CSHL's COVID/SARS CoV2 Rapid Research Meeting Reports V

Brianna Bibel, CSHL School of Biological Sciences

The COVID/SARS CoV2 Rapid Research Reports is a series of meetings organized by Cold Spring Harbor Laboratory (CSHL) that brings together scientists from around the world to discuss the very latest research on the novel coronavirus SARS-CoV-2 and the disease it causes, COVID-19. The series began in June 2020 and continues with the sixth installment this July.

Brianna “Bri” Bibel is a fifth-year graduate student in the CSHL School of Biological Sciences studying biochemistry and structural biology in Leemor Joshua-Tor’s lab. Bri is the scientist behind the popular blog, The Bumbling Biochemist and Instagram account @thebumblingbiochemist. Her style of explaining basic biochemical experiments is approachable, and appreciated by science teachers and young students alike.

After a well-deserved break for the fourth CSHL COVID meeting which was held in Suzhou time, Bri returns to guest write for us, reflecting on the fifth installment of the meeting series:

• A short primer on viral mutation

• The B.1.1.7 variant

• The D614G variant

• Immune response and disease severity

• Antiviral therapeutics

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It’s been over a year since SARS-CoV-2, the coronavirus that causes the disease COVID-19, was discovered, and scientists are still learning new things about it daily as labs across the world dedicate themselves to unlocking the virus’ mysteries. Whether it’s determining if so-called “Variants Of Concern” are truly concerning (and if so, why), teasing apart why some people get much sicker than others, or discovering potential therapeutics to stop the disease in its tracks, there’s much to be learned. But of course, knowledge is only useful if it’s shared, and science thrives best when it isn’t siloed. CSHL’s Rapid Research Reports meetings continue to provide an opportunity for coronavirus researchers from around the world to come together, present their findings, get feedback and ideas, and maybe even strike up new collaborations.

Neil Ferguson, Imperial College London

The latest Rapid Research Reports meeting, the fifth in the series, was held virtually in January. It started with the topic that was on all our minds and in the headlines – the new coronavirus “Variants Of Concern” or VOCs. Imperial College London’s Neil Ferguson got us up to speed on what was known at the time about the B.1.1.7 variant first identified in Britain. Most of us had heard about B.1.1.7 only recently but, according to Ferguson, genomic surveillance data indicates that it actually emerged last August or September, likely in the UK, before gaining wider ground during the winter months. Unlike in the United States, where genomic surveillance has been lacking, the UK’s surveillance infrastructure was well-situated to detect when B.1.1.7 started to gain dominance. One of the reasons scientists were concerned was that it did so very quickly: By the time of the meeting, the variant was already over 50% frequency in the UK.

A short primer on viral mutation

Viruses mutate all the time because each time they replicate their genetic information (genome), there is a chance for typos. We call these typos mutations, and when you get a mutation you get a viral variant. This might sound scary, but most of the time these variants are inconsequential (or they make the virus less fit, so it can’t survive). Thus a variant, in and of itself, isn’t scary.

Sometimes however, a virus acquires a mutation that makes it more fit (e.g., allows it to spread more easily), and epidemiologists are always on the lookout for evidence of that happening. Often this evidence initially comes from genomic surveillance – scientists sequencing the RNA of random viral samples discover that a growing proportion of the samples they test are of a certain variant. When this happens, they might give that variant the “VOC” label.

Many of these VOC’s turn out to be coincidences, often the result of genetic founder effects whereby a viral variant is able to spread more widely because it “gets there first” and/or has more opportunities to spread, such as if a person sick with it attends a crowded event. In those cases, the mutations in the variant aren’t causative of the variant’s wider spread, they’re just “passenger mutations.” But the reason VOCs are concerning is because there really can be something to be concerned about – some mutations really do matter.

“...a variant, in and of itself, isn’t scary. Sometimes however, a virus acquires a mutation that makes it more fit...and epidemiologists are always on the lookout for evidence of that happening.”

