Scientist Stories: Jie Sun, Respiratory Viral Infections

This is episode 43 of the Immunology Podcast, Respiratory Viral Infections with Dr. Jia Sun. Hey everyone, this is Dr. Jason Goldsmith and Dr. Brendel Raud. Welcome back to the Immunology Podcast, where we have conversations with immunologists. The Immunology Podcast is brought to you by Stem Cell Technologies, a global biotechnology company supporting life science research and fostering communication and collaboration in science. If you enjoy the Immunology Podcast, rate us and leave us a review. We're always looking for feedback on how the podcast can be improved and for suggestions on guests. Today we have Dr. Jie Sun from the University of Virginia on the podcast to talk about his research studying respiratory immunology under homeostasis and disease conditions. Almost like we've had a viral pandemic lately or something.

Yeah, yeah, a lot of work.

We've also got our usual roundup of recent highlights in immunology news coming up, but First, exciting news.

Immunology 2023, which is the annual meeting for the American Association of Immunologists, is taking place from May 11th to May 15th, 2023, in Washington, DC, and early bird registration opens today. Abstract submission is also open until December 19th, and those who submit can also apply for an AAI travel award. Don't miss your opportunity to be a part of the world's leading annual all-immunology meeting. Visit www.immunology2023.org for more information.

Well, I know where I'm going.

Very exciting. Have you been to AAI meetings before?

Actually, I don't think I have. I usually stayed on the gut side of immunology, but I'm looking forward to it.

Yeah. Yeah. Me neither. It was too far away on the other side of the pond, I guess.

Does it count as the pond if you're all the way that far on the Atlantic, or you can only say pond if it's in like, you know, Britain or something?

Good question.

Like, is it the pond?

If you're in California and you're in Amsterdam, there's a pond, there's a park, there's a smaller park in between. Yeah, I guess, I guess it's a pond. Oh, this is the city of an understatement. The pond actually is a huge badass ocean.

You know, we have 5 oceans now, right? Did you see that?

You know, we always had 5 oceans?

We had— we know they added one.

Which one?

Yeah, so there was, you know, the Indian, the, the Arctic, the Atlantic, the Pacific. But there's a 5th ocean, right? Sorry, there's a 5th one. So apparently the— there's a part— the Antarctic in a section near Australia, if I get this right, is its own ocean now because it has its own— like, apparently there's a definition of what makes an ocean an ocean. All right, it's all connected, right? Like, it's not like the water from one ocean doesn't touch the water of the other ocean. So, so there's something about what defines an ocean that I do not know, but like, it has to do with like currents and salinity changes and temperature and stuff. It's like an isolated ecosystem. And apparently there's an Antarctic Ocean.

I mean, I'm not surprised, but weren't there like 7 oceans?

Those pirates were around the 7 seas. Yeah, the 7 seas, right? But you know, I think a couple, I think a couple of oceans got split up. Into some multiple seas.

I really hope that no geographers or geologists are listening to the podcast because they would probably be—

if you know what makes an ocean an ocean as a definition, let us know because we don't know what we read on the interwebs.

Let's stop making fools of ourselves. Why don't— let's talk about things we do know, like papers.

All right, well, do you have one that's— do you have a car T-paper you want to talk about to start with?

Uh, no, actually no CAR-T papers this time, but I do have a nice paper that is kind of a follow-up to the paper I talked last time.

So, all right, so you can count as knowing that because I had to jump far afield this time for me for at least one of my papers.

All right then, show me.

All right, you want me to show you? All right, well, I will talk about more things I only kind of know about, which is the paper Circulating c-Met Expressing Memory T Cells. Cardiac Autoimmunity. First author is Silvia Fonti, last author is Federicia M. Marelli-Berg. Came out in Circulation on the 23rd of November.

What a way to butcher those names, you know, my hideous American accent.

Um, that being said, I know what CMET is, I know what memory T cells are, I've treated cardiac autoimmunity. And yet combined together, this one was an interesting paper to try to wade through just because it got very in-depth on things that I don't think about very often. So long and short, one of the cardiac— we all think when we think about heart, we think about heart attacks, but there's other stuff that happens to hearts too. One of these is myocarditis, which is inflammation of the heart. And one of those is autoimmune myocarditis. And there's this— of autoimmune, there's a subset of— there's this phenomenon by where often after auto— where your heart can have dilated cardiomyopathy. So it kind of blows out and doesn't do so well afterwards because then it doesn't pump as well. And so there's idiopathic cardiomyopathy, dilated cardiomyopathy, IDCM. But they find that in those cases— but, and they also— so they have that disease, and there's also idiopathic, meaning we don't know where it comes from, right? Right. That, that, that's the whole joke of it makes you an idiot as a condition, right? So you have this idiopathic dilated cardiomyopathy, then you have autoimmune known myocarditis. Well, most cardiomyopathies, dilated cardiomyopathies, happen after when you see it, they happen— many of them occur after autoimmune myocarditis. So the autoimmune inflammation leads to a dilated cardiomyopathy. But they've also found in recent years that if you just go and look at an idiopathic one, like after the fact or with some type of imaging, or if you take their blood cells, they have a lot of the same immune markers as autoimmune. And so there's this question of how much of idiopathic is just undiagnosed subclinical autoimmune myo— carditis that then leads to the symptom of dilated cardiomyopathy, which they label idiopathic because the autoimmune part wasn't as obvious.

So that's obvious. Would be like cells infiltrating.

