103 - Dr Amy Proal - Viral Persistence in Long Covid

Episode 103 of the Long Covid Podcast is a chat with the fabulous Dr Amy Proal, a microbiologist researching viral persistence in Long Covid. We chat through what brought her to this research as well as taking a bit of a deep dive into the topic.

Review Paper: https://www.nature.com/articles/s41590-023-01601-2#MOESM1

PolyBio Research Foundation
recent paper on SARS-CoV-2 reservoir in LongCovid/PASC

For more information about Long Covid Breathing courses & workshops, please check out LongCovidBreathing.com

(music credit - Brock Hewitt, Rule of Life)

Transcript

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Jackie Baxter  
Hello, and welcome to this episode of the long COVID Podcast. I am absolutely delighted to be joined today by Dr. Amy proal, who is a microbiologist at Poly bio Research Foundation, working on collaborating on infection associated chronic illness, which includes long COVID of course now, so a very warm welcome to the podcast today.

Amy Proal  
Thank you so much. Thanks for having me on.

Jackie Baxter  
I'm so excited to dive into all of this. Before we kind of dive deeply into all sorts of complicated stuff, perhaps you would just give a little bit of an introduction to yourself and what you actually kind of do?

Amy Proal  
Sure. So as you said, I'm a microbiologist, but I'm also the president and the Chief Scientific Officer of PolyBio research foundation. And we're a nonprofit research organization. And what we do basically is work with different academic teams at different institutions across the world. So for example, Harvard Medical School, or Stanford, or the Karolinska Institute in Sweden, and we collaborate with teams studying the chronic consequences of infection at those sites. 

And we pull them together to build collaborative projects where we can work on what we feel are gaps in the field, or projects that involve really advanced technologies that we feel we could build more effectively if we work together as a group. And we build those projects. And then we actually find private funding to make them work. And so we're working together. 

And as part of that, we formed specifically for the study of long COVID, what is called the Long COVID Research Consortium. And we're a specific group of researchers working to study the root cause drivers of the  long COVID disease process, with a focus on the persistence of the sars-cov-2 virus in tissue, which is also called viral reservoir. And I can get into that later. 

And we're not only studying that, but generally speaking, a lot of our research is going in that direction into that possibility of basically what it is, is that people don't fully clear the virus after they've gotten the initial infection. And a small amount of virus sticks around in different tissue body sites. And that, you know, failure to clear the virus and to continue to have it in a tissue reservoir site could be driving a lot of the inflammatory, the clotting, even other components of the disease process. So that's one of our main focuses.

Jackie Baxter  
Fantastic. So this all existed before long COVID came along. So - like what brought you into this kind of field, initially?

Amy Proal  
you're right, we actually started probably by about two years before COVID started. And the reason that we formed the organization then was because we, and unfortunately, so this is when COVID began, we unfortunately knew from day one, as soon as we knew that people are getting infected with the SARS cov 2 virus, this novel virus, that this subset of patients were going to get chronic symptoms that would probably never resolve, or might not resolve. 

And we knew that because we were studying what we call infection associated chronic disease, which is the understuded yet very important field of understanding that most if not all, well studied pathogens, viral, bacterial, and even fungal, are associated with a chronic syndrome in which yes, a subset of patients just don't get better and keep developing chronic symptoms. So those include the herpes viruses like Epstein Barr Virus, those include Borrelia, and pathogens that are bacterial pathogens. That's the Lyme pathogen. 

So we were already building and executing research projects to study patients with those other infection associated chronic conditions, some of whom get diagnosed with ME/CFS - myalgic encephalomyelitis chronic fatigue syndrome, which is a diagnosis in which patients often after a viral or bacterial infection, develop common symptoms and meet the criteria for this disease ME/CFS. And we were working on better understanding what was making those patients sick.

Jackie Baxter  
Yeah, because I think this is something that I I suppose I'm lucky in some ways to have never really fully understood, until long COVID came along. But as you say, chronic symptoms following an infection is not something that is new to long COVID, or to COVID itself. You know, it's been around for many, many decades, I suppose beforehand. 

And I think for a lot of people who have developed long COVID, it's been quite an eye opening experience and quite a humbling experience, I think to to realize that there have been many, many, many people who have been kind of living this sort of nightmare for a lot longer. So yeah, I think it's, it's definitely a very sort of necessary area to be working in, isn't it? But definitely not one that many people certainly have been fully aware of, I think.

Amy Proal  
right? It's growing, and a lot more people are coming into the space to study these patients, which is amazing. You know, definitely, I would say post Ebola syndrome, for example, got a decent amount of attention. So even after Ebola, which is, you know, very severe disease, often the epidemics are in Africa, a subset of those patients develop chronic symptoms and a chronic syndrome. 

And there's been some interesting research on the persistence of the Ebola virus in those patients. So even before the COVID pandemic started, and even when the pandemic was beginning, one of the interesting findings in that space of people developing these Ebola chronic syndromes, was that they were retaining the genetic material of the virus in these reservoir sites, like eye tissue or even in semen or breast milk. 

And I'll be honest, that's a little bit - that knowledge is a little bit of what set our group early on to the topic of SARS cov-2 persistence in patients with long COVID. Because we were looking at, for example, Ebola. So there's a lot of knowledge out there, but yeah, it's not that well communicated, we need a lot more attention on it for the average person.

