Scientist Stories: Eleanor Fish, Interferons as 1st Responders

Okay, we're just at about 10, so we'll go ahead and get started. Good morning, good afternoon, and good evening to everyone joining from around the world. My name is Sabrina Welch, and I'm the Director of Programs and Operations at the Human Vaccines Project., and I'll be your moderator for this webinar. The last week in the COVID vaccine space has been very interesting and gives us lots of reason to be hopeful that multiple COVID vaccines are going to be available in the next year. Both Pfizer and Moderna announced data from their Phase 3 trials in the past week, and Pfizer just yesterday announced that they completed their case accrual. In the Pfizer trial, the primary efficacy analysis demonstrates that the vaccine was 95% effective against COVID-19 beginning 28 days after the first dose. 170 confirmed cases of COVID-19 were evaluated, with 162 observed in the placebo group versus 8 in the vaccine group. Moderna's mRNA-1273 vaccine also appears to be protective at 95% based on the interim analysis, and we'll know more, I would think, in the next week or so once they reach full case accrual. Initially, both companies estimated that the efficacy data would be available in the beginning of next year, But because of the surge of cases in the U.S., both companies were able to recruit cases faster and get a quicker read on how effective the vaccine candidates are. Again, this is a sobering reminder of the current state of the pandemic, both here in the U.S. and globally. Today we'll hear from a researcher who's been studying SARS for years before the COVID-19 pandemic began, and most recently her lab is focused on investigating the therapeutic effectiveness of interferon treatment for Ebola virus disease and now SARS-CoV-2. Just a quick note before we begin. The information presented today includes some pre-published data that is currently under peer review. At the request of the author, we ask that you not reproduce or disseminate the data presented. This session will be recorded and available on our website and our social media channels for you to review. Today's speaker is Dr. Eleanor Fish. Dr. Fish is a fellow of the American Academy of Microbiologists and a fellow of the African Academy of Sciences. Dr. Fish has received many international awards acknowledging her scientific achievements and has published more than 170 peer-reviewed scientific papers in international journals.

Dr.

Fish studies the interactions of cytokines, specifically interferons and chemokines, with their host-pathogen interactions— oops, sorry— with receptors in normal and diseased tissues and cells. One focus of Dr. Fish's research is the investigation of host-pathogen interactions at the cellular and molecular level specifically in the context of viruses and interferons. Her research activities span basic science wet lab research to small animal models of disease and translating these findings to clinical utility. In today's lab meeting, Dr. Fish is going to talk about global virus outbreaks and using interferons as first responders. During the presentation, please send me your questions using the Q&A function in Zoom. I'll ask our speaker a broad selection of your questions after she's finished the presentation. We'll have about 25 minutes for discussion, and I hope you'll take the opportunity to ask your questions about the data presented today. It is now my pleasure to introduce Dr. Eleanor Fish from the University of Toronto. So welcome, Dr. Fish. Good morning. I'll stop sharing my screen. If you'd like to take over and show your slides, that would be great. And you are still on mute.

