If COVID Could Talk … But It Does, at the Cellular Level

What goes on on the cellular level when it comes to COVID-19? To find out, Infection Control Today® reached across the Atlantic Ocean to the University of Bonn in Germany. Joachim L. Schultze, MD, is a professor of genomics and immunoregulation at the Life & Medical Sciences Institute at that university. Schultze is also the coordinator of the German COVID-19 OMICS Initiative, which seeks to integrate biological and science data with social sciences and the humanities. Schultz’s research focuses on understanding the range of severity of COVID-19 symptoms through an analysis of single cells. His latest research appears to indicate that proteins such as TLR3 and IFN I and II may be useful indicators of disease severity and/or an area to explore for possible therapies. His research also seems to indicate that the heterogeneity of COVID-19 may have to do with three factors: the host, the environment, and the virus itself. Schultze seems to have ruled out the virus. In a wide-ranging discussion with ICT®, Schultze also discusses the significance of neutrophils in immune response to COVID-19, the immune system’s role in COVID-19 long-haulers, and how the single-cell analysis that can be useful in the diagnose and treatment of COVID-19, can also be used to diagnose, treat, and potentially prevent other kinds of bacterial, viral, and fungal infections.

Infection Control Today®: Your research focuses on understanding the range of severity of COVID-19 symptoms through an analysis of single cells. So, in layman’s terms, could you explain to this beat reporter where your research has led you?

Joachim L. Schultze, MD: When the first wave hit Germany, we basically decided that the knowledge that we have in immunology, as well as in genomics and single-cell technology, we should basically use to understand this new disease, which is basically due to the SARS-CoV-2 infection. And at that time, there were some early reports on classical techniques used in the clinics, looking at blood samples that the immune systems seem to be awkward. And depending on how severe the disease was, it seemed to be different kinds of things. It looked very heterogeneous. And that’s why we thought that modern technologies, single-cell technologies, that allow us to dissect the immune system cell by cell would basically help us to understand this heterogeneity. We teamed up with clinicians and other partners and university hospitals in Germany, but also Europe, to get a sufficient number of patient samples that we could analyze the immune cells. We mainly focused many studies on the blood compartment. But with some colleagues, we also looked into the lung. And I had a couple of questions. The first one is, what is the dynamics of the disease over time when the symptoms started, what is the difference in the immune response when you have mild symptoms versus severe symptoms, and maybe also find early signs that tells us that people are at risk. And we wanted to know whether there are certain aspects concerning the two different arms of the immune system. Is there something happening in the innate immune system versus the adaptive immune system that would be associated with severe disease. And that’s what the starting point was. And that’s when we basically looked at many, many patients then by these modern single-cell technologies.

ICT®: You published your findings. Which journal did you publish in?

Schultze: We had several papers that we have published in the meantime. Our first study was published in Cell last summer where we actually focused on the innate immune system, particularly the so-called myeloid cells. These are mainly monocytes and granulocytes in blood because we had the idea already there were some clinical observations that neutrophils were

