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0:16
Hello everyone and welcome to today's webinar which is entitled Influenza mRNA Vaccines: Mechanisms and Methodologies. This is the first in a series of four webinars being brought to you by Jen and the CRISPR Journal, that will cover some of the latest advances in RNA research and technology. This webinar series is being made possible through proud sponsorship from TriLink BioTechnologies.
0:47
I'll be your host for today's event i'm Jeff Buguliskis Technical Editor for Genetic EnGineering and Biotechnology News, which has been at the vanguard of the life sciences industry for more than 40 years now, bringing you the latest information on the newest tools and technologies that can aid your research endeavors. Now after more than a year of hearing about mRNA Vaccines regularly in the news and in the scientific literature, and then more recently being able to
1:15
finally witness their power and versatility, the scientific community as well as the general public are much more convinced about expanding their usage beyond just covered vaccines. Prior to the coronavirus pandemic mRNA Vaccine research was well underway for use in combating other human diseases like cancer, HIV, and influenza, a virus our guest for today's event knows quite a bit about and he's going totell us much more about his laboratory's work
1:45
on a universal influenza vaccine program that utilizes some cutting-edge methodologies.
1:54
But before we dive into the data let's learn a little bit more about today's guest speaker. I'm delighted today to welcome Dr. Norbert Pardi who is an Assistant Professor of Microbiology at the University of Pennsylvania. Where his research interest is the development of mRNA based therapeutics with a particular focus on new generation vaccines. Norbert has a strong background in biochemistry and genetics and has been a PENN since 2011, when he
2:26
joined as a postdoc in the Weissman Lab where he continued working with his mentor mRNA innovator Dr. Katalin Kariko. During his tenure at PENN, Norbert explored the development of a novel therapeutic delivery platform using nucleoside modified mRNA in lipid nanoparticles or LNPs.
2:48
And used it to generate highly effective vaccines targeting various pathogens, now this pioneering work is something he's going to talk a lot more about in just a few moments.
3:01
But first before we begin the webinar, I want to remind the audience that we'd love to hear from you in our Q&A session, now Q&A works a lot better if you send in questions during the event you don't have to wait until the end of the webinar you can send your question in at any time during the presentation, all you need to do is type your question into the ask a question box and then hit submit. Norbert's going to be on hand to try to tackle as many of your questions
3:30
as possible. Okay now that all the housekeeping is complete i'll hand things over to Norbert.
3:40
Thank you very much for the kind introduction Jeff, good morning everybody in the U.S good afternoon everybody in Europe, it's really a great pleasure to me and to talk about my work today, my lab is primarily interested in the development of messenger RNA-based vaccines against infectious diseases but we are also involved in the cancer vaccine field we have several research programs that really focus on the better understanding of the mechanism of action of messenger RNA-based
4:14
vaccines. Today I'm going to tell you about our Universal Influenza Vaccine program that also uses the nucleus modified messenger RNA maxim platform and in the second part of my talk uh i'm going to share you some pretty recent data on the mechanism of action of mRNA based vaccines.
4:38
So we published the development of nucleoside modified and purified messenger RNA lipid nanoparticle vaccine platform roughly seven years ago let me very briefly tell you how we make these vaccines so the messenger RNA is transcribed from a plasmid DNA template that has the entity encoding sequence it has some special optimized five prime and three from UTR elements and the poly tail we do transcriptional capping uh so the CAP1 structure which is basically the clean CAP uh
5:12
it's added to the five prime end of the messenger RNA during the in vitro transcription reaction once the RNA is made it undergoes a stringent purification procedure we use cellulose purification to get rid of double standard RNA and other types of contaminants that would otherwise be recognized as pathogen associated molecular patterns and they would induce unbounded immune responses we replace uridines with vampire studio uridine in the RNA sequence to further increase
5:43
protein production from the messenger RNA and decrease immune recognition of the messenger RNA. Once this purified and modified RNA is made it gets formulated into lipid nanoparticles this platform for infectious disease vaccine development and this is actually the platform that is used in the very successful COVID-19 messenger RNA Vaccines developed by Moderna and Pfizer-BioNTech. So let me tell you about our universal influence vaccine program as
6:13
you all know influenza virus infection causes significant morbidity and mortality every year that can result in hundreds of thousands of deaths annually and tens of millions of cases the issue is that currently with seasonal flu vaccines often provide very low level of protection in some years when the vaccine strains match with the circulating influenza virus change the effect effectiveness of these vaccines can be 50 or 60 percent but in some years when
6:48
the vaccine strains do not match well with the circulating influenza virus things the vaccines provide significantly lower level of protection it can be as low as 10 percent or so so there is an urgent need for the development of broadly protective influenza vaccines that protect from many different implants of virus strains including potentially pandemic strains.
7:14
So the messenger RNA lipid nanoparticle vaccine platform has many advantages over other vaccine platforms one of the most critical advantages is the enormous flexibility of this vaccine platform that allows the co-formulation of several or sometimes many antigen encoding messenger RNAs into a single vaccine formulation, so we take advantage on this, in this vaccine program we want to target several parts of the influenza virus including the stock or stem region of the
7:50
major surface protein hemaglutinin of influenza virus the reason why we really want to focus on the stock or stem because it's very established that it can induce broadly protective responses, we also want to include the neuraminidase (NA) the second surface glycoprotein of influenza virus because it's also well documented that it's a fantastic antigen and any specific antibodies can use a high level of protection, matrix protein 2 M2 it's also a good vaccine target it's a
8:26
critical part of the influenza virus it causes an ion channel this is what we want to use in our program. So this is a beautiful ongoing collaboration between my lab at the University of Pennsylvania and several people at the icon school of medicine at Mount Sinai in New York City.
