With COVID, When Can Schools Re-Open?

As a pediatrician, I’ve seen plenty of cases of Kawaskai disease during my clinical years in the 1960s-70s-80s.

—D

July 21, 2020

With COVID, When Can Schools Re-Open?

Every school year, children catch illnesses at school, and so do teachers and parents. Occasionally an outbreak causes schools to close for a time, say during a flu epidemic that is causing a lot of absenteeism. But never before have all schools been closed for months—even though COVID-19 has the unusual feature that children seldom either get sick from it or transmit it to others.

The CDC reported 14 deaths involving COVID-19 in children age 5-14 between Feb 1 and Jul 11. There were 2,173 deaths from all causes in that age group in a population of 41 million. As seen in the graph below, deaths in older age groups rose sharply after Mar 21, reaching a peak on Apr 18, and have been declining since. A recent surge of “cases” (positive PCR tests) has not so far caused a bump in deaths — which have returned to the baseline of expected mortality. By this metric, the pandemic is over.

Scary reports of “child COVID syndrome” are probably Kawasaki disease, which has been associated with many viral syndromes. There are about 5,000 hospitalizations per year attributed to this, and the number of cases has not been increasing.

Some teachers are reluctant to return, and some parents are also hesitant to send their children back to school. But if it is not safe to re-open schools now, when will it ever be safe? Who will believe it is safe if children are forced to wear masks and stay 6 ft apart?

The hiatus in schooling could lead to far-reaching changes. Parents may be more aware of what their children are learning—and not learning. Taxpayers may ask: if online learning works, why do we need so many teachers? Parents who find they are able to work from home and avoid an exhausting, expensive commute may want to keep doing that—and home-school their children. Several states report a sharp uptick in interest.

Giving parents more choices could be a positive outcome. But the long-term consequences of keeping children in fearful isolation will worsen, the longer the uncertainty continues. It is possible that the disruption has not prevented a single COVID-19 death.

For further information on COVID-19 diagnosis and testing, see Civil Defense Perspectives, January 2020, and on the surge or “second wave” see AAPS News, July 2020.

Executive Director, Association of American Physicians and Surgeons (AAPS)

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Advances in Technologies for Therapeutic messenger RNA (mRNA) Delivery

As a follow-up to an earlier GEITP email about “mRNA delivery, attempting to create vaccine production,” this excellent review [see attached] is very thorough and complete. 😊 Current clinical efforts include development of vaccines, protein replacement therapies, and treatment of genetic diseases. The latest advances in clinical translation of mRNA therapeutics have been made possible through novel developments in the design of mRNA manufacturing and intracellular delivery methods. Broad application of mRNA is still limited, however, by the need for improved delivery systems.

The translatability (process to create a protein from the messenger RNA) and stability of mRNA, as well as its ability to activate immune cells (immunostimulatory activity) are the key factors that require optimization for specific therapeutic application. Increased translation and stability can be affected by many regions of the RNA: 5’- and 3’-untranslated regions (UTRs) are required for recruiting RNA-binding proteins and microRNAs (miRNAs) in the intact cell, and these UTRs can profoundly affect translational activity. Modification of rare codons in protein-coding sequences — with synonymous (i.e. do not change the translated amino acid) frequently occurring codons (so-called ‘codon optimization’) — can result in order-of-magnitude increases in expression levels. Modification of the 5’-mRNA cap can also enhance mRNA translation, by inhibiting RNA decapping and improving resistance to enzymatic degradation. The importance of immunostimulation (by chemical modification of RNA bases) can depend on the application (and, in some cases, it may actually improve performance, as in the case of vaccines). Most importantly, methods and vehicles for intracellular delivery remain the most major barrier to the broad application of mRNA therapeutics.

Intracellular delivery of mRNA is generally more challenging than that of small oligonucleotides (proteins having relatively small numbers of amino acids) — because it requires encapsulation into a delivery nanoparticle — in part due to the significantly larger size of mRNA molecules (1000-15000; i.e. 1 to 15 kilobases, kb) as compared to other types of RNAs [e.g. small-interfering RNAs (siRNAs; 20-27 bases) and antisense oligonucleotides (ASOs; 21-28 bases)]. For those interested in more of the history of RNA therapeutics, and details of the mechanisms involved, please see [the attached] review. 😊

DwN

Mol Ther Apr 2019; 27: 710-728

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COVID Immune Responses Explained

This interview — with immunologist Dr. Akiko Iwasaki — seems to these GEITP pages to be a very worthwhile update on COVID-19, possible mechanisms of its infection, possible vaccines, and gender/age/genetic differences in response to the SARS-CoV-2 virus. Therefore (warning: it is quite a long read), let’s share this informative interview with everyone. 😊
DwN

COVID Immune Responses Explained

Eric J. Topol, MD; Abraham Verghese, MD; Akiko Iwasaki, PhD
August 21, 2020

Eric J. Topol, MD: Hello. I’m Eric Topol and I’m with Abraham Verghese for a new edition of Medicine and the Machine. Today it’s a special treat to have a chance to talk with Akiko Iwasaki, professor of immunobiology at Yale. Welcome, Akiko. Great to have you with us.

Akiko Iwasaki, PhD: Thank you very much, Eric. I’m delighted to be here.

Topol: Akiko’s background is quite fascinating. She came from Japan to Canada, finished her baccalaureate and PhD at the University of Toronto, was a post-doc at the US National Institutes of Health, and she’s been at Yale for 20 years. She is a Howard Hughes Medical Institute scholar and National Academy of Sciences electee. She has become my go-to immunobiologist, through Twitter, her writings, and her videos. In fact, toward the end of July, she posted an Immunology 101 on YouTube that is a must-see.

Before COVID, the most precious talent-people were the data scientists. Now it’s become the immunologists. Akiko, I know you spend a lot of effort on innate immunity and the interferons (IFNs), but can you give us all a broad view of the two main components of the immune response to SARS-CoV-2?

Iwasaki: You are right. Immunology has become quite relevant with this pandemic. And I’m trying to not only do research on immunity to COVID, but also to communicate to and educate the public about the immune system and how it works.

Our immune system consists of two different layers. The first layer is the innate immune system, which acts within minutes of infection to provide kind of a rapid response. This doesn’t require any specificity; it is engaged after any kind of infection. But this innate activation is important to triggering the second layer of the immune system, which is the adaptive immune system. In the adaptive immune system, the key players are B cells and T cells; they eventually acquire specificity and memory, which are the basis of the vaccinations. Having those T- and B lymphocytes that are specific to a particular pathogen and provide a memory response in the long term is very important.

Topol: One thing that came up recently was this kind of scare whereby some said that the antibody response that was tested in a few different series of patients who recovered from COVID seemed to be abating over time. That may have been a false alarm. Maybe you can put that in context.

Iwasaki: I don’t blame people for becoming worried about this because the longevity of the immune response is what we are counting on for protection of the whole population. That’s what vaccines are supposed to do. If you follow COVID-infected patients over time, their antibody levels do seem to wane to some degree within 2-3 months. But that is not a cause for alarm because that’s what happens when you get infected or when you become immunized for the first time. The antibody levels peak within the first couple of weeks and then eventually come down over a few months.

That’s okay because you still have memory B cells specific to that antigen as well as a T-cell immune response to the viral antigen. So the second time you’re exposed to the same virus, you can mount a rapid, specific, and robust immune response. It’s likely that you won’t feel anything the second time you’re infected. It will be a pretty mild or asymptomatic infection.

I want to make that clear, that waning antibody levels alone is not a huge cause for concern. The second point I want to make is that with vaccines, we usually give booster vaccines, which stimulate a much more robust and long-lasting immunity. That immunity should be sustained for years. So again, having a waning antibody response to natural infection shouldn’t be a huge concern for going forward with vaccination.

Topol: That’s very reassuring. There are so many different dimensions where immunology comes into play here. One of the areas that’s especially interesting — and it’s somewhat related to your recent paper in Nature where you characterize the different responses in people with moderate vs severe disease — is this deficient IFN response in some people, and whether or not that can be boosted early with some type of IFN. It’s complex, though, because there’s not just one type of IFN, and the timing is kind of like Goldilocks — you have to get it just right. Where do we stand? I know several clinical trials are ongoing with type-I IFN.