Well, like, if they— well, well, they're not going to take your heart and do that, right? Because it's your heart, you're alive. But like, if you feel sick with autoimmune myocarditis, then you have a clinical presentation. If you feel sick with the dilated cardiomyopathy, that's your clinical presentation. So they're thinking a lot of this idiopathic, I don't know what caused it, was subclinical autoimmune myocarditis. So that's kind of the background of this paper. And what they find is that when they look at both of these populations, but then take normal control or compare it with an infarction, which has immune cell migration, but in, but doesn't have like necessarily the same features, that a couple of things pop out. The first is that there's this T-cell marker, CMET,. And those tend to be much higher in these autoimmune states, and they produce a lot, and they tend to produce multiple cytokines, IL-4, IL-17, IL-22. And then they took the cells out and looked at autoimmune mediated proliferation. So they took human antigens from people with these diseases and found that they actually, the immune cells from both of them will rapidly expand in the presence of cardiomyosin, which is, that's an autoimmune autoimmune process. And so that's the big thing here is that they're able to identify these different cell populations are distinct and there's some overlaps in mechanisms of inflammation. So there's some c-MET, but it's not quite the same c-MET positive cells in heart attacks. It doesn't necessarily— this triple positive high cytokine doesn't respond to autoimmune antigens and expand. And so they show that this autoimmune component's pretty specific. And then they go into an experiment, and this is all in humans. This is in human samples. Peripheral blood macrophages taken from people with these diseases. And then they go and they looked at Sjögren's as a negative control, which is an autoimmune disease, but it's not cardiac typically. And then they went and, um, then did a mouse model that they published on before of autoimmune myocarditis. And then they used a c-MET inhibitor and they show the same pattern. And also that the, but the cytokine profiles are a little different from these cells. Partially because they produce interferon gamma, but in humans those cells didn't. And then they show also though that a c-Met inhibitor reverses the effects. So if you do this induction model and you do a c-Met inhibitor on top, it doesn't work. You can't induce the— you get much less autoimmune myocarditis in this experimental model. And so, you know, it did a couple cool things. It shows c-Met is a player here. It shows a c-Met inhibitor is enough to stop at least an experimental system. This disease state. And it shows that these cells seem to be pretty responsible even for things that aren't, that we think have autoimmune component but aren't known, that idiopathic condition. And that's what I think's really neat is that's really pointing to that theory that a lot of this idiopathic stuff is just stuff that was not diagnosed because they weren't sick with the inflammation part of the disease.

Sneaky immune system behind all of these diseases. What was not clear to me is So C-Met is a transcription factor. Uh, does, do they, do they know how it kind of correlates with this identity? Does it?

Is it? Yeah. So it's driving some of the downstream cytokines.

Ah, okay. Does that allow cytokines? Only cytokines or like some chemokine receptors or something that gets these cells to the, to do?

They look at transmigration for chemokines somewhere. Um, but they don't go super in-depth on it. They really mostly focus on cytokines, but it's not immune ones like IL-17, IL-22.

Okay. I mean, makes sense that many of the times you find these cells that you weren't necessarily expecting and they can be quite problematic. I wonder if this gives, uh, like if by, for example, Uh, you can, you can do c-Met inhibition as a therapy for patients with this idiopathic, um, that's the idea, right?

Or, or even autoimmune.

Yeah. Because if you treat them with like steroids, for example, does it help? Do they mention that?

So they don't, in people they haven't done it, but in mice it did. So in the mouse inducible model, if you block c-Met, you get, you get rid of this. Uh, but they have, there's no therapy for autoimmune. Myocarditis, we just give people steroids.

Yeah. Okay. Steroids kind of help for a while.

Yeah. I mean, if you're coming in with rip-roaring autoimmune myocarditis, we immunosuppress you, right? With just, you know, just, you know, bazooka blast.

Let's hope we don't have to do that in the future. Talking about immunosuppression, I'm going to talk about the opposite vaccination again, about vaccination against with HA, uh, hemagglutinin proteins for influenza. If you might remember from our last episode, I talked about the potential of using the stock of HA, uh, as a source of antigens for, uh, for vaccination. And that the paper I, I discussed, they had this, uh, particular nano— nanoparticles that were very good at initiating an immune response. And they used these stocks, and they could actually kind of achieve some usable kind of universal influenza vaccine with this approach. And here I'm going to present another approach, another try at making a universal influenza vaccine. And I thought it was very, very nice, a little bit different. In this case, instead of looking for an antigen that is widely present, they're just looking for a platform that allows a lot of antigens at the same time to be administered. And then you can just generate a multiple immune response against all of the potential HA molecules of the commonly identified influenza strains. So, The paper is a multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes published in Science. First author Claudia Arevalo from the lab of Scott Hensley at the University of Pennsylvania. And what they did is they kind of went for a brute force approach. And they thought, well, how can we vaccinate people with all 20 known lineages of influenza A and B viruses. And they figured that not with current, with the most commonly used platforms for generating seasonal influenza vaccines, which usually are inactivated virus that are made in chicken eggs. And it's very, very time-consuming, very complicated. And usually they only include up to 4 different HA molecules for the 4 predicted to be dominant strains of the season. You can also make vaccines with many of the antigens, for example, like protein antigens. But in their case, they went for what it is now a very well-proven technology, which is messenger RNA in— and in lipoparticles. Encapsulated in LNPs. And so basically they took these 20 different hemagglutinin molecules, they encoded them in mRNAs, and they generated a multivalent vaccine. And basically they tested it. And they did challenges in mice. They also did some experiments in ferrets, which are kind of a better a closer model to human situation. And they had really interesting results. And I think it might actually— they might be onto something. So they basically had doses of a total of 50 micrograms divided between 20 different recombinant— sorry, 20 different mRNA for the 20 different HA. And they immunized the mice with that. And they also compared it to, for example, what happens if you would immunize them with 50 micrograms of 20 recombinant proteins instead. And unsurprisingly, the mRNA vaccine could really elicit antibody responses against all 20 HA, much better than the vaccinated with just the proteins did. And And this was also the case even if the mice had been previously exposed to, for example, H1N1 virus. They were— they could show that this does not affect the generation of antibody against other variants. There was no kind of any disruption of the normal antibody response because it was dominated by one particular previously encountered influenza vaccine. And they show that they can really neutralize viruses within this cocktail. If they select— they did experiments with the H1N1 virus and also H3 viruses. And they show that if you treat the mice with this vaccine, you can really nicely mediate protection. And they also, in the case of H1N1, they had different strains. One of them was antigenically similar to the one that was in the vaccine, one that was antigenically distinct. And although— and so the way they vaccinated the mice and then they challenged them with either the antigenically similar or the distinct strain, in both cases they offered protection. But it was, in the case of a related strain, it was almost complete protection. The strain was almost undetectable in the lungs of the mice. And even with an antigenically mismatched strain, they could protect the mice from severe disease and death. So, this showed that it was a very good response that was very, very useful for the mice. Interestingly, they see that this is mostly guided by antibodies. Because if they depleted the T cells after the vaccination and they challenged the mice, the mice still survived. So, and that was mostly— and they did other experiments, which they concluded it was mostly because of either neutralization of the virus, especially when you have antigenically similar strains, or antibody-dependent cellular cytotoxicity, ADCC, in the case of kind of mismatch strains that you still had protection. Not immediate, but a little bit delayed, but still enough to protect from disease. And in the end, they did this prime-boost approach and they tested it in ferrets. So similar to the COVID vaccines, they did an initial vaccination with 60 micrograms of combined HA mRNAs. And then they had a 28-day booster. And then basically they challenged the mice and they showed that really the best vaccination protected the ferrets from weight loss, from the symptoms, and from death. And although the viral titers in the nasal swabs were similar, they— the vaccinated ferrets really had a faster viral clearance and none of them died from the disease. So I think it's very interesting because I've been wondering how long it would take to get— now that we've shown that mRNA vaccines can be so potent for COVID, why are we not looking into this for influenza that is infamous for being kind of complicated and variable and we have to go to a different vaccine every time. And so maybe this will pave the way, and I'm sure other people are also researching this, uh, to choosing a different format for influenza vaccines.