Just unmuting myself and I've got this glitch on my, uh, okay, got rid of that glitch. That's always fun. Yeah, I know, Macs have this thing now that suddenly your pointer can disappear. I have no idea why. Let me now proceed and, um, great, and we can see your pointer. Great, I'm just going to go to here and get rid of some of the stuff that I don't need to see. Bear with me, um, one more second. All right, I'm now happy to start, and thank you for inviting me to share some of the research that I've been involved with, specifically related to SARS coronaviruses. So what my topic this morning is going to focus on, SARS coronaviruses,. But what I wanted to at the outset indicate that the notion of pandemics is really not new to us. You can see from this particular slide there have been many outbreaks of many different virus infections that have spread around the globe and can be considered as both epidemics, outbreaks, and indeed pandemics. I've highlighted a number here, whether it be chikungunya virus, which I would suggest is something we should keep our eyes open for, West Nile virus, which we became aware of, which is now in the northern hemisphere, Zika virus, yellow fever virus. Most recently, we had Ebola virus in West Africa, a number of different virus outbreaks. Indeed, HIV can be considered a pandemic with more than 40 million people infected worldwide. But what we're going to focus on this morning are the coronaviruses, and certainly in 2002 we had SARS-CoV, which we've now called SARS-CoV-1, with over 8,000 cases worldwide and some 774 deaths. In 2012, we had Middle East Respiratory Syndrome, which has actually spread to a number of countries about 2,500 cases and 912 deaths. But most significantly, SARS-CoV-2 has had a massive footprint around the globe with over, with 56,728,000 cases as of today and some 1,357,809 deaths. So the footprint of SARS-CoV-2 is huge globally and certainly has permitted us to glean a lot of information about coronaviruses, how they infect, and look at potential options for targeting for interventions. So our first line of defense against any and all virus infections is our innate immune response, and those first 24 hours, those first days are absolutely critical and determine the outcome to that infection. And what I hope to convince you in the ensuing minutes is this is certainly dependent to a large extent on a type 1 interferon response. So as you are all aware, we have in place a nonspecific surveillance system of pathogen pattern recognition receptors that allow us to identify molecular patterns associated with pathogens, and whether whether these be Toll-like receptors on the surface of cells, in endosomal compartments, or helicases in cytoplasmic areas, such as the ones I've identified here, MDA5, RIG-I, or the NLRP system. There are a number of these pattern recognition receptors which allow us to recognize these molecular patterns, This invokes an incredible signaling cascade, and what I want to point out is that they all converge on the production of type 1 interferons, interferon alphas, and interferon beta. And why is that? It's because interferon alphas and interferon beta have receptors, IFNAR1 and IFNAR2, that are ubiquitously expressed. So every cell that has the potential to to be infected by a virus, and there are many— when one considers this, whether it's the respiratory tract, whether it's a cardiotropic virus, a hepatic virus, whether it's a virus that enters through the skin, pretty much every cell in our body has the potential to be infected by a virus. So it's important that those cells express receptors that will respond to a biologic that will trigger inhibition of that virus. So interferon receptors are ubiquitously expressed, on all cell types, and they invoke, once activated by the interferons, an incredible signaling cascade. Now, we've learned for many years about the JAK/STAT signaling cascade, but in collaboration with my colleague Leon Plattanius at Northwestern, we've elaborated and elucidated a number of other non-canonical signaling cascades associated with MAP kinases and the PA3 kinase pathway that are all involved to invoke that interferon response, which results initially in histone modification, then the alteration, the transcriptional activation of genes, and I've listed a number of here that we know are associated with an antiviral response, and at the same time, interferons will signal such that there's a change in mRNA translation, so the rapid production of those proteins that are involved in that antiviral response. So this scheme serves to illustrate that in addition to the direct antiviral response invoked by interferons, there are other responses. So that direct antiviral response, as I've mentioned, that invokes the transcriptional activation of a number of genes and their gene products will target many different facets of viral replication for all viruses, whether they be DNA viruses, RNA viruses, enveloped or not, and regardless of the cell type which they infect. So some of those gene products limit the ability of viruses to enter the plasma of the cell via the plasma membrane. They limit the ability viruses to uncoat their envelope. They will degrade the viral DNA or RNA, or they will prevent the assembly of proteins to form the virion, and they will also prevent the egress of viral progeny. At the same time, if the viral burden is huge, interferons will induce apoptosis of those cells. So there are direct antiviral effects which target many different stages of a viral replicative cycle. So as I've mentioned, they will target all viruses, and at the same time we have this ability of interferons to— which is often overlooked— their ability to activate an immune response. So interferons will induce the recruitment by a secretion of a number of chemokines at the site of infection of specific immune cell subsets that I've highlighted here, and they also will be involved in clearance of the viral infection, whether they be CD8-positive cytotoxic T cells, whether they be macrophages, natural killer cells, and neutrophils. They're all involved in adding to this viral