8:47
So our Universal Influence of Vaccine program has basically four parts the overall goal of the program is to target influenza A and influenza B viruses the reason for that because these they are responsible for disease in humans. So influence of a viruses can be further divided into influenza A group 1 viruses and influenza A group 2 viruses and we want to develop a vaccine against influenza B viruses we are pretty much done with these programs and right now we are working on the
9:22
truly multivalent universal vaccine formulation. Today I'm going to tell you about our influenza A group 1 program it's published and our influenza B paper has recently been accepted for a publication. So let's start with the influence A group 1 studies. Like I told you we included several influenza virus antigens in the combination vaccine so we targeted the hemaglutinin in glycoprotein of influenza will influenza A group 1 virus and
10:00
this was a stem targeting construct that's why we call it mini HA and we used Y type sequences from a michigan 2015 H1N1 strain the reason why we picked this because this is a recent vaccine strain so we use the neurominidase the M2 and also nuclear protein from this virus we decided to include nuclear protein in this vaccine, because it's very established that
10:28
this component is very conserved so maybe it can induce probably protective responses and it can and then be specific uh cytotoxic TSA responses can contribute to vaccine protection.
10:43
So the vaccination strategy was fairly simple animals received the single immunization with the 20 microgram mRNA LNP Vaccine for each component and four weeks later we bled the animals and challenge them with influenza virus and monitor the weight loss and survival before we started the studies we wanted to make sure that each of the messenger RNA components in the combination vaccine works very well, so we
11:18
performed an ELISA to measure antigen-specific IgG titers uh in mice that were immunized with the monovalent vaccines or the combination vaccine or fire from lucifer is including RNA which is basically an irrelevant formulation and we were very pleased to see that we could induce antigen-specific immune responses with the monovalent vaccines and also with the combination vaccine and we did not see a significant decrease in antibody responses in use by the different
11:50
components of the combined vaccine there was a little decrease in and two specific antibodies in the combined vaccine compared to the monovalent vaccine but it was really not a big issue.
12:05
So um we started the challenge studies uh with the heterologous strain it's called IBR 180, the reason why we picked this virus for challenge was that unfortunately the A/Michigan 2015 is not lethal in mice and really wanted to use a virus that kills the animals and you can see that the any component of the vaccine it's basically identical with any in the IVR-180 but all other components are pretty different particularly the HA component.
12:39
So animals received a single immunization with the monovalent vaccines or the combination vaccine or the luciferase negative control vaccine and four weeks later they were challenged with five times the AD50 IVR180 virus this is what you can see on the left hand side and you can appreciate all the monovalent vaccines and the combination vaccine in used protective immune responses animals survived viral challenge and it was we were very pleased to see that the neuraminidase
13:13
vaccine and the combination vaccine induced protection without weight loss in these animals, in the next set of studies we use the very high challenge those 500 times LD50 this is what you can see on the right hand side and we were very pleased to see that the na vaccine and the combination vaccine in use protection even against these very high challenge dose. So next we wanted to learn a little about the mechanism of protection and
13:44
so we did a serum transfer study in mice animals received two immunizations four weeks apart and four weeks after the second immunization animals were blood and it was actually terminal bleed and
13:58
serum was transferred to naive mice and a couple of hours post serum transfer these animals were challenged with the IVR180 virus and again the looked at body weight loss and survival over time and as you can see pretty much all vaccines except or all anybody's from different groups induced protection except the NP specific antibodies this is not very surprising I told you before that the main reason why we have NP in the vex in the combined vaccine is that it can induce
14:36
potent cytotoxic T cell responses. So what we learned from this serum transfer study is that the protection induced by this vaccine is primarily mediated by antibodies. Next we wanted to learn a little more about the mechanism of protection in use by these antibodies so we performed the micro neutralization assays using serum from immunized mice again immunized either with the monovalent vaccines or the combination vaccine and it turned out that only the
15:06
neuraminidase component can induce neutralizing antibodies this is again not very surprising uh because uh each is stem it's reported that each is stem-specific antibodies and M2 specific antibodies often act through antibody dependent cell or cytotoxicity activity and this is exactly what we could measure we performed ADCC Assays using serum from base immunized mice and it really turned out that M2 specific antibodies and HG stem specific antibodies also have ADCC activity.
15:42
We looked at the rule or we looked at the induction of T cell responses by the NA and the NP vaccine animals received a single immunization intradermally and 12 days later animals were sacrificed and we looked at antigen-specific NA NP specific T cell responses CD for insidious T cell responses and in splenocytes so the um any specific responses are shown in the green bars the NP specific responses shown the purple bars and the take home is that both vaccine induced antigen
16:22
specific CD4 and CD positive T cell responses and here in the G panel you can appreciate that NP specific um responses particularly a CD8 these responses were particularly strong we could measure high levels of interferon gamma producing NP specific seed positive thesa so most probably it was a it was a good decision to include the NP component in our combination vaccine. Next we perform the dose DE-Escalation study and try to determine the lowest protective dose induced by
16:58
the neuraminidase vaccine or the combination vaccine you can appreciate that even when we immunize mice with very low amounts only 0.05 micrograms so basically 50 nanogram any vaccine or combination vaccine we could still measure antigen-specific, antigen-specific antibody responses and in the lower panels you can see that when we challenge these mice with the IVR180 virus particularly in the case of the combination vaccine you can see it on the right lower
17:34
part of the slide as little as 50 nanogram vaccine a combination vaccine after a single immunization could protect mice from losing weight so this is a really really important vaccine formulation.