Yeah, I feel like universal influenza, if we can get it right, will be out in about 5 6 years. They've been really working on it for a while now. All the new technologies are suggesting some paths forward that have been a struggle before. But like, I think part of it is, we've been talking about before, is what antigen site do you use, right? Yeah, it's been, been a struggle.

Well, in this case, they, they take all the hemagglutinins from all of the 20 strains in one vaccine. So there's less choosing. You can just use them all. Why not?

Why not all? All your hemagglutinin belong to us.

So yeah, one vaccine to rule them all. Ooh, super bad.

One antibody to bind them.

Yeah, one antibody to bind them. And one macrophage to get rid of the, kill the virus or something.

All right. I will talk about— now this paper combines a variety of different loves of my life together into a Voltron of nerdiness. I will probably also butcher names here though in advance. So this is in Nature Communications coming out on now. They don't have— Nature doesn't do it by month. It is issue 13, article number 7197 in this year. It's Kinetics of mRNA Nuclear Export Regulate Innate Immune Response Gene Expression. First author is Diane Lefebvre. Last author is Alexander Hoffman, but it also cites the UCLA Ribonomics Group, which has a bunch of people on it. So this is a really deliciously nerdy paper. It involves kinetics modeling, which makes me happy. Well, differential equations are a way of life.

Oh, wow.

Yes. What it looks at though is they take from bone marrow derived macrophages from mice simulated with LPA, right? So immune stimulated. Mm-hmm. They look at chromatin associated RNA-seq versus nucleoplasm RNA-seq versus cytoplasm. As we think about it, right? CARNA is just made but not spliced. Nucleoplasm RNA is being spliced and have the poly, you know, the caps and tails thrown on, right? Then you have your cytoplasmic RNA. And they look at the same gene across all of these, and they're trying to understand based on quantity, right, at 1 minute, 5 minutes, 10 minutes, at different time frames. And I can pull up the time frames here. So they, they look at it at up to 120 minutes afterwards. So they do 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 90, 120 minutes RNA-seqs and all these fractions. And then look at the genes at each— in each fraction over time to understand how long it takes a gene, an mRNA, to go from the chromatin to the nucleoplasm to the cytoplasm. Constant immune stimulation.

Sorry, of which cell type again? Uh, macrophages.

Okay, primary macrophages from mice. So an immune— yeah, it's an outstanding— and then they model this to describe the kinetics of this. So the export rate, the, you know, the on rate, off rate, the bulk export, because they're gonna be transported back in. So they do all this kinetic modeling, and then they have a model, and they test fit the model against the third cohort. So they did two cohorts of data and then tested the model against the third cohort. So what they find is actually what's really interesting. So they look at then what happens to these different genes, cuz there's some genes. So they have this concept of the chromatin to cytoplasm export rate, how fast it goes from one end to the other is very gene specific and varies up to 100-fold. And then for less than 5% of synthesized, and, and then for some genes, less than 5% of synthesized transcripts arrive in cytoplasm as mature mRNAs. While others are highly exported very quickly. But here's a couple crazy things. So one, you would wonder if the half-life or how much mRNA of something you have in the cytoplasm is related to how good it's being exported, but that's not true. Export rates don't actually determine time gene responsiveness, but they're complemented by gene decay rates. So what you have is that there's— that what ends up happening is that genes that come out really hot and fast from the side of— from the nuclei— nucleus, right? If that gene is something that's high on, high off, it will also have a very fast mRNA decay rate, right? So this boom is going to get sucked up real quick. The ones that export less tend to have lower mRNA decay rates because you want that mRNA when it finally gets out to do something. The other thing they find is that the more splicing you have to do and the longer the mRNA, the longer it sits in the various spots before getting out of the cytoplasm, particularly at the nucleoplasm where all the splicing happens, right? Then guess what? Cytokines don't have a lot of splice sites, have relatively short RNAs, and they're the ones that burst the most. But then they have a very fast decay rate. And so they show all of this in this paper. But the cool thing is that this export rate isn't what drives the half-life. There's decay rates that match.