Iwasaki: As you say, type-I IFN has to be delivered at the right time with the right dose and the right type. What’s coming out, including from our own study, is that a prolonged level of type-I IFN, especially late during disease, may be associated with a worse disease outcome. So giving COVID patients recombinant IFN late in disease is probably not going to be a good idea. Whereas using recombinant IFN as a prophylaxis against infection, or if you can catch the patients very early during the infection and give enough IFN to shut down viral replication, those strategies hold promise. I’m waiting to see what happens in the clinical trials. But I think the early timing and giving enough dose to block the virus replication is going to be key going forward.

Topol: A paper just came out in Nature Reviews Immunology that reviewed four small ongoing trials and that multicenter study from China. All of them look quite promising. If you were to predict the future, do you envision that we all could have an inhaler with an IFN that we would take at first diagnosis or exposure? Do you think that’s a possibility?

Iwasaki: As long as we can give the right dose without toxicity, that may be the future, especially in a preventive setting. For example, if your family member was diagnosed with a viral infection, you could potentially treat the rest of the family members with prophylactic IFN, and healthcare workers or people who are exposed to high-dose virus on a daily basis. That’s what happened in China; they gave an inhaled IFN to healthcare workers and none of them were infected. So this may be a good thing to do in the future.

Verghese: Akiko, you have a lovely way of explaining very complicated things in a simple fashion. I’m an infectious disease physician, but I must say, the way cytokines and the cytokine storm are described is so bewildering. Can you help make sense of what we should be paying attention to in all the various cytokines and why?

Iwasaki: I hate to add to this bewilderment, but the typical cytokine storm that has been reported includes IL-1, IL-6, tumor necrosis factor, these sort of acute innate cytokines. But in our recent report, we also found cytokines that belong to completely different kinds of immune responses also coming up, like those that are dedicated to fungal response or helminth response — type III and type II immune responses. So in severe cases, the immune system kind of looks confused and disoriented, generating all types of cytokines that are also causing some sort of storm. I think COVID disease may start with the typical cytokine storm but then extend to different types of tornados and hurricanes and all kinds of misdirected immune responses.

Topol: Everything gets dysregulated. It seems as though people, for the most part — unless they have other hits such as a pulmonary embolism or something else — the cytokine storm is the principal cause of death in terms of the actual mechanism. So far, the only thing we have to block it is dexamethasone. But that’s not having exceptional efficacy. We need to do better on that whole process.

Another big topic right now is “long COVID.” You’ve taken a systematic approach to the whole spectrum of the disease, from mild to very severe or critical; long COVID seems like the next chapter. Very little immunologic work has been published to date. But with the joint pains, profound fatigue, and many other signs and symptoms, it looks like an immunologic condition. What are your thoughts on this?

Iwasaki: I absolutely agree. Long COVID is quite mysterious in terms of what’s driving such long-term disease. Also, the symptoms in these patients appear to shift during the course of post-exposure to COVID. I have three hypotheses to explain it, but it could also be a combination of these. The first hypothesis is that a reservoir of virus is hiding somewhere that’s activating and reactivating periodically to cause these types of responses. The nasopharyngeal swabs that we currently use to test for the virus are unable to pick up those kinds of reservoirs.

The second hypothesis is related to this. Perhaps it is not reservoirs of infectious virus but bits and pieces of viral RNA or protein that are being retained somewhere in the body that are activating an immune response against the virus and causing these shifting and prolonged symptoms. The third hypothesis is that the infection generates an autoimmune disease. Perhaps the virus is mimicking self-antigens or virus infection, being so inflammatory in this case, and is eliciting autoreactive T and B cells. We are trying to understand which of these possibilities is true. But perhaps all of these things are happening at the same time.

Topol: The autoimmunity may not be so difficult to address, but how would you get at a virus reservoir or the bits of RNA that are somehow creating or sustaining the hit?

Iwasaki: Some of the insights are the result of autopsy reports of patients who’ve had COVID and passed away. We don’t have a lot of insights from the “long-haulers” in terms of autopsies. But at least the investigation of those autopsies after acute infection is revealing infection in many places in the body, including the lung, obviously, but also the gut and many other places. Autopsy results will reveal whether remnants of virus or potentially infectious virus are hiding in these organs.

Topol: That is something we need to prevent. It seems to be not uncommon. To your point about the retention of virus, many of the cases I’ve reviewed remain PCR positive weeks and months later. So there’s something that’s consistent with that hypothesis for sure. Somewhat related is the multisystem inflammatory syndrome in children (MIS-C). It is obviously a variant of Kawasaki’s, but it’s different with respect to somewhat older kids, perhaps more heart and other organ involvement. And that does appear to be an inflammatory, autoimmune type story. What are your thoughts about that?

Iwasaki: In order to understand whether autoimmunity is involved, we need to identify autoantigens. I know that a lot of groups are working on it, but currently we don’t really know the relationship between COVID exposure and the development of autoantibodies or even autoreactive T cells that may cause these types of inflammatory diseases. So identification of the culprit antigen is going to be key.

Verghese: You have an elegant paper on a mouse model for COVID, which would be a wonderful way to tease out the separate elements of this disease. Talk, if you would, about developing that model and what you see as its future applications.

Iwasaki: Thank you for bringing that up. The mouse model we’ve created is a very easy-to-use and versatile model where we transduced the mice with adeno-associated virus (AAV)-encoding human ACE2. Its versatility comes from the fact that you can use any kind of background animal — whether it’s knockout, transgenic, or reporter mice. You simply have to transduce the mice with AAV for 14 days and until the expression ACE2 becomes fully developed. Then you can infect these animals with the human SARS-CoV-2.

That publication reflects the beginning of what we have done. We are looking at all kinds of immune response players, different cell types, different cytokines, and their relationship to protection against this virus, and also pathology that results from immune activation. In that first paper, we were able to examine the role of the type I IFNs, going back to Eric’s question, and showed that, at least in the mouse model, type I IFN appears to be incompetent for blocking the virus replication. If you look at the viral titer of animals that are either wild type or IFN-receptor knockout, the titers are not very different on different days of infection. What is different is the pathology we see in the line. Type I IFN induces a lot of chemokines that attract leukocytes into the lung. Unfortunately, it’s sort of fueling the fire by recruiting these leukocytes into the lung, and at the same time, not being very competent in blocking the replication of the virus.

This sort of mimics what we found in human patients, which was this long-term, smoldering type-I IFN response being associated with mortality and length of hospital stay. In the animal, we’re seeing the same thing: endogenous IFN is not very competent to block the virus replication and instead it’s leading to pathologic effects. This is the first insight we can see in parallel, from human to mouse and mouse back to human. Now we’re looking at other types of immune responses and whether they’re also contributing to pathology vs protection.

Verghese: Do you think this is a problem with the level of IFN or is it a qualitative defect in IFN’s ability to handle this particular virus?

Iwasaki: It is probably a mixture. We’re probably not getting a robust IFN-induced response because SARS-CoV-2 encodes numerous evasion mechanisms to block the induction of IFN and even IFN receptor signaling altogether. That’s why when we knock out the IFN receptor from the host, we don’t see much of a difference in bioreplication. It speaks to the ability of this virus to evade and sort of suppress the IFN response. That’s why having a recombinant IFN treatment early might make sense.

Topol: As long as it isn’t overridden somehow by this very elusive, difficult virus.

One of the things you’ve been uniquely championing is that we’re not paying enough attention to the mucosal immunity, the immunoglobulin A (IgA). Almost all of these 200 vaccine programs are working on spike protein shots or some component of the virus to provide the antibody and T-cell response. But there’s a whole other path, which would be to get to mucosal immunity. Maybe you could tell us more about the IgA response and why you think that’s not being given enough priority.

Iwasaki: Because of the nature of this pandemic, people are trying to develop a vaccine as soon as possible, which is important for us to return to regular society. So I think the first generations of vaccines will be the ones that elicit the most robust neutralizing antibody in circulation. That has a huge importance for preventing further spread of this virus. But given a little time and more effort, people are also starting to look at mucosal vaccines and potentially delivering the same kind of vaccine through the nose instead of into the muscle. When those vaccines are tested side by side, I believe we’ll see a difference in providing a sterilizing immunity in the mucosa vs inhibiting disease after exposure. I’m not discouraged by the fact that these vaccine companies are developing a systemic vaccination. But in the future, I think we should be looking more into mucosal vaccines.

Topol: Do you think they would be complementary? The vaccines that are in rapid development, phase 3 trials, are not going to prevent the infection or achieve sterilization but will modulate the response, whereas the IgA antibodies could actually block the infection in the first place. Could they be paired at some point?