clearance. And at the same time, we've learned that interferons, type 1 interferons, specifically interferon beta, is very important to the polarization and activation of B cells to become antibody secreting. So the point of this is to reiterate that type 1 interferons are very pleiotropic in the context of their direct antiviral effects and their abilities to recruit and activate immune cells to clear virus. And now this brings us to the heart of my lecture, which is talking about coronaviruses. And as I mentioned, in 2002, 2003 in Canada, we had this outbreak of SARS-CoV-1, which resulted at the end of the outbreak with some 8,098 people infected worldwide and some 700 74 deaths. At the time of the outbreak, there was no vaccines and no approved antivirals. In common with all the viruses that have co-evolved with us, SARS-CoV-1 encodes in its genome a number of factors that very specifically block an interferon response, and this again should inform us of the importance of interferons in viral clearance. And what I show you here are these factors: nonspecific protein 1 and 3, open reading frame 6, and the M protein. And they're involved differentially in inhibiting interferon production through IRF3 and IRF7 binding. They limit interferon mRNA translation, and certainly there's been some data to suggest that they will inhibit the downstream signaling from interferon binding to its receptor very specifically in the context of STAT1 phosphorylation and its nuclear translocation. So cognizant of the importance of interferon, certainly in the context of this virus which blocks its interferon production at the start of the outbreak, we undertook some in vitro studies to look at the ability of interferons and the treatment of choice, the drug of choice, ribavirin, that was being used. This is an in vitro study using human lung cells, and what I show you is that with increasing concentrations of ribavirin, there is absolutely no inhibition of virus. However, when we used a novel synthetic interferon, and this is interferon alpha con1, and it is a consensus of all the 14 interferon alpha subtypes,. So the interferon alphas are 165 amino acids. It's a protein, 165 amino acid proteins. So at each point, at each amino acid along that 165 amino acid stretch, we took the most frequently represented amino acid amongst the 14 and we generated a consensus interferon. Anecdotally, we've subsequently shown it's the most potent., and it's now available clinically and has been approved. So this shows you that with increasing doses of interferon alpha CON1 up to 5,000 international units per mL, we see good inhibition, in fact complete inhibition of virus, where up to 2,000 micrograms per mL of ribavirin, which is a massive dose, we see no inhibition of virus. So this prompted us to undertake a clinical study, not a randomized controlled trial, but a clinical study in one of the hospitals in Toronto during the outbreak. And the idea was to treat— the intent was to treat patients with interferon and compare that with standard of treatment at the time, which was corticosteroids. So patients had to be within 10 days of symptom onset. They had to have progressive radiographic deterioration over the preceding 48 hours, which involved more than 20% of their lungs, and progressive deterioration of their respiratory status also over the preceding 24 hours. In the upper panel, I show you a series of chest X-rays from an individual who received only corticosteroids, and you can see that progressively from day 9 of symptom onset, this lung became more occluded, more opaque, and they had severe respiratory issues. Those patients that received interferon alpha CON1, from onset of symptoms onwards, you can see that their lungs progressively cleared. And in fact, if you look at time to 50% resolution of the chest X-ray abnormality,. With the interferon group, this was 4 days versus 11.5 days with the control group. We also looked at the effects of interferon on a number of clinical parameters. Obviously, because this is a respiratory disease, saturation— oxygen saturation is an important measure of lung function. And what I show you here is that those patients who received corticosteroids alone, their oxygen saturation dropped. Usually around about 93% is when you'd start introducing nasal prongs, continuous flow, and as it progresses worse, you'd go to mechanical ventilation. And you can see with the interferon-treated group, their oxygen sats remained very good. Another measure of this disease of SARS was a spike in creatinine kinase, and it transpires that those patients who had severe acute respiratory distress syndromes had significant spikes in creatine kinase. And again, I show you this— the control group versus the interferon group, which did not have a spike in creatine kinase. And in fact, when we subsequently looked at gene expression analysis of peripheral blood mononuclear cells of those patients who either did not receive interferon, or those who did, we observed an increase in a number of genes, um, such as defensins associated with an interferon response, reinforcing that the better resolution of disease was indeed associated with an interferon response. So now let's switch to SARS-CoV-2. So as I mentioned, there are some 50, 56 million cases right now around the world with over 1,350,000 deaths. At this point in time, we know that type 1 and 2 pneumocytes, alveolar macrophages, and endothelial cells can all be infected by this virus. Whereas the fatality rate was pretty high with SARS-CoV-1, about 10%, right now the global fatality rate is about 2.7%, and certainly in jurisdictions where dexamethasone is being used now, that fatality rate is coming down. As I mentioned to you before, there are a number of factors encoded in the genome of these coronaviruses. In fact, we know across all the coronaviruses there are about 10 factors which limit an interferon response, For SARS-CoV-1, these are the ones identified, and for SARS-CoV-2, these are the ones that are involved. And again, they're associated with, with blocking interferon production. We also know that for those