Very interesting. I like that they see a different pattern for cytokines than other, um, mRNAs. And I hope— do they mention also, like, I know that mRNAs also have a bunch of binding sites for proteins that interact with these transcripts. Do they mention that?

Yeah, so they look at transcription factors. No single transcription factor seems to like, well, if you bind to this transcription factor like NF-kappaB, are you slow or fast? So they look at that and don't find anything. It generates gene structure and sequence motifs, not epigenetic signatures.

Okay. But I mean the transcripts themselves, like the mRNA molecules, there's like, there's like RNA binding proteins that also interact with transcripts in the cytoplasm mostly, I guess.

So to examine if RNA binding proteins play a role in regulating the effective transport rate, we test for richer number of motifs. Yeah. Some were correlated with a higher effective rate., but some were on the 3 prime end were associated with a lower rate. So it depends.

It's not universal. Oh, wow. Something in biology depends on the situation.

But you know, it depends on which one it is. So QK1, which is RNA binding motif, that made it faster. Okay. YBX1, which I don't know anything about either, made it slower. All right. And these proteins tend to be involved in mRNA splicing, transport, and decay.

Okay. I guess also, well, I wonder if you have longer RNAs and then— Longer RNAs make you slower. Make you slower. More splicing makes you slower. And is there anything that is particular to macrophages? Because I assume that these things have been studied in other cell types.

I'm pretty sure this type of work has never been done before at all, I believe. What?

Okay.

That's quite interesting. Or at least what leads with the author say. I mean, think about it. They take hundreds of runs of cells at various data points and fractionate into 3 parts each cell and then run RNA-seq on each of those parts.

Yeah, that seems like a very, um, delicate operation you need to do there. And I guess macrophage, why do they choose macrophages of all cells?

Because it's an easy, I don't know, they didn't say, but my guess is because it's an easy immune cell to extract from mice and play with.

Stimulate? Probably. I don't know, they get stuck into plastic and stuff if you're not careful.

That's great. You stick them to plastic, you stimulate them, and you lyse them with trizol or whatever you're going to do for your export, right? That's true. Trizol, in case— because you'll get everything. But you lyse them with your fractionation buffer on the plate.

Sounds good to me.

True. Well, if you're the author of a paper— or a B cell, or heaven forbid a neutrophil that you're going to look at, and it's going to—

oh yeah. I would not do this with neutrophils. You look at them funny and they explode. So like, obviously wrong choice for this. Well, if you're the author of this paper and you want to justify your choice of macrophages with, uh, we just had a lot of those, please do comment on Twitter and let us know.

Tweet at us, why macrophages?

Yeah, why macrophages? Anyway, so very nice story. I'm glad that you got to geek out with modeling or formulas in this biological world. And I'm going to close our session today with also from a cool story, also from Nature Communications. First author, Judith Svensson-Alvarundt. Oh my gosh.

Alvarundt. Did you just butcher a name, Brenda?

Yes, I just did. And I'm sorry. I apologize. From the lab of Joshua Brody at the Icahn School of Medicine at Mount Sinai in New York. And the paper is called Expanding Cross-Presenting Dendritic Cells Enhances Oncolytic Biotherapy and Is Critical for Long-Term Antitumor Immunity. So, this paper was really a tour de force. My congratulations to the authors. So I'm going to try to keep it high level because there's tons of details. But basically, in this paper, they show very nicely, very elegantly, what are the benefits of combining an approach for tumor immunotherapy that combines the kind of induction or kind of upregulation of cross-presenting dendritic cells using FLT3 ligand, which is known to upregulate conventional dendritic cells. And together with an oncolytic virus that in a way targets tumor cells and releases the antigen, releases enough stuff for these cross-presenting dendritic cells to actually present. So, The one— in the kind of the— they introduced this topic saying that there's been a lot of kind of either/or approaches in which you are either inducing dendritic cells with FLT3 ligand treatment. So, this is a treatment that has been used in clinical trials. And there are patients being treated with FLT3 ligand to upregulate their dendritic cell counts. There's also have been approaches with oncolytic viruses that in the hopes that they will kind of partially lyse the virus, reduce, lyse the tumors, reduce tumor load, and maybe release enough of the antigens for the T cells to recognize and get trained with. But here they kind of try, why not both come in, in a situation? Why not doing them at the same time? So, They have this, this, they have these oncolytic virus, Newcastle disease virus, NDV. And they basically have do a lot of experiments in which they treat mice with that have a B-cell lymphoma. And they inject this B-cell lymphoma with NDV. And they show that when you combine this together with mice that had been pretreated with D3 ligand, this actually works synergistically to improve the antitumor response. So, they showed that on the one hand, the virus infects the lymphoma cells. So, on the one hand, it's killing some cells. So, that's good. But on the other hand, it also upregulates those infected cells, upregulates expression of costimulatory molecules of MHC I and II. And very importantly, for the whole system to work, they upregulate interferon responses. And all of this together really activates the dendritic cells to take up the tumor fragments and present them. And they can become very good at doing this. So, they see an upregulation with the FLT3. They see an upregulation in conventional dendritic cell subtypes. And you know that, for example, cDC1s are known for being good at cross-presenting. And they see particularly a good increase in this population. And then when they get access to the antigen from the lysed virus and the immune— an interferon type 1 response environment, this really potentiates their ability to cross-present. And these really help. So, they have this system in which they have tumor cells that express GFP, and they have Jedi mice, which have TCRs that recognize an epitope from GFP. So, that's kind of cool. So, they have used this system to show that they're— you have specific responses against this, what would be a neoantigen in a way, because GFP is a non- not normal antigen in wild-type mice. So, you can really induce responses. They also have experiments in human cells where they show also that they can induce DC activation and tumor antigen uptake by infecting the tumor cells with NDV. And this is substantially increased when you pretreat this, in this case, mice, or you get this DCs from patients that have been pretreated with FLT3 ligand. And they also show that in a situation which you have other tumor neoepitopes, they do exome sequencing of these tumor lines. And they also show that they can induce this combination therapy, can induce neoepitope-reactive CD8 T cells, other— to antigens other than the very obvious GFP antigen that they are introducing in this tumor site. Yeah, cell line. So, overall, I think it's very, very cool, kind of double punch approach. And a lot of it has already been tried for one or the other. So, this might be maybe a way of really helping generate long-term. They also look into the long-term protection. This induces more long-lived responses than either or either only, for example, only treated with oncolytic virus. So I really thought it was a cool approach to immunotherapy, tumor immunotherapy using oncolytic viruses.