17:51
Finally immunized groups of mice with the monovalent vaccines the negative control vaccine and the combination vaccine and this was a single immunization and we challenged these mice with the different influenza A viruses and we monitored weight loss and survival in these various groups and the take home as you can see all mice in the negative control group died after influenza virus infection and the monovalent vaccines induced variable levels of
18:23
protection against some by that some monovalent vaccines were pretty protective against some viruses but not against everything and you can see the blue bars demonstrate that the combination vaccine induced very high level of protection so basically there was a little weight loss after challenge but it was also pretty minor uh it provided broadly protective responses. So the take home from this part of my talk is that nucleotide modified mRNA-LNP vaccination elicits
18:55
strong antibody responses to multiple influenza antigens and the combined vaccine protects mice from little challenge with diverse influenza challenge strains. So uh let me tell you about our influenza B studies uh the manuscript that describes these studies has very recently been accepted for publication it's not out uh yet uh but i'm gonna tell you about the data, so our approach was very similar we targeted the HA NA and NP and M2 we made monovalent vaccines
19:28
and we made a combined vaccine formulation in this particular case we use the full length hemaglutinin in because at the time of these studies uh stage a stem targeting influenza an influence of BHA stem targeting construct was not available so we used the full length ages and actually the vaccine the combined vaccine has two has the reason for that is that influenza B viruses have two distinct lineages the Yamagata
20:01
lineage and the Victorian lineage so we picked one HA from each of these lineages we use the HA from the B uh Phuket virus it's a Yamagata virus and HA from a big B Colorado strain it's a Victoria strain so we use these two full length has and the NAMP and M2 component from the B Colorado virus we made monovalent vaccines and they made the pentavalent combined vaccine and used the loados one microgram from each antigen and the high dose five microgram from each antigen
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in the studies and again the luciferase here was the irrelevant negative control vaccine in all of these experiments animals received intradermal immunizations prime boost immunizations so one or two doses and we looked at the antibody responses and then we did several challenge studies using both Victor Victoria and Yamagata lineages and also the ancestral influenza B strain. So
21:03
we were very pleased to see that similar to the group one studies a single immunization with the monovalent vaccines and also with the combination vaccine induced antigen-specific antibody responses as measured by ELISA and again it was great to see that the in the pentavalent formulation there is no significant decrease in anybody responses against each of the vaccine components and the second immunization significantly boosted the immune responses as expected.
21:41
Then we looked at the quality of anybody is induced by the nations and we could measure neutralizing anybody response against the neurominidase component this is not surprising and we could also measure lasting antibodies against the HA component of these vaccines this was also expected because in these studies we use a full-length hemaglutinin we expected to induce utilizing antibodies against the head domain of hemaglutinin,
22:11
so this is exactly what we see neutralizing antibodies and type C and then type any antibodies both in the monovalent and in the combination vaccine and these responses were boosted after a administration of a second vaccine those. We could also measure uh antibodies with antibody dependent cellular cytotoxicity activity we could measure it in the anti HA and anti NA antibodies after a single immunization and interestingly anti M2 and anti N and
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P-specific antibodies also had some ADCC activity after administration of the second vaccine dose.
22:52
So basically the take home from the studies that immunization with the influenza B vaccine antigens can induce functional antibodies next we performed a bunch of challenge studies again the groups of mice were immunized with the monovalent vaccines or the combination vaccine and then challenged with the ancestral influence of B strain or various Victoria and Yamagatan viruses I know that this is a pretty busy slide the take home is that the
23:26
the monovalent vaccines induce variable levels of protection so many times even if the animals were protected there was some weight loss M2 and then P vaccines induce really variable levels of protection sometimes they were not protective at all against some viruses but the most important finding is that the mono that the pentavalent vaccine even in the low dose so one microgram for each component induced protective immune responses so no death and many times minimal
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weight loss, so these vaccines are really truly rudely protective. To demonstrate that
24:08
the combined vaccine can induce a sterilizing protection we immunized groups of mice with the monovalent and the pentavalent vaccine and then sacrificed one group at three days I'm sorry and then four weeks post immunization we challenged these mice with the Victoria and the Yamagata strain and then three days later three days post infection groups of mice were uh sacrificed and six days six days a post infection another group of mice
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was uh sacrificed and we looked at uh viral titers uh in the lungs of these animals by pluck assay and the take home is that the combination vaccine again the monovalent vaccines in used variable levels of protection sometimes they could not protect from viral replication in the lungs in some cases they were more protective the HA and the NA vaccines worked uh somewhat better uh than the anti or the M2 vaccines but the most important finding
25:14
that the pentavalent vaccines and again even the low dose could induce sterilizing protection three days and six days post infection against Victoria and Yamagata virus so again this was really really great news to see this sterilizing immunity. Similarly to the group one studies we performed the dose DE-Escalation study we determined the lowest protective dose and
25:43
we only use the combination vaccine in this particular experiment so animals receive the single immunization with the five microgram 0.5 microgram uh or 50 nanogram five and five nanogram combined vaccine or the luciferase negative control we looked at ELISA titers and again we could still measure uh titles after immunization with only a five nanogram combination vaccine and then we challenged these mice with the
26:12
BEAM Elijah train and looked at morbidity and survival and the take home is that as little as 50 nanogram combination vaccine could induce protective immune responses in this in this challenged animal so it's really highly protective after administration of a single dose. We also perform the passive transfer study to see if anybody's can induce protection from viral infection and in the top panels you can see the animals received two immunizations four weeks
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after the second immunization we collected serum from these animals on the left hand side you can see the ELISA titles measured in the donor serum so every single vaccine in use antibodies the M2 specific antibodies had a pretty low level and then we transferred the serum from immunized mice to naive mice and challenged them a couple of hours post serum transfer before challenge they were blood and we measured anybody titles in the recipients here SARA as well and you can
