in context with the peptide that comes from the endocytosis. At that point, that is not sufficient to induce an immune response. The antigen needs to be presented through the MHC class I, but it also needs, for the CD4, but it also needs another part to be effective, which which is a CD40 on the surface of the membrane as well. And those CD40, their expression increased tremendously when the APCs are stimulated by pathogen-associated molecular pattern, like TLR4, TLR5, all those TLRs. So if you do not have that signal from the pathogen-associated molecular pattern, this does not induce an effective immune response. And also, and this is the third signal, which is cytokine-induced. When you have that phenomenon that occurs, then you have a transient cytokine induction that arises that impacts on the CD4, which on that side, T cells allow for expression and increase in expression of the CD40 ligand, which is a counterpart of the CD40 on the antigen-presenting cells. And this is those two that are key to induce persistence and, and strong antibody response. So now if we go back to the mRNA, here we look at— so you have two types, the unmodified and then the modified nucleoside, which is purified. Those are the— this is what is in the licensed vaccine, so we will focus on those. So they are fully purified, the nucleoside is modified you do in that context, you, so you have production and expression of native antigen which is presented and exposure of the peptide. So you do get CD4 and CD8 response, and you should have a high antigen, you have a high antigen production, so you should have a high immune response in return. When you are in the context of unmodified, then the, you do have high production of type 1 interferon, and this is known to have a negative impact on the, the expression of the antigen. And that, by design, you will still have a CD8 immune response, but your antibody humor response will be tremendously decreased. So there's a schizophrenia here between what happens with the unmodified and purified mRNA and what you see with the modified purified mRNA, because today we have seen that actually the CD8 response of those vaccines is good, but the humoral immune response really certainly can be improved. And the question really can be, to that low response, is it because it's not purified enough, it should be modified correctly? Is it not purified enough? Is the level of antigen not sufficient? And is it not sufficient because you have a loss in translation, whether it's in the endosome, or when you do the injection, the LNP will, uh, will transfect any cells you come across. So that can be myocyte, there can be a lot of different cells, and maybe they are suboptimum. Or is it the lack of the first signal through the pathogen-associated molecular pattern? In that context, there are several ways you can, you can work, improve another immune response, and this is already done. You can increase the LNP antigen presentation targeting, and this is referred here. And that, that can, in particular, that can be structure. And there's a lot of work that has been done, especially by Bob Langer, on the architecture of the LNPs, the surface that can help for interaction with membrane. So specific targeting of receptors on the membrane that should allow for not only them coming too close to the membrane, but also by interaction with the receptor being integrated. And those, those work on are being executed, and that should not only increase the targeting of the APCs, but also the transfection and the efficiency of the current platform. And for that, indeed, you can work on the property of the substance you're going to use, really, then the architecture, the shape is critical for especially the shape that you will take when you interface with the membrane and the lipid use and the targeting moiety. This is a whole area of research and work, and that part certainly can improve LNPs not only for vaccine and but also for therapeutic. If it's the lack of signal one, With the current platform, you can enhance or target immunomodulation. You can either have co-expression of immunomodulator, and that can be done through different mRNA that are used for your, your system. And that has been done with DOTAP cholesterol, where you have here mRNA that express the type 1 interferon., and you do have MPL moiety that have been inserted in the structure. On the other hand, you're in the N-modified, then it is self-adjuvanted through different type of toll-like receptors. So you can here play on the type of immune response you can, you can induce by shifting from one type of TLR receptor to another. You can also add exogenous adjuvants. So let's say you you don't want to disrupt the LNPs that you have already done because it's stable, they protect the mRNA. So the— you need to completely abrogate the adjuvant effect of the NLP itself, and you increase the innate immune response through classical adjuvant that you mix with your formulation. That has been done in the context of a cationic nanoemulsion which is based on MA59 and a self-replicating mRNA. Sorry, RNA, and that in mice, and that did work well. So this is a structure, you have an emulsion like you would have for MA59, which at the surface are quite a few of the constituents you find on the LNP that ensure the stabilization, the recruitment, as well as the ability to protect the mRNA. You can include those pathogen-associated molecular patterns within specific effector cells, you can include those, and you will induce an immune response which is specific to what you, you look. And this is actually what exists for the CureVac vaccine, which is an unmodified mRNA which is complexed with protamine, a polycationic peptide, and that protamine acts via TLR7 signal. So here in that context, you target specifically a TLR7 response. So there are ways based on either what exists today to improve the immune response, one specific arm of the immune response, or to capitalize on existing and not classical adjuvants, or use alternative molecules for which you know what are the immunomodulatory properties and use them as an adjuvant complementary mRNA. So what is next? Well, I think current mRNA technology has clearly demonstrated its value, and especially for fast emerging response. There's no doubt about that. Their strengths, which is to induce yet immune response in naive individuals in the context of COVID actually, they seem to target to be recruited at the level of the lung where they are needed the most, which is certainly an added value. But their weakness, which is the low quantity and persistence of antibody versus recombinant adjuvant vaccine, as well as the reactogenicity that can be explored. They have to be addressed, and actually they have been highlighted because of the never-before-reached amount of vaccinated people in such a short time that has allowed to see really fast their strengths and weakness and allowed to work on them to improve that platform as well. And I think that today it is possible to combine existing adjuvant technology to new mRNA platform, and hopefully that combination will allow to reach the best of both worlds.