If we look at where we are with immunotherapy now, that was this pipe dream probably 15, 20 years ago, where we are now talking about this next generation stuff. And it's exciting to see what's coming up now at that translational science front that really is going to tell us where therapy is going to go. It's just fascinating, all these new technologies and leveraging biology to deal with other biological problems, right?

Yeah, it's great. And when it works, it's even better. When you actually get to treat patients successfully, it's very— must be very rewarding to be part of these projects.

Exciting times. All right. Well, we're going to be speaking to Dr. Jae Sun at the University of Virginia in just a moment. But before we get to that, learn about the features of air-liquid interface cultures, a physiologically relevant model system for in vitro infection systems, and how researchers are using them to study a variety of viruses, including the novel coronavirus, at stemcell.com/covid-and-ali. And as someone who's done a lot of ALI in my life in recent years, I can endorse ALI work. It works very, very well for a lot of these things.

Hello everyone. We are talking today to Professor Jee Sun. He's Harrison Professor of Medicine at the University of Virginia, and his lab research mainly the immune response to viral infections. And what I think is very important, the role of the immune response in the aftermath of these infections and the chronic conditions that they can trigger. So very excited about our conversation today. Thank you so much for joining us.

Thank you for having me. I'm really excited to be here.

Thank you for coming on board. All right. Well, I'm going to go with the first one here. Very broad question. What's it been like these last few years being, you know, involved in some of the science that is important for all of humanity in a very acute way?

Yeah, that's a good question to start. I have to say it's really challenging but also exciting. So I have not just for myself, In the past 3 years, I feel I only take a very short break, vacation. Otherwise, I always working in my office. Even during the worst time, we don't know what's our future going to be. We don't have vaccine. We don't have treatment. 2020, I was working all the time. So, initially, we want to get into COVID research really badly because I feel like I was studying respiratory viral infection for like 15 years. And right now, came with a major pathogen, I was really interested in to study it. And of course, it's bad to the humanity, to the society. But as a respiratory— people who study respiratory viral pathogenesis, I was very interested to study it. And so, we initially, mainly because we were in Minnesota at that time, I was still at Mayo Clinic. A good thing for Rochester is a small city. Good thing is we don't have much of COVID cases, but, and it's coming late. So, we did not involve in a direct COVID research in the first couple of months. But I was like always on top of it, try to collaborate, discussing with friends, colleagues. And so, it's been, you know, challenging and exciting time. And, you know, so in general, in the field, so many people involved in the COVID research. And the science have been moving forward so fast. And essentially, we have to update our knowledge daily. And that was because the whole, you know, field, everyone is rushing to study it. And that's, you know, we have a great outcome with the vaccine and treatment. And a lot of knowledge been learned.

I mean, I remember those, remember those days that every day was something new. And of course, at the beginning, really not knowing anything about it. It's like, is how pathogenic is it? How is it really that contagious or not? Or how, what are, what are the long-term consequences of infection? When are we going to get the vaccine? It must have been, yeah, such an exciting in a way, exhausting in another way. And, but, uh, you clearly, you, your lab did help our understanding of this disease. And I want to talk about that a little bit more. More, and also your studies on mucosal immunity, also some of your studies on kind of long COVID and the long-term consequences of COVID particularly on the immune system. But I just want to quickly touch upon something very, also very now, because at this moment we're still, of course, struggling with COVID COVID is not gone and there's new, this new strains that seem to also be able to evade immunity. But there's also now a resurgence of other viruses that we had a little bit forgotten about in the whole, uh, 2020, 2021, which is influenza and RSV, that often you don't think a lot about these viruses in general. Um, I am very excited to talk to you and ask you, what is your read on the current kind of tripledemic? Is it as bad as we're thinking? And how does this, from your knowledge, is there anything out of the ordinary that we should be paying attention? And does this relate to the previous COVID pandemic?

Yeah, that's very interesting. Right now, we saw this like a surge of 3 viruses, essentially RSV. Normally, we particularly for adults, we handle it very well. Not a major problem. But, you know, RSV and the flu infection are always there. You know, every year we do have epidemic for flu and RSV, particularly in the wintertime. And flu this year came a little bit earlier. So why this happened? That's very good question. We don't know why, whether this is related to, you know, in the past couple years, we kind of through masking, social distancing, essentially maybe the society are not exposed to flu or RSV. And maybe we could have some like a waned immunity. As a general in the society. And now with people getting socialized together and less people wearing masks, could it be the virus start— we have suddenly become more vulnerable to the virus. But of course, because flu keep mutating, we in some years maybe flu just are better in evading our pre-existing immunity, they're going to go better. The good thing is usually the U.S. flu epidemic following the Australia curve. The Australia curve this year also seems initially started with like a bad flu season, but then seems quiet down very quickly. So it'll be interesting to observe whether the U.S. flu incidents are going to quiet down in somehow earlier than other years this year.

Yeah.

RSV, it's interesting question. I guess, you know, again, you know, RSV always are there. And I feel, you know, since COVID we kind of the society probably a little bit more cautious with all the viral infections spreading. Maybe we are just more aware of it. And then on top of that, maybe particularly when we suddenly getting socialized, getting together again, it's really— and as I said, because of the waning immunity, the RSV could start to really spread more than the other years.

Some people have suggested that part, in a similar way to measles, COVID can also result in a battered immune system with reduced counts of CD8 cells or naive CD8 cells in blood, kind of this aging of the immune system. I think maybe in the— I would like to ask you about that in the larger context of what are the sequelae from a COVID infection? What are the things that you're looking into that are particularly problematic? And whether there is any support for this idea that there's some specific reduction in the immune response in the kind of the potential of the immune system to react to other diseases afterwards?

Yeah. So, that's a good question. People, you know, have all these different ideas. You know, when the— this acute severe infection, you know, definitely really is a big challenge to our body's immune system. Our study mainly focusing on the lung per se. So, you know, that idea early on saying maybe COVID could be, you know, immunosuppressive because it's really reduced the blood lymphocyte count. That notion has been challenged a little bit, particularly because if we look at it, you know, in the tissue, the T cells seem rarely— because of the inflammation in the tissue. Presumably, the T cells have a preferential migration to the tissue and may partly explain why there's a diminished lymphocyte count. And also, at the same time, Because of the infection inflammation, we're probably going to have more acute myelopoiesis and, you know, generating more monocytes and neutrophils, which may also contribute to, you know, decrease of lymphocyte percentage in the blood. So, some of these effects can last, you know, really long, even after virus being clear both in human as well as in animal models. And these, you know, altered immune response may contribute to some chronic symptoms in patients. And nowadays, we can also observe in animal models. So, whether this COVID is going to induce, like, immunosuppressive— long-term immunosuppressive— actually, causing increased infection of other virus like influenza or RSV could somehow explain why we have these surges of two other respiratory viruses. It's a good question. Personally, I would believe maybe the effect could be moderate. I cannot imagine it would have like huge effect on, you know, after recover from COVID would have a huge, you know, really set up the susceptibility of the host to flu or RSV infection or other viral infection. But it's a good question. I don't think anybody and have a clear answer when you look at this.

So one of the things you study is the sequelae, or long-term consequences, of viral infection. And long COVID has been something people talk about a lot, but I've seen contradictory papers on this to an extent about how much it's a phenomenon specific to COVID and how much of it's a phenomenon that's actually much more common in a lot of viral infections, and everyone's just paying a hell of a lot of attention to COVID these days, and so we all think about it there. So, I wonder if you could comment on that some. And in particular, is Long COVID really unique to COVID, or is there more something that's true for a lot of viruses we're paying attention to today?

Yeah, that's a good topic. So, for me, I do— first, I do believe, you know, many acute infections could lead to long-term consequences. And this has been, you know, investigated before. Not get a lot of public attention. For, you know, some of those systemic infections definitely can do that. Specifically for respiratory viral infection, you know, like flu infection or prior MERS, SARS-CoV-1, even RSV infection have all been recognized that can lead to long-term consequences to the host, either through damaged tissue or through some kind of chronic inflammatory response in the host could cause some symptoms to the host. So, first, I believe some of these non-COVID symptoms may not be unique to non-COVID. A second part. So, some of them may be unique because unlike flu or RSV, usually viral replication are very much restricted to the lung. You know, we know now COVID, SARS-CoV-2 virus can potentially spread to other organs, particularly in the gut. And this infection in other organs may also causing damage or, you know, leaves some viral remnant or antigen there, chronically stimulate our body's immune response or inflammation, may contribute to, like, a multi-organ or systemic effect. Normally, like, a flu infection or RSV infection seems to lead to a long-term effect more often in the pulmonary respiratory tract. But, you know, with long COVID, we Nowadays, we noticed essentially this, a lot of our systemic bed and also chronic sequelae in actual pulmonary organs. So, in short, I believe first, you know, meaning a killing infection can cause chronic sequelae. And second, there may be some unique property of this non-COVID or unique etiology of non— and symptoms of non-COVID.

I think that's really so important to understand. I particularly am very curious about the kind of the tropism of the virus, whether this idea that COVID is more systemic than other respiratory viruses that we're used to before. And how that really affects our understanding of the chronic disease that they can leave behind. When it comes to the lungs, um, what do we understand about the mucosal immunity that we get? You also have, uh, you also studied what kind of mucosal immunity we can get from vaccination and how protective that is against newer strains that come out. Maybe do you want to maybe comment a little bit about that, about this publication? I think it was very interesting regarding mucosal immunity against SARS-CoV-2.

Yes. So, yeah, we get into these. Firstly, you know, right now we have lots of study on in terms of really established both the viral infection or vaccination, like particularly mRNA vaccination, could induce a pretty robust immune response in the blood, in the circulation. We get into this topic at the time we were thinking maybe there's a knowledge gap in this because ultimately COVID spread, or SARS-CoV-2 virus spreads through the respiratory tract. We In order to really assess how pre-existing immunity induced by either infection or vaccination in protection against later contraction with, you know, second exposure of SARS-CoV-2, we wouldn't need to really assess the mucosal immunity. And in my lab, mainly interested in the cellular immunity, the T-cell response and the B-cell response. So, we saw that, you know, in order to study the mucosal cellular immunity, we really have to directly assess the lower respiratory tract and the lung so we can get enough cells to study that. So, what we found— so, we What we decided to do is we want to do bronchoalveolar lavage. And to obtain the fluid and the cell component from the lower respiratory tract and the lung of infected individual as well as vaccinated individual too. And then we also get the blood. Samples to try to compare side by side how infection and vaccination going to induce this differential circulating and mucosal immunity. What we found is that— so, the vaccination, particularly mRNA vaccination, rarely induce strong circulating immunity, which is demonstrated by many other groups. However, this vaccination seems does not induce a strong mucosal immunity. We call it a suboptimal mucosal immunity. Essentially, we can find this spike-specific IgG response in the lung. But not IgA. And also vaccination really induced very moderate or sometimes minimum cellular immunity, the T-cell and B-cell response in the lung. So, we conclude that unlike prior infection, which can induce robust both arms of the adaptive immune, both the cellular and the humoral immunity, particularly IgA response. This vaccination really induced only an IgG response, which presumably comes from the blood, but not IgA. So, IgA is usually produced at the site of infection or at the mucosal site. And also does not generate a good B-cell and T-cell response at the site of viral entry. So, we conclude vaccination alone, intramuscular vaccine, current vaccination alone, probably does not induce strong mucosal immunity, therefore cannot stop virus from entry.

Yeah. I think we've discussed in this podcast a lot about nasal vaccination, or so I guess that you probably are also a supporter of a nasal priming, for example, strategy to push up those mucosal immunology or those, yeah, specific mucosal numbers of T cells or B cells or a particular Ig subtypes in patients with, or people treated with maybe a different type of vaccination?

Yeah, definitely. We think the ultimate, you know, the best vaccine, if we can develop a mucosal one, if we can have an ideal mucosal vaccine, can induce a very strong IgA response as well as strong mucosal resident memory B cell and resident memory T cell response, we could ideally— once the virus— once the host contracted the virus, we could theoretically immediately block the virus infection to achieve this sterile immunity and sterile protection against viral entry. Regarding the IgA response, you know, some clinical studies start to emerging that the mucosal IgA response seems is a better prediction against the breakthrough Omicron infection, but not systemic IgG or system mucosal, even mucosal IgG response. Somehow, the IgA in the mucosal surface is more protective against those variant of concerns than the IgG response, either in the mucosal surface or in circulation. So, I would be a strong supporter to, you know, have a vaccine can induce very good mucosal IgA and cellular response.

[Speaker:JOSHUA_ELKINGTON] So, we've covered several papers that show that you know, the system of like a primary series that's— that typically is one of these mRNA vaccines, and then they do a booster that's nasal, and that's antigen, or antigen coupled with, um, nanoparticles, or a lipid particle mRNA. The different papers have done different things, but they all show this massive— you kind of actually want the priming from an IgG response to then help with the IgA later. And then these nasal vaccines go in and then give you that IgM mucosal response. So it seems like everyone's pointing the same direction, right? You're saying it, all these papers are showing it, there's tons of mouse data this works really well. Why haven't we already done this in people to this point? We popped out mRNA vaccines very rapidly. Why not just get the nasal thing going and get this as a booster in 2024 or 2023 even? Do you know at all why there's a holdup on taking this step to a mucosal vaccine?

Yeah, that's a good question. You know, so far the animal studies really suggesting mucosal vaccine or mucosa, particularly mucosal booster. We, yeah, we do think maybe with mRNA priming in the periphery and mucosal booster probably is a better strategy in terms of induction of immunity, as well as supported by other people's work. In terms of why we still haven't got there, I think there's a number of reasons. First, so right now, we just don't have, you know, many mucosal vaccines on the market. People are kind of skeptical. With the live attenuated influenza vaccine, that's the only, like, vaccine on the U.S. market. It worked well in children, but not working very well in the adult, presumably because of the adults already have a preexisting immunity against the live attenuated viral infection. So, the virus don't infect very well. Therefore, it does not induce a very good genomic immunity. And therefore, the protection would not be good in the adult. So, this may scare some people in terms of, you know, this not so successful mucosal vaccine on the market to majority of the individuals. The other thing that, you know, there are many also practical issues, I guess people may question. So, if we have already have a good vaccination on the, you know, current mRNA vaccine on the market, which is to protect us from severe disease, even though it does not provide so good protection against, you know, mild infection. Or spread of the— does not, you know, really generate this so-called herd immunity to really cease the infection spreading. But, you know, people still get protection from hospitalization or from death. Maybe we don't need a mucosal vaccine. You know, there's some other, you know, practical reasons. For instance, you know, how are we going to— in order to develop a mucosal vaccine, we need to really— because a current assessed, you know, vaccine efficacy is sometimes, you know, through indirect assessment of blood antibody response. So, in order to assess the efficiency of mucosal vaccine, we need to assess the mucosal immunity. So, in human, it's just been harder to do. And also, you know, with different delivery strategies, with potential safety concerns, I guess these all being the potential hurdle or challenges of mucosal vaccine. So that being said, we do have like two adenovirus-based mucosal vaccine approved against SARS-CoV-2 in India and in China. And particularly with the data I saw in China, it seems the adenovirus-based mucosal both vaccines seems to generate a very good immune response in both systemically as well as in the mucosal surface. So, you know, we still don't know the real-world efficacy of that vaccine, but maybe that would give us a lot of information whether this route in a mucosal delivery of a vaccine would be really a promising route., or not.

Um, that's very interesting. I didn't know that there were already approved mucosal vaccines in India and China. Uh, I guess in the case of China in particular, they probably could use, say, um, a booster of a, a vaccine to help out their vaccination, uh, the vaccination percentages. I hope that they, if it works, that they maybe— is it easier when it comes to like Rolling out a nasal vaccination, is it something easier to do logistically, do you think, than having to inject? Have people like that are trained to apply injections? Would that have also a result in a different way of administering this vaccine?