Iwasaki: I think so. But if the systemic vaccines are providing a robust blocking of disease, that would be a great first start. By the way, sterilizing immunity is not the goal of most vaccines.

Topol: Some of the vaccines in trials are claiming it in their papers, in non-human primate models. It doesn’t need to be achieved. But it’s interesting how it’s being claimed right now.

The geneticist George Church recently took a mucosal vaccine via nasal spray. And it was criticized because it is like the Russian vaccine — there’s no safety profile. But George is quite a pioneer. It would be interesting to get actual results from the crew of people who are testing that vaccine.

One of the controversial issues right now is what’s been called effective herd immunity, a natural immunity that is being debated. Most experts agree that you need to get 70% or 80% of the people to be immune to establish herd immunity. But recently we have seen cases in the United States — particularly in states that were hard hit, such as Texas, Arizona, and Florida — where, for some peculiar reason, it looks like their infection numbers are going down, and not just because there’s less testing, which is another confounding issue. That has led to some people theorizing that maybe this 20% of people who are infected is providing a lower amount of spread. What is your sense about this? Is it an explanation for reduction in cases?

Iwasaki: First, I have to say that I’m not a mathematical modeler. I can only provide my insights as an immunologist. It’s a little dangerous to rely on herd immunity at this point to open up society because herd immunity requires a significant proportion of the population to be immune to the virus. Even if there is a community in which 20% of the people have immunity, we don’t know how long this lasts because they acquired it through a natural infection. We just don’t know enough about the protective level of antibodies and T cells in people who recover from this infection to know how long such a protective immunity will last. Also, every individual may be slightly different. I believe it’s premature and dangerous to depend on those numbers without a vaccine that can be distributed throughout the population.

Verghese: You know, before we leave innate immunity, I have what may seem like a naive question, but I know you’re an expert in mucosal immunity. Early on, some kitchen-sink wisdom suggested gargling and using nasal rinses as a preventive, because if the virus is going to attach, that’s the first step. Is there any logic to that? Might that be a strategy before we actually have immunity at a local level? The strategy would be to somehow block virus attachment to receptors in the nasopharynx.

Iwasaki: I’m not sure how much virus saline nasal rinses would actually get rid of. However, I am a proponent of humidity. We’ve published a mouse model looking at the role of humidity in our respiratory tract and immunity to influenza virus. We saw that a 40%-60% relative humidity helped the animal to be able to remove the virus from the respiratory tract through mucociliary movement. Whereas if you kept the mice in 20% humidity, which you will find indoors during the winter months, those animals did poorly because they were not able to clear the virus. So there is a natural mechanism for removing the virus, which we can take advantage of, by maintaining the relative humidity at certain levels.

Topol: Getting back to the vaccine front, there’s been a lot of consternation about what the goal should be. Some vaccine candidates produce relatively little T-cell response, particularly CD8 T cells, whereas others do. What would you envision as the ideal response, if you were to look at a vaccine and try to project that it’s going to achieve a durable protection? What’s your sense about that?

Iwasaki: If a vaccine can elicit very high levels of neutralizing antibody, that would pretty much block the spread of the virus in the person. T-cell immunity may not be needed at all, meaning CD8 T cells. Of course, to achieve that kind of antibody level, you needed to have CD4 T-cell help. So by default, such a vaccine would have elicited very good T–helper cell immunity. But whether you need robust CD8 responses and neutralizing antibodies to confer protection is an open question. I’m not sure we need both of them. If you only saw the CD8 T-cell response, you would not achieve a very rapid clearance of the virus, because antibodies are needed to really block the spread of the virus. CD8 T cells are great at picking off the virus-producing factories, but they’re not going to prevent the infection altogether. In terms of importance, it’s the high neutralizing antibody titer, and if there is a robust CD8 response, that’s sort of icing on the cake.

Topol: Most of the vaccine candidates have quite a good profile for neutralizing antibody response, so that’s encouraging. Can you make any inferences from the SARS epidemic and what worked then, since there’s a similar structure? Certain people who had SARS apparently still have signs of immunity to SARS now.

Iwasaki: Several vaccines were studied during the SARS-CoV-1 outbreak. Some vaccines worked really well and others, unfortunately, induced an inadvertent disease enhancement. For example, the double-inactivated SARS-CoV-1 virus vaccine elicited an antibody-dependent enhancement (ADE) type of response. But I’m encouraged that even these kinds of inactivated vaccines against SARS-CoV-2 are inducing pretty robust neutralizing antibody responses with no evidence of ADE. Thus far, none of the vaccine candidates out there have reported any major adverse effects or ADE type of effects.

Topol: Antibody enhancement is a paramount concern. Do you believe that, or an immune complex disease. won’t be a big issue going forward, or at least only on a very rare basis?

Iwasaki: That is my hope. The first two clinical trial phases haven’t reported any of these effects; however, it’s really during the phase 3 trials that we find out if there are adverse effects in rare cases and why. That’s why it’s so important not to rush that process.

Topol: We’ve noted a big gap between male vs female risk for COVID-19. Can you tell us what you think is causing that difference?

Iwasaki: We’ve been actively looking at sex differences in immunity to COVID-19. So far, we’ve found that male patients who develop severe COVID tend to have very low T-cell activation, whereas female patients who develop severe COVID have elevated innate immune cytokines. Thus, there seems to be a different way in which women and men respond to the virus. The lack of T-cell immunity in men is interesting, because when we look at age on the X axis and T-cell activation on the Y axis, we see an age-dependent decline in T-cell activation in men, but not in women. So this may have something to do with why there is more severe disease in men who are infected.

Topol: There seems to be a preponderance of women in the long-haulers with COVID. Have you made any connections with that?

Iwasaki: It’s tempting to speculate about a link between autoimmunity and these long-haulers, because the vast majority of autoimmune diseases have a preponderance in women vs men. Of the three hypotheses I listed, the autoimmune disease could be occurring in women and that may be contributing to the long-hauler disease. But without any data, I don’t really want to speculate.

Topol: I guess we’ve asked you to speculate a lot, which is fun. It’s great to hear your views.

I want to ask you about kids. We talked about MIS-C but we didn’t talk about this whole controversy now with schools reopening and that children are less likely to manifest disease. Perhaps their transmission is less. There’s been a lot of confusion about all of this. What are your thoughts about the unknowns in children as compared with adults? And is there a difference between younger and older kids?

Iwasaki: There definitely seems to be an age gradient of symptoms associated with exposures to SARS-CoV-2. But the symptoms do not relate well to whether they’re infectious. Children can have a high titer of replicating virus in their noses even without symptoms. Whether they become spreaders without knowing they are infected is a real possibility. I have two children and they are dying to go back to school; they’re so sick of spending the entire summer with their parents. So I totally get it. And it’s important for their mental health for them to be with other children. But I do worry about their ability to transmit the virus, even if they don’t have symptoms.

Topol: That brings up the question of their prior exposure to common cold coronaviruses and that children may have preexisting T-cell immunity. Perhaps that’s happening more in children than adults, in part, because there’s the temporal gap between when adults may have been exposed to the common cold before coronaviruses and when children were exposed. You’re in New Haven, Connecticut. Things are pretty quiet there, in terms of spread. Do you feel better about kids going to school and a lack of a transmission chain in a place like Connecticut?

Iwasaki: As of now, we have a low number of cases, which is wonderful. But a lot of travel occurs within the country from states with high case numbers to those with low case numbers, even though there is a kind of quarantine. So it’s difficult to know when we would feel safe because spreading may be occurring without really knowing about it. It takes a couple of weeks for the numbers to actually come up. I’m definitely feeling better than I was in April, but at the same time, I still take as much caution as possible.

Topol: Will you have your kids in virtual classes or will they physically go to school?

Iwasaki: They would be very upset if I said they can only go virtual. Right now, the school is planning to open and they will have in-person classes. But it’s a fluid situation. I’m keeping my eyes on what’s going on right now.

Verghese: I just wanted to follow up on a personal note. Initially, in March, all the research labs were shut down and gradually they’re trying to open. But with all the challenges of having people in the same space, how have you managed your research lab? You’ve been incredibly productive. Tell us about the challenges of being at work.