27:19
appreciate it obviously compared to the donor serum there is a decrease in anybody titles and again for our serum transfer animals were challenged with the uh
27:35
with the Victoria virus and we measured morbidity and mortality and it turned out that any and age specific antibodies can induce protective immune responses against particularly the serum from the animals that received the combined vaccine was really highly protective NP and then two specific antibodies did not induce a very high level of protection from viral infection so the take home from this experiment is that
28:07
the protection in use by this influenza B mRNA vaccines is primarily antibody mediated. So the conclusions from this study is that most of the monovalent vaccines influenza B vaccines protected mice from infection after a single vaccination minimal or no weight loss was observed in mice immunized with the pentavalent vaccine a very low dose only 50 nanograms single immunization can protect mice from influenza B infection and the pentavalent vaccine formulation induce broadly
28:39
protective responses after a single immunization. So I'd like to thank the contribution of many of my uh great collaborators uh Jamily and Matthew significantly contributed to the group one studies Hiromi and Choba from my lab contributed to the influence of these studies Rafael Nakagawa and Alec Frane were absolutely key in the group one studies uh from uh Mount Sinai and Megan Mcmahon really spearheaded the influenza B studies from Sinai and of course aquitas therapeutics
29:18
they are very long-term collaborators they provided the lipid nanoparticle formulations. So let me change gears now and talk about pretty different uh topic I would like to share some uh data on the mechanism of action of nucleus I'd modified and purified the messenger RNA lipid nanoparticle vaccines, as I told you this vaccine has two basic components the modified and purified RNA and the lipid nanoparticle and it's very well established that the platform
29:49
works very well both in preclinical models and also in humans as it turned out in the context of COVID-19 mRNA vaccines but we know very little about the mechanism of action of these vaccines so we really don't know how but signaling pathways are involved in immunosensing for example of mRNA-LNP vaccines and I do believe that this is a very important it's very important to learn more about it because once we understand how these vaccines work
30:23
we should be able to come up with even more potent mRNA vaccines. So number of years ago we asked a very simple question what provides the adjuvant activity for nucleus modified and purified mRNA LNP vaccines it's a very simple question but actually we don't know the or we didn't know the answer so all vaccines have some kind of an adjuvant activity in for example in viral vaccines either live or inactivated virus it has an inherent adjuvant activity the components of
30:55
the virus can stimulate immune responses in a subunit protein subunit vaccine we need to use adjuvants such as alum or MF59 to be able to induce strong immune responses if you use an unmodified RNA vaccine we do know that the RNA has inherently joint activity so it is recognized by various immunosensors but the nucleoside modified RNA was really designed not to induce
31:25
strong inflammatory responses uh it's an important question and it was really important to figure out what is the adjuvant component in this in these vaccines so to give a pretty quick answer I put together a very simple uh experiment. So I used sublime protein sub unit vaccine and the protein was a recombinant PR8 hemaglutinin in a protein so this was basically an influenza vaccine and
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boxy mice received a single intramuscular immunization with 10 microgram protein either protein without any adjuvant or protein mixed with empty lipid nanoparticles or five translucent ferris including messenger RNA lipid nanoparticle construct four weeks and eight weeks after a single immunization animals were blood and hemaglutinin inhibition titers were determined in these serum samples and it turned out that as expected the HA protein without an adjuvant did
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not induce a measurable HA titles but the empty LNP event in order luciferism mRNA-LNP when the
32:39
vaccine induced pretty high HA titles and there was no difference in the HA titles between the empty LNP adjuvanted and the mRNA olympia educated the protein vaccine so the take home from this experiment was that the lipid nanoparticles have uh adjuvant activity uh so we repeated these studies a little later and we added a comparator a HA protein subunit vaccine adjuvanted with the autovox it's a very similar agilent MF59 which is basically a licensed FDA approved adjuvants, so
33:19
similarly to the previous study animals received a single immunization with 10 microgram HA protein
33:26
without an adjuvant with the other exaggerant with empty LNP or the luciferase amarillomp and we followed the HA titles homoglutenation inhibition titers for 20 weeks and as you can appreciate the uh LNP adjuvanted vaccines or a vaccine or the mRNA-LNP adjuvanted vaccine induced higher level of HA titus compared to the otterbox adjuvanted protein subunit vaccine.
33:52
So um it's very it's value established we and others showed several years ago that nucleus had modified them mRNA-LNP vaccines can induce potential follicular helpers responses what anti-follicular helper cells the follicular hair process are activated CD4 positive T cells that help to form and maintain germinal centers within the pisa follicles germinal centers are the micro anatomical sites of uh antibody affinity maturation so if Tfh cells provide
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help for B cells they provide signals IR4 IOS IO21 and they drive anybody affirmative maturation they also contribute to long live plasma cell formation and visa memory formation, so essentially if you want to develop if you want to develop a vaccine that can induce strong antibody responses you need to find a platform that can induce the potential follicular hypersar responses. And again it's well established that nucleotide modified our mRNA and P vaccines can induce strong
35:01
TSFA responses and in the subsequent studies we used TFA cell responses as a readout, so um we immunized mice with the hemaglutinin protein subunit vaccines adjuvanted either with other box or the MTRMP and they used HA mRNA-LNP vaccine as a positive control again I told you that we didn't know that it couldn't use uh Tfs responses the question was if LNP adjuvanted protein subunit vaccines can induce also strong Tfs responses.
35:36
In all of these studies animal received the single immunization with 10 microgram protein
35:43
and animals were sacrificed uh 10 days post immunization and the TFA response is measured in the draining lymph nodes of these animals in the B panel you can see the total number of Tfh cells and you can appreciate that LNP adjuvanted vaccines induced higher numbers of Tfh cells compared to the other box adjuvanted vaccine and the deeper now you can see the hemaglutinin uh specific T follicular hyper cell responses and the difference is really striking between
36:17
the LNP and juventus protein and mRNA compared to the other box adjuvanted protein vaccine so again LNP adjuvanted vaccines can induce strong T follicular high pressure responses we also looked at germinal center B cell responses so GC B cells uh in these animals again in the lymph nodes and take home was very similar LNP adjuvanted protein vaccines and mRNA-LNP and mRNA vaccines can induce stronger GC B responses compared to the otherworks adjuvanted protein vaccine.