Thank you. Thank you so much, Dr. Garçon, for that really fascinating presentation and the history and the future all covered so eloquently. Thank you so much. I think we'll jump in. We have some specific questions we can address and maybe then some broader ones as well. But we'll start with someone who asked if you could specifically comment about the combination of alum and CpG and the efficacy as an adjuvant combo?

Yeah, so alum CpG, the first time alum CpG was combined in a vaccine, that was done by Coli, and not Coli Professor, but Coli Biotech, where they added— that was in the early, mid-'90s. They actually, they took Engerix, hepatitis B and aluminum, and they added CpG to that formulation. And interestingly, what was seen at that time, so it was mid-'90s, I think, the response was much faster. So the antibody response went very high, very fast, and was stable for quite some time. The alum alone induced a response that was shorter, was smaller,. And other adjuvants like emulsion, or emulsion essentially, were reaching the same level of antibody that CPG but didn't go as fast. So this is quite a good formulation for sure. I think what has stalled the use of CPG as an adjuvant, and that has been— it is actually in an hepatitis B vaccine licensed today by Dynavax— is the hypothetical safety risk linked to the fact that— and that's what I said earlier— those molecules act on specific receptors. The expression of cells, immune cells, in humans or across species can be different. And in the context of TLR9, there are receptors on memory B cells. So the hypothesis was that if you have somebody that has an autoimmune disease, he has memory B cells against that specific antigen, and And if CpG can reach those cells, then you will have a flare of the autoimmune disease. That has not been seen, and that has not been shown either with the Dynavax vaccine. So that has slowed down the use, but it's a great molecule. Yeah, yeah.

So we have a question about the Bharat Biotech COVID-19 vaccine, which uses a toll-like receptor, TLR-7/8, agonist IMDG absorbed to alum, Algel IMDG. And the question was, is this also an example of combining a structure that would have a combined effect?

That's exactly that. Yes. But this is a recombinant antigen. It's not an mRNA, as far as I know. Right. Yeah, that's exactly that. You have aluminum on one side and the TLR7/8.

Yes.

Okay, okay.

So then we have a question which I guess is kind of like the million-dollar question, but what is the best adjuvant to give to increase the persistence of the response, the duration?

The adjuvant that will be able to impact on the, the germinal cell in draining lymph node. So there are some that can do that. I don't think you can say that will be— again, that's not for one-size-fits-all. I think it will— I think the best of the best for one specific arm of the immune response, if it does have a reactogenicity which is not acceptable, it's not a solution. So I think one must— we should rather look at what is critical to have persistence, and that's creation and persistence of germinal cells. So the one that can do that will be certainly the champion. And for that, right now, the people are trying to have adjuvants that go directly into the draining lymph node. And that's a question of the size, the shape, the charge.