Iwasaki: I personally haven’t been at my workplace for a long time (just like Dan Nebert), in order to provide enough space for members of my lab to work. We did shut down quite aggressively in mid-March. The only type of research that was allowed was COVID-related research, so even though the university shut down, my laboratory kept going. In fact, they were working harder than ever, trying to study immune response in real time. My lab has been working hard but it’s also following guidelines of physical distancing and de-densification. We couldn’t have all the members of the lab working at the same time; it had to be one person per bay, so it did slow us down in that way because we weren’t able to all be there. Fortunately, everyone practiced physical distancing and used personal protective equipment, so no one was exposed to the virus during that time.

Topol: Let’s leave the pandemic for a bit and learn more about you, Akiko. You’ve had an amazing career. Your father was a physicist, your mother was an activist standing up for women’s rights. How did they influence you in other things that happened in your career to get to where you are today?

Iwasaki: They had an enormous influence on me. Growing up in Japan, seeing how much my mother had to struggle to even keep working, really taught me the importance of speaking up and of believing in yourself as a woman, to be able to state the problem and to address it head-on. My mother is an activist. She is soft-spoken and the most gentle person you’ll ever meet. But even with that sort of personality, she exhibited this strength. I definitely was influenced by that and I tried to emulate that in my life. Sex-based discrimination or racial discrimination unfortunately happens everywhere. So she taught me to be proactive and vocal about it without having to be extremely loud.

Watching my father through my childhood was interesting because I told myself that I’m never going to be a scientist; he’s always reading journals and thinking about science, and what kind of life is that? Then eventually I became that person. I’m afraid that I’m deterring my daughters from pursuing science because I do the same as my father. Science is difficult, but it’s also the most exciting thing for me to do. I can’t imagine another job for which I get paid to do what I love thinking about.

Topol: Well, all the things that happened along the way created a phenom. You’re a great educator as well as a great scientist. We’re pleased to have had a chance to visit with you today.

Verghese: This has been a real eye-opener for me — a clear way to understand the immune system. Thank you for being with us, and good luck with your continued research.

Topol: I recommend to everyone that, if they’re on Twitter, they follow Akiko Iwasaki (@VirusesImmunity) as their number-one source for really useful information. And if you’re not on Twitter, you ought to get on it, because she’s got a lot to offer.

Akiko, thanks so much for taking the time to join us. We’ll follow your work and your group very closely, because I know you’re going to unravel and deconstruct a lot of the unknowns we have today.

Eric J. Topol, MD, is one of the top 10 most cited researchers in medicine and frequently writes about technology in healthcare, including in his latest book, Deep Medicine: How Artificial Intelligence Can Make Healthcare Human Again.

Abraham Verghese, MD, is a critically acclaimed best-selling author and a physician with an international reputation for his focus on healing in an era when technology often overwhelms the human side of medicine.

Akiko Iwasaki, PhD, is an immunobiologist at Yale University and the Howard Hughes Medical Institute. Her research focuses on immunity against viruses at the mucosal surfaces. She is particularly interested in educating the public about the immune system and how it works; she is also an advocate for improving the culture of science and for students in science.

Posted in Center for Environmental Genetics | Comments Off on COVID Immune Responses Explained

A Systematic Literature Review of Whole Exome and Genome Sequencing Population Studies of Genetic Susceptibility to Cancer

This extensive review [see attached] describes the use of both whole-exome sequencing [WES; i.e. protein-coding genes only; spanning ~30 Mb (megabases, million bases)], plus whole-genome sequencing (WGS; 30,000+ Mb, i.e. the entire haploid genome). The application of NGS technologies has been successful in revealing complex somatic mutational signatures associated with different types of cancer — a disease that is (by definition) a result of somatic mutations. The estimated cancer heritability is ~33% for overall cancer, and its unexplained component has remained high (e.g. 91% of cancer cases tested negative for known mutations in a large gene-panel testing study). In addition to the heritability hidden in current array-based studies and likely detectability in using larger sample sizes — it has been postulated that the missing familial heritability may reside in rare variants of high or moderate/low penetrance, which are now potentially detectable by NGS technologies.

Authors [see attached review] therefore conducted a systematic literature review, evaluating the degree of success and limitations in identifying germline cancer susceptibility variants using WES and WGS. Given the transition of genomic discovery research from candidate genes (historically of limited success), the high cost of WES/WGS methods, and their specific challenge with sifting through millions of variants — this review focuses on the effectiveness of WES/WGS studies (and not on other NGS gene-targeted approaches) to identify novel variants and genes involved in cancer risk.

This review provides selected study-related characteristics, technologies, and methodologic details for 186 WES/WGS-related publications (out of a total of 6,339 unique articles) with the goal of informing the design of future studies. Authors also discussed the research needs and opportunities that could further advance the discovery of cancer susceptibility genes or variants. It should be noted that — although the 186 selected articles were not chosen on the basis of their focus on rare versus common variants, nor on their focus on low versus moderate/high penetrant variants, because more cost-efficient approaches (based on genome-wide genotyping assays) exist to study common variants, whereas NGS technologies are necessary to study rare variants. These GEITP pages believe the list of genes and genetic pathways shown in Table 3 was most informative. 😊

Authors found that variability across studies — on methodologies and reporting — was considerable; most studies sequenced few high-risk (mainly European) families, used a candidate-analysis approach, and identified potential cancer-related germline variants or genes in a small fraction of the sequenced cancer cases. This analysis, however, does highlight the importance of establishing consensus on standards for the application and reporting of variants filtering strategies. Authors also describe the progress in identification of cancer-related germline variation to date. Authors believe that their findings point to the untapped potential in conducting studies with appropriately-sized and ethnically-diverse families and populations, in which results across studies can be combined and expanded — beyond a candidate-analysis approach — to advance the discovery of genetic variation that accounts for unexplained cancer heritability. This overview should be a valuable resource for anyone interested in the heritability of cancer susceptibility. 😊

DwN

Cancer Epidemiol Biomarkers Prev Aug 2020; 29: 1519-1534

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Why and when was lactase persistence (LP) selected for? Insights from Central Asian herders and ancient DNA ..

To answer your question, Doron, the authors simply raised a philosophical (perhaps anthropomorphic?) question that cannot be answered, i.e. why Mother Nature has chosen this (genetic) form of evolutionary adaptation (for most northern Europeans) — instead of the “simpler” (at least, in our tiny human brains) “cultural adaptation” — is her secret. 😉

To answer the question from “Anonymous” — this is not a naïve question; it’s an exciting question.😊 Regulation of LCT gene expression is a fascinating story. The LCT gene is transcribed into messenger-RNA (mRNA), and then translated into a protein called “pre-pro-lactase”; this remains anchored in the endoplasmic reticulum (ER) membrane. Several subunits of pre-pro-lactase are cleaved off. The immature protein dimerizes (i.e. attaches to another copy of itself) within the ER. Then a transport vesicle, containing pro-lactase, splits off the ER and travels into the Golgi apparatus; once there, the “pro” subunit prevents degradation and ensures proper folding of lactase into its mature quaternary structure. Finally, a vesicle containing mature lactase travels to the external brush border membrane of gastrointestinal (GI) epithelial cells (enterocytes) — where the enzyme functions, to break down dietary lactose.

In most mammals (including ~65% of humans), levels of LCT gene expression in enterocytes decrease dramatically after weaning; this is because mammals do not typically consume milk after childhood — thus, maintaining enzymes to digest milk is unnecessary (it is ‘energetically wasteful’). Age-dependent lactase regulation occurs at the level of transcription. Transcription factors (TFs) are proteins that bind to a specific piece of DNA (usually 4-20 base-pairs), influencing a gene’s transcription frequency; once bound to DNA, a TF either attracts or repels the molecular machinery necessary for transcription. TFs often attract other TFs — to form large transcription complexes. TF “activators” bind to specific enhancer sites (on the DNA), and participate in initiating transcription by binding to RNA polymerase and other proteins used in transcription (this type of TF therefore increases expression of a gene; other TFs influence the probability and frequency of transcription by binding to TFs at enhancer sites). Enhancer sites can be far away from the start of a gene — but DNA can form large loops, allowing distant enhancers to come into contact with the transcription complex (this increases the frequency of transcription of the gene and, by extension, increases the expression of a gene).