36:53
The immunized groups of mice uh the HA other box and HA LNP protein subunit vaccine and let them sit for a while uh for several months and then to do and then we looked at the plasma cell responses and memory B cell responses so we really wanted to wait until we can measure these longer term responses and uh it turned out that the LNP adjuvanted recombinant HA vaccine induced muscular hemaglutinin specific plasma cell responses and memory B cell responses
37:29
compared to the other box adjuvanted protein subunit vaccine, so again LNP juventud vaccines can induce stronger T follicular hyper cell germness enterprise plasma cell and normally visa responses than the other oxygen HA protein vaccines.
37:48
So we in the next set of studies uh we wanted to demonstrate that uh not uh we can we that LNPs can be used with other protein antigens as well so not only with the influenza hemaglutinin so we repeated this studies with uh with another protein subunit vaccines and this was the
38:10
receptor binding domain of the spike uh glycoprotein of SARS-CoV-2. So the experimental setup was very similar mice received the single immunization 10 microgram RBD protein with mLNP 10 microgram RBD protein with other box or
38:29
10 microgram RBD mLNP and in the a panel you can see that um we measured the antigen specific IgG titers by ELISA and you can appreciate the LNP adjuvanted vaccines shown with the green and
38:49
pink induced higher levels of RBD specific IgG compared to the otterbox adjuvanted protein vaccine shown with the with blue we measure the utilization uh neutralization ability of antibiotic vaccine use antibodies and again other box protein vaccines did not induce measurable levels of neutralizing antibodies but the LNP adjuvanted RBD vaccine and mRNA-LNP
39:24
RBD vaccine induced pretty high levels of neutralization titled as measured by um episode virus neutralization essay, similarly to the aha studies we found that uh LNP adjuvanted protein and mRNA vaccine used higher level of D follicular hydrocele responses, germinal center visa responses, plasma cell responses, and memory B cell responses compared to the other box adjuvant in the protein subunit vaccine the RBD vaccine so the take home message from this part is that uh
39:57
this that LNP can be used uh with uh not only with RNA but also with proteins to induce uh strong antibody responses uh and cellular immune responses and the LNP component of the vaccine mRNA vaccines provides very strong adjuvant activity, so we went on and we wanted to determine which component of the LNPs lipid nanoparticle is responsible for the adjuvant activity, the lipid nanoparticles that we use have four different components and the probably the
40:34
most critical component is the so called ionizable cationic lipid that is critical for the safety feature of lipid nanoparticles and it also plays a role in the uptake of mRNA-LNP vaccines, so um they asked our collaborator aquitas therapeutics that provide this lipid on the particles for us to generate a lipid nanoparticle empty lipid nanoparticle with the ionizable lipid empty lipid on the particle without ionized or lipid this is shown with brown you can see that
41:06
the size of these particles are roughly similar the empty LNPs without ionizable lipid are a little bigger and so we repeated our immunization studies and immunized mice with the recombinant HA protein adjuvanted with the empty LNP bit ionizable or MTL and LNPs doweled ionizable lipid and we measured hemoglotonation inhibition titers and we were very surprised to see that if we use an empty LNPs without ionization basically it loses all the adjuvant activity so we could not measure
41:40
any HA titers uh four weeks or eight weeks post single immunization with these vaccines so the ionizability is critical for the adjuvant activity of LNPs, next we looked at the um the cytokine and chemokine uh profile induced by uh the a induced by other box adjuvant the LNP adjuvant
42:07
and they have used LPS as a positive control animal has received a single immunization uh with a single injection with this compounds and and then we looked at several cytokines or actually many cytokines and chemokines 4 hours or 24 hours post injection in the draining lymph nodes of these animals the take home message is that the empty LNP with ionizable lipid and the mRNA-LNP could induce again a very important production of several cytokines and chemokines and we did not
42:42
see it with the empty LNP without dionyson lipid and the other boxing use actually lower uh amount from this from the cytokines and chemokines, IL-6 was particularly interesting for us because we did measure high production of IL-6 by eLNP and luciferasamar nail LNP and IL-6 is a key regulator of TFA cell biology in mice so we were particularly happy to see this and we wanted to measure we wanted to take a closer look at the role of IL-6 in mechanistic studies.