But that's what's thinking right now. Right, right. Well, it's interesting because I always— people always described adjuvants as sort of these, you know, sort of like magical elements that were added to things. But it's very clear, the mechanisms are very clearly defined and, well, to some degree.

Let's say that if you're talking about pathogen-associated molecular patterns, that's known. I mean, this is— and that's a great— that's the flip side of the coin of innovation. It's research, and all those data that are generated in research feed really innovation from the beginning to the end, either to test an idea or support the idea at the end. And the Single molecule, yes, it's known, the pathway is known. It's when you start to present them differently, to combine them, then it can change because it can— it's called pharmacokinetics and pharmacodynamics. They can go to different organs differently, and that can change the way they work. So it's—

that's the beauty of this.

Yeah.

So we have a question about whether scientists are mixing adjuvant emulsion with mRNA-LNP or absorbing mRNA on emulsion?

So the only, the only one I'm aware of is the one I showed, that the one from, uh, that is a cationic lipid based on MA59. So it's a squalene, and, uh, and there's quite a lot of different things that has been used to decorate the surface to stabilize it, allow for the mRNA to be there. And actually, it's a self-amplifying mRNA, that's is being used.

Yeah, yeah. We have one here which I think is more of a comment than a question, but it's saying that in vivo it is actually quite difficult to target specific cells, mainly APCs, and to improve vaccine efficacy and reduce mRNA vaccine local and systemic reactogenicity. So I think it's maybe just commenting on the, the ultimate goal may be more difficult than, uh, than it sounds.

So yes and no. Yes, and because then what you need is to go back to the initial purpose of mRNA, to bypass production. So I have an expression system, produce a protein, purify, etc., and have that only as a mini factory in the individual organism that brings the cargo that is then produced and processed by the individual. The difficulty there is to be able to stabilize it in a way that is not reactogenic, and being able to remove every single signal in the mRNA strand that can be an immunomodulator, TRAS7, 8, 9, and doing that without impacting the strain that will produce the antigen you're interested in. So I won't say it's impossible, but it's certainly a challenge.

Right, right. Yeah, makes sense. Can you comment about the importance of cationic liposome for the mRNA vaccine world, which was not seen or used for the adjuvanted vaccines needing more neutral liposomes, and what maybe the advantages and drawbacks of both formulations?

So the drawback of the cationic liposome is that it's very toxic. And I mean, when you start— I mean, when I started working on liposome, I won't say how many years ago, but First thing you're told is never ever use cationic liposomes. They are very reactogenic because they have a tendency to break down membranes. So it's like what you saw with QSNA1, everything leaks out and you have necrosis. Yet in the context of mRNA, you need to stabilize your mRNA, which is negatively charged, and there's not many ways. And the fact that this cationic positively charged lipids. Lipids stabilize your mRNA within your structure, but you do have that cationic lipid as well in the membrane. And that's why PEG is interesting, because PEG creates a space between the, the charge and yet outside of— you have that layer of PEG that does not allow that that LNP to come in contact with a membrane. And actually, that's the way it works. For the LNP to work, the PEG has to be shedded from the surface, and that's what happens when it's secreted and when it's in a special environment. So it's a pretty neat design. You have what you need to stabilize your mRNA, yeah, but you can't pick and choose. It goes to the the mRNA, but it's also in the membrane. But you have something else that sheds that positive charge from the cells because you have a barrier which is creating this peg. Right. I'm not sure if I'm clear, but— Yeah, yeah. It's complicated.

No, it's not complicated. No? Yeah. So we have a question about the combining of different types of COVID vaccines and whether, you know, if there are different COVID vaccines with different adjuvants associated with them. What do you think about sort of using an mRNA for a prime and a Novavax protein-based vaccine for a boost where you have different adjuvant possibilities?