Joachim L. Schultze, MD

very high and lymphocytes were very low, so we get lymphopenia. And they basically built also a ratio saying that if the ratio is toward neutrophils that you have a severe course. And so, what we did try to answer is, is what kind of neutrophils are these? It’s very interesting because many labs in the world are using peripheral blood mononuclear cells when they look at blood. And usually, what you then do is you delete also the granulocytes because by this way of isolating the cells from the blood of patients, you’re losing the granulocytes. And we had the idea that based on the clinical observations that it would be a good idea to look at the granulocytes as well. And so, we used technologies that allowed us to do that. For example, for the single-cell RNA sequencing technology, we knew that a technology based on arrays and the one we used was the Becton Dickinson Rhapsody technology. But the cells are very nicely kept in this system so that we could capture also their information. And that was very good because over time we could see that in these patients with severe COVID-19 we had not only more of these granulocytes, but we had also some that changed their program completely. An interesting point is except for the absolute experts in neutrophil biology, not so much was known about single-cell RNA sequencing of the cells because many people always said these cells are not transcriptionally active anymore. And so, they are not so interesting. But we found even in healthy people that there are different subtypes of neutrophils on a transcription level. And in COVID, they changed completely. We found suddenly cells that were very inflammatory. But later during the disease, they would look more like cells that can suppress other cells, so kind of suppressive functionalities. And they could only be uncovered by these high resolution, single-cell technologies. Otherwise, we would not have seen that. And we not only did that in one cohort. We actually did that in two cohorts in one study. We had validation within our own project from the beginning. And I think that was one of the strong points early on in the COVID pandemic that also convinced the editors at Cell that we had really nicely validated. Many studies at that time had looked at a few patients only at one cohort, and that is sometimes prone to bias because you just take the samples that you get from the patients you have and then you cannot generalize it. We basically circumvented that by having two cohorts, finding the same in these two.

ICT®: You mentioned that severe COVID-19 … what are some of the practical applications of your research? Will this help physicians in the future or near future be able to determine who might be more susceptible to severe COVID-19?

Schultze: Yes. We also complemented the studies in the meantime with technologies that are more clinically feasible. So now, we know from our single-cell studies which cell types are changed and the granulocytes have played an enormously important role. But you can also do blood testing, for example, on transcriptome level on old blood, and we did this as well and then could correlate that directly. And what we can see in these studies is that you also see this granulocyte signatures in the blood. And what we found out, that there is a molecular phenotypes. So, you're not having one kind of COVID, but we found molecularly in the immune system, in the peripheral blood, that there's at least five subtypes of this disease. And what you then can do, is you can do, for example, drug prediction. So that’s what we did on these. We basically say, which drugs are known based on previous work where people had screened drugs for changes these drugs can do in transcriptomes, which of the transcriptome changes that we see in COVID could be returned back to normal by these drugs that have been tested. And what we predicted early on is that a subgroup of patients with severe disease would benefit from steroids. We knew that last Easter already, so a time when people really were searching heavily for treatments. And what was interesting to see then when clinical colleagues, particularly also in the UK, started these large trials also looking for drugs that could be used and they treated patients in a large trial with corticosteroids. Their finding was that none of the mild symptom patients benefited from corticosteroids really and the subgroup of patients with severe COVID. And that’s exactly what we predicted from these kinds of studies. We wouldn’t have said already because of the molecular phenotypes that it’s not all severe patients but only a subgroup and the clinical observations are in accordance. So now, we have to bring this together. I think it’s a good example of taking these high resolution, high throughput technologies identifying molecular phenotypes, and then tailoring better potential treatments. What we want to do in the future with precision medicine. In a way, I think, all of these findings will help us in the long run to find better drugs and better treatments for these patients.

ICT®:What do you think may be the immune system’s role in these occurrences of long haul COVID-19?

Schultze:The concept of long COVID is also to really understand that this is a heterogeneous syndrome. It’s just basically in the beginning saying that there are people that have long-term effects, but we now already know that there are different reasons for that, because you have those that had a very strong and severe disease and then take extremely long to basically get back to normal. So, that’s one basically big group of patients with long COVID. There’s another one where you had mild symptoms and then maybe even no symptoms for a while, and then symptoms come back like fatigue, and so on, and there’s mixed versions of this. So, there might be a heterogeneous picture. And again, in this situation, we think that all of those have to do with changes and reprogramming of the immune system both in the adaptive immune system as well as in the innate arm. So, I know that worldwide studies are ongoing to better understand what happens there and what are the changes that we seek to find, again, potential therapeutic or even preventative strategies for those that might have a mild COVID and might be prone to get these fatigue life syndrome diseases. If we understand what the role of the immune system in these patients is, I think we can very much help them to reduce their symptoms. I’m pretty sure that the immune system plays an enormous role.