43:21
So first we immunized the white type mice with the recombinant HA plus other works and recombinant HA plus empty LNPs and we looked at the kinetics of IL-6 production so we looked in the draining lymph nodes of these animals so we measured it at four hours eight hours 12 hours 24 hours and 48 hours post immunization in or post injection in groups of mice and the take home is that there is a very strong IL-6 wave after immunization with the LNPs adjuvanted hemaglutinin
44:00
vaccine the peak is at four hours and then there is a gradual decrease and basically that is almost none two days after the immunization. To further uh investigate the role of IL-6 we performed the uh immunization experiments in IL-6 knockout mice so animals were immunized with the HA LNP or the HA mRNA-LNP vaccine and the Tfh cell responses were measured 10 days after a single immunization and it turned out that
44:33
the Tfh cell responses are really blunted in IL-6 knockout mice compared to the control by type mice so this is evidence that there is probably some evidence that a LNP news IL-6 is critical for the induction of strong T follicular helper cell responses by LNP adjuvanted vaccine forms both RNA and protein we obtained very similar data in IL-6 knockout mice when we looked at germinal center visa responses again center visa responses were blunted in IL-6 knockout mice
45:09
after HA protein and LNP or HA mRNA-LNP immunization, to provide some more evidence for the critical role of IL-6 uh if you performed an experiment in a white type boxing mice um animals receive the
45:29
treatment with an anti IL-6 antibody or a isotype control antibody 24 hours before immunization with an HA mRNA-LNP vaccine then they receive this immunization with the HA and P vaccine and we looked at the T follicular herpes responses and it's very clear that the Tfh cell responses are blunted when they block IL-6 with the block in anybody, so again the take home is that LNP and use IL-6 is very important for the induction of strong T follicular herpes response in mice, next we
46:09
wanted to learn a little more about the sensing mechanisms as you know there are many uh RNA sensing receptors in different cellular compartments and we can sense us different forms of single standard RNA we can sense double stranded RNA so we've we really wanted to learn a little more about it and this is ongoing work i'm going to show you just two or three slides about our initial studies but we are still working on the
46:38
better understanding of mRNA-LNP sensing by our immune system and how these vaccines can induce an immune response so in the first set of studies we use the nebs knockout mice so mevs is a common adapter for RIG-I, MDA-5 and many other receptors that can sense RNA different types of RNAs and actually it turned out that when we immunize maximize or control white type mice with HA protein plus empty LNP or HA mRNA-LNP vaccines and look at T follicle hyper cell and germinal center
47:17
visa responses we could not see any difference between the control mice and the maps knockout mice so the take home is that mRNA- LNP vaccines are not sensed by receptors that act through maps. We looked at the role of another adapter important adapter molecule MyD88 it's a common adductor for all like receptors that can sense RNA and
47:44
the experimental setup was very similar so animals they see the single immunization with the HA LNP, or HA mRNA-LNP and LNP and we looked at the follicular helpers germinal center visa responses uh 10 days after a single immunization and it turned out that we could really could not measure strong or significant differences uh when they used between the maps mice and the control mice when we used the aha plus empty LNP but we did see a blunted Tfh cell response and germinate visa
48:18
response and the immunized mice with the HA mRNA-LNP, so the take home is that most probably the mRNA component of mRNA and P vaccines is sensed by receptors one receptor or more receptors
48:31
that act through the MyD88 adapter. So these are ongoing studies and I really hope to learn more about the sensing mechanisms in the near future, so to wrap up the second part of my talk
48:45
the LNP component of modified mRNA-LNP vaccines is a strong T follicular hyper cell B cell and humeral immune response inducing adjuvant this is I believe a very important discovery and others also have very similar findings two beautiful papers were published in nature immunology from iron immune and the polypulan translab and then with similar findings we also found that LNPs can be combined with protein subunits to create potent
49:14
novel vaccines so it's a very good adjuvant that works not only with the mRNA but also with protein the ionizable lipid component is well LNPs is responsible for the adjuvant activity of LNPs LNP induced IL-6 is a major driver of theophase differentiation and finally LNPs are not sensed by either reguy or MDA-5 or receptors that directly or indirectly rely on MyDD88 but the RNA component or the degradative products of RNA are recognized by two like receptors and maybe other receptors
49:47
that act through MyD88 so uh I'd like to thank um uh or people who contribute to today's studies uh Matthew Pine and Hiromi Muramatsu from my lab several people from the wise men's lab and I'd like to mention Nikhil Alochi because she her group particularly Emily Bettini
50:08
was absolutely key in their studies Emily and Michaela performed all the Tfh cell and GCBs studies so uh they were really important and again I'd like to thank everybody else in this uh everybody else for their contributions so thank you very much for listening and I'm very very happy to take questions thank you.
50:36
Norbert really truly amazing work you're doing and the data is really really compelling so, I'm really looking forward to your future endeavors, as well as hopefully being able to take a universal influenza vaccine sometime very very soon to the audience the Q&A is coming up in just a few moments so if you have a question for Norbert about his presentation which it looks like many of you do already if anyone's sitting there thinking about asking
51:04
a question don't hesitate all you need to do is type it into the asset question box and hit submit.
51:12
Okay since we have a ton of questions already, I'm going to start the Q&A in just a moment but if you still have a question don't hesitate just type it in and hit submit, all right bear with us for just a moment as we transition into the Q&A session.
51:30
All right thanks everyone for joining us for the Q&A session Norbert again great presentation really appreciate that we have a bunch of questions here for you so let's try to get through as many as we can the first one we have an audience member states if we formulate A 10 to 12 mRNAs or if we formulate 10 to 12 mRNAs in a single mRNA-LNP vaccine can we induce potent responses against each component in humans?
51:59
Hi everyone it's a great great question um so uh what we found in um animal studies that um it's possible to induce very good immune responses against 10 to 12 mRNAs encoded the antigens when we use them in a single vaccine I think in humans the major concern is the dose because if you have 10 to 12 components this means that most probably using the current formulations and thinking of the Pfizer or the Moderna vaccine we cannot really
52:43
use more than 5 to 10 micrograms per component and I think the big question is uh if you can induce strong enough immune responses with the let's say 5 to 10 microgram messenger RNA so far uh it seems that the in at least in those vaccines that we have tried so far that different mRNAs um do not fear it do not interfere with each other so this is not a concern but again the big question is the dose because I doubt that we can use uh again based on the COVID-19 mRNA vaccines I
53:22
doubt that we can use more than 100 microgram mRNA um in um uh in a single formulation and again if you know in a covet vaccine it's only the spike mRNA but if you if you want to use maybe 10 of them then again the big question is is easily that the relatively low dose is sufficient to induce strong enough immune responses you need to test it in uh in human trials.