I'm all for it. No, and actually there has been clinical trial that has been done, and today most of the recombinant adjuvanted vaccines are seen as boosters. I mean, I don't know you, but there's a lot of people that I talk with or talk to me. One shot of mRNA is good, two, that's really scratching, three, you really itch, and there's many people that aren't ready to get a fourth dose. And that's the first thing. And the second thing is all the data that have been generated so far show that the recombinant adjuvanted vaccine give titers that are 10 to 20 to 50 higher. So it may not be more persistent, but because you— and we don't know that yet because we don't have enough data— but because you start 100, 500 levels higher and they decrease at the same speed, you will be protected longer. So yes, prime boost in that condition is certainly a good idea. Right.

So we have a question about, you know, the growing interest in manufacturing vaccines more globally, particularly with mRNA. And what about adjuvants? What are the manufacturing requirements for adjuvants, and would it be possible to see that being manufactured in Africa, for example?

Actually, it starts to be manufactured in many different countries in Asia, and it's not that complicated. The manufacturing, the constraints are the same than for any biological product, I mean, or any drugs. You have to comply with the regulation. And, but it's, they're fairly simple to produce. I mean, it's easier to produce an adjuvant. Now, I'm not talking about the immunomodulator molecule, that can be more complex. But when you do your adjuvant, which is a fine bundle, it's not complicated. And it is more and more being produced in other countries. Especially with the lapse of patents that's starting to happen.

Yeah, so we have a question on whether the lack of mucosal or oral mRNA vaccine technology right now is a result of poor adjuvant solutions.

Well, there's no adjuvanted mucosal immunity right now with recombinant antigen, so I don't think we should blame mRNA for that. It's a different completely different domain. And there's quite a lot of work being done, and in part also with the better understanding of the immune response which is induced and the important role of Th17. And you know which cell type, so you can try and see what can stimulate them. And but where, although the Mucous immunity is certainly what you consider the holy grail because it's not an injection. But if you look at the amounts that you will need to— I mean, you have to target a little bit less in your guts to have the immune response induced. And so it's complex. There's quite— there is the nose is here. I mean, it's more— nose is much more constrained. There was a drawback on the nose at the time that there was a flu vaccine that was developing in Switzerland, and with an adjuvant which was an enterotoxin, which was very potent as inducing a mucosal immune response. There was an adverse event that occurred, and that pretty much killed the vaccine. And my experience for at least all the adjuvants that I've tested in intranasal for internalism purpose. That one, that enterotoxin, was the best to work, but it was toxic. It was creating— that was going on a nerve, olfactory nerve, and that in that case there were cases of what's called Bell's palsy, so facial paralysis. So that pretty much stopped— the year of the nose didn't end well. But now It's clear also that you, through intradermal administration, you can induce mucosal immunity. So that's an area that is being explored. It's much more easier to go to the, into the dermis, and you can use much less.

So, well, thank you so much. I think we, we can probably keep you for another hour with all the questions we still have on adjuvants, but I mean, it's such a fascinating topic, and we are very, very fortunate to have such an expert here to talk with us about it. So thank you so much for presenting today and taking the time and sharing your expertise.

And thank you also.

I just hope it was clear. Yeah, it was absolutely clear. Thank you again to all the attendees too for participating in today's webinar and for submitting such great questions for our speaker. And I will just let you know that our next Global COVID lab meeting, which will be held on Thursday, July 28th. Our speaker that day will be Dr. Judy Lieberman, Endowed Chair in Cellular and Molecular Medicine at Boston's Children's Hospital and a professor of pediatrics at Harvard Medical School. And she will present on antibody-mediated monocyte and macrophage infection, inflammasome activation, and pyroptosis in SARS-CoV-2. And if you're interested in more research on COVID-19, please sign up for the HVP COVID Report, a newsletter providing insights from experts around the globe and highlighting the latest scientific articles and data. Finally, please visit our website and follow us on LinkedIn where you can find the full webinar series. And with that, I'll say thank you again for participating today. Stay safe, and we hope to see you again on the next Global COVID Lab Meeting. Thank you so much.

Thank you, Dr. Garçon. Bye-bye. Bye-bye.