ICT®: Did it challenge you a lot that this is so new, that COVID was so new, never seen before? And even as you just talked about looking at long-haulers, I mean, it hasn't been around that long. We don’t really know how long long-haulers can suffer from the after-effects of getting COVID, right?

Schultze: But what I’ve found interesting despite the fact that it was not organized worldwide in a completely organized fashion, I have to say that science has done an enormous job in the last 14, 15 months, and the technologies that we have at hand and that can be applied to patients these days allowed us to answer things much faster. And so, combined with the communication that we have in science worldwide, you could basically team up in larger groups. That’s what we did in Germany, for example, with this German COVID-19 OMICS Initiative where we have more than a hundred labs and more than 45 institutions now and that worked together on things. That’s the good side of things that when this happens you suddenly can answer questions much more quickly because we have experts for everything in the team and you can organize yourself better. We have basically protocols across different hospitals and can deal with patients in a similar fashion. And with the long COVID, they started already in the first wave. And now, since we have 14 months into this, if you started from day one to think about potential long-term effects, then you can actually design your observational studies along this way and that happened. And if you share that information that you’re doing it with your colleagues, others can do the same. And then, of course, we have enough numbers of patients that we can really identify what’s happening there. So, that’s the good side. Unfortunately, it didn’t happen everywhere. So, I think there’s still space for improvement, but bottom line is I think we never learned so fast with a new disease compared to COVID.

ICT®: I think you mentioned machine learning, and that might be a little bit off topic, but has that played any part in your research and what is it actually?

Schultze: Machine learning means that we’re using large data. And instead of having us looking at the data in a more manual fashion or with the classical statistical approaches, there are new tools out there where we basically give a certain algorithm, certain data, and the algorithm learns structures in this data. That’s the machine learning. And then we have additional independent samples and you’re trying to find the same structure in the data and these other data. That’s what's the field of machine learning. And part of that can be done by algorithms that are now following principles of the brain, mainly with so-called deep neural networks, and that is artificial intelligence. It’s weak artificial intelligence. It’s not the kind of it can do everything. It is just specialized algorithm that can do one thing very good, namely recognizing patterns in big data. And it’s interesting, not only in immunology but mainly also for other things in fighting the pandemics, many groups around the world have tried to use AI to find patterns, whether it was x-rays or CT scans, but also behavioral patterns to see whether we can learn about this disease and its circumstances. I have to say it’s a very early field. And I’m pretty sure for the next pandemic AI will play a much more important role because here many people jumped on it. And then sometimes the studies were very small, and that’s a problem for AI because you need enormously large data to really be successful, to really have good recognition of structures and data. And so, the first wave was super enthusiastic last year. But in the meantime, we also have more critical people that looked at the results and see how valid they are. And here we have to say there’s a little bit of backlash at the moment because when the studies are too small, it’s sometimes not possible to validate that. I would say we’re in the situation that the enthusiasm is still there, but we’re now also getting more realistic. What we have done there is a little bit different. We’re working on aspects that we can generate larger data sets so that we can bring more people together. Again, the idea of teaming up, and that’s what’s going on at the moment to circumvent the problem of too small data sets in machine learning for this. And again, there you can use medical data. So, we are using transcriptomes and also x-rays to find out if the data sets are large enough that you then can use this new technology to learn more about diagnostic potential for

ICT®: In your publication, you stated that the heterogeneity of COVID-19 may have to do with three factors, the host, environment and the virus itself, and you seem to have ruled out the virus. Can you elaborate a little bit on that?