53:53
All right great thanks Robert um next question we have this uh pretty interesting one an audience member says the current egg-based flu vaccine has about 40 percent protection do you think mRNA vaccines can reach 90 protection like covid using uh the mRNA
54:12
this is an excellent question excellent point at the moment I don't think that this is a this is a realistic expectation uh influenza virus is a pretty complicated vaccine target there are several issues but one of the most important issues is that
54:34
the most of us have pre-existing immunity against influenza and it's pretty well documented that
54:44
pre-existing influenza specific immunity can have very often a detrimental effect on vaccine-induced immune responses uh and I think this is a main issue uh right now and uh and I'm really not sure if mRNA vaccines can really overcome this this problem I think we can uh definitely come up with the at least as good uh mRNA vaccines as as egg-based flu vaccines uh maybe even better and I think we still have like a big advantage and this is the uh
55:23
the ease of production and it's very easy to uh come up with a modified RNA vaccine uh the production of these vaccines are much simpler again uh if for those people so for most of us who have resisting immunity uh against flu uh I really don't think that ninety percent uh efficacy is uh at least in the current uh platform that we use I don't think that we can reach it
55:50
it would be very interesting to see um how for example universal mRNA based influencer vaccine would work in very young children you know young children who have not who do not have uh pre-existing antibodies it would be really interesting to see if he can induce uh let's say using this uh using the multi-component uh universal influenza
56:15
that I just talked about it would be interesting to see how those vaccines would work
56:21
in again in this population without pre-existing antibodies.
56:28
All right thanks Robert uh next question can you compare same circular RNA and current traditional linear mRNA uh with respect to production and safety and efficacy?
56:42
it's a great uh point uh I think in terms of production uh these are the mRNA so all of these are uh synthetic platforms uh so I think uh
56:57
COVID-19 pandemic really showed us that it's possible to produce these vaccines in very large uh quantities I do not think that this is a this is an issue with any of these um platforms maybe one consideration that the self amplifying RNA it contains not only the antigen of interest but
57:19
also other components so these rnas are usually pretty long so I can imagine that it may cause some issues with production I'm not sure about that
57:33
in terms of the safety and efficacy so the the circular RNA is still a relatively new uh platform uh so we do not have any uh human data on that based on animal studies uh non-human primary studies and most studies it seems that it works pretty well and the people didn't see any uh
57:57
any major adverse events after using circular RNA but again this is only uh preclinical work we have some data some human data with the self-amplifying RNA and it seems that this is a very uh promising platform but um most probably uh it needs to be optimized further optimized for humans because it works very well particularly in mice it also works in non-human primates but based on some recent publications and at least in the context of COVID-19
58:34
mRNA vaccines uh it turned out that they did not work that well in humans and they need to understand why uh this was happening uh and we have um probably the uh the most uh the vast majority of the data it's it's the non-replicating RNA both unmodified RNA and nucleoside modified RNA so for the nucleoside modified RNA I think the answer is pretty
59:02
simple uh the human trials phase three trials provided evidence both the Pfizer and the Moderna trial that the nucleotide modified RNA is it's pretty safe and uh
59:15
very effective at least in the context of COVID-19 vaccines the unmodified platform
59:24
was not as good as the nucleus I modified but I can imagine that
59:29
it is possible that we can increase uh both the efficacy and also the safety profile of this
59:37
of the of the unmodified RNA vaccine maybe that cure that is a good example they publish their phase three data the copied vaccine COVID-19 vaccine and it was not as good as the nuclear site modified RNA but again there is probably still some room for improvement just one more sentence for the nucleus i'd modified RNA for the safety I think it's safe but
1:00:02
many people experienced reactogenicity so for the future generation of uh
1:00:10
next generation of nuclear modified mRNA-LNP it will be very important to
1:00:16
to decrease these adverse events after adverse events after uh vaccine administration.
1:00:24
All right thank you Norbert, uh next question is what um what antigens are taken in combination vaccine and do you need a combination of mRNAs or one mRNA encoded encoding all antigens?
1:00:40
Let me start with the second part of the or the second question um so we uh we make uh individual messenger RNAs uh if you talk about the flu vaccine so we make the messenger RNA and coding the HA mRNA-LNP M2 components and then uh the combines and they could combine these messenger RNAs before formulation to liquid nanoparticles so we don't use a single messenger RNA encoding all of these vaccine components but instead we make individual messenger RNAs mix them together
1:01:17
and then formulate them into lipid nanoparticles. All right thank you for that uh next question we have uh this is a very good question uh interesting one as well too and they wanted to know if you have if you attempted intranasal immunization? Yeah that's a that's a great point so uh at the moment the the messenger iron lipid nanoparticle vaccines that we make are really not optimized uh for intranasal delivery, so we uh we do not uh we do not use them
1:01:50
um but uh I do know that there are some laboratories uh that try to
1:02:00
generate formulations uh that are really optimized for intranasal delivery and I'm pretty confident that in in the coming years uh there will be a great progress in this field as well so again great question we are we will probably need to wait a little more uh but again I my expectation is that in a year or two there will be mRNA-LNP optimized for intraness or delivery.
1:02:36
So as a follow-on question that I think you started to address this but the next question we have is what was the rationale for immunizing mice via the intradermal route? Yeah that's a great point uh so um so the reason is that when we started this when we started uh to test uh messenger RNA lipid nanoparticle vaccines we used we tried many different types of routes of administration so we tried subcutaneous we tried intraderma
1:03:07
we tried intramuscular and then it turned out that infradermal is at least in our hands it seems that intradermal is the is the best way to go don't get me wrong intramuscular and also subcutaneous
1:03:23
immunizations also work well but for introdermal we had to use a little lower dose and still induce the same level of immune response compared to intramuscular, in most studies we started to switch um to switch back actually to intramuscular because this is the most common route for uh vaccine administration uh again we probably need to use a little higher dose for intramuscular to get as good responses as with the introduce most probably the reason for that and the original
1:04:01
rationale was that we were thinking and uh thought that okay the skin is a really uh the skin is full of uh antigen presenting cells uh longer house cells uh different types of dendritic cells and and we thought that uh if they do intradermal immunization we can uh we can regional cells and we can induce a pretty important immune response and actually this is this is what we see uh but again in a muscular is just uh is almost as good as infradermal.