Several TFs — that regulate the amount of lactase mRNA an enterocyte produces, over the course of its life — have been identified; these TFs bind to DNA ~14,000 base-pairs (bp) upstream of the LCT gene, within an intron (non-protein-coding region) of the upstream neighboring gene, MCM6. Thus, most research (concerning evolution of LP in humans) focuses on mutations not in the LCT gene, but rather on mutations in enhancers within introns of the MCM6 gene. Several single-nucleotide variants (SNVs) are associated with LP — all of which increase or decrease a TF’s ability to bind to DNA within specific response elements. For the most-thoroughly studied SNV 13,910 bp upstream of the LCT, a thymine base (T) has been substituted into the DNA sequence in place of a cytosine (C); this C>T mutation increases the binding affinity for the TF, POU2F1 (POU class 2 homeobox-1; formerly called ‘Oct-1’). POU2F1 acts as an activator (i.e. increases the transcription-complex binding to the promoter, thereby enhancing production of lactase mRNA).

Regulation of a gene via TF-binding site(s) in a neighboring gene is not that uncommon. For example, the human NFKB1 and SLC39A8 genes are located adjacent to one another on Chr 4q24, and they exhibit reciprocal regulation. NFKB1 (which is a TF) can activate SLC39A8, which results in enhanced influx of zinc (Zn) into many cell types; this leads to the coordinated NFKB1-mediated transcription of other inflammatory-factor genes. The Chr 4 g.102532378C>T NFKB1 intronic variant represents an expression quantitative-trait locus (eQTL), which causes decreased SLC39A8 mRNA expression in monocytes and macrophages; reciprocally, the SLC39A8-mediated higher Zn levels stimulate NFKB1 gene transcription, functioning negatively to down-regulate pro-inflammatory responses [via Zn-mediated down-regulation of IκB kinase (IKK) activity]. This example reflects a negative feedback loop involving SLC39A8 that directly controls innate immune function — through coordination of Zn metabolism and NFKB1 gene transcription.

Dear Anonymous, this might be a longer answer than you really wanted to know.😉 But regulatory interactions of neighboring genes along the same chromosome — are one of my favorite topics to read and learn about. 😊

DwN

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Reactive oxygen species (ROS) regulation of RAS and vulva development in the nematode (roundworm), C. elegans

This topic for these GEITP pages is a good example of hysteresis (in biology, this is a non-linear dose-response curve, where low doses are ineffective, a higher dose is beneficial, and a higher-yet dose is toxic). Examples of gases involved in signal-transduction pathways include nitrous and nitric oxide (N2O and NO), hydrogen sulfide (H2S), carbon dioxide and monoxide (CO2 and CO), hydrogen peroxide (H2O2), and oxygen, ozone, singlet-oxygen and superoxide (O2, O3, O. and O2•-). Each of these is essential to life at some concentration in living cells, ineffective at lower doses, and of course toxic at higher doses. Singlet-oxygen, superoxide and hydrogen peroxide are examples of reactive oxygen species (ROS), which are critically important signaling molecules; ROS studies in the intact organisms are often hampered by their potential toxicity. In the roundworm Caenorhabditis elegans, development of the vulva is regulated by a signal transduction cascade — which includes LET-602ras (the worm homolog of mammalian RAS, an oncogene in the GTPase family), MPK-1 (the worm homolog of ERK1/2, mitogen-activated protein kinases), and LIN-1 (an ETS proto-oncogene transcription factor).

Using C. elegans vulva development as a model, authors [see attached article] show both mitochondrial and cytoplasmic ROS act on the worm homolog of RAS — by way of a redox-sensitive cysteine (at position 118 in the protein, i.e. C118). However, in contrast to what is observed in cultured mammalian cells, it was found that C118 is oxidized by hydrogen peroxide, rather than by superoxide or nitric oxide, and that its oxidation inhibits, rather than activates, this pathway. Furthermore, authors show that regulation of LET-602ras (by its oxidation) does not act through MPK-1 to affect vulva development. In addition, authors identified two additional ROS-signaling pathways that promote vulva development: elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-602ras, and downstream of

LIN-1, whereas NADPH oxidases (BLI-3, ortholog of human dual oxidase-1; and DUOX-2, dual oxidase-2; these exhibit heme-binding activity and peroxidase activity), and their redox-sensitive activator CED-10rac act in yet another parallel pathway to promote vulval formation. CED-10rac is orthologous to human RAC1 and RAC2 (small GTPases), which participates in cell migration, embryonic morphogenesis (e.g. vulva development, and generation of neurons.

In detail, authors show that the pro-oxidant paraquat (PQ) (as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS), inhibit the activity of LET-602ras on vulval development. In contrast, the anti-oxidant N-acetylcysteine (NAC) and loss of sod-1 (encoding superoxide dismutase) — both of which decrease cytoplasmic H2O2 — enhance the activity of LET-602ras. CRISPR replacement of C118 with the non-oxidizable serine (C118S) stimulated LET-602ras activity, whereas replacement of C118 with aspartate (C118D) — which mimics a strongly oxidized cysteine — inhibited LET-602ras; these data indicate that C118 is oxidized by cytoplasmic H2O2 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibited LET-602ras. This finding contrasts with results in mammalian cells where it is mostly nitric oxide (not found in worms) that oxidizes C118 and activates RAS. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-602ras acts on an as yet uncharacterized MPK-1-independent pathway. Elevated cytoplasmic superoxide was found to promote vulva formation independently of the C118 in LET-602ras and downstream of LIN-1. Lastly, authors discovered a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Therefore, there are at least three genetically separable pathways — by which ROS regulates vulval development.

How is this study related to gene-environment interactions? This study shows that endogenous (intracellular) signals of gases/chemical agents are really no different from foreign environmental signals — in promoting genes that function in up- or down-regulation in signaling cascades. In this case, the signal transduction pathways are critically important to organ morphogenesis in the developing embryo. 😊

DwN

PLoS Genet Jun 2020; 16: e1008838

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RNA vaccines: a novel technology to prevent and treat disease

This information is a completely new concept to these GEITP pages(!!). I recall (in the late 1970s/early 1980s, when I was still in kindergarten 😊) our lab had tried to inject large quantities of isolated mRNA — into the peritoneal cavity of mice, hoping to get a response in liver (e.g. translation of the mRNA into a specific drug-metabolizing enzyme protein). These experiments did NOT work, the reason being that the ubiquitous nature of RNAases (enzymes that rapidly degrade the RNA at room temperature or body temperature of 37 Co) very quickly destroy the injected RNA. This article explains that scientists have now found novel methods by which they can prevent that degradation and, hence, use a specific RNA to be translated by the host’s cells into an antigen, which then can result in antibody formation in the host.

Antigen-presenting cells (APCs) are a heterogeneous group of immune cells that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes (e.g. types of T cells). Classical APCs include dendritic cells, macrophages, Langerhans cells, and B cells.

This is a very exciting area of new research in the field of vaccine formation. And it offers great promise for development of a vaccine to the dreaded SARS-CoV-2 coronavirus sooner than later(!!!). Clinical trials (with large numbers of volunteers, i.e. 30,000 or more) are already underway. 😊

Dwn

MAY 5, 2015

RNA vaccines: a novel technology to prevent and treat disease

by Alexis Hubaud
figures by Anna Maurer

Vaccination is key to preventing disease and has been a major advance in public health to eradicate epidemics like smallpox or polio. Vaccines work by mimicking an infectious agent, and by doing so, train our bodies to respond more rapidly and effectively against them. A new class of vaccines, “RNA vaccines”, has recently been developed. RNA vaccines rely on a different way to mimic infection. Compared to previous vaccines, this method is more robust, more versatile, and yet, equally efficient. Therefore, the RNA vaccine technology holds great promise to prevent and treat a wide range of diseases, such as influenza or cancer.

Have you heard about RNA vaccines? This technology recently made the news when the Bill & Melinda Gates Foundation invested $53 million in the German company, CureVac, which specializes in the development of these vaccines [1]. In this article, we will discuss how RNA vaccines work, their main advantages compared to traditional vaccines, and their applications in diseases such as influenza and cancer.
How do RNA-based vaccines work?

Vaccination is the process in which substances called antigens are introduced artificially into the body to stimulate the immune system, the set of cells that protects the body against infections [2,3]. Those antigens are generally infectious agents – pathogens – that have been inactivated by heat or chemical treatment so that they will not cause disease, or they can also be purified proteins from the pathogens. Exposing the body to antigens leads to the production of molecules specifically directed against them, called antibodies. Antibodies create a memory of a specific pathogen (“acquired immunity”) and enable a more rapid and efficient response to a real infection with an active pathogen.