The B.1.1.7 variant

At the time of the meeting, scientists were still trying to figure out whether the B.1.1.7 mutations mattered. We call B.1.1.7 a variant but it actually contains numerous variations compared to the original reference genome (the sequence of the initial sample from Wuhan). These variations include, most concerningly, mutations in the Spike protein. Often abbreviated as simply “S,” the Spike protein juts out from the viral membrane and helps the virus latch onto our cells by binding to cellular ACE2 receptors. Certain antibodies made by the immune system in response to SARS-CoV-2 infection or vaccination can bind to S in such a way as to block that docking and thus stop the virus from infecting cells. Therefore, scientists are always worried that the S protein might evolve in a way that prevents these neutralizing antibodies from binding, allowing the virus to escape the immune system. Thankfully, Neil Ferguson told us there was no evidence of such immune escape with B.1.1.7, so antibodies invoked by vaccines or natural immunity from prior infection should still be able to stave it off.

Furthermore, Ferguson told us that one S mutation in B.1.1.7 was actually fortuitous for epidemiologists because it made the variant easily spottable in the lab without requiring full-on genetic sequencing. This is because the mutation involves a deletion in part of the S gene that’s looked for by one of the diagnostic tests commonly used in the UK. The test, a PCR test, works by using short DNA probes called primers to look for several regions of viral genetic information. Because the S mutation contains a deletion in one of the primers’ binding sites, that primer would give a negative result, but the test’s other primers would give positive results so the person would still be determined positive for the virus. Using data from genomic surveillance and from the frequency of “S dropout” in the PCR results, Ferguson estimated that B.1.1.7 was about 40-70% more transmissible than the reference strain.  

The D614G variant

Another S mutation that scientists have been watching for much longer is a genetic typo leading to an amino acid change, D614G. The “D614G” nomenclature means that the RNA mutation causes the 614th amino acid (protein letter) of the S protein to be swapped from an aspartate (D) to a glycine (G). Aspartate is a big and negatively-charged amino acid whereas glycine (G) is tiny and neutral, so scientists have long suspected that this swap could affect how the protein functions and/or is processed. At the meeting, we saw evidence that this is indeed the case.

The CDC’s Bin Zhou told us that this amino acid change enhances the virus’ ability to replicate and spread, which likely contributed to D614G becoming the dominant version of S currently circulating globally. First, he showed results from experiments using purified proteins which demonstrated that D614G has enhanced binding to ACE2 compared to the original reference version. I had seen similar studies done before, but the next things he showed us I had never seen, and they were really cool! Zhou did competition experiments in which he pitted the D614G-containing virus against the original under different conditions and saw which won out. He started with cell culture experiments – when he treated cells with equal amounts of D614G and original virus and then observed which became dominant in the cell culture, D614G won every time. Even if he started with 9 times more of the original, the D614G variant was able to gain ground. But does that correspond to real, in vivo, effects? Zhou showed results from competition experiments in hamster and ferret models of SARS-CoV-2 infection as evidence that yes, it does: In the animal models, D614G outcompeted the original and was able to spread better from animal to animal.

Sophie Gobeil, Duke University School of Medicine

But why? Could hints be in the spike’s structure? Sophie Gobeil, a postdoctoral researcher in Priyamvada Acharya’s lab at Duke University School of Medicine, thinks so. She presented a poster of work she and colleagues had done characterizing the effects of D614G at the structural level. Using cryo-electron microscopy (cryo-EM), they got high resolution looks at the original and D614G versions of the S protein. One thing to know about S is that it’s a homotrimer, meaning it’s made up of three identical subunits working together. Each of these subunits contains a receptor binding domain (RBD) which, if in the “up” conformation, can bind to cellular ACE2 receptors, allowing the virus to dock onto our cells. Gobeil found that, compared to the original S, the D614G S protein was more likely to have an RBD in the up conformation, and thus was more “ready” to bind ACE2. The amino acid change is far from the RBD itself, which Gobeil says indicates local disturbances to accommodate the glycine led to ripple effects elsewhere in the protein – a phenomenon biochemists refer to as allostery.

Although the amino acid swap-out is far from the RBD, it’s near the furin cleavage site. Furin is a sequence-specific protease (protein-cutter), and the furin cleavage site is a location on the protein that contains the sequence furin likes to cleave. Gobeil presented evidence that the D614G change enhanced cleavage by furin. Why might that matter? Antoni Wrobel, a postdoctoral researcher in Steven Gamblin’s Lab at the UK’s Francis Crick Institute, has some ideas – and data! Wrobel showed us how this cleavage loosens up S in a way which primes it for fusing with the cell’s membrane and dumping the virus’ contents into the cell. 