1:04:37
All right thank you for that uh this next question you may have covered some of this already uh remembered in the first question but an auditory relationship if you could compare dosage and safety profile of egg cell and mRNA based flu vaccines? Yeah that's uh that's also an uh um an excellent point uh so um in terms of the dose if I remember well uh fluzone which is an inactivated influenza vaccine the quadrivalent flu zone contains four different inactivated virus
1:05:15
strains and if I remember well it contains 15 miles 1 5 15 microgram HA per uh per component
1:05:26
so I think it's kind of comparable to an mRNA this through vaccine because we talked about it earlier that I think the um those the mRNA vaccine those is somewhere between 30 to 100 micrograms so they are I believe in the same in the same range one important note is that it's kind of hard to compare these doses because uh in in case of the inactivated flu vaccine we talk about uh protein dose and in case of the messenger mRNA those and we do not know exactly how much protein
1:06:04
is made from the messenger RNA but anyway they are roughly in the same in the same range in terms of the safety profile the these inactivated flu vaccines have a very good safety profile and they've been using them for uh for decades, for the messenger RNA I mentioned this earlier that the nucleus had modified messenger RNA vaccines messenger and lipid nanoparticle vaccines have an
1:06:35
acceptable safety profile but I do believe that we need to optimize these vaccines and somehow we need to reduce the reactogenicity and retain the immunogenicity of these vaccines I believe it's going to be it's going to be possible to do that.
1:06:54
All right and next question we have non-december says although LNP provide adjuvant function does it cause most adverse effects in mRNA vaccines?
1:07:12
So the answer is most probably uh yes uh but we need to uh we need to um uh we need to further investigate it uh so that's something I touched uh a minute ago that most probably the reactorgenesis and actually it's still a question if the reactogenicity and immunogenicity
1:07:38
of a vaccine go hand in hand uh I really hope that this is not uh this is not the case because uh if so then it will be complicated too very hard to reuse the reactogenicity without losing the immunogenicity uh, anyways I do believe that uh the most probably the LNP component needs to be optimized to reduce the uh adverse events however I have to mention too that uh based on our studies our published studies and others also found had similar findings
1:08:13
it seems that the messenger RNA component or the degradative products of the antigen encoding RNA
1:08:23
is also sensed by the immune system we do not know if it specifically causes any uh uh adverse events or not uh it's kind of hard to dissect uh you know this comes from the RNA this comes from the LNP but again my guess is that it's primarily the repeat on the particle.
1:08:44
All right thanks for that the next question we have uh it says adjuvants like AS-1 and CpG which are currently used in many protein-based vaccines which are relatively superior to advex what are your views on these adjuvants compared to LNP as an adjuvant. So excellent point um so I completely agree uh it will be very important to compare these adjuvants to uh to the lipid nanoparticle adjuvant I've never seen actually I
1:09:19
there was there is a couple of that there are a couple of papers uh there uh published by Harry Friedman's lab from PENN he used olum uh plus CpG um with protein vaccines uh and uh and mRNA-LNP vaccine so it's not directly comparable because the wood comparison would be a CpG plus protein or or LNP plus protein so there is no to my best knowledge uh
1:09:52
there are no or very few papers that looked at it personally I've never looked at it but again this is a great suggestion and that's something that we need to do uh in the future uh just one more very important uh point uh that it's not gonna be easy to do that or it's gonna be probably a pretty big study because uh but we need to emphasize that different LNPs have different um uh like level of adjuvant activity so some LNPs are much better than others so this and that's why
1:10:26
again I can imagine that you can find LNPs that are not as good as uh CpG or CpG alum or even uh
1:10:36
other uh potent adjuvants uh currently with eduards but I can imagine that some LNPs are are better than any other currently used or unknown vaccination again these studies need to be done but this is a great point. So Norbert as a follow-up to that what components of LNP confer adjuvanicity? So based on uh excellent question based on our studies uh we see that uh if he and this is what we published in the in the immunity paper uh that if we leave out the
1:11:14
ionizable lipid component of the of the lipid nanoparticle then we lose the adjuvant activity so most probably the ionizability component is critical for the adjuvant city but
1:11:32
I would but I can also imagine that it's possible to uh to play around with other components and increase the adjuvant effect of LNPs again this is something that we are very actively investigating at the moment.
1:11:52
All right and I think we have time for one last question here uh an audio server says where do dendritic cells come into play when handling mRNA vaccines?
1:12:02
Another fantastic question um so at the moment uh we still don't know uh uh how so what what happens after uh mRNA administration so uh it's possible that uh the messenger RNA
1:12:21
lipid nanoparticle uh traffics to the uh let's say the the draining lymph nodes and in the draining lymph nodes uh um in the draining lymph nodes the cells take it up and translate it and it induces an immune response but it's also possible that the dendritic cells take up the RNA
1:12:42
somewhere in the periphery at the site of the injection in the skin or in the muscle and then they produce protein from the RNA and they basically migrate to the draining lymph nodes and induce an immune response at the moment we really don't know how uh mRNA-LNPs reach the uh the draining lymph nodes so again maybe or if it's also possible that both uh
1:13:11
both scenarios happen so the analytics us take them up and bring them to the lymph node or they just traffic to the lymph nodes without without dendritic cells so some uh some
1:13:26
it would be nice to put together some studies that look at it.
1:13:31
All right well thank you Norbert and with that we've come to the end of our webinar. So i'd like to remind everyone that the webinar will be archived on the gen website uh at jenningsnews.com for up to a year so if you missed any parts of it you can watch it again or feel free to follow the link to any of your friends and colleagues, I like to thank Norbert again for his wonderful presentation and I'd like to thank you the audience for your attention and very thoughtful
1:13:55
questions, a very special thanks to TriLink for sponsoring this webinar, uh hopefully we'll see you again at another webinar in the near future keep an eye out for the next webinar in this series uh which is coming up in a few weeks everyone have a great weekend stay safe and healthy.
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