individuals who have any mutations in genes associated with an interferon response, or who have autoantibodies to interferon, or low serum interferon levels at the start of their disease, they are at higher risk of developing a severe disease. What I show you here is, is published work from Dr. Chew's group where essentially he has been able to demonstrate that the replication of SARS-CoV-2 is much higher, the replication rate, than SARS-CoV-1. And these panels below reiterate again that there is a failure to induce a good interferon response, whether it be to SARS-CoV-1 or SARS-CoV-2, and whether this be looking at the interferon alpha response over time, the interferon beta response, or the type 3 interferon lambda response. So I just want to remind you again about this whole process of SARS-CoV-2 infection and the immune response associated with it. So in this upper panel here, I show you how the virus enters the cell through ACE2 and sets up viral replication. And at the outset, these alveolar macrophages will start to secrete chemokines, which will recruit a number of immune cells to the site of infection with obviously the intent to clear the virus. And indeed, if there is a normal healthy immune response, you get appropriate CD8 T cells, CD4 T cells, macrophages clearing the virus. However, if there is an aberrant or failed immune response, then there is an excessive infiltration by monocytes macrophages, and T cells. We observe a systemic cytokine storm, pulmonary edema and pneumonia, and widespread inflammation and multi-organ involvement. So that is the severe disease. So this has prompted looking at various stages of the virus life cycle and at the various clinical outcomes to see where there might be interventions. And we're all familiar with the strategy to, to block receptor with ACE2 inhibitors. We're familiar with the use of lopinavir, ritonavir, remdesivir, a number of, favipiravir, ribavirin. To date, remdesivir is the only approved drug. The others have not shown tremendous efficacy at all. The use of inhibitors of this cytokine storm, and indeed the use of JAK inhibitors, baricitinib, to limit this inflammation, and dexamethasone also. What I want to remind you is that there are 3 phases of the disease. There's this early viral response phase, this pulmonary phase, and this severe life-threatening hyper-inflammatory phase, and you can see the viral phase really is somewhat limited in terms of opportunities to block virus. So certainly remdesivir has utility over the whole viral response phase. Interferon alphas and interferon beta at the same time, it's really important that they be used during the viral response phase because we want to knock down the virus, and the JAK inhibitors and dexamethasone have utility later on in the disease. And it's important to appreciate that one would not look at using these early on in infection. For the JAK inhibitors, this is going to block any residual interferon response. If you block the JAKs, you absolutely limit an interferon response, or indeed a number of other cytokines. Kinds, and dexamethasone, which is an immune suppressant, that allows for the virus to absolutely take off. So one should be very cognizant of the timing of the particular therapeutic intervention. Based on our SARS-CoV-1 studies and all the data that were emerging about this virus and its ability to block an interferon response, response, but we decided to, at the very start of the outbreak, undertake an exploratory study in Wuhan, China, one of the hospitals, Union Hospitals. And what we did was we recruited 77 SARS-CoV-2 positive hospitalized cases that were treated either with nebulized interferon alpha-2b 5 million units twice daily, Arbidol, which is a broad-spectrum antiviral and that was pretty much standard of care across the hospitals in Wuhan, 200mg tablets 3 times a day, or a combination of interferon plus Arbidol. All these patients throughout their time in hospital had throat swabs for PCR confirmation of viral presence. They had CT scans looking at their chests. We monitored temperature, oxygen sats, and a number of blood biochemistry parameters, as well as cell counts and cytokine levels. Of note, no patient exhibited signs or symptoms of end organ dysfunction, and no patient developed respiratory distress requiring prolonged oxygen supplementation or mechanical ventilation. What I'm showing you here is a chest X-ray of a 68-year-old woman with fever, worsening shortness of breath. RT-PCR swab was positive 3 days prior, and this is actually somebody from Toronto. And the chest X-ray findings in common with SARS-CoV-1 show peripheral airspace consolidation in both the upper and lower lobes. So in our patient cohort, what I want to draw your attention to is that we did not see this prolonged spike in fever that we had anticipated. So you can see that patients, you know, normal temperature is 98.6, and the maximum we got was in a handful of patients, 99. So we did not see these symptomatic spikes in fever. And also, to reiterate that this was a mild to moderate group of cases, we saw that their oxygen saturations for all the cases was pretty good. If we now look in this slide at a number of biochemical parameters that were evaluated, and the dotted lines show the upper limits of normal. You can see that, yes, there were fluctuations in these levels amongst all the cases, whether or not they were treated with Arbidol, interferon alone, or interferon plus Arbidol, and that's the red plot. There were fluctuations, but they were all, generally speaking, when one considers from onset to symptoms, days from onset of symptoms, they all fell within an acceptable range.. And likewise, when we looked at blood cytokines and PCT levels, procalcitonin levels, among the various treatment groups identified here, again, there were some fluctuations, but nothing that was dramatically out of the range of normal. Again, reinforcing that these were moderate cases of COVID-19. When we looked at peripheral blood cell populations amongst the various treatment groups, and again, The arrows shows you— the dotted lines show you the normal ranges. You