If the D614G variant is more easily cleaved by furin, as Gobeil showed, then it would then be primed more often, as Wrobel showed, and this could help explain some of the increased transmissibility Zhou saw in his challenge studies. It was great to see different avenues of research from researchers around the world coming together to help piece together this scientific puzzle!

“...different avenues of research from researchers around the world [came] together to help piece together this scientific puzzle!”

Immune response and disease severity

Speaking of puzzles, one of the most puzzling things about SARS-CoV-2 is that it affects people so differently, with some developing severe pneumonia while others don’t even know they’re infected. To investigate why this is the case, scientists have dissected the immune system response in patients spanning the entire spectrum of disease severities, and at the meeting we got to hear from some of the immunologists leading this charge.

“Patients with COVID have high levels of...autoantibodies against many cellular proteins, including the immune system’s own proteins.”

James Heath, a scientist at the Institute for Systems Biology, told us about a deep, longitudinal analysis he was doing on the blood of around 200 COVID patients with a full range of disease severities. His group collected blood samples from patients at time of diagnosis, 1 month later, and 2-3 months later, then performed as many experiments as possible to try to determine everything that was in the blood: Proteomics to study the proteins present, metabolomics to look at small chemical products of metabolism, even single-cell secretome tests to see what signaling molecules cells released. Heath fed all that data into his computer and asked it to look for trends. When he did this, he found distinct signatures of mild vs. moderate disease, and also saw evidence of a lingering immune response in patients with moderate to severe COVID months after the original infection.

And that’s not the only evidence of immune system dysregulation seen in COVID patients. One thing that became apparent early on in the pandemic was that many of the sickest people were suffering not from the viral attack itself per se, but from their own immune systems. We usually think about antibodies as being “good,” such as anti-S antibodies binding to the Spike protein. But not all antibodies are good – they’re only good if they target things that are “bad.” Antibodies against one’s own proteins are called autoantibodies and they can cause the immune system to attack the body, destroying cells and tissues. Evidence from a number of labs shows that patients with COVID have high levels of such autoantibodies against many cellular proteins, including the immune system’s own proteins.  

Aaron Ring, Yale School of Medicine

Because there are so many different proteins these antibodies could be binding to, it has been hard to measure and characterize them, but Yale’s Aaron Ring told us about a system he developed to do just that. Named Rapid Extracellular Antigen Profiling (REAP), the basic idea is to get yeast cells to make human proteins and display them from their surface. More specifically, each yeast cell displays a different human protein and contains a corresponding DNA barcode, which Ring can sequence to infer what protein was displayed. He then mixes a patient sample with the yeast cells. If that sample contains antibodies targeting human proteins, those autoantibodies will bind to the yeast cells displaying those proteins. He can then isolate the antibodies and any bound yeast, and use DNA sequencing to see what human proteins the antibodies were stuck to.

Using this system, Ring confirmed and expanded the findings of other researchers showing that patient samples contained high levels of autoantibodies targeting proteins on the surface of immune cells. He presented evidence that these autoantibodies lead to the depletion of specific populations of immune cells, contributing to immune system dysfunction. He also found autoantibodies against proteins that are prevalent in a variety of tissues known to be affected by COVID, including tissues of the vascular system, heart, lungs, and central nervous system. The autoantibody signatures were different for different patients, and he’s following up on these findings to determine whether levels of these tissue-specific autoantibodies are associated with specific symptoms. He’s also tracking autoantibodies from samples taken over time to figure out whether they may be implicated in post-COVID syndrome (aka long COVID). Because the technology is so new, he doesn’t yet know how much of what he sees is specific to COVID as opposed to being a general viral response, but he’s eager to test samples from patients recovering from other viruses in order to find out!

“[Teams of scientists] are hard at working tyring to discover new antiviral therapeutics, especially ones that can be taken on an outpatient basis.”

Antiviral therapeutics

With all the talk of vaccination in the news, it’s easy to forget that there’s a whole other pharmacological aspect to confronting the pandemic which we will still need as long as the virus is around – namely, treatment. But teams of scientists around the world aren’t forgetting. Instead they’re hard at work trying to discover new antiviral therapeutics, especially ones that can be taken on an outpatient basis. At the meeting, we got to hear some of the latest prospects.