Jackie Baxter  
Yeah. So, I'd love to dive a bit more into viral persistence or viral reservoirs. Because you mentioned the - I think you said the herpes virus, and that's one that does remain in the body and can get reactivated, I think, because that's something that we've been hearing about, and I think the chickenpox one is the same? So are we talking here about - is that the same thing as that? Or are we talking about two sort of sides of the same thing? Or how does this work?

Amy Proal  
That's a great question. And so the herpes viruses are perfect examples of persistent pathogens. By definition, once you've gotten infected with a herpes virus, for example, Epstein Barr Virus, or human herpes virus six or seven, you retained that virus in your system for life. And most of us actually, most of the human population actually has at least one herpes virus that they're harboring. The thing there, though, is that the virus can persist in a person in what's called a latent or dormant form, where it's basically really not doing anything. It's and the immune system is keeping it in check. 

Or, over time, depending on other situations, other infections, the immune system, all kinds of other variables. There's a lot of research now showing that the herpes viruses for example, Epstein Barr Virus can activate in certain people. And they're, in fact new tissue or new sites, or create proteins that can start to dysregulate a lot of different human signaling pathways in a way that can most definitely drive disease. 

So for example, Epstein Barr Virus is now increasingly tied to the MS or multiple sclerosis disease process, where several teams have done some really important work showing that in the MS brain, basically an antibody part of the immune system that targets human tissue in the brain. The reason that happens is because the immune system is trying to target an Epstein Barr Virus protein, and that protein has a similar size and shape to a protein or structure that's a part of a neuron protein involved in the MS disease process, so it sort of hits that too, and that collateral damage starts to drive MS disease. 

So overall, that understanding that you have these pathogens that can stick around and get into sites like the brain, like the spinal cord, in other words, not just patient blood, but basically throughout a patient's tissues depending on the disease, is much more documented with the herpes viruses, which are DNA viruses. 

Now the interesting thing about SARS cov two persistence and even Ebola, which I mentioned before, and some of the other viruses that are also being studied for the same phenomenon, is that they are RNA viruses. So SARS cov Two is a single stranded RNA virus, it basically just means it has like a different chemical backbone than the herpes viruses. So there's some differences there like, yes there are all viruses but the RNA viruses, for whatever reason, had just not been studied as much as the herpes viruses for their ability to persist and drive chronic symptoms. 

There's, I'll be honest, it's kind of an assumption because no one studies at that much. The idea that you get infected with an RNA virus like SARS Cov 2, or you know, like Zika or something like that, that's another RNA virus. And in those cases, it clears -  once you're done, you have your kind of your main symptoms go away. The virus also left your system and it's not there. And so if people have chronic symptoms afterwards, well, I don't know there's something else to find out. 

But now, in the last couple of years, there's been this surge in actually studying those RNA viruses and saying, wait a second, can they also stick around after acute infection? Is it possible that they too, can persist in what we call a reservoir site? And that can be part of these chronic symptoms that people experience after RNA virus infection?

Jackie Baxter  
Right! That's so interesting. And it seems really strange to me. I mean, again, I'm coming at this from the perspective of very much not a scientist, that one subset of viruses can be so studied, and yet the other ones are virtually kind of like, ignored and kind of like brushed away.

Amy Proal  
It's true that, and this is true of a lot of things in science. Sometimes, it'll seem like something is not the case. It'll be like, I haven't heard that. That's, but one of the reasons you haven't heard it is because no research team actually just studied it to see if it could happen. Right? So that is an issue with RNA viruses. And that's part of why they haven't been studied this way is that, you know, I don't know, I don't have a perfect answer. 

But one of the research verses, for example, that's part of our consortium team is named Diane Griffin, and she's at Johns Hopkins. And she's one of my idols, because she's been studying RNA virus persistence, is one of the few people really just honing in on this trend for years. And part of her work even looks at in mice, how measles virus, which, for example, is just not a virus again, that people think about in a persistent way, can actually, in these mouse models, stick around after acute illness and also persist. 

And even kind of change the way it acts. in the way that it you know, kind of the way it's genetic material, it will sometimes it will actually deliberately mutate itself and modulate the way the immune system recognizes it to put in other words like, these different RNA viruses even have unique mechanisms by which they're able to evade the immune system so that they don't get recognized and eliminated. 

And so she showed that with measles, and it really goes to show that, yeah, if someone starts to look at these RNA viruses, there's this big capacity for the fact that there may be contributions from their persistence to conditions that we're just not even sure about yet. It's an ongoing area of inquiry.

Jackie Baxter  
Yeah, I mean, it's so interesting, isn't it, and it makes them sound incredibly clever, which I think maybe maybe they are, which is kind of scary, isn't it?

Amy Proal  
They are extremely clever. This is the thing with pathogens, is pathogens, just all of them. That's, you know, I'm a microbiologist, so I studied pathogens, and that's why I became a microbiologist. Because when you start looking at pathogens, they are all so crafty. And they have so many different mechanisms by which they can drive disease, in which they can, you know, sort of evade immune detection. It is remarkable, in fact, that's what interests me and that's one of the primary parts of what we study, is the proteins made by many pathogens, viral or bacterial, they can directly interfere with how our human genes are expressed. 

So for example, the human genes that set you up every day, that are programming what you're doing, these pathogens can often get into the center of your cell. And they can actually create proteins that interfere with how those human genes do their functioning. So they can hack our human genes, which is pretty crazy, just the problems they can cause. And that is profound. 