Vaccination has been central in diminishing or eradicating multiple infectious diseases, such as smallpox or polio. However, producing vaccines is a long and complex process, and it has been difficult to implement vaccines against certain pathogens. Thus, designing new vaccines remains a major challenge for public health. To answer this challenge, there have been many improvements to designing vaccines, such as using computational prediction. Development of nucleotide vaccines based on DNA, and the related molecule RNA, is another promising area of progress in the field [4].

In each cell of a living organism, DNA is the molecule that contains the genetic information of the organism [5]. It is composed of a series of four building blocks, whose sequence gives the instructions to fabricate proteins. This process requires a transient intermediary called messenger RNA that carries the genetic information to the cell machinery responsible for protein synthesis. As an analogy, one can see the DNA as a cook book in a library: the recipe is stored here but cannot be used. The commis, or chef’s assistant, first makes a copy (the RNA) of a specific recipe and brings it to the kitchen. The information is now ready-to-use by the chef, who can add the ingredients in the order specified by the recipe and create a cake (the protein).

Figure 1: RNA vaccine technology. An RNA is injected in the body (left). This RNA encodes the information to produce the antigen, which is a protein from a pathogen, that will stimulate the immune system. Inside the cells, the RNA is used to synthesize the antigen, which is exposed to the cell surface (middle). Then, a subset of immune system cells recognizes the antigen and triggers an immune response (direct response and long-term memory) (right).

For a classical vaccine, the antigen is introduced in the body to produce an immune response. However, in the case of DNA- or RNA-based vaccines, no antigen is introduced, only the RNA or DNA containing the genetic information to produce the antigen. That is, for this specific class of vaccines, introduction of DNA and RNA provides the instructions to the body to produce the antigen itself (Figure 1). They can be injected in various ways (under the skin, in the vein or in lymph nodes) and then they can enter our body’s cells. Those cells will use the RNA sequence of the antigen to synthesize the protein [2,6]. After this step, the mechanism is similar to classical vaccines: the antigen is presented at the surface of a subset of cells and triggers the activation of specific cells of the immune system (Figure 2).

The ways in which DNA and RNA vaccines work are similar in many ways, and some of the common steps are described above. However, RNA vaccines have some distinct advantages. One is that RNA-based vaccines appear to perform better than DNA-based vaccines. Another is that they are also safer, as injection of RNA presents no risk of disrupting the cell’s natural DNA sequence. To continue our kitchen analogy, disruption from DNA is like inserting a foreign ingredient in an existing recipe, which can change the resulting dish [2]. Injecting RNA, on the other hand, is like temporarily adding a new recipe in the cook book while keeping old ones untouched, and therefore will not result in surprising changes to existing recipes.

Figure 2: Disease prevention. Vaccination with RNA induces a primary response (top) by instructing the body’s cells to produce an antigen that is presented to the immune system. This activates specific cells, which create a memory for this antigen. Later, when the real pathogen is present (bottom), those cells recognize the same antigen and react rapidly and strongly against the infectious agent (secondary response).
How are they produced?

With the considerable progress in DNA sequencing, it has become relatively easy to determine the genome sequence of pathogens. RNA can thus be produced in vitro, i.e. outside the cells, using a DNA template containing the sequence of a specific antigen. Creating a RNA vaccine also requires some engineering of the RNA to achieve a strong expression of the antigen [4,6].

This is a much simpler process than the culture of virus in eggs. Egg cultures, the more common way of producing vaccines, can provoke allergic reactions; the in vitro production of RNA avoids this possibility. Producing RNA vaccines is also less expensive than producing the full antigen protein [4,6,7].

Another advantage is that the production of RNA-based vaccines is more rapid compared to production of traditional vaccines. This rapid production could be a major advantage in face of sudden pandemics. Moreover, RNA-based vaccines may be effective against pandemics because they also provide more flexibility to prevent or treat pathogens that are rapidly evolving [8,9]. For instance, influenza vaccines have to be tailored each year to specific strains that are most likely to cause disease in the coming season. However, these forecasts have not always been accurate, such as during the winter of 2014-2015, making the influenza vaccine less protective. The World Health Organization estimates it takes approximately five to six months to produce an influenza vaccine, whereas the company CureVac claims that RNA-based vaccines could be manufactured in less than two months at a lower production cost, making it possible to respond to epidemics even as they develop. Therefore, RNA-based vaccines offer a comparatively simple and rapid solution to unpredictable, rapidly evolving pathogens.

While injection of simple RNA can elicit an immune response, RNAs in this form are prone to a rapid degradation. Current vaccines are fragile and can lose their efficiency when exposed at freezing or high temperatures, and must be stored at 35-45°F (2-8°C)[4,6,10]. Thus, preserving the cold chain is a major hurdle for the implementation of vaccine campaign. Fortunately, scientists have found ways to combat this RNA degradation. For instance, they can change the sequence of RNA to make it much easier to store. Furthermore, other molecules can be added to bind the RNA and protect it. Such engineering enables the storage of RNA vaccines at room temperature for at least 18 months. This feature precludes the necessity of maintaining the cold chain, making RNA vaccines particularly practical for developing countries.
What is the current state of the research?

This new exciting technology could be applied to many diseases, and pharmaceutical companies are making major investments in that area. RNA vaccines are still at the pre-clinical or clinical stage, but have yielded promising results. Below, we will explore two examples with the most advanced results: RNA vaccines to treat cancer and RNA vaccines to prevent influenza.

In the field of cancer immunotherapy, “cancer vaccines” take advantage of the expression of specific markers by cancer cells to direct the immune response and attack the tumor. RNA vaccines against prostate cancer, melanoma, and lung cancer (non-small cell lung cancer) are currently in clinical trials. For instance, six different RNAs against proteins produced in excess in tumor cells were used to formulate a vaccine against lung cancer. By taking advantage of the flexibility of RNA vaccine production, scientists can thus produce a vaccine with different antigens which is consequently better at targeting the tumor cells [11]. In the case of the prostate cancer vaccine, a preliminary study showed that injection of those RNAs foster an immune response in most of the patients. Whether this production of antibodies is sufficient to slow down the tumor progression remain to be determined.

Interestingly, because of the versatility of RNA vaccines, they could be tailored to fit the antigen repertoire of each patient tumor. Tumor cells are very different between patients, and this variability is an ongoing an issue for cancer treatment. An ongoing clinical trial is testing whether RNA vaccines may be effective for addressing variability in melanoma patients: in the trial, each tumor was first sequenced to identify its unique antigen repertoire, and then, a RNA vaccine is tailored to each tumor (Figure 3). This study shows that RNA vaccines could play a major role in this growing field of “personalized medicine” [7]. Moreover, these tailored, on-demand vaccines are practical – the company BioNTech claims that it could be manufactured in 5 months [12]).

Figure 3: Disease treatment (example of personalized cancer immunotherapy). The DNA from the tumor cells is first analyzed (top) to identify antigens specific to the patient’s tumor (Antigens A,B,C). Secondly (middle), a personalized vaccine comprising the specific RNAs for those antigens found in the analysis is injected to direct the attack of the immune system against the tumor (bottom).

RNA vaccines are also being developed to prevent infectious diseases. A vaccine against rabies is currently in clinical trials, while vaccines against influenza, HIV or tuberculosis are still at the research stage. Published results with the influenza vaccine [9] showed promising protection in mice. Indeed, injection of RNA coding for different proteins of the influenza virus induced the production of antibodies, and when the mice were later exposed to the virus, all survived. Similar immune response was observed in ferrets and pigs. All these observations in animals point to a potential use in humans.

The field of RNA vaccines is still nascent. However, their production is flexible and rapid, and recent studies indicate they could be effective against a wide range of infectious diseases and cancers. While their clinical potential in humans remains to be firmly established, RNA vaccines appear to be a promising technology worth watching out for.