John Chodera, Memorial Sloan Kettering Cancer Center

One of my favorite talks was from John Chodera, a computational chemist at Memorial Sloan Kettering Cancer Center. Chodera told us about the “COVID Moonshot” project, which uses crowdsourcing to develop an inhibitor of the SARS-CoV-2 main protease (MPro). SARS-CoV-2 has many proteins, but it makes them as a long chain of connected proteins called a polyprotein. In between different proteins in the chain are protease cleavage sites, and the virus gets our cells to make the specific protease that recognizes them; block that protease and the virus can’t separate its proteins and thus can’t replicate. The goal of the COVID Moonshot project is to develop molecules that can to do just that.

I’d actually been following this project for a while, so it was great to hear a talk from one of the leaders. The basic story is that scientists at the Diamond Light Source in the UK soaked crystals of MPro with a compound fragment library (basically little drug-parts). Some of those fragments bound to MPro and the scientists used x-ray crystallography to solve crystal structures showing, at the atomic level, where those fragments bound in the protein. They then uploaded their data and asked volunteers to try to connect and/or modify the fragments to design drugs that could bind better and hopefully inhibit MPro. The team used an AI tool called PostEra to predict which designs would be easiest to make, then selected the most promising designs, synthesized the molecules, and tested them. In their first round, over 350 designers submitted a total of more than 7,000 molecules and the team synthesized and tested over 800 of them, finding promising leads that they then followed up with optimization.

Amanda Hargrove, Duke University

We also heard talks on other potential treatments that I hadn’t heard of before. For example, Duke chemist Amanda Hargrove told us how her lab is developing small molecules that target the coronavirus’ RNA. We often think about RNA as long strands, but those strands fold up into functional 3D shapes. SARS-CoV-2 has multiple stem-loop folds that are crucial for viral replication, and Hargrove is working to create molecules that specifically bind those structures and inhibit replication. The work she presented was based off of knowledge she’d gained while doing similar experiments to target HIV RNAs; in addition to potentially treating COVID, the knowledge she gains from her current work can likely be used to help treat other diseases with some tweaks of the molecules’ chemistry. RNA-binding drugs are definitely an exciting field to keep an eye on!

Speaking of keeping an eye on things, I can’t wait to see what’s in store at the next Rapid Reports meeting!

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This post is part of a series, and here is a summary of the first, second, and third COVID meetings.

The next installment of the CSHL COVID/SARS CoV2 Rapid Research Reports meeting series will take place on July 7-8, 2021.

CSHL's COVID/SARS CoV2 Rapid Research Meeting Reports III

Brianna Bibel, CSHL School of Biological Sciences

The COVID/SARS CoV2 Rapid Research Reports is a series of meetings organized by Cold Spring Harbor Laboratory (CSHL) that brings together scientists from around the world to discuss the very latest research on the novel coronavirus SARS-CoV-2 and the disease it causes, COVID-19. The series began in June 2020 and continues with the fifth installment this week.

Brianna “Bri” Bibel is a fifth-year graduate student in the CSHL School of Biological Sciences studying biochemistry and structural biology in Leemor Joshua-Tor’s lab. Bri is the scientist behind the popular blog, The Bumbling Biochemist and Instagram account @thebumblingbiochemist. Her style of explaining basic biochemical experiments is approachable, and appreciated by science teachers and young students alike. Here she guest writes for us, reflecting on the first three installments of the CSHL COVID meeting series.

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The third COVID/SARS CoV2 Rapid Research Reports meeting was pretty doggone cool! And not just because we got to see videos of doggies being cute… Well, they weren’t only being cute – as University of Pennsylvania’s Cynthia Otto explained in one of the highlights of the August 25-26 meeting, they were being trained to sniff out the scent of COVID-19. As with the first two of this meeting series, the third meeting (virtually) brought together scientists from around the world to discuss the very latest findings about the novel coronavirus that’s captivated everyone from hard-core virologists to random grad students like me who, before this all struck, couldn’t recognize a coronavirus if it hit me in the face with its Spike protein!

What was especially great about this third meeting was that, unlike the first two meetings which featured only invited speakers (which, don’t get me wrong, was really awesome!), the third meeting featured speakers selected from submitted abstracts. Because of that, we got to learn more about viral proteins that haven’t gotten nearly as much attention as Spike, but which also offer tantalizing therapeutic targeting potential. Other exciting additions to this meeting were a virtual poster session and a roundtable discussion on convalescent plasma therapy. Here’s some of what I found the most fascinating.