can see, yes, there were fluctuations and some increases above normal from onset of symptoms throughout the course of their disease, but nothing that was dramatically indicative of a problem. Certainly there's some areas in platelets that were noteworthy. However, what we did observe was that interferon treatment accelerated viral clearance. So in this left-hand panel, I show you the 3 different plots associated with Arbidol treatment, interferon treatment, and Arbidol plus interferon. And in the right-hand plot, what I've done is I've consolidated all those cases that received interferon.. And you can see that there is an accelerated viral clearance that is statistically significant. And when one considered— when we adjusted for comorbidities, for sex, and for age, the effects of interferon treatment remain significant. When we looked at that time for the effects of interferon treatment on some known biomarkers of inflammation, namely IL-6 and C-reactive protein, what we can see here is that for those individuals who did not receive interferon, there was that characteristic spike in circulating IL-6 levels. Yet those patients who received interferon treatment, their IL-6 levels remained very low. And this is just a reminder, this is a log scale here. Similarly, with C-reactive protein, we saw that interferon treatment maintained, ensured low levels of circulating C-reactive protein. And again, when we adjusted for comorbidities, sex, and age, interferon treatment effects remain significant. Now, as I mentioned, this is a respiratory disease, so all the action at the outset of the disease is in the lung. So we have gone on to look at lung abnormalities and the effects of interferon treatment on these lung abnormalities. So what I show you here is some representative CT images that are scored based on lung abnormalities. So 0, there's no lung involvement. Each lung lobe is scored up to a level of 5, with 5 is greater than 75% percent lung abnormality. And in this patient here, you can see this ground glass pattern, which gives a CT score of 7. In this individual, there's considerable consolidation, a CT score of 11, and this is quite profound in both of these lobes, are giving a CT score of 16. So what we did is we looked at those cases that were treated with Arbidol alone or those that were treated with interferon either with or without Arbidol. And what I want to draw your attention to is that prior to treatment there really was no difference in the CT scores of all the cases that we looked at. And indeed on day 1 of treatment when one wouldn't expect to see any change in the lung abnormalities we also see no difference here. However, over the course of treatment, what I hope you will appreciate is that those patients who received interferon, their CT scores, their lung abnormalities remained low, whereas those that did not, they had worsening lung abnormalities, and the p-values are quite significant. In fact, if one looked at the maximum CT score The final CT score or the percent change, I hope you'll appreciate that interferon treatment limits and accelerates resolution of these lung abnormalities. What we then went on to do was to see if we could find clinical parameters that were predictive of CT scores or worsening lung abnormalities. So independent of treatment, we took all the patient values and we looked, for example, as I've listed— as I've shown you here, a number of potential correlates. So for example, we know that CD8-positive T levels— T cell circulating levels here are highly predictive of whether the disease will progress. So if you have good CD8 levels,. Now you have low CT scores, but lower CD8 levels, worse CT scores. Likewise, we know that platelet dysregulation leads to increased coagulation and severe disease, so low platelet numbers, lower CT scores, higher platelet numbers, higher CT scores. And the two biomarkers of inflammation, IL-6 and CRP, Again, low levels, low lung abnormalities, worsening scores, worsening levels, higher CT scores. The question then is, what are the interferon effects on clinical parameters that affect these CT scores? So we looked at— we only considered those circulating or clinical parameters where prior to onset of treatment there was no difference amongst all the cases, whether or not they received interferon treatment. And I show you here for CD8 levels, whereas in the absence of interferon treatment, we can see that those CD8 levels remain relatively low. Interferon treatment absolutely increases, recruits, activates CD8-positive T cells. Likewise with IL-6, when one considers prior to treatment, no difference,. And yet on interferon treatment, we fail to see this spike in IL-6. And finally, when one looks at another pro-inflammatory indicator, TNF, once again we see that interferon treatment will suppress the activation of those TNF levels. So one of the major issues associated with interferon has been that there's going to— it's going to invoke a cytokine storm.. And what I hope these data have shown you, that far from that, whether we looked at SARS-CoV-1—I didn't show you the data—or SARS-CoV-2, if you treat during the viral phase, you do not invoke a cytokine storm, and if anything, you limit that inflammatory response. So in summary, cognizant of the limitations that this was a very small cohort, it was non-randomized and the treatment arms were of unequal size. Our data provided evidence that interferon treatment accelerated viral clearance and limited lung abnormalities. It reduced IL-6 and CRP levels. Age, comorbidities, and sex did not negate the interferon effects, and these findings are the basis for ongoing randomized controlled trials. So I would like to suggest that, as I've mentioned, type 1 interferons are active on all cell types, they're effective against all viruses, and we should be considering them for the treatment of acute virus infections where I hope I've demonstrated to some extent that there is no cytokine storm and we didn't see severe adverse events. And with that, I'd like to acknowledge all those who contributed to the SARS-CoV-1 study and the SARS-CoV-2 studies.

And open up, I'm receptive to any questions you may have. Thank you. Dr. Fish, thank you so much. This is really interesting and a little bit different from, from previous presentations we've seen. This is the first interferon kind of focused one. So thank you so much for sharing your expertise with us. I'm going to take a couple of questions. If you can keep your slides up just in case the attendees have specific data questions, that would be great. Okay. Okay. Let's see. And guys, please remember to send your questions in the Q&A function so that I can ask them to Dr. Fish. Okay. First question: In the Wuhan clinical trial, was treatment randomly allocated?

And if not, how were treatments assigned? So it was randomly allocated, but as I mentioned, it wasn't a randomized randomized control trial. And the allocation was based on the attending physicians.

So they made the decision whether to treat with interferon or not. Okay. And a question: What is Arbidol and how is IFN administered?

So dose and frequency, if you can comment on that, please. Okay. So Arbidol is a broad-spectrum antiviral. It's it's been in use, it's a tablet, it's been used for many years. I think all the emerging data, certainly against SARS-CoV-2, is that it's pretty ineffective. So to our advantage, it's actually, for me, we compare it to an ineffective therapeutic. And what was the question? It was twice daily, no, 3 times daily, I think it was. Let's just go back to that slide. I've got it in front of me, just bear with me and I will tell you exactly what the dosing— here we go. So it was 200mg tablets, 3 times a day, and the interferon we used, it was nebulised. So you take a solution of interferon alpha 2b, 5 million international units, you nebulise it, and then you use a face mask to inhale the interferon.

Okay. And it was twice daily we did that. Twice daily.

Were patients treated on the same day post-diagnosis of the infection or post-infection? That's a good question. That's a very good question. So you'll appreciate it's much the same here that somebody has symptoms. They then go to get tested for COVID and post-confirmation, they might start a treatment. In certainly in Canada, in the US, this doesn't happen until they arrive in hospital. In Wuhan, as soon as somebody had a positive PCR, they were immediately admitted to either one of the hospitals or one of the temporary shelters and started treatment. So the time between PCR and treatment start was was very short. The time between onset of symptoms to start of treatment varied, and we've adjusted for all of that.

That's really interesting. Okay, um, let's see, a couple of questions on CT scores.

Did the CD4 T cell percentage also associate with CT scores? Uh, we're examining that right now, but the most profound one was actually the CD8. And let's just go and have a look at that Again, look at that slide. I don't think I've shown the one there which has CD4s. Let me just put it up. I don't think we've got that one. No. So, yeah. So I haven't got that here, and we're evaluating that right now.

These are the ones that were the most significant, obviously. Okay. Great.

I don't know that I know this acronym.

Why is interferon administered only BID? I'm sorry, what can you say that again?

Why is interferon administered only BID? If that person could just clarify what BID stands for. It's twice daily. You know, we— this was the— so if anything, the question should be asked, why was it interferon used twice daily as opposed to once daily? And the reason is that the, you know, when I was shown how they were going to use how they were going to use an inhaled interferon. So again, I just want to say that an inhaled interferon in a respiratory infection is absolutely appropriate because you're targeting the airways and that's where the virus is. So I would argue this is the best option for treatment. The problem was that the system that was available for us at that time, and this was at the very start of the outbreak, and the numbers were huge in Wuhan, was a mask system where you certainly covered the nose and the mouth, but a fair amount of the interferon got ingested. So that's why we recommended twice daily. So even though we had 5 million international units that were being inhaled, we knew that a fair amount was being ingested. So coming in twice daily would ensure you got a better level of interferon in you airways. And indeed, in subsequent studies where people have been looking at interferon around the globe and treatment options, it's usually 10 to 12 million international units per day.

I like to think that's based on our study, but, but who knows? Yeah. So here's a question that seems fairly obvious after your statement that you just made.

Is it available in the US? And if not, why? And another place right now, there's, there's a huge study that I'm a part of that's being run by the NIAID called ACT-3 where patient— actually, we've stopped recruitment now. We've got over 9— close to 1,000 patients where hospitalized patients were treated with either remdesivir or remdesivir plus interferon. And we're going to wait and see what the outcome is there. But so you have to do the clinical trial before you get approval. My hesitation is that the outcome might not be that great because we were including patients who were in later stage of disease, and as I mentioned to you, the interferon is really only going to have utility early on. So that's ongoing now, we'll have the results of that. The Cynogen group in the UK used an inhaled interferon interferon, and their results are very promising. They just published those in Lancet Respiratory Medicine. There have been a couple of publications from a Cuban group where they're using intramuscular and sub-q injections with interferon, also very good results. And if I can just get your patience for a moment, I'm going to show you a study that we're just about to start hopefully tomorrow. Oh great. Santiago, Chile, whereas I, you know, I keep stressing it's really important to catch individuals early on in their disease. So what we're actually doing is we're doing a post-exposure trial. So we're going to go into households, so the first index case in a household confirmed positive will be this is prior to hospitalisation, as soon as they get a PCR positive within 72 hours of symptom onset, they will be started on interferon treatment. At the same time, all those eligible individuals between the ages of 18 and 80 in a household that have been exposed to this contact but are PCR negative will also receive interferon. Those that are ineligible We're still going to monitor, so we're going to at various times post-onset of treatment, we're going to be sampling these individuals in their households to see one, if the index case that we've caught very early on hopefully will clear virus quickly and will not go to hospital. Whether the treatment eligible post-exposure contacts, whether or not they— this interferon treatment protects them from getting infected, or if they get infected, whether they get a reduced severe disease. Yeah, and whether those that are ineligible— so the elderly, the very young, pregnant women— by this protection, that's got this treatment in the household, whether they avoid them getting infected. So this whole trial is about preventing transmission.

So I think this is a really promising trial. This is a really elegant study design too. It's nice you kind of have controls at every possible measure. So that's really nice. I was curious, for the index cases, are you working with doctors' offices to try and identify, you know, the minute they have a positive PCR, you offer enrollment into this type of study?

Yeah, so the Santiago— Santiago, the city of Santiago in Chile has a you know, as soon as the PCR is positive, there's a centralized registry. So we're working with that centralized registry. And that records, you know, the names, the addresses, the ages of every individual.

So we'll have all that information and then be able to contact the households, get their consent, and proceed.

What a great solution that we could be doing in the US, but we're not. Well, this is a randomised controlled trial, and the beauty of it is you can see by day 11, which we know that in the context of infecting somebody you have this, this window when they're infectible. So within 11 days we're going to know whether these household contacts have become infected or not. So we're going to have a result very, very quickly. So in contrast to the standard trials that go out to, you know, 29, 30 days, we'll start to see whether this is having any efficacy fairly quickly. Okay, so you think it's about 11 days between when they might become infected? So these are the 3 times we dose them, so they're getting 3 doses in total. Okay. Pegylated interferon, so 3 doses. But my point is, the index case, if somebody's going to get infected in the household, it's going to be within 5 to 7 days of exposure. Right? So we're going to see very quickly, yeah, day 16 at the latest, whether we're getting transmission.

That's a great study design. A couple of questions now on this new trial.

What defines ineligible for treatment? It seems like you've got an age cutoff and pregnancy. Age is an obvious one. Young children and very senior adults we're concerned about. That's usually in clinical trials, your cutoffs. Pregnant women, that's a cutoff. Also, if somebody has a comorbidity that has been demonstrated with interferons, because they've been clinically approved for decades—epsi, multiple sclerosis—we know what individuals are contraindicated.

So if you have certain kidney abnormalities, liver abnormalities. Great. And in this study, how will the INF be administered?

Is it the same nebulizer they were using in the other study? For this particular study, we're using interferon. It's going to be a subcutaneous injection. As I said, this is a pegylated interferon, so we have high circulating serum levels for up to 5 days. So that's the 3 doses. But there has been interest from, as I've said, you know, the UK group are interested in following up on this trial using—

coming to Santiago and using their inhaled interferon. So— Great. —it's next on the list. Okay. I think this refers back to the other study. Have you immunoprofiled the patients in more detail during the treatment to see what subsets of CD4 and CD8 T cells are more prominent on the IFN treatment?

No, that's all ongoing. The challenge here was, first of all, the challenge is that this is in China, so for me to get— I can't get any samples. That's, that's a no-go. Right, okay. It's a standard hospital, it's not a teaching hospital, it's not a research hospital, so there is a limit to what they were able to do in their clinical labs. So we're still exploring if we can get some other analysis done, but it might My gut feeling is what we've got is what we've got. Yeah, sure. The difference is in the ACT-3 trial in the US, we're going to have a lot more data analysis.

So I think that probably knocks out this next question. If you've looked at the antigen specificity of T cells that arise, so anti-spike or other anti-COVID-2 recognition.

So again, I'm in control of the Santiago trial where we— if I tell you the extent of the analysis that we're going to do on peripheral blood. It's huge. Great. We're going to tease out a huge amount of data from this very well-controlled trial, utilizing collaborations with some stellar scientists all around the globe in terms of transcriptomics, in terms of looking at their immunophenotype.

So just Be patient. Yeah, definitely. That's a running theme in a lot of these analyses. A question about— I'm not familiar with this one— the Synergen company in the UK. How does interferon alpha compare with the beta version as used by Synergen in the UK?

Are you familiar with this? So the reality is, and if you can cast your mind back to that first, one of the first slides I showed you interferon alphas and interferon beta bind to a receptor. The reality here is that they all bind— both the— all the interferon alphas and interferon beta bind to the same cell surface receptor complex. There is a subtle difference in how interferon beta interacts with this receptor compared with the interferon alphas. Interferon beta has a more sustained binding compared with interferon alphas. And yet, in all the studies we have done in my group over many years, one can get exactly the same signaling output. So if you dose appropriately, the outcome is identical. And so I, I hear, you know, why did we— why did the WHO recommend interferon beta and not interferon interferon alpha, there's no difference. The reality is we have way more experience with interferon alphas in terms of viral infections, so clinically it would have been more appropriate to go with interferon alphas because if there would have been severe adverse events, we'd know how to treat them. You'd know what to do, yeah, sure. But in answer to the question, I don't think it makes a difference, and it's going to be availability. Right now, interferon beta is being used for MS, So there are a number of companies that have stocks available. The interferon alphas, because of their failure, or because of the Gilead direct antivirals, no longer used for hep C, so production manufacturing has been cut back dramatically. Oh wow, that's interesting.

So in actual fact, availability may determine what, what ends up being used. Sure, sure, sure.

And how long is the half-life of interferon alpha Yeah, so, yeah, so, you know, the half-life is about 20 minutes. So, so with the pegylated interferon, so you have to daily dose, you know. With the pegylated interferons, you know, I didn't show you the data in healthy adults, you can show that you can maintain good serum levels that will invoke an antiviral response for, you know, the doses we, we're planning to use.

For up to 5 days, and that's why we've got 5 days. Gotcha. Okay, um, a question on the Lancet paper. The inhaled results published in Lancet appear to be very promising. Is a similar trial likely to be conducted in the U.S.

using this drug or this method of application? Well, as I've said, I've been a strong advocate that for respiratory diseases, whether it be coronaviruses or influenza viruses, the smartest approach would be would be to target the respiratory tract, because if you can shut down the virus there, you're not going to get spread. And also, if you, you provide a good dose to the lungs, you're going to limit viral replication and hopefully get viral clearance before you get that inflammatory response. So unfortunately, the ACT-III trial is with a subcutaneous injection of interferon. That doesn't mean we might not see positive results. Similarly with our post-exposure trial, you know, we want to get this up and running as soon as possible, and we're using a sub-q injection. I'm in discussion with various groups to see if we can develop the kinds of inhalers that you use for asthma that you would be able to do this in the community rather than in hospital. So that's, that's the plan moving forward.

I think the utility will be for respiratory diseases that we use an inhaled form. It would be interesting in that case to work with maybe pharmacists and community point-of-care type testing where you could provide it really rapidly once they've had that test. That would be— yeah, that sounds really innovative. So a question now on the efficacy of interferon. It's seen at the early stage, it looks like it's preventing viral replication, and the early stages of contracting the disease. Is there any efficacy on some of the post-effects of the disease, like the effects of the organs, lung damage, repair?

It's sort of too early to know, I would think. Yeah, and I, you know, the reality is in some, you know, there have been some publications which show that, you know, later on in the inflammatory, the hyper-inflammatory, that a severe stage of the disease, there are good levels of interferons, and yet they're not making anything better. So, you know, one might argue that once that infection becomes systemic, you actually don't want to be treating with interferon because then you might indeed be promoting chemokines that will invoke a cytokine storm. So there's a rate limiter, there's a certain cutoff point where you actually wouldn't apply that? Yeah, I mean, I think it's contraindicated to use interferon late in disease.

I mean, I think there's that's— if data are going to emerge.

And late in the disease, time-wise or just based on symptomatic presentation? Like if someone— so in this disease, they're ordinal. So, you know, if you come in with, you know, just mild symptoms, it might be ordinal 1, ordinal 2. Once you start requiring continuous flow oxygen, then into mechanical ventilation, into a hyper-inflammatory stage, they've all got— they've been classified and characterized based into ordinal. So I would say beyond ordinal 5, once somebody's in the ICU and ventilated, it's absolutely not appropriate. Okay.

Dexamethasone is the treatment of choice at that point. Yeah, sure, sure, sure. Okay, we're just about at the end of our time.

Any final thoughts or comments you'd like to leave with the group before we wrap up? Uh, no, just thank you again for having the opportunity to share results and hopefully get folks optimistic that there will be yet another treatment hopefully coming down that will complement the utility of vaccines.

Yeah, we really need a suite of approaches, and this seems like a really promising one. So that's really exciting. Thank you so much, Dr. Fish, for sharing your insight with us and this exciting data.

It's, it's great to see.

Thank you for your time. My pleasure. Okay, and thanks to everyone for joining and asking questions. These lab meetings are really interactive and we really appreciate your engagement, so thank you for joining and asking your questions. I invite you to join us 2 weeks from today on the next Global COVID Lab Meeting, which is scheduled on the 3rd of December. Our next speaker will be Dr. Dara Duffy from the Institut Pasteur, who will talk about immune variability to infection with SARS-CoV-2. And if you're interested in more research on COVID-19, please sign up for the HVP COVID report. In this newsletter, we present insights from experts all around the globe and highlight the latest scientific articles and data. And finally, please feel free to visit our website and follow us on LinkedIn. We'll post a recording of this presentation there for you to review. And with that, I'll say thank you again for joining. Stay safe, and we hope you'll join us again in 2 weeks on the next Global COVID Lab meeting. Thanks, everybody.