That's an interesting question. You know, first of all, I have to Make a disclosure, I'm an immunologist.

I'm not an expert.

Yeah, of course. Or this logistic. You know, we do know some people are afraid of needle injection. So maybe a mucosal delivery or nasal spray, mucosal delivery of vaccine could be good for those people who are afraid of needles. But, you know, also have some challenges in terms of the mucosal vaccine. So, for a needle injection, we can give people exactly the same amount of antigen or the dose of mRNA. For mucosal delivery, either nasal spray or inhalation or nasal drops, one problem is it's hard to control the dose, right? And some people, maybe if we have nasal drops, some people maybe just don't take it. And nasal spray, Yeah. So, that's also some practical issues. How are we going to standardize it? You know, everybody getting the same amount of antigen could be a potential issue.

But—

So, sorry. So, going back to what you're— I think a better topic of discussion, and also continuing on the research that your lab does, There's also, I think also what I find very interesting is what it, our understanding is on the relationship between age of a patient and the kind of the age of the immune system of a patient and how this patient responds to respiratory diseases and the potential for long-term, long-term consequences of infection. So maybe that's also something very interesting. We do know that as We age, our immune system changes quite dramatically, particularly certain effector T-cell subsets that are becoming more and more prevalent when we measure at least in blood. But I assume that this would also be the case in mucosal surfaces. So what do we understand about the role of aging on our response to mucosal diseases or mucosal infections?

Yeah. So, aging is a major risk factor for, you know, mucosal infections. You know, as you just alluded to, you know, aging, you know, strikes so many challenges to the immune system. And we have immune senescence. Generally, it's just not so good to generate like a de novo immune response against a pathogen, both in terms of innate as well as adaptive immunity against viral infection. So, in general, what we observed, both like in animal models as in human. So, in aged host, it really takes time to generate a good antiviral immune response. So, the antiviral immune response is just not so speedy. So, that caused a problem to restrain the virus. So, virus could replicate higher and spread more to the organs, so causing more injury. On the other hand, aging, once the— it'll take time to generate a good immune response. Once the immune response generated in young, Individuals, once the virus has been contained, we want this immune response to be downregulated because we want to shut off the immune response to potential dangerous and to resolve the inflammation and repair the injured tissue. In aging, once the response has been generated, it's also taken longer time for them to— for the inflammation or immune response to resolve.. And so, the tissue can heal. So, aging just not induce a very speedy and a good response and also cannot shut off the response timely. So, would causing more injuries. And the tissue may be not heal very well. And then this could lead to some chronic sequelae of the tissue and our entire body and may lead to the chronic sequelae.

See, Brenda, it's not fun getting old. I was about to make that same comment, but I thought it would be too ageist of me, but I'm glad you did. So take care of yourself, Jason.

Take care of yourself.

No more infections. With those two kids you have, hard.

Oh God. Yeah, little petri dishes. Yeah. All right, well, we could talk for quite a while, but we, we are limited on the time that we're supposed to just hang out. And I know we could probably talk viruses forever, uh, but that being said, we always like to wrap up the podcast by asking kind of a fun question, get to know something about you other than your views on virology and immunity. And so that being said, If you could answer any single scientific question, regardless of your expertise or your field, if you could just get this answered, what would the question be?

What would it be and why? Yeah. So I, if I can choose another thing to do in the term of science, I feel I really want to do synthetic biology. I feel it's really cool to create a like a cell or organism from scratch, you know, kind of, you know, act like a god to really create something. That would be really cool and fun to do. Of course, I know, you know, the entire field are not there yet. That's, it's challenging, but that's why it's interesting to do.

I guess the field is really open for the big questions of what makes cells life. But I mean, they have made some, there are some like minimal genomes and they synthesize them from scratch and they put them inside some kind of empty cell or something.

Well, we're getting there. There's also, I think, self-replicating ribosomes to an extent now, RNAs that'll self-replicate. But then doing that de novo is the question. So we can make it so then if you put it in primordial soup, it'll make more of itself even outside of a cell.

Fun. So it was just starting the, the whole ribozyme world.

Well, right. They think that's the first enzyme, right? And that's the first sign of life as a ribosome that could replicate itself. They think, they think, okay. Because that, that's the minimum. That's the smallest unit. Yeah. Is something that can make more of itself that is a something.

Well, I also like synthetic biology, but I think I like it more from the from the point of view of like simplifying, uh, the biology through synthetic programs and like these people that do these kind of circuits, uh, analogous to, to like electrical circuits, but with signaling and things like that. And they have these gates and like, like by using logic and then if not gates. And that's really fun because it's like a little software thing, but it's actually in a cell that lives.

But I just like that. So one day we'll have computers in us, and we'll also have biological computers, and then the entire world will just be The Matrix. Well, thank you very much for coming on and sharing with us. Uh, do you have any open positions for postdocs or grad students you want to advertise here before we get going?

Um, yeah, maybe for a postdoc position. Yeah, um, as you guys probably heard Nowadays, it's really harder to recruit the postdocs. So we're kind of constantly recruiting postdocs. It's, you know, that's like a challenge for almost every colleague I know.

I've heard that. Apparently, it's because evil businesses like mine keep hiring all the people.

I was about to say, Jason is the reason. He is the example. Well, then postdocs alert. And I hope that you get to find the right colleagues in this environment.

All right. Well, thank you again for coming on.

Thank you so much. That brings us to the end of our show. Don't forget to subscribe to our newsletter at www.immunologypodcast.com to get the show notes, including an episode summary and links to all of the interview and roundup papers. You can also reach out to us on Twitter @immunopodcast or via email at info@immunologypodcast.com. With feedback, or maybe if you want to suggest a guest. See you next time.