Structural biology, molecular biology, and immunology

As with the previous two meetings, this one was broken up into sections by topic and first up was “Host-virus Interactions/Structure.” There were some great talks looking at which animals are likely susceptible to the virus based on the genetic similarity of their ACE2 receptors to our ACE2 receptor (which we know is the way this coronavirus is able to dock onto our cells). Minks definitely are susceptible to getting and spreading the disease, as we learned about from Wim van der Poel of Wageningen University, who told us about his work studying outbreaks of SARS-CoV-2 amongst farmed minks in the Netherlands. In his studies, he was able to genetically trace outbreaks amongst people, minks, and even wandering cats!

Yogesh Gupta, UT Health San Antonio

Those “host-virus interactions” talks were really interesting but, as a student in a structural biology lab, I was particularly excited by the “structure” half of the session. At the second meeting we heard a lot about the Spike protein and RdRp, the RNA-dependent RNA polymerase which the coronavirus uses to copy its genome. At this meeting, more of the coronavirus’ dozen or so Nsps (non-structural proteins) got their time in the spotlight. Yogesh Gupta of the University of Texas and Karla Satchell from Northwestern University each told us about their labs’ work studying the structure and function of the nsp16/nsp10 dimer. These two Nsps work together to help disguise the coronavirus’ RNAs from our immune system by adding a small chemical adornment called a methyl group to the viral RNAs’ cap, making the viral RNAs look more like host RNAs. Both labs had promising early findings on the potential to target these proteins for therapeutics, and Satchell explained how her work was open access so that other groups can use the crystal structures and biochemical data her lab has generated to come up with ideal drugs. You can learn more in their publications here and here.

The second session, “Coronavirus Biology,” continued the theme of “evading the immune system.” Nsp16/nsp10 helps the coronavirus evade innate viral RNA sensors, but our cells also have intricate signaling networks to pass along warnings from a number of other hints that something’s amiss. Often these pathways lead to the addition of sugar groups called ADP-ribose onto proteins. University of Kansas’ Yousef Alhammad told us about a specialized section of the coronavirus Nsp3 protein, called a macrodomain, which removes this distress-signal from proteins, effectively erasing the cell’s attempts to trigger protective pathways. 

Silvia Rouskin, Whitehead Institute

One of my favorite talks of the meeting was by the Whitehead Institute’s Silvia Rouskin. The coronavirus genome contains “recipes” for making different viral proteins, and the virus gets the cells’ protein-making complexes, ribosomes, to use these recipes to make their proteins. But instead of making each protein separately, the ribosomes make some of the viral proteins as continuous chains or “polyproteins” which the virus then cuts into individual proteins using its viral proteases. Rouskin told us about a way the virus can choose which proteins to make when by altering the shape of its RNA. A region of the coronavirus genome folds up into a knotty structure called a “frameshift element” that’s able to stall the ribosome long enough that it slips, causing it to backtrack a letter and add extra proteins onto the polypeptide chain its making. Rouskin used a technique she developed called DMS-MaPseq to figure out which RNA letters were bound to other letters inside of infected cells. This allowed her to find that the structure of a coronavirus’ frameshift element inside of cells is different from that predicted based on the structure of shorter, isolated RNA. And there isn’t just one structure - she showed that the RNA took on various different shapes (alternative conformations) with different propensities for inducing frameshifting. The ratios of the alternative conformations the RNA took depended on the cellular context, hinting that certain intracellular cues might help the virus regulate which proteins to make at which times.

“...even never-hospitalized people who have “fully recovered” from COVID-19 have some strange things going on with their immune system.”

In the third session, “Pathogenesis and the Immune Response” we got to hear from medical doctors, immunologists, and epidemiologists monitoring the generation and stability of immune responses to SARS-CoV-2 infections. In a really fascinating talk by a medical student from the University of Alabama at Birmingham named Jacob Files, we learned how even never-hospitalized people who have “fully recovered” from COVID-19 have some strange things going on with their immune system. When he examined blood samples from patients over time, he found signs that immune cells called T cells were still working harder than would be expected since their virus was presumably cleared. At this point, they still aren’t sure what the significance is, but definitely something to keep an eye on. See more in this article in the Journal of Clinical Investigation.