But they can also then - I wrote a paper about this with my colleague, Mike VanElzakker, who's at Harvard Med, who also works with me at Poly bio - if they get into the center of your cell, they can also directly hijack your mitochondria. So the energy powerhouse of every cell that's helping it to create all the energy substrates that the body needs to function. Viruses, by definition, and this is crazy, actually must hijack the metabolism of the cells they infect, in order to replicate. They - what they do is they get in there and they pull substrates, like proteins from our own mitochondria, so that they can build new versions of themselves. That is how they do it. 

So inevitably, when you have a viral infection, your mitochondria function is modulated so these pathogens, they're not just for example, driving inflammation, they're literally hijacking and hacking our gene expression, and metabolism, all kinds of functions and once you start to realize the scope of that, you really understand the scope to which they can drive chronic disease processes, and that's why it's been - it's always ridiculous to me when someone says for example, like oh Long COVID. It must be psychosomatic or something like that. That is so categorically ridiculous to me. 

Because if you actually understand these viruses like SARS cov two, and the number of ways, the sheer just number of ways in which they can drive chronic disease, to jump to something absurd, like, oh, it's the person's thinking or something. It's hilarious. It's just that those people who are saying I just don't get viruses, you know, I mean, so. So in other words, there's there's huge precedent for the fact that people develop chronic symptoms after infection, because it's part of what these pathogens do. They don't want to leave your body, they don't, you know, like, that's, why would they? And so they have all kinds of ways that they stick around. 

Jackie Baxter  
Yeah, and it's just wild that it can just go all the way down to the sort of like, genetics and stuff that is literally the like, bits of Lego, isn't it, that makeup like us, and our sort of very, very basic level. And, again, this has taken me back to my sort of like school level biology. But you know, as soon as you start mentioning mitochondria, we go, oh, mitochondria, that's the powerhouse of the cell, because that's what we all learned. But then you realize, well, actually, if you're like, powerhouses aren't working, then you're not going to work. 

But I don't know if you have an answer to this, but why is it that some people just seem to be fine? And some people are very much not fine? Like, if this virus can get into the sort of the building blocks of us, then what makes some people able to, I don't know, fight it off, and some people not?

Amy Proal  
That's a great question. And I don't have a perfect answer. But I definitely can think of some factors. And first, you know, I want to say when I'm talking about the persistence of the SARS cov two virus in tissue as a driver of long COVID, we don't have enough evidence yet to know that that's happening in every single person who gets chronic symptoms after long COVID. So we do, and we see increasing evidence that it seems to be happening in a good number of people. But long COVID is such a broad diagnostic label that certainly there can be different things happening. 

And some people, maybe some people, as you've seen, there's some evidence that Epstein Barr Virus, that virus I mentioned, before could reactivate. And that could be part of the process, or there could be microbiome issues, like the organisms that are in are these kind of ecosystems, collective communities in our gut and mouth, there can be other issues. We just think that the persistence of the virus is where we're seeing the most evidence leading to us in long COVID. And the one we're going to continue to study most robustly for now. 

But overall, let's say we're thinking about why someone might not fully clear that SARS cov two virus after infection, in other words, someone does hang on to the virus and have an issue where it sticks around in the tissue of their gut or the tissue of their lungs. There are some factors that could determine why that might happen in one person and not another. 

And definitely in the state of the immune response at the time when the person is infected is a major variable. We have a number of studies looking at antibody responses and people who go on to not clear the virus because for example, there are antibodies that neutralize pathogens like SARS cov two. And there may be differences in the level to which the virus gets neutralized in people who do and do not develop long COVID. In other words, there could be issues with the immune response, that are unique to people who don't fully clear the virus or who develop chronic symptoms. And we need further study on that front. But that's certainly one of the possibilities. 

The interesting thing is that the immune response, and how it's acting at any given time, can be impacted by the pathogens that a person already has. So one interesting thing to think about is that, for example, if someone already has Epstein Barr Virus, let's say that other virus, and that virus is an issue, that virus might be and it does - we know for example, that the Epstein Barr Virus or the herpes viruses modulate the immune response often as part of the way that they survive. 

So there was a team, for example, in Sweden that showed that twins that had cytomegalovirus, which is one of those herpes viruses had very different immune responses, and were expressing very different parameters of the immune system, than twins who didn't have the virus. And what that means is when you get the next infection, you will actually respond somewhat differently to it because your immune system is already being shifted a bit from the fact that you have the previous pathogen. 

And I think that trend is interesting to think about in Long COVID. Because when you talk to patients as you know, there are often people who describe different what I call hits in their cases. So while their symptoms largely started mostly after SARS cov two infection, if you start to talk to patients that say, you know, I did have a really bad case of mono - which is caused by Epstein Barr Virus - when I was younger. Or I did have an issue where I had problems managing colds for a while, or I did have an issue where I was exposed to mold, which is another, for example, situation where you have mycotoxins that could hold down the immune response. 

And, and there are cases in which people might actually be building up sort of hits that affect the immune response. So when they get that infection with SARS cov two, now they're no longer able to clear it or handle the virus as well as someone else who didn't have those other exposures or infections in their case. That's one possibility. 

And then also, it goes into our human genetics. So we all, as I mentioned before, you know, have these human genes that make us who we are, but they also control parts of our immune responses and our systems. And we all have differences in our human genes. And so there can be variations of human genes that impact how the immune system can function in a given person. And people might just be unlucky enough. That's why we have a large genetic components to include it in our studies of the virus, to understand if there are, you know, variations in the human genome that impact parts of the disease process that might then impact how SARS cov two infection happens. 

And that even could extend for example, beyond - it could, let's say an impact something like clotting, for example, you probably know the research showing that patients with long COVID have the fibrin amyloid microcloud. So Resia Praetorius' work right? Those little fibrin amyloid deposits, which do, by the way seem you know, they've done an experiment showing that the spike protein can feed those deposits. So certainly, like the formation of those micro clots could be connected to the persistence of the virus. But overall, there are some known human gene variants where people just are predisposed to increase clotting issues. 

And so for example, let's say if you were unlucky enough to have one of those variants, where your blood already clots a little bit more than other people's, you might just be unlucky enough to have more of a problem with the micro clots that might form from the spike protein. And that becomes part of the issue as well. So all these sort of flow on effects from the virus, the things that it does, if your human genes are not set up well for those issues, you kind of might have these, you know, problem areas, where you become more susceptible.

Jackie Baxter  
Yeah, that's interesting. And when we were talking earlier about how chronic symptoms following infection is like, very much not a new thing. Do you think this kind of process of long COVID and the process of MECFS - Do you think that's the same? Or very similar? Or is that one of these areas that we still just really don't know?

Amy Proal  
I think it could be similar. I definitely don't think it's the same. There are times when I hear people say long COVID is MECFS. And I do find that to be confusing, because MECFS, when we've been working with patients with that diagnosis for a while, the way that you get that diagnosis is you fill out a questionnaire and you meet a certain number of symptoms that include autonomic dysfunction, post exertional malaise, different flu-like symptoms. You can likely get - and we know that, we see that enough in our studies - to the point of developing, you know, getting that diagnosis for those symptoms from multiple different sort of start points, right. 

So most of us studying MECFS, don't think there's a single thing happening with every patient, either. We think there's common pathways, perhaps affected in patients that lead to a common set of symptoms. We think the same thing with long COVID, which is that not every patient likely has the exact same thing happening, but there's again, common pathways and common things impacted that lead to common sets of symptoms, that can overlap with the symptoms of MECFS. 

In the cases of long COVID, though, we want to make sure to separate them out to a point from the you know, you could be a long COVID patient and get an MECFS diagnosis. And that's fine, of course, like. But I personally would still, as a researcher, and someone also designing clinical trials, would want to keep that long COVID name to the greatest extent possible as well, because as we're studying, for example, that a lot of long COVID cases could result from that during the SARS cov two virus, I need to know that your case started with SARS cov two infection. 

And if that happened and you are infected with SARS cov two whether or not the virus even persisted in you, maybe even if it didn't. That means your body was exposed to the SARS cov two spike protein which is really unique in the kind of illness that can drive. So for example, the spike protein does have pretty specific effects on the blood vessels and ony the clotting system and on platelets and stuff like that, that are fairly unique to what it does as the sars cov two spike protein. 

So there's a point where, even if long COVID patients have similar symptoms to MECFS, there may be biological differences in the nuances of those symptoms because of the specific proteins and everything that Sars Cov 2 makes. That doesn't mean though, and then that's not complicated - that there can't be kind of common pathways, that the different pathogens that initiate cases of MECFS and the SARS cov two virus impact in patients with either a long COVID or MECFS diagnosis to get to shared symptoms. 

So, and one of those that we look at is actually the vagus nerve. So the vagus nerve is this really important nerve that branches throughout the whole body into all the major trunk organs of the body. It's really highly innervates the gut, so a lot of communication from the gut is modulated by vagus nerve, but it gets into the lung and all these other organs. And then it connects to the back of the brain in an area called the brainstem. And there it relays signals from the body to the brain, including a lot of signals about inflammation in the body that impact how the brain then goes on to signal. 

So we have always thought with patients with MECFS. If you look at the symptoms of the brainstem, that area of the back of the brain that the vagus nerve enters, there are nerve bodies there that control the development of flu like symptoms in patients. They're also nerve bodies in that region that control autonomic function like that ability to move correctly from sitting to standing, like that kind of tone of autonomic function. There's also nerve bodies in that same area that control pain and nausea. 

And a lot of like, if you start to look at this region of the brain, the brainstem, and what's controlled by it, many of the symptoms controlled there are the symptoms of MECFS. So if you have, let's say, an issue with the vagus nerve, and there's inflammation, kind of driving problems with that region, you could get those MECFS type symptoms. And we've always thought like, this is just kind of an example. But maybe in a patient with MECFS, a herpes virus, or a different virus like an enterovirus, it's another RNA viruses that are involved in cases of MECFS, could infect or dysregulate the vagus nerve. And you would get those flu like, autonomic, nausea, pain type symptoms in a patient. 

And then in patient with a long COVID diagnosis, maybe the SARS cov Two virus can infect or dysregulate the vagus nerve, and you would get, you know, those same kind of pro-inflammatory flu, autonomic symptoms. So in other words, what could be happening is different pathogens, could be dysregulating similar pathways in these patients. And that's why we're seeing the commonality in symptoms. That doesn't really mean that long COVID is MECFS. It more means that the pathways and the processes, and part of what's happening to patients may greatly overlap.

Jackie Baxter  
It's not a simple answer, is it? But that absolutely explains why there's commonalities and overlaps and things I think between the two, yeah. But I totally take your point about it being really useful, at least to know whether it was COVID or not, when you're studying it, because it could make a difference?

Amy Proal  
We do - it is very useful for us, because the long COVID diagnostic label is really broad, right, which is fine. But it encompasses everything from, you know, any sign, symptom, event after COVID, which makes the patient group really just heterogeneous. And same with people who meet the MECFS diagnostic criteria. It's actually a pretty broad group of people who can meet that, right. 

So when we're trying to design a study or a clinical trial, let's say we were trialing an antiviral, for example, there's some trials of Paxlovid. And we're going to increasingly try to trial more antivirals coming up now for the possibility of the reservoir that patients might not clear. If we're trialing an antiviral and it's a SARS cov two antiviral, we definitely want to make sure that we're bringing in the people with SARS cov 2 onset into that trial. Otherwise, it could just get too messy. So if people keep Long COVID it just helps us sort of with that classification of where to put people in studies and trials.

Jackie Baxter  
Yeah, definitely. I hadn't really thought of that, but it seems really obvious now. And I'd love to dive a bit more into kind of like your - maybe your research and like, this is a really broad wide open question. So feel free to take it wherever you like, but like yeah, what is the research that you're involved in?

Amy Proal  
So what we're trying to do is, as I've mentioned a couple of times now, the main thing that we're trying to look at is whether or not patients with long COVID clear the sars cov 2 virus. And if they don't, this is what makes it hard, is - you would think and hope that it would be easy, like, Okay, well, they could do a blood test, and then they'd still have the virus, and then we'd know. But it's not that simple, because as a general rule, when pathogens persist after initial illness, they tend to clear from the blood, which is just a dumb place to be - like, if you're a pathogen, you do not want to be in the blood, because that's where all the immune system cells are, and everything that's going to target you is there. 

So they don't really stick around there - pathogens, in simple terms, they hide. And that's where they go into tissue, where they're more protected. And that's what we mean by reservoir, in a way, that's like reservoir, it's kind of like this little site where the pathogen is hiding out, and the immune system is less able to target because it got into this tissue site. 

The problem there, then, is that we can do blood tests on you and we can look for the virus and stuff and you won't have it if you're a long COVID patient. You could interpret that to say, oh, no, they don't have the virus anymore. And people do do that, which can be confusing. If we want to then show that people might still have it, we have to get tissue samples. So we have to do - and that is a big part of our long COVID consortium Research Program - is a big part of it are tissue biopsy studies. 

So we're working with teams that are collecting tissue samples from patients. So there's a team that's getting gut tissue from patients via colonoscopy with long COVID. And then we can look for the virus and all kinds of other immune dysfunction in that intestinal tissue sample. There's another team we work with, it's getting lung tissue via and, you know, a bronchoscopy procedure from patients with long COVID. And then we can study that lung tissue, right, there's another team getting tissue from the lymph nodes of patients. 

And so a big part of our program is going out of the way to - and they're harder studies to design, they can take longer to do because it takes a while to get the permissions to be able to do the procedures on the patients and to have people undergo. And we are so grateful, honestly, to the patients that participate in those studies, because it can be obviously it's a bigger deal to undergo colonoscopy or something to get your sample than it is to do a blood draw. But despite that extra effort, we think it's very worth it to get these additional tissue types from patients. And that's a big, big, big part of our program. 

And when we do get the tissue samples, a big part of our program, is not just to look for the SARS cov two virus, but to know, once we have that valuable sample to go beyond that, and also just use a lot of the new - there's a lot of computer based tools we can use now in science, that can pull sort of the genetic material out of the tissue sample, and tell you a lot about what all the cells of the immune system are doing in that tissue, what most of the human genes, how they're being turned on and turned off in that tissue. 

So we can get all that additional information. And then we can correlate that with whether or not we find the virus there. And that actually helps to start answer some of your questions to say like, well, maybe when people still have the virus in their intestinal tissue, this part of the immune system isn't working. We can pull those trends together, because we're doing that additional work. So we have a lot of like, pipelines on those tissue samples. 

And then we also work with several teams, for example, the other big part of our program is using imaging. That's another way, so we can put patients into scanners like very high resolution scanners. And we can use, depending on the team. It's a radio lag. And it's like we take something that we want to know what's happening. And like, for example, there's a team at UCSF, that's part of our consortium. And what they want to know is in a long COVID Patient, are their T cells activated? These part T cells of the immune system. 

So they take this little very safe radio particles. And they put attached to it, a little bit of antibody that will bind to activated T cell, if it finds it. And they put it into the patient and then that diffuses throughout their body, and then they put the patient into an extremely high resolution scanner. And the scanner can map every time that that like little particle with the thing that recognizes the activated T cells happens, and so you can get a map basically of where the T cell activation is happening throughout the patient's entire body and brain, from the spinal cord to the lymph nodes. 

And so that team did recently publish - it's a preprint. A study in long COVID that we supported via Polybio - where they found in patients with long COVID, increased T cell activation by that imaging method in the gut wall, in the spinal cord, like I said before, in the lymph nodes. And that it goes to show you that like, we have to be looking at what's happening in the full bodies of these patients and not just their blood samples. It was a really, really compelling findings to make that clear. 

And in some of those same patients that did that T cell imaging, they also got gut biopsy samples, from several of them. And in all of them, interestingly, they still had the virus, and the spike protein part of the SARS cov two virus in their intestinal tissue, in one patient up to 676 days post initial infection, which was just the longest time that they did the analysis. It wouldn't even necessarily stop there. So those are the kinds of studies we're doing too, where we're even trying to combine the biopsy work with the imaging work so that we can really just start to correlate and say what's happening in patients tissues, body and brains. 

And from that there's, you know, a couple other flow on parts, but we're doing also, for example, a study where we can image fibrin deposition in patients with long COVID, again, throughout the body with fibrin being like one of the clotting things like the microclots and everything, that can be driven by the spike protein, so that we can actually see if that's happening in the lungs, in the tissue of patients. And also neuro imaging studies of inflammation in the brain, other things like that. So tissue biopsy studies, and imaging, and those kinds of things are kind of a key part of our product.

Jackie Baxter  
Yeah, I mean, this is all sounding almost kind of like space age. It's just it sounds so cool. You're talking about the little, the antibody that you put in that sort of pings around the body and activated the, showed the T cells and the virus in the cells. And going back to what we were saying earlier about things like the herpes virus, for example, remaining dormant in the body. Would they show up on one of these kinds of scans? Or would they not show up because they're dormant? Because I'm just trying to work out if the, you know, when we're seeing the COVID virus still there - is it like still alive? Is it dormant? Is that still to be confirmed?

Amy Proal  
That's a good question. That's like a key question that all of us want the answer to. In the study I described by the way, there were two parts, one was just showing the activation of T cells without like, we don't know if they're responding to virus or not, we suspect they are. And then the biopsies were in the same patients but separate and that's where the virus was found. So like the T cell activation in the virus in the gut samples are in the same patients, but we don't necessarily yet know if the T cell activation in areas like the spinal cord was from the virus because we couldn't get - they couldn't get biopsy samples from that area. 

But to answer your first question, that method was specific. With the T cells, it could be applied to other types of patients. But the same team is doing similar imaging, where they have an antibody that binds to the spike protein, that study is ongoing. In that case, that can help them basically map reservoirs of where the virus might still be throughout the body. You can, and this is one of the things the team really wants to do, do that for the - they've done it for the HIV virus. 

So the way this team at UCSF started was a couple years ago, they tagged an antibody for the HIV virus and mapped HIV reservoirs through the same imaging method they're now using in long COVID, in HIV. And the next virus, they're not doing it for SARS cov Two, in this long COVID study. But from there, like they could go on and this is one of the things that they want to do. No one's even done it with Epstein Barr Virus. But yes, you could - you could take an antibody as long as you can find one that reliably binds an EBV, Epstein Barr protein. You could do one for the Lyme disease bacteria and do an antibody. 

So yes, one of the things that we're trying to do as part of our long COVID program, is use the SARS cov two virus to really push the boundaries of how technologies can be used in the study of general viral infection, so that even if we complete the long COVID sars cov 2 study, we have the whole setup ready to turn it now into another virus or another pathogen, and something like that's a big part of what we focus on in the way we design studies. It has that in mind, yeah. 

And then your second question about like, is the virus active or not? Is it replicating? That is still part of - we're beginning soon, and we'll begin a phase 2 sort of second studies that are part of this consortium program and that's a big focus of that program. This answer, which is not perfect, is that it can be different from patient to patient. Some people might just kind of have the genetic material and it might not be doing much. In some people, though, maybe it is going through periods where it activates and makes protein, and then does that for a bit, and then activates and makes protein - that does seem like might be happening in some long COVID patients at least. Because one of the findings that is important in the long COVID space, when it comes to the persistence of SARS Cov 2 is that a growing number of teams are finding the spike protein of the virus in blood. 

And now, not to confuse you like, they're not finding the virus in blood - that's less likely to be there. But the spike protein is being found in blood. And what we think is happening there is that the virus is in like a tissue reservoir site, let's say like in the gut lining and the tissue, and that the protein basically just comes off the virus in simple terms and leaks into the blood, where it can be measured. So like the virus is hiding in the tissue, but the protein is leaking off it and getting into the blood. 

And if that's happening, like there's a couple of teams now that have found the spike protein in long COVID patient blood, like up to 12 months, even in one study now, I think it will be published soon if it's not, 16 months after initial infection. And it's hard to explain how the protein would be there in the blood, like 12-16 months after infection, if the virus wasn't replicating somewhat. 

Because if you had spike protein in your blood for that long, the body would start to clear it. I mean, it would start to get rid of it. So if it's still there, it strongly suggests that it's being seeded by virus that is at least going through periods where it's active enough to be like making more protein sometimes, making more protein sometimes. And in that sense, we do think that there's some evidence that the virus is still going through periods of replication in patients with long COVID.

Jackie Baxter  
And could this tie in with the kind of relapsing-remitting nature of long COVID?

Amy Proal  
Yes, we do think so. Because one of the things that makes studying SARS cov 2 persistence just logical in patients with long COVID, is the relapsing remitting symptoms that people often have. So another one is the fact that a lot of patients with long COVID don't even have really severe acute cases, they actually have mild or asymptomatic cases, and then they actually start to get worse and worse and worse and worse over time. 

So in that patient, too, it's like, well, if long COVID, and that could be were caused by just inflammatory damage, or something that happened during acute COVID. That's hard to explain in someone who didn't really even have symptoms, right? It makes a lot more sense that if a person has ongoing symptoms that actually almost get worse over time, that something is still happening or still there. And honestly, the persistence of the virus in tissue would be a very straightforward reason that they just continue to have ongoing symptoms that even maybe get worse over time, even when their initial disease was mild. 

And is the same thing too, when, you know, some of those patients, like you said, have, you know, periods where they're worse, or they feel a little bit better, but then they crash. Because, yes, like, pathogens change their activity. That's part of what they do. They might be like, like we said, creating more protein, and that might escalate symptoms, and then maybe the immune system does control it a little bit, and you go through a better period, but then maybe you're under more stress, and it just breaks out again, you know, yes. In other words, the relapsing remitting nature of symptoms is very consistent with the activity of something like a pathogen that can change its behavior and the proteins that it makes over time.

Jackie Baxter  
This is kind of blowing my mind, which I knew it was going to. Amazing. So should we maybe dive a little bit into the recent paper, which I think came out - was it last week? I think anyway, I'll drop it into the show notes for anyone who wants to read it in its entirety. But can we talk a little bit about that?

Amy Proal  
Sure. And that paper is a review article. So what the point of that paper was, is that if you look at all the authors on the paper, those are many of the different researchers working together as part of our consortium. And we are doing, as I mentioned, already, a lot of the tissue biopsy work and the imaging work. And as our different groups start to uncover more evidence about the persistence of sars vov 2, we wanted to write a paper to just provide clarity on the topic for the rest of the research community. 

And that's what that paper is - it's us coming together to say, look, you know, basically reads like, there are good and, you know, different possibilities for long COVID but one that really is starting to build evidence for is the persistence of the virus in tissue and then we walk through in the paper, it's almost like just I was talking to you today, we walk through the studies that have found it so far, there's a chart in the paper that goes through, like kind of just documents different studies where people have found it, you know, in what tissue. 

For example, there is a really interesting study that found sars cov 2 in the tongue tissue of people who have loss of tastes, it's such Yes, this is the really interesting part. It was I think it was like up to 163 days, they did the study or something like that, in every patient who had loss of taste, they found the virus in the tissue of their tongue, associated with kind of immune dysfunction that seemed like it could be driving taste problems. 

And so it's like, in the paper, we went through that, like, it's been found in the olfactory bulb of the nose with people of loss of smell, and it's been found. So we, we go through the sort of evidence of what sort of builds a case for persistence, you know, at least occurring in some patients right now. And then we go into the mechanisms by which we think and are actually beginning to be shown that the persistence of the virus could drive symptoms of disease. 

And that gets into, like I mentioned before, one of them is just like, if you don't clear the virus and Spike protein, like I said before - it continues to leak in the blood or be produced, that could see the formation of the fibrin microclots that are being found. It could also keep damaging the blood vessels. So we kind of show that there could be like coagulation problems afterwards. 

We talk about how, for example, if someone doesn't clear the virus from their gut, maybe the lining of the gut then can get more worn down, like a leaky gut, because the virus is driving inflammation, because it's not getting rid of it. And then that could cause like, problems with the other bacteria and microbiome in that area. And like leakage from leaky gut of bacteria in the blood. So we go through sort of these all these different mechanisms by which the virus, not, you know, clearing and being in a reservoir could drive problems. 

Then we go in the paper into considerations - like kind of what we just talked about, which is, do we think it's replicated? Do we think it's active and go literally through what we're talking about, we talk you know, about like, other viruses and what we know about them and their activity and this kind of thing. And we, in that part of the paper, we actually kind of say, we need more more research. 

And then we document how we think this should continue to be studied. And we put in one of the boxes, like of the paper, like what we think are the essential research questions needed to move forward in the field to keep going on this. And those are actually, as part of Phase Two that we're now beginning, those are the questions that our next studies are designed to answer. 

And it's kind of like, it's a statement to say like, this is what we're thinking, this is what we're at, we think this matters. And we do then, at the end too, emphasize that, if this is the case, and we see a lot of evidence in this direction, we really, really, really, really need to accelerate clinical trials of antivirals and other therapeutics that might be able to clear a reservoir on patients. 

And we think that it is time to just begin those trials. And so we mentioned there are some Paxlovid trials already happening. But we also mentioned that momonoclonal antibodies, for example, coould be trialled, or combinations of antivirals, because honestly, with other viruses that persist, sometimes one antiviral is not enough. In other words, you sometimes have to like use one antiviral, and then target it with a second one, and even sometimes add a third drug in there to really you know, get to the virus so that it can't cause problems. 

So we emphasize the potential need for combination therapies, and just kind of up the argument to really continue clinical trials in this area, which we hope, honestly, that the NIH pays attention to - I mean, we have some private support and things where we're going to begin to do some of those ourselves. But part of it is just a general call to bring awareness to the fact that we need these clinical trials to really happen.

Jackie Baxter  
Yeah, definitely. Because yeah, that was going to be my next question was - right, you know, say we have the evidence for this. What do we actually do about it? Because I guess that's the kind of the answer that people want, isn't it? And yeah, clinical trials - that's the answer, basically?

Amy Proal  
Yeah, we need more clinical trials. And like I said, I think - we actually have one of the researchers in our consortium is working on this. And she's using sort of tissue models that she infects with the virus and then she can treat with different drugs to figure out sort of combinations of what can work. It really is going to matter that we think intelligently about the design of the clinical trials so that we can try to combine drugs that might make sense in a patient. 

For example, we might even down the road want to combine an anti-viral clinical trial with a drug that supports, let's say, part of the immune system that could also help maybe boost, in a sense, the immune response towards the virus and trial those at the same time to see if it's the combination of the immunomodulatory drug and the antiviral there really gets people further. 

So we need not just clinical trials, but we need to be able to think through them intelligently. And to a point, we need the buy in of companies making the antivirals - that's been an issue in this space. And we've been doing meanings for a long time. There are some antivirals that are generic, for example, Truvada, that's an antiviral that has shown activity. Well, it's used with HIV, but it's also shown activity against Epstein Barr Virus, and also with hepatitis, which is another one. So we are going to try to start a trial of Truvada at Mount Sinai, and in that sense, that drug is actually - we can just get it, it's it's a generic drug, so it's available. 

But in the case of some of the drugs that we want to trial, like remdesivir, some other drugs, we need the company making it to at least donate the drug, if not maybe support part of the trial. Because, you know, I mean, if the drug became used in long COVID, it would be something that they would sell as a treatment for long COVID. So we're trying to encourage them to think that that is a good idea, that they should come in and bring their drugs into the space, and give us as the scientists and clinicians the opportunity to try them. So that's part of what we're hoping is communicated.

Jackie Baxter  
And like, I guess, like, this is probably something you can do with all of this data that you're kind of gathering, is to work out and kind of map, what antivirals would work in which areas or which combinations would kind of work well together rather than, you know, the opposite effect, I suppose. And that's something that you're able to kind of work out, is it?

Amy Proal  
exactly, that's part of why we have designed our program to have people working, for example, just on the tissue biopsy studies, to just know if patients with long COVID still have the virus. But we also have people working in models at the same time to try to already figure out what combinations of drugs might make sense. 

For example, that researcher who's at University of Pennsylvania, she treats with possible therapeutics, she treats intestinal tissue, but she also treats lung tissue, and then she has endothelial tissue. And it literally may be that depending on what tissue type the virus is in different drugs or combinations might be more important for that area of the body. So we do have research on that at the same time. 

And then we're also working on just the infrastructure of clinics as well. So for example, I'm beginning to work with David Putrino at Mount Sinai to start a new clinic there, where our goal will be to take all the findings from this and really say like to what extent can we begin to put this into the treatment of patients and build a model, a working model, for how we can most implement some of this information into clinical care and make that a very open model, so that maybe other groups that they want, can copy us. And we can sort of build up the structure of clinics that can actually start to do more of the treatments and stuff out the findings. So that's kind of like our multi pronged approach of like, yes, trying to do all that at the same time, so that we can make the best decisions on how to move forward.

Jackie Baxter  
Yeah, because I guess like, you know, it's great having all this data and all this knowledge and thinking, you know, I've got this massive spreadsheet that tells me all of these different things, I'm sure it's a lot more complicated than that. But like, if you can't actually turn it into something that's useful, and is gonna give you results, then it's kind of like, you've got all this data and nowhere to go kind of thing, isn't it?

Amy Proal  
You are exactly right. That is that - it literally is giant spreadsheets of data that no, you're not wrong. Like, it's that and that is the thing is, we definitely need that information, because the nuances of that information, really do help us make the right kinds of clinical trial and treatment decisions. And that matters, because we don't want to put people through something that doesn't work. And then that loses energy because it didn't work. And then the pharmaceutical industry gets concerned, you know, in other words, like it really matters that we kind of like make the right choices, because that's going to help with like a lot of like, the interest and people coming into this space. 

But to just sit on that data - No, like that's the whole reason that for example, I am so excited. And I've already started to work with David Putrino at Sinai where we work with you know, I'm now part of all of that sort of larger picture of the clinical care that's part of that clinic, and why we're prioritizing clinical trials as part of our phase two of this because yeah, there's no way that we can just sit here and I think all of us even as scientists are, like need to feel that clinical trials are happening and that treatments and things are moving forward as part of our work. I think we would go a little crazy if we didn't feel that way, honestly. So  that is undoubtedly the whole reason for doing everything, is just to get the information so that we can make it something that works that actually has meaning.

Jackie Baxter  
Yeah, yeah, definitely.

Amy Proal  
You know, take a look at our paper. And also, on our site on the PolyBio site, you can see some of the projects I mentioned, where we wrote out some of the descriptions of what we're working on, and we'll keep updated that way. And but I hope that maybe, you know, who knows, in whatever, a couple of months or so maybe I can come back and give some updates.

Jackie Baxter  
Yeah, absolutely. Yeah, I would be so excited to get you back. Cool. Well, thank you so much for giving up your -  I was gonna say evening, but it's probably not evening for you - part of your day, to talk to me. It's been absolutely fascinating. And I feel like I need to go back and listen to this all again so I actually take some more of it in. So thank you so much. And yeah, good luck with the rest of the research.

Amy Proal  
Thank you, no, thanks for having me on. It's fun to talk about it. I like to talk about it and try to explain what we're up to. So thanks very much for having me.

Transcribed by https://otter.ai