Alexis Hubaud is a PhD student in Developmental Biology working at the Brigham and Women’s Hospital / Harvard Medical School
References

[1] Press statement from the Bill and Melinda Gates Foundation and CureVac
http://www.gatesfoundation.org/Media-Center/Press-Releases/2015/03/CureVac-Collaboration
[2] Introductory video about vaccination http://www.pbs.org/wgbh/nova/body/immunity-and-vaccines.html
[3] Vaccination ingredients from the NHS (UK National Health Service) website http://www.nhs.uk/conditions/vaccinations/pages/vaccine-ingredients.aspx
[4] Review about RNA vaccines- Schlake et al. RNA Biology (2012) 9(11):1319-1330
[5] Introductory video about synthesis of proteins from DNA and RNA http://www.pbs.org/wgbh/nova/body/cellular-factory.html
[6] Review about the CureVac vaccine – Kallen et al., Human Vaccines and Immunotherapeutics (2013) 9(10):2263-2276
[7] Review about RNA-based therapies – Sahin et al., Nat Rev Drug Disc (2014) 13 :759-780
[8] News article about the use of RNA vaccine against Influenza
Making a Flu Vaccine Without the Virus – http://news.sciencemag.org/2012/11/making-flu-vaccine-without-virus
[9] Scientific article on a RNA vaccine against influenza
Petsch et al. Nat Biotech (2012) 30(12):1210-1216
[10] Website from the company CureVac, which specializes in RNA vaccine http://www.curevac.com/
[11] Scientific article on a RNA vaccine against non-small cell lung cancer – Sebastian et al., BMC Cancer (2014) 14 :748
[12] Website from the company BioNTech, which specializes in RNA vaccine http://www.biontech.de/

RNA vaccines: a novel technology to prevent and treat disease

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How dangerous is COVID-19? A Swedish doctor’s perspective

How dangerous is COVID-19?

A Swedish doctor’s perspective

By: Sebastian Rushworth

August 11, 2020

I want to preface this article by stating that it is entirely anecdotal and based on my experience — working as a doctor in the emergency room of one of the big hospitals in Stockholm — and on living as a citizen in Sweden.

As many people know, Sweden is perhaps the country that has taken the most relaxed attitude towards the COVID-19 pandemic. Unlike other countries, Sweden never went into complete lockdown. Non-essential businesses have remained open, people have continued to go to cafés and restaurants, children have remained in school, and very few people have bothered with face masks in public.

COVID-19 hit Stockholm like a storm in mid-March. One day I was seeing people with appendicitis and kidney stones, the normal things you see in the emergency room. The next day all those patients were gone and the only thing coming into the hospital was COVID-19. Practically everyone who was tested had COVID-19, regardless of their presenting symptoms. People came in with a nosebleed and they had COVID-19. They came in with stomach pain and they had COVID-19.

Then, after a few months, all the COVID-19 patients disappeared. It is now four months since the start of the pandemic, and I haven’t seen a single COVID-19 patient in over a month. When I do test someone because they have a cough or a fever, the test invariably comes back negative.

At the peak three months back, a hundred people a day were dying of COVID-19 in Sweden, a country with a population of ten million. We are now down to around five people dying per day in the whole country, and that number continues to drop. Since people generally die around three weeks after infection, that suggests virtually no one is now being infected. If we assume around 0.5 per cent of those infected die (which I think is very generous, more on that later) that means three weeks back 1,000 people were getting infected per day in the whole country, which works out to a daily risk per person of getting infected of 1 in 10,000. And remember, the risk of dying is at the very most 1 in 200 if you actually do get infected. And that was three weeks ago.

Basically, COVID-19 is in all practical senses over and done with in Sweden. After four months.

In total, COVID-19 has killed under 6,000 people in a country of ten million. A country with an annual death rate of around 100,000 people. That makes COVID-19 a mere blip in terms of its effect on mortality.

That is why it is nonsensical to compare COVID-19 to other major pandemics, such as the 1918 pandemic that killed tens of millions of people. COVID-19 will never even come close to those numbers. And yet many countries have shut down their entire economies, stopped children going to school, and made large portions of their population unemployed in order to deal with this disease.

The media has been proclaiming that only a small percentage of the population have antibodies, and therefore it is impossible that herd immunity has developed. Well, if herd immunity hasn’t developed, where are all the sick people? Why has the rate of infection dropped so precipitously? Considering that most people in Sweden are leading their lives normally now, not socially distancing, not wearing masks — there should still be high rates of infection.

The reason we test for antibodies is because it is easy and cheap.

Antibodies are in fact not the body’s main defence against virus infections. T-cells are. But T-cells are harder to measure than antibodies, so we don’t really do it clinically. It is quite possible to have T-cells that are specific for COVID-19 and thereby make you immune to the disease, without having any antibodies. Personally, I think this is what has happened. Everybody who works in the emergency room where I work has had the antibody test.

Very few actually have antibodies. This is in spite of being exposed to huge numbers of infected people — including at the beginning of the pandemic, before we realised how widespread COVID-19 was, and when no one was wearing protective equipment.

I am not denying that COVID-19 is awful for the people who do get really sick or for the families of the people who die, just as it is awful for the families of people who die of cancer, influenza, or an opioid overdose. But the size of the response in most of the world (not including Sweden) has been totally disproportionate to the size of the threat.

Sweden ripped the metaphorical band-aid off quickly and got the epidemic over and done with, in a short amount of time, while the rest of the world has chosen to try to peel the band-aid off slowly. At present, that means Sweden has one of the highest total death rates in the world. But COVID-19 is virtually over in Sweden. People have gone back to their normal lives and barely anyone is getting infected anymore. I am willing to bet that the countries that have shut down completely will see rates spike when they open up. If that is the case, then there won’t have been any point in shutting down in the first place, because all those countries are going to end up with the same number of dead at the end of the day anyway.

Shutting down completely in order to decrease the total number of deaths only makes sense if you are willing to stay shut down until a vaccine is available. That could take years. No country is willing to wait that long.

COVID-19 has at present killed less than 6,000 in Sweden. It is very unlikely that the number of dead will go above 7,000. In an average year, 700 people die of influenza in Sweden. Does that mean COVID-19 is ten times worse than influenza? No, because influenza has been around for centuries while COVID-19 is completely new.

In an average influenza year, most people already have some level of immunity because they’ve been infected with a similar strain previously, or because they’re vaccinated. So, it is quite possible, or in fact very likely, that the case fatality rate for COVID-19 is the same as for influenza, or only slightly higher, and that the entire difference we have seen is due to the complete lack of any immunity in the population at the start of this pandemic.

This conclusion makes sense of the Swedish fatality numbers — if we’ve reached a point where there is hardly any active infection going on anymore in Sweden, in spite of the fact that there is barely any social distancing happening, then that suggests at least 50 per cent of the population has been infected already and has developed immunity, which is five million people. This number is perfectly reasonable, if we assume a reproductive number for the virus of two: If each person infects two new people within a 5-day period, and you start out with just one infected person in the country, then you will reach a point where several million are infected in just four months. If only 6,000 are dead out of five million infected, that works out to a case fatality rate of 0.12 per cent, roughly the same as regular old influenza, of which no one is the least bit frightened, and for which we don’t shut down our societies.

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Evolutionary success of cancers due to their induction of failed DNA repair and errors during DNA replication ???

How a cancer evolves and how mutations are generated — are highly intertwined processes, and nearly impossible to observe directly (instead, scientists are usually restricted to making inferences about them, using data from a single snapshot in time after a cancer has formed). However, when the two DNA strands (that form the double helix) are considered independently, authors [see attached article & editorial] showed that, for a cell that has undergone DNA damage, such a snapshot provides intriguing information. Analysis of cancer genomes has led to identification of many driver mutations and mutation signatures, which illustrate how environmental mutagens cause genetic damage and increase cancer risk. The numerous patterns of mutations identified in cancer genomes reflects both temporal and spatial heterogeneity of exogenous and endogenous exposures, mutational processes, and germline variation among patients. A previous study of diverse human cancers had identified 49 distinct single-base-substitution signatures — with almost all tumors showing evidence of at least three such signatures.

This is a complicated story, … so bear with me. 😊 Authors reasoned that a more controlled and genetically uniform cancer model system would overcome some of the limitations as to how a cancer evolves and how mutations are generated. By effectively re-running cancer evolution hundreds of times, authors aimed to explore oncogenesis and mutation patterns at high resolution (and with good statistical power). Using a single dose of diethylnitrosamine (DEN) in postnatal-day-15 (P15) male inbred C3H mice, authors chemically induced liver tumors. For comparison and validation, authors replicated the study in the divergent CAST mouse strain.

DNA resembles a ladder, with the ‘Watson’ and the ‘Crick’ strands; these are fused together by ‘rungs’ of two complementary nucleotide base-pairs [either cytosine (C) paired with guanine (G), or adenine (A) paired with thymine (T)]. When a cell divides, each daughter cell inherits either the Watson or the Crick strand from the parent cell; this provides the template from which the other (complementary) strand is then replicated. Damage to a base can trigger a repair process — but, if repair is not swift enough, the damaged base might be mispaired with an incorrect base during the next round of DNA replication. At the next round of cell division, when a daughter cell with such a mispaired base prepares to divide, the base complementary to the mispaired base will be added to the newly synthesized strand. This leads to a double-stranded mutation at the base-pair corresponding to the original damaged base [see this in the fantastic illustration shown in the editorial].

Standard practice for genome sequencing is to consider mutations — without paying attention to which of the strands was originally damaged. However, when a chemical change occurs that damages a base, creating a site referred to as a “lesion”, this lesion is on only one of the two DNA strands of the affected base-pair. Authors were clever enough to see that (because the ‘parental’ Watson and Crick strands of an original cell that underwent DNA damage are separated into different daughter cells) when the cell divides, two cell lineages can be tracked individually by following the unique pattern of mutations, on each of the parental strands, that the lesions generate.

Whole-genome sequencing (WGS) of 371 independently-evolved tumors from 104 C3H mice— revealed that each

genome had ~60,000 (approximately 13 per Mb) somatic point mutations (which is a level similar to that found in human cancers caused by exogenous mutagens — such as tobacco or UV exposure). [Insertion–deletion (indel) mutations and larger segmental changes were rare.] Point mutations were predominantly (76%) T→N, or their complement A→N, changes (where N represents any other nucleotide; this is consistent with the long-lived thymine adduct O4-ethyl-deoxythymidine being the principal mutagenic lesion). Known driver mutations were in the EGFR–RAS–RAF (epidermal growth factor receptor/GTPase-activating protein/proto-oncogene, serine-threonine kinase) pathway — and usually mutually exclusive; similar results were replicated in CAST mice.

Authors found that most mutagenic DNA lesions are not resolved into a mutated DNA base-pair during a single cell cycle. Instead — DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations — resulting in the

chromosome-scale phasing of subsequent mutations. Authors showed that DNA replication, across persisting lesions, can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity. The phasing of lesions — enabled the authors to accurately measure strand-biased repair processes,

quantifying oncogenic selection, and fine mapping of sister-chromatid-exchange events. Lastly, authors showed that, in human cells and human tumors as well, lesion segregation is a unifying property of exogenous mutagens — including UV light and chemotherapy agents. This study has profound implications for understanding the evolution and adaptation of cancer genomes. 😊

DwN

Nature 9 Jul 2020; 583: 256-270 & editorial pp 207-209

COMMENT:

Thank you, Dave. Pondering this question for a while, my first thought was to do the study in an established cell line in culture. Or freshly prepared trypsinized hepatocytes which are then grown in culture. However, I always worry about artifacts in “established cell lines” and when cells are placed in culture — even for very short periods of time.

Consequently, the best experiment I can think of — is to treat the intact mouse with whatever (e.g. dietary substance, chemotherapeutic drug, or DMN, etc.). Although “liver tumors” would arise many weeks after the tumor initiator is given, any initial “distinct tumor mutational pattern” is likely to appear within the first few days after the tumor initiator is given. Hence, one would need to sac mice at several time-points (e.g. 0, 12, 24, 48, etc. hrs after exposure to tumor initiator), isolate a substantial number of single hepatocytes, and perform whole-genome sequencing (WGS) on each single-cell sample (yes, this is not trivial; this would be a very expensive and time-consuming project). ☹ If anyone has a better idea, speak now or forever hold your peace. 😊

DwN

From: Dave
Sent: Friday, August 7, 2020 6:39 PM

Fascinating study — and an excellent summary of this very complex paper. Do you think it will be possible some day (maybe soon) to distinguish a tumor mutational pattern that would allow an assessment of a specific ‘environmental chemical’ contribution, separating this from a random ‘background’ mutation [e.g. mutations from dietary substances vs chemotherapeutic agents vs dimethylnitrosamine (DMN) mutations]?

David

Posted in Center for Environmental Genetics | Comments Off on Evolutionary success of cancers due to their induction of failed DNA repair and errors during DNA replication ???

Identifying and Characterizing Stress Pathways of Concern for Consumer Safety in Next-Generation Risk Assessment

Historically, safety assessments of ingredients present in consumer products (e.g. cosmetics, food) have relied on apical endpoints derived from animal testing. However, ethical and regulatory considerations on excessive animal use, in addition to scientific need to use more human-relevant data, have led to emergence of next-generation risk assessment (NGRA). NGRA is an exposure-led and hypothesis-driven approach, wherein safety assessments are conducted in a tiered manner — using detailed information on levels of consumer exposure to the ingredient — together with appropriate new approach methodologies — including in silico, in chemico, and in cell culture approaches.

For exposures in which systemic toxicity is predicted to be significant, physiologically based kinetic (PBK) models can be used to simulate distribution of the substance throughout the body (i.e. bioavailability). Output from such models can be combined with high-throughput cell culture assays where toxicity biomarkers of concern, and concentrations at which they are perturbed (i.e. the point of departure [PoD]), are identified. Developing suitable high-throughput assays for different toxicity outcomes remain a major challenge within NGRA. In particular, studies have suggested that many substances are associated with nonspecific toxicity modes-of-action — leading to cellular stress or mitochondrial toxicity — which, in turn, are associated with various organ toxicities.

Perhaps the most comprehensive datasets (looking at general bioactivity of substances) are those from U.S. EPA Toxcast and U.S. federal cross agency Tox21 programs. Analysis of these datasets revealed that there is a disproportionate increase in positive assay responses at concentrations that coincide with cytotoxicity and cell stress. In that analysis, it was generally not possible, however, to distinguish between specific stress responses triggered by a chemical at sub-cytotoxic concentrations (which may have subsequently led to cytotoxicity at higher concentrations, or later time-points), and cell stress events that coincided with, and potentially occurred as a consequence of, cytotoxicity (referred to in this article as a “cytotoxic burst”). This dilemma limits the degree to which data could be used to develop a hypothesis on a potential mechanism-of-toxicity. As such, developing a suitable set of assays and analysis to unravel these events is an ongoing challenge in the field of NGRA.

The objective of this work [see attached article] was to develop and evaluate a cellular stress response panel that could form part of an early tier screen for identifying substances that could, at relevant exposure levels, be associated with adverse effects caused in humans. The panel consisted of biomarkers covering the key cellular stress pathways already identified, together with mitochondrial toxicity and various cell-health parameters. To evaluate the suitability of the panel for chemical risk assessment, authors generated data using two sets of benchmark chemicals. The first set included chemicals that, at defined human exposures, are known to cause adverse systemic effects due to cellular stress in a subset of exposed individuals.

The second set included chemicals that, at relevant human exposures, have not been associated with adverse systemic effects related to cellular stress. A key principle of NGRA is that the various sources of uncertainty (e.g. identifying positive biomarkers and estimating the associated PoDs) should be robustly characterized. To this end, a novel concentration-response model was developed. The authors’ approach used Bayesian statistics, which allowed for uncertainties in the model outputs (i.e. PoD estimates) to be quantified in a probabilistic manner. Using the stress panel — together with the statistical approach described herein — it was possible for the authors largely to distinguish between chemical exposures that are associated with adverse health outcomes and chemical exposures that pose a low risk for the consumer.

The cellular stress panel — comprising 36 biomarkers representing mitochondrial toxicity, cell stress, and cell health [see Table 1 of attached article] — were measured predominantly using high content imaging. To evaluate the panel, authors generated data for 13 substances at exposures consistent with typical use-case scenarios [see Table 2 of attached article]; these included some that have been shown to cause adverse effects in a proportion of exposed humans and that have a toxicological mode-of-action associated with cellular stress (e.g. doxorubicin, troglitazone, diclofenac), and some that are not associated with adverse effects due to cellular stress at human-relevant exposures (e.g. caffeine, niacinamide, phenoxyethanol).

For each substance, concentration response data were generated for each biomarker at 3 time-points (Figures 3 & 5). A Bayesian model was then developed to quantify evidence for a biological response, and if present, a credibility range for the estimated PoD was determined. PoDs were compared with the plasma Cmax levels associated with typical substance exposures. These data indicated a clear differentiation between “low risk” and “high risk” chemical exposure scenarios. Developing robust methods to characterize the cell culture bioactivity of foreign chemicals is an important part of non-animal safety assessment. The results presented in this work show that the cellular stress panel can be used, together with other new approach methodologies, to identify chemical exposures that are protective of consumer health. 😊

DwN

Toxicol Sci July 2020; 176: 11–33

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