Roundtable discussion on convalescent plasma therapy 

Cynthia Otto’s coronavirus-sniffing dogs may have stolen the show the first day, but the highlight of Day 2 for me was the roundtable discussion on the use of convalescent plasma (CP) therapy for patients with COVID-19. The session came just days after the FDA issued a controversial Emergency Use Authorization for this treatment, in which the cell-free part of the blood (called the plasma) from recovered patients is infused into sick patients. The rationale behind this strategy is that, among other things, anti-SARS-CoV-2 antibodies in the recovered patients’ sera can help block the virus from infecting more cells and doing more damage in the sick patient’s body.

Arturo Casadevall, Johns Hopkins School of Medicine

In the U.S., CP is a highly politicized issue but Arturo Casadevall from Johns Hopkins School of Medicine urged us to look past the hype and counter-hype to examine the data. And if there’s one person who knows that data well, it’s Casadevall. He led the early push for the testing of CP, which as he explained was no easy task. Nevertheless, he was able to put together a network of scientists and doctors who established an “expanded use” (EU) program through the Mayo Clinic. The EU program, though not set up as a controlled clinical trial, did allow doctors around the country to administer CP to severely ill patients – which they did in droves. In fact, the success of program had the downside of making it harder to enroll patients into controlled clinical trials, which made it harder to get the data needed to definitively say if CP is helpful. However, Casadevall showed us convincing signs based in part on analysis of data from the EU program that CP likely is helpful for some patients, especially if administered early and at “high doses” (sera containing high levels of protective antibodies). Read more about the analysis here.

“...one of the main reasons Casadevall is urging researching on CP [is because it] can be more easily deployed in developing countries.”

The other scientists on the panel, including Paul Bieniasz, Adrian Hayday, and Stanley Perlman, seemed optimistic about the potential of CP as well, but all stressed the need for more data – and from large randomized control trials so we can tell if effects are due to things like chance or the placebo effect. Since the treatment seems to help some people but not others, research is underway to try to figure out why this is and whether doctors can target the treatment to those most likely to benefit.

Speaking of benefitting, CP has the potential to potentially benefit large numbers of people who otherwise might go untreated. CP is often seen as a “stopgap measure” for addressing an emerging pandemic, before more targeted treatments are developed. But even once more targeted treatments (such as purified monocolonal antibodies) are available, they likely won’t be available to everyone; one of the main reasons Casadevall is urging research on CP is that, unlike extremely expensive and less stable monocolonal antibodies, CP can be more easily deployed in developing countries. The sincerity with which Casadevall spoke of his desire to help these underserved populations and the pain he felt over the polarization of the issue were palpable and stuck with me. I look forward to seeing the results of the controlled trials that are currently underway, and I hope that CP really is able to help patients around the world.

Although nothing can truly replace the experience of an in-person meeting, the third COVID/SARS CoV2 Rapid Research Reports meeting showed, once again, that virtual meetings can bring together scientists and allow them to disseminate their research to wide audiences as well as engage in fruitful discussions. It was an immense privilege to participate.

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This post is part of a series. Go here for a summary of the first COVID meeting and here for the second meeting.

The next installment of the COVID/SARS CoV2 Rapid Research Reports series will take place on January 26-27, 2021.

CSHL's COVID/SARS CoV2 Rapid Research Meeting Reports II

Brianna Bibel, CSHL School of Biological Sciences

The COVID/SARS CoV2 Rapid Research Reports is a series of meetings organized by Cold Spring Harbor Laboratory (CSHL) that brings together scientists from around the world to discuss the very latest research on the novel coronavirus SARS-CoV-2 and the disease it causes, COVID-19. The series began in June 2020 and continues with the fifth installment this week.

Brianna “Bri” Bibel is a fifth-year graduate student in the CSHL School of Biological Sciences studying biochemistry and structural biology in Leemor Joshua-Tor’s lab. Bri is the scientist behind the popular blog, The Bumbling Biochemist and Instagram account @thebumblingbiochemist. Her style of explaining basic biochemical experiments is approachable, and appreciated by science teachers and young students alike. Here she guest writes for us, reflecting on the first three installments of the CSHL COVID meeting series.

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The second COVID/SARS CoV2 Rapid Research Reports meeting, held (virtually) July 21-22, 2020 was enlightening, but also a reminder of how much there is still to learn, and a lesson in “why things are so complicated.” At the first meeting, we got the basics down, and at this second meeting, scientists starting packing in the nuances and details they were finding as they dove deeper into the biology of this trickster of a virus. We got to hear from scientists approaching SARