Scientist Stories: Akiko Iwasaki, Immunology and Antibodies

Hello, I'm Richard Young, your host with Facundo Batista and Lena Fayn for MIT's course on COVID-19, SARS-CoV-2, and the Pandemic. We have evolved an immune system that throughout history has seen many pathogens, but not SARS-CoV-2. We're learning that patients with COVID-19, the disease produced by this novel coronavirus, have numerous shared symptoms, but there can be striking differences in clinical disease from one patient to another. What's responsible for the heterogeneity in this disease? Today we have the pleasure of hearing from Akiko Iwasaki, who is one of the world's experts in viral immunity on this topic of clinical disease heterogeneity and how the immune system plays a role in this. Dr. Iwasaki is the Waldemar von Zedwitz Professor in the Department of Immunobiology and a professor in the Department of Molecular, Cellular, and Developmental Biology at Yale University. She's also a principal investigator at the Howard Hughes Medical Institute. Akiko, thank you for joining us today.

Thank you very much, Dr. Young, for the kind introduction. I'm very happy and excited to tell you about the heterogeneity of COVID-19 COVID and whether immune system has something to do with that. So, I'm going to share my slides. So, today I'd like to talk about immune response to SARS-CoV-2 from the point of view of human immune response to this virus and what, what difference in immune responses we see depending on the demographic factors. Sorry. So SARS-CoV-2, by now you know very well, is a member of the coronavirus family. And we also have related coronaviruses that cause the common cold, the regular human coronaviruses that are circulating all the time, but they cause seasonal infections. And there are those zoonotic coronaviruses, including SARS-CoV-2, cause much more serious infections and disease. And but all of these viruses have in common structures like this where the spike protein of the virus is what's needed to enter the host cells by binding to host receptor. And this is also the target of a lot of antibody and T cell immune responses. And there is inside this envelope is the the nucleocapsid protein that coats the viral genomic RNA, and it has a rather large genome of over 30 kilobases of RNA. And then there are matrix proteins and envelope proteins that also become target of immune detection. As with all viruses, SARS-CoV-2 hijacks the hosting host cells to replicate itself and to infect other cells. And so this SARS-CoV-2 spike protein binds to human ACE2 and allows entry into the cell, injection of genomic RNA, which can produce both viral proteins and be used to produce more of those genomic copies that will eventually become enveloped again to infect other cell types. The ACE2 is expressed by multiple different cell types, many of the respiratory epithelium as well as intestinal epithelium, have the expression of ACE2, as well as other cells like neurons. So as of today, what do we know? Well, since the pandemic began, enormous amount of research has been focused on understanding SARS-CoV-2 and its infection and disease. Many studies have reported that antibodies to SARS-CoV-2 wane within 3 to 4 months, but there are other reports showing that this is much more long-lived. T-cell immunity also is generated and are maintained in most patients who recover from SARS-CoV-2. And people who have recovered from the common cold coronaviruses do get reinfected with the same virus within the year. So it is possible that with the SARS-CoV-2 as well, there may be reinfections. And we have We have already seen some rare cases, extremely rare cases of reinfections that have been reported throughout the world. And these reports keep coming out. But there are really no clear patterns as to what predisposes someone to reinfection at this time. The symptoms are either better or worse the second time around. Again, not really clear what dictates the outcome of the reinfection. And vaccines are needed to achieve herd immunity that would ultimately provide a population-based protection against this infection. And just yesterday, we heard from Moderna about the incredible efficacy in the interim results that they've press released, shared, which is 94.5%. And there is also last week Pfizer reporting over 90% efficacy. So these are just amazing news for immunologists and for everyone in general that these mRNA-based vaccines, which have never been used in humans before, have such amazing efficacy to date. And obviously we'll have to see more numbers to be reported, but these are very good interim reports thus far. So before I get into the SARS-CoV-2 infection, I wanted to touch on the seasonality of respiratory infections. When we think about these respiratory viruses, many of them have very strict seasonality, mostly kind of ramping up in the beginning of the winter and really peaking in cold winter months. And we began to address why this might be. And so looking at the seasonal factors that are, you know, such as temperature, humidity, and sunlight, whether they have any impact on the host immune responses. And there's evidence coming out showing that indeed many of these things do impact the host in addition to virus. So as you may not appreciate, that 90% of our lives are spent indoors, and this is really the indoor environment that matters with respect to transmission and disease. And so if you look at the indoor environment that in the wintertime, what you see is that you have very low relative humidity because you're importing cold, dry air from the outside, heating it to 70 degrees. That allows basically drop of the relative humidity to below 40% sometimes down to 20% humidity. And what that does is it allows for the virus to become stable, so the viability of the virus is increased, as well as the airborne nature of the virus is maintained. So there's a lot more airborne virus at low humidity. In addition, we've shown recently that when you house mice in relative humidity of 20%, they're much more susceptible to influenza-mediated disease compared to those that were housed at 50% humidity. And this is because the dry air dries out the mucociliary clearance mechanism in the airways, allowing the virus to latch onto the epithelium and replicate there. And once that happens, humidity— low humidity also impacts innate immune defense such that the virus can no longer be controlled by the innate immune defense in the dry air.

Environment.

So in addition to the biophysical characteristics that are controlled by temperature and humidity, the host immune response is indeed quite affected by humidity. And a few years ago, we've also shown that temperature also controls innate immune response to rhinovirus, and that RIG-I dependent interferon response is impaired at the temperature that you would find in the nose during the winter months. So, just reducing the temperature to 33 degrees Celsius from 37 degrees Celsius core temperature, we see a drop in the innate immune defense. So, there's something to think about going into winter, you know, how do we deal with these infections and whether there are non-pharmacological interventions that we can provide ourselves to better withstand these types of infections. So getting into the topic of today's lecture, I wanted to kind of remind people that there's a large variety of disease outcomes from this one single virus. So you have many— most of the infections are asymptomatic to mild infections, and some people get moderate disease that requires hospitalization and severe disease leading to lethality. So there's a wide range of symptom outcomes. In addition, there is the difference in the duration of disease. Some people have acute disease and they recover completely, while others have long-term diseases, so-called long haulers, who are suffering from over 100 days of symptoms. Some, some of them are pretty severe from the virus.. And so what gives this kind of heterogeneity, and does the host immune response have to do with any of this? And so one of the, you know, things that we are looking at is the sort of the age of the patients, of the subject, people who are getting infected, as well as comorbidities, you know, obesity. Of course, aging is a very key factor that controls disease outcome. And so today I want to talk about, you know, what's the difference in immune response in moderate to severe disease and also discuss sex differences in immunity with this virus infection. And before I go on, I kind of wanted to touch on the fact that we also have an ongoing study where we are looking at potential neural invasion by this virus, and this is a autopsy samples from a person who died of COVID-19, whose frontal cortex actually has neurons that are infected with this virus. And you can see clearly these spike protein antigens expressed in the neurons. And so, in addition to inflammatory consequences of this infection, there may be direct infection of neurons and other cell types, such as cardiac myocyte and so on, that might result in direct damage to certain organs that are involved. And so when we think about antiviral defense, the first thing that the immune system does is really this innate immune defense, which is triggering of type 1 and type 3 interferons. And so every, pretty much every cell type in the body is equipped with innate sensors such as those indicated here, Toll-like receptors within the endosome that detect RNA, as well as RIG-I and MDA-5. These are sensors in the cytosol that detect RNA presence in the cytosol, and this recognition event will allow signaling to synthesize proteins such as type 1 and type 3 interferons, as well as cytokines. And once the interferons are secreted, they can bind to the receptor on the neighboring cells to trigger interferon-stimulated gene production. And there are hundreds of these interferon-stimulated genes that combined confer protection against further infection, and some of these ISGs are important to drive adaptive immune responses. And again, before I go on to the actual data discussion, I wanted to also point out that the immune system is complex in that a single naive T cell that interacts with antigen-presenting cells such as dendritic cell can adopt multiple different fates. So, for instance, naive T cells that are stimulated with dendritic cells that secrete these different first-order cytokines can be programmed to become T follicular helper cells that help B cells secrete antibodies. They can also become Th2 cells that are very important for anti-parasite immunity, and Th1 cells that are important for antiviral and antibacterial immunity, and Th17 cells that are important for antifungal defense. And then you can also have regulatory T cells to dampen down an excessive immune response. This all starts by the interaction of naive T cells with dendritic cells, and dendritic cells secrete these kinds of first-order cytokines depending on the, you know, pattern recognition, the pathogen-associated molecular patterns that they're detecting through their sensors. And so this is a very important link between innate recognition to adaptive immune responses.. And so what I'm going to show you today is that with COVID we're seeing a very mixed misfiring of the immune response that basically belong to all of these categories. So in March, we began to study human immune response to SARS-CoV-2, and this began as a collective collaborative effort between multiple laboratories. Over 100 people are involved in these studies. This was initiated by the biorepository creation by Dr. Albert Ko in the School of Public Health, where he and, like I said, a large number of people contributed to collecting samples from patients. And we had set up 3 cohorts. One was the inpatient cohort, in which we recruited people with COVID coming into Yale New Haven Hospital, and we, we collected various biospecimens throughout the time course of their stay in the hospital. This allowed us to look at longitudinal immune responses to SARS-CoV-2. We also had this second cohort of outpatients which have milder disease who didn't require hospitalization but nevertheless were COVID-positive, and we also are collecting samples from this cohort. And third cohort is the healthcare worker group, which we are collecting, you know, again, biospecimen from these volunteers who provided a critical control to the other cohorts that are indicated here, as well as we were able to detect, you know, in real time the COVID PCR positive samples from the nasopharyngeal and saliva from this healthcare worker group, which allowed us to alert them to the status and for them to seek proper clinical care, as well as isolating themselves from the patients. So this served a multipurpose biorepository group.. And this is called the impact group. So what we did was we collected, like I said, many different biospecimens. Today I'm going to talk about mostly the peripheral blood mononuclear cells from these patients that were admitted to the hospital. And we have done some flow cytometry to analyze what kinds of cells are in these peripheral blood and what can we learn from both the cytokines and T cells about the status of their disease. And so the study design is to recruit patients that have different clinical scores into this study where we gave clinical scores based on the supplemental oxygen that the patients required during hospital. So all the way from none being score of 1 to mechanical ventilation score of 5 and death score of 6. And so we were able to study 80 moderate disease patients and 33 severe disease patients and 108, um, uninfected healthcare workers. So one of the first things that we began to notice is that the T cells were really decreased in patients who had COVID. And so this is like looking at T cell in the peripheral blood that goes from this— these are the healthcare workers that go from this level to here in moderate, which are the navy, and the severe, which are the pink labels. And conversely, we saw increases in monocytes with disease, as well as increase in these low-density neutrophils that come into the peripheral blood mononuclear cell prep, as well as eosinophils, which are very strange because Eosinophils are important in helminth infection, like worm infection. But we are starting to see these types of cells coming up in patients with COVID And there are all kinds of cytokines that are elevated, including IL-1, IL-6, interferon alpha. This is the antiviral cytokine that I talked about, as well as many, many chemokines. And so So, just to sum up what we found overall is that, so, if you look at the healthy PBMC, these are the kinds of compositions that you might typically find. However, in the COVID PBMC, we were starting to see very reduced T cell percentages as well as increase in monocytes, neutrophils, and eosinophils. And there's also a drop in dendritic cells that may interfere with adaptive immune responses. And so what's different about the moderate and severe patients? Turns out that one of the things that are different about these patients is their viral load. So the severe patients were unable to control the viral load over time compared to the patients who were moderate, had moderate disease that eventually cleared the virus in 20 days post-symptom onset. So, the severity is associated with the failure to control viral load from the nasopharynx of these patients. And what was interesting is that these antiviral cytokines that are supposed to control the virus are actually elevated as a result of increase in viral load. So, on the x-axis, I'm showing you the viral load in the nasopharynx in log scale here. And then on Y-axis is the interferon levels, and you see there is a positive correlation between viral load and interferon alpha, interferon gamma, which is a type 2 interferon, which is also antiviral, but instead of controlling the virus, the virus is driving these types of interferons. And similarly, we see inflammatory cytokines like TNF and TRAIL come up as a result of increasing viral load. And we took this a step further and asked, were, you know, are some of these interferon levels correlated with mortality? And so what we did is we took the levels of cytokines in the plasma from patients and asked, uh, what were the levels of these cytokines within the first 12 days of symptom onset in patients who died of this disease, which are these pink and those who were discharged from the hospital live. And so what we saw was that in the deceased patients, their levels of interferons were indeed higher in the first 12 days of symptom onset compared to those that were discharged from the hospital. And similarly, IL-1 receptor antagonist, which is indicative of IL-1 receptor signaling, were also higher in the discharged patients compared to those that died. And there were some other cytokines that also had trended to higher levels in the deceased group. So there seems to be this cytokine misfiring that's occurring in severe COVID patients. So in addition to the interferon that I showed you, there are these other types of cytokines as well as cell types that keep going up in severe patients, which are pink lines here, compared to those that have moderate disease. So everything from type 1 immune response to type 2 anti-helminth immune response to type 3 antifungal immune response, everything is becoming elevated or at least maintained highly in the COVID patients who had severe disease.. And things like IgE, which we normally don't see at all in healthy people unless you have allergy, are also— were also coming up in the severe patients. So this was a very unexpected feature of COVID is that we are seeing essentially every type of immune response being elevated. And so what do these mean? Well, we've done some unsupervised clustering analysis to which revealed 3 clusters of patients. The first cluster of patients, so the darker the color, the higher levels of cytokines they have in their blood. The first cluster of patients had enrichment in the, in the signature A, which contains many tissue repair growth factors such as PDGF, EGF, VEGF, IL-7, which is a growth factor for lymphocytes, and so on. The second cluster of patients had in addition an enrichment in this second signature, which mostly consisted of chemokines, even though there's IgE and other things in here, as well as this mixed cytokine responses that had type 1, 2, and 3. And the third cluster of patients had the worst— I'm sorry, the highest levels of type 2, 3, and mixed cytokines. In their blood. And so what is different about these clusters of patients? Well, their age isn't significantly different between the three clusters, even though there may be a slightly elevated median age for the second and third clusters. Length of hospital stay was also not dramatically different between these clusters. However, what was different was their disease trajectories. So, the first cluster of patients that had mostly, mostly these tissue repair growth factors, they were able to control disease and were, were able to recover from infection, whereas the second and third clusters of patients, they developed worse disease and some of them died of this infection.. And there was more increase in coagulopathy that was measured also. So, just by this unsupervised clustering analysis, it basically segregated patients into those that recover in the first cluster, which have tissue repair proteins in their blood, and then second and third clusters who go on to develop worse disease. So we took all of these measurements and asked for biomarkers for COVID-19 mortality. And this revealed that the worst sort of, or the most strongly associated biomarker for mortality was IL-18, which is interesting because IL-18 is a cytokine that's released as a result of inflammasome activation. Inflammasomes are these intracellular complex that forms in response to different types of infections. And it sort of hints at the fact that these inflammasomes may be triggered highly in the patients that have died of this infection. And second in line is the interferon alpha, which is an antiviral cytokine. As I mentioned, it's supposed to control viral infection, but instead what we're seeing is that it's highly associated with mortality.. And you can go down the list all the way to the most protective factors such as CD4 T cells activated. And so we are starting to see patterns of immune responses that correlate with mortality versus those that are correlated with protection. So to conclude this part of the talk, what I've shown you is that the moderate versus severe patients, what may distinguish these are the persistent viral load that's found in the severe cases. And the viral load is what seems to be driving the interferon response and not the other way around. Increase in this interferon alpha as well as some of these inflammatory cytokines within the first 12 days of symptom onset are predictive of mortality, and that there are 4 cytokine signatures that can distinguish patients who recover versus who develop worse disease. And finally, in patients who develop worse disease, there are these, all these types of cytokines and cell types being engaged. And it's likely a maladaptive response. We don't know why these factors are engaged all at once and/or how. So, just to kind of pause a little bit and think about what we've learned so far, this is a textbook, you know, example of a good immune response. If you have a viral load that's coming up, but you also have rapid onset of large amount of interferon response, this is supposed to protect us. There is rapid viral clearance and mild disease. So this may be happening in young people, or perhaps in children, or in cases where you have very low viral exposure. What we're seeing in the hospitalized patient may be this scenario in which there is interferon coming up, but it may be delayed with respect to the viral load. By the way, what we're measuring in hospital is really after the virus has peaked, so probably timeline after this time point. So we don't know what happened in the first few days of infection because no one has symptoms at that time. But just sort of inferring from what we found here, it's probably what's going on in the patients that we see, um, or this delayed interferon scenario in which the interferon is induced but it's too late or too little to control the infection early on, leading to severe disease. And this may be what's happening in older adults or high viral exposure settings.. And then there is this other extreme of interferon deficiency in which either you have genetic mutations in interferon pathways or neutralizing antibodies to interferon. These are two studies in Science that was published by Jean-Laurent Casanova's group, which showed that there are people who really lack or very low levels of interferon. And in these cases, you have like virtually no interferon, virus can replicate to high titers, and that might cause severe to life-threatening disease. However, we can counter these things by, I think, and this hasn't been proven yet, but by early treatment or even pre-exposure prophylaxis with interferon or interferon-inducing agent. And what that does is to quickly supply the interferon that's necessary to squash the viral load early. And if this can be achieved early enough, I think we'll have a viral clearance and milder disease. Now, all of these things are just hypotheses at this point, but it's important to draw, um, some of these hypotheses early on so that we can design a better clinical trial for, um, going forward for prevention or treatment of disease. So, in the remaining time, I'd like to speak about sex differences in immune response. So, from very early on, we've started seeing that male sex is a risk factor for severe disease with COVID-19, as well as mortality. And this was reported from China, Europe, US, and many other places where there was sex differences that were being reported for hospitalization and mortality. And so this is not the first time there have been reports of sex disparity in infectious diseases. This is a table that is from Dr. Sabra Klein's group who did a review on this topic, showing that there are some infectious diseases in which there is male prevalence, male dominant prevalence or incidence, and also there are male or female dominant severity in infectious diseases. And what's interesting to know is that MERS-CoV, the sort of related beta coronavirus to SARS-CoV-2, has both increased male prevalence as well as increased male severity. And there are obviously, you know, other viruses that have female dominance in severity as well. So why would this be? We wanted to understand if there's any immunological component to this sex difference. And so what we did is we designed a study in which we will interrogate the sex differences in immunity, and we focused on the baseline analysis, which means that we we analyzed patients' immune responses who were not in the ICU and who had not been treated with any immunomodulatory agents such as tocilizumab or high-dose corticosteroids prior to the sampling. And this was important to us because we wanted to understand the natural immune responses that occur in male and female patients that was not affected by any of these immunomodulatory treatments. And so, in this cohort, we had 39 patients, 17 men and 22 women, and we followed these patients over time to see what happens. We obviously also had some healthy healthcare worker controls, as well as we had extended this analysis to a larger cohort with all stages of disease. So what we found, first of all, is that there was no statistical difference between the viral load in the NP swab or the saliva in the male or the female, a male versus the female patients. So the male patients are in orange and female are in purple. And also we did not see from this first sampling any difference in IgG against spike 1 or IgM against spike 1 between male and female. We also saw many different chemokines and cytokines that were elevated in both sexes, such as CCL5 shown here and CXCL10. You see this dramatic increase in COVID patients of these types of chemokines, but there were no difference between male and female patients. However, we did see a couple of innate immune factors such as IL-8, which is important for recruitment of neutrophils, and IL-18, as I mentioned, the top mortality-associated biomarker from the longitudinal study, were elevated in the male patients compared to the female patients. And we also saw with respect to different cell types that were also elevated in the male patients, these inflammatory monocytes compared to the female patients. And what was interesting is that if we plotted the level of CCL5, one of the chemokines that attract these inflammatory monocytes, versus on the y-axis the non-classical or inflammatory monocyte percentage, we see a nice correlation positive correlation between these factors in only the male patients but not in the female patients. So, there appears to be a sex-dependent difference in the inflammatory monocyte, both the percentage as well as potentially their recruitment controlled by CCL5. And we, conversely, we saw elevated activated T cells in female patients. Patients. For instance, we saw increase in activated CD4 T cells, CD8 T cells, as well as terminally differentiated CD8 T cells in the female over the male patients. So, now that we have this baseline data, we asked if these data can inform us about future disease trajectory in these patients. So, what we now did is to divide the patients in both sexes into those who had stable disease, which are the open circles, and those that had progressed to worse disease, which are these filled circles. And what we saw is that only in the male, there is an increase in age of the patients who did worse with this disease, whereas we did not see such a difference in the female cohort. And similarly, with the BMI, was elevated in male patients who had deteriorated from this disease compared to female patients with no significant difference. And what was striking to us is that male patients, so I told you male patients in general, they don't develop very good activation of T cells, but those patients who did worse with disease had very little activation of T cell. These are sort of the healthy healthcare worker level of sort of quiescent level of T cell activation that we see, and male patients who had this very low level of T cell activation belonged to the worst disease group compared to the male patients who had stable disease. And this can be seen across the different types of analyses that we've done. Both the activation of T cells, differentiation to terminal differentiation of T cells, as well as interferon gamma secretion from T cells, were all reduced in male patients that had worse disease outcome, whereas there was no difference in the female counterpart. And conversely, however, we also detected some factors such as TRAIL, CCL5, and IL-15 that were elevated in the female female patients who did worse with this disease compared to the male patients in which there were no differences. And finally, what we found is that older men had lower T cell response. So this is looking at patients across different age on x-axis and the activation of CD8 T cell on the y-axis or interferon gamma from CD8 T cells on the y-axis. Axis. And what we saw is the age-dependent decline in male patients in their ability to stimulate these T cells, whereas there was no age-dependent decline in female. If anything, female, they continued to go up with respect to these T cell activation markers. So, to conclude this part of the lecture, what I've shown you is that we knew that men develop worse disease outcome from this infection. And what we saw was that there were two factors, IL-8 and IL-18, that was elevated in the male patients. And female patients developed better T cell immunity at the first baseline analysis. And male patients who developed very poor T cell immunity were the ones that developed worse disease.. And women who developed some of these inflammatory cytokines also were in the worst disease category. And finally, there was age and sex-dependent differences in T cell immunity in that T cell activation really declines in male patients with age but not so in female patients. So, kind of combining all the literature out there about sex differences in COVID infection and immunity, What we've found so far, combining with what I just told you, is that there may be a significant difference in both the entry receptor, the ACE2 expression, which is downregulated by estrogen, as well as some of these innate sensors such as TLR7 being higher in females, IRFs, the factors that drive these inflammatory and cytokine responses, also elevated in female, as well as some of the type 1 interferon were elevated in female. And there have been, as I mentioned earlier, neutralizing antibodies against type 1 interferon that was found mostly in older male. So this may also be different in, in between the sexes. And then I showed you that some of these inflammatory cytokines are elevated, IL- IL-8 and IL-18 in the male. And ultimately, in terms of adaptive immunity, females were able to stimulate activation of T cells at the first sampling time point, whereas other studies have shown that over time there are, there's elevated antibody in male patients compared to female patients, and that may reflect the disease severity. Immunity that we see in the male patients. So we're learning a lot about the— what differences there might be in infection and immunity between male and female. So I'm going to end here by thanking the amazing group of people that I have the privilege of working with. I already mentioned about the IMPACT team that is headed by Dr. Albert Ko, but it's a really close collaboration with others such as Saurabh Omar, who is the director of Yale Global Health Institute. Charles Delcruz and Shelly Farhadian are physician scientists who are really recruiting and working and treating these patients. Nathan Grubel is a molecular virologist who is tracking the viral mutations across all these patients. Craig Weiland, a virologist that we collaborate with, as well as Aaron Ring, who does a lot of these antibody analyses, which I didn't have time to talk about today. And these are the lab members that have contributed just enormously to not only the research that I talked about, but also implementing testing for PCR, especially in the early days of the pandemic. And these are the other people who contributed to the studies that I discussed today. And I'm I'm going to end here by thanking my amazing lab members who really stepped up to the challenge of doing this COVID-19 research. All of us were doing something else before the pandemic, and they really shifted their focus to studying patients' immune response as well as developing new mouse models to study coronavirus infection and immunity.

So, I'm going to end here and be happy to take questions. Thank you so much, Dr. Iwasaki. The students have a number of questions. One is, your findings about immune misfiring in severe cases, can this be leveraged to improve treatments for patients?

For example, with, you know, the excess type 1, 2, 3 cytokines, is this easy to monitor in the clinic? Right, so that's an interesting question because, you know, these biomarkers, we don't just want it to be an academic exercise. We want this to inform future treatments. So there are many, many clinical trials that are ongoing with respect to both treatment with interferon, recombinant interferons, as well as treatment with some of these JAK inhibitors that will interfere with the interferon signaling pathway. And so interestingly, Interestingly, you know, again, this timing of treatment really, really matters. My suspicion is that early or even prophylactic treatment with recombinant interferon will benefit people. And there have already been some early analyses from clinical trials showing that inhalation of interferon beta have a protective value. And then later on when these misfiring is occurring and there is— it's the disease is no longer driven by the virus but the immune system, that's when you want to, you know, treat with JAK inhibitors or some other interferon modulatory agent.

So I am looking forward to the results of these clinical trials. And Antonio Diaz has noted this seems like a promising approach, but have these interferons been used previously to treat or prevent other diseases?

Right, so interferon, interestingly, have been— recombinant interferon have been used for many different types of diseases, chronic hepatitis infections, as well as for multiple sclerosis. Interestingly, people are using interferon because some of these interferons, type 1 interferons, actually have impact on blood-brain barrier And fortifying that, for instance, you know, with interferon might be beneficial for the multiple sclerosis. So there's many different uses of interferon, and obviously we're sort of repurposing the interferon for COVID-19 right now.

But yes, there have been a lot of trials and safety data available. You mentioned that there were some unusual features to this immune response in some patients, for example, when eosinophils are detected. Are there other examples of pathogen infection where cell types meant to deal with one type of infection actually get induced for another type?

Right. So you can find many examples, you know, not this comprehensively, but there are many other examples in which, for instance, eosinophils have been seen the original SARS coronavirus mouse model, in some of the studies in older mice, they detected this, you know, type 2 inflammation, which was only in older mice and not in younger mice. So I think it's interesting to keep in mind that, you know, many of these misfiring features are seen in older people and Many of the patients that are very severely sick are in older group. So age, sex, and all these other factors do contribute to that.

But yeah, so there have been reports of eosinophil-mediated inflammation for SARS-CoV, the original SARS-CoV. You mentioned that older males have lower T-cell responses.

Are there other aspects of immunity that generally just decline with age? Right, so there have been many studies that have shown, like prior to COVID even, decline in adaptive immunity. There are fewer naive T cells to stimulate de novo immune responses. There's also a decline in type 1 interferon. So we've done this study in which we isolate monocytes from people over over 65 of age versus those that are 20 to 30-year-old. And the one of the biggest difference we see when we infect those monocytes with influenza is the decline in interferon, type 1 interferon, as well as type 3 interferon in these monocytes from older people. So innate defense is definitely being impaired with age.

We didn't see sex difference in that setting, but age definitely impacts innate and adaptive immunity. I guess women experience autoimmune diseases more frequently than men.

In your view, is that an indicator of a more robust immune system in females than in males? Right. So, there are many factors, both in terms of sex hormones as well as X chromosome-linked genes that are, you know, important immune genes that are on the X chromosome. I mentioned one of them, Toll-like receptor 7. But so, these are definitely elevated in female over male. And that may be predisposing women to autoimmune diseases like lupus. And at the same time maybe conferring some of this protective immunity against this virus infection. And what, what's interesting to point out, speaking of the heterogeneity of infection, is that a lot of these long hauler phenotypes that we're seeing are women of younger age, average age of 44. 80% are women. So the So this kind of hints at the possibility that long hauler diseases may be driven by an overt immune response and potentially even autoimmune disease. So we are kind of looking into that now to see if there's any link between long hauler phenotype to autoimmune disease or other inflammatory conditions.

Given sex differences in mortality and the observation that ACE2 receptors might be downregulated by estrogen.

Could hormone therapy be used to treat COVID-19? Right. So there are actually estrogen therapy that's ongoing. I don't know the results yet, but there are clinical trials already ongoing to test these hypotheses.

And so I look forward to what they find out. And more generally, Have sex differences with other diseases been used to change treatment strategies for men and women?

It should, but one of the things that I think we need to highlight is that the sex differences are not well studied. For instance, the vaccine studies or treatment with a variety of different drugs, there really aren't a great data set for sex differences because that's not something that people are studying. And I think it's very important to really include sex as a variable going forward so that we, we can have a better treatment for male versus female.

And on that important note, Kiko, I want to thank you again for presenting this and giving us new insights. Insight into how the immune system may be contributing to these patient-specific differences.

Thank you so much.

Thank you very much.