Schultze:That’s a good point. I mean, we’re talking worldwide about mutations right now in the virus and then the mutants that have different infectivity. For example, the binding to the receptor is now an issue. And of course, we’re also talking about the first mutants out there might also lead to more severe disease. So, that is basically development that we just saw in the last couple of weeks that were unclear last year and were actually not seen in the early mutants, like when the Wuhan original virus was exchanged by the first mutant as D614G, which basically happened last year already from February to April and May and that led to a higher infectivity because of better binding to AC2 that is known now, but there was no indication that the disease itself changed. Now, with some of the new variants of concern, particularly the B.1.1.7 first found in the UK, there is now indication that there might be not only infectivity but also more severity, and therefore, also a higher mortality rate, but we still don’t know exactly why this is the case. It could simply be that this mutant still has just more virus that the host has to cope with. So, I would say, a little bit more uncertain than a couple of weeks ago is that there is a chance that there might be a role for the virus, but the indication is not yet good enough. So, we have to have more data on that. Having said that, when you look at the epidemiology and then also the immunological findings that we have now and look at the host immune system, then we have indication that the people that are getting more severe disease are elderly and people with co-morbidities. They have an altered immune system very often. And so, I think the reasons for a different disease course is still mainly in the host. Now, while there might be an additional component now coming from viral mutants, the host still dictates mostly the severity. I think that's just as of today.

ICT®: In layman’s terms, what do you mean by the host?

Schultze:The host meaning that basically everybody of us has a certain status in the immune system, and depending on your comorbidities or your age, and so on, you have a different status of inflammation. You might have activation that a young person doesn’t have. That’s one thing. And of course, there’s a third component, which I forgot so far or didn’t mention yet, is there are also some people that have a genetic predisposition based on certain mutations in defense genes, for example, in the interferon system that has also been shown so that the pathways are not working as they do in the normal population. For example, there is less type I interferon response, and that might be also something that leads to a worse outcome because the system is extremely important for the defense against SARS-CoV-2. And there’s another aspect which has been shown that some other patients apparently have B cells that produce auto antibodies against neutralize interferon alpha. In some studies, it has been shown that those patients with severe disease are enriched for those that have higher numbers of these auto antibody producing cells and have high levels of these auto antibodies against interferon alpha. So, we have already some indication what mechanisms are underlying these problems of the immune system in severe COVID, but there’s more to be discovered yet.

ICT®: Most of the experts seem to agree that they’re worried about the variants, but they say that the vaccines will have at least some effect against virus. Do you agree?

Schultze: I’m not a vaccinologist at the moment, but I’m following this very closely because I think that’s important for everybody and also for interest for an immunologist. So, far the experiments that have been reported for the main vaccines that are on the market in the US or in Europe, in other places in the world, all of them seem to be still functional against the current variants. There is difference in strength that some of the new variants have, the immune response after the vaccine is not as strong, particularly in the neutralizing antibodies against the virus, but it’s still sufficient to ensure that you’re not getting a severe disease or even a lethal disease, so that’s reinsuring. But it also told us that mutants have to be followed and have to be monitored so that we’re not missing something that some mutations that might escape the vaccines that we have today. I also see that it’s very important, and this happens more and more, and in more and more countries, that we are sequencing viruses from patients and from infected individuals so that we have a good idea of what is currently happening across the globe concerning the mutation rate of SARS-CoV-2.

ICT®: Is there something that I didn't ask you that you would like people to know?

Schultze: I would say that an important aspect, what we had learned in this pandemic, is that very good science can help to answer important questions of this disease much faster than we had the chance ever before because of better communication, better connection between scientists worldwide, but also technologies and the application of new technologies like single-cell sequencing, et cetera, to patients directly. So, that is a very positive development. I think there’s two things that we have to learn more. Because we have this incredible development in science, we need to make sure that the right information is given to our decision makers, to politics, as well as to the society that they understand, and we have to educate because it’s complicated what we are finding. It was complicated to us and it’s still sometimes puzzling us. We have to better communicate. And I think, what we’re doing right now, these things have to be done so that we can show to people there’s good things happening. And if we wouldn’t do the science, we would not be able to develop these vaccines so fast, but we have to make sure that everybody understands what we’re doing and what the consequences are so that we can have decisions in society, in politics, with the decision makers that help us all to get through this much faster. And here, I think everything that we can do to even further optimize that is a good thing to do.

This interview has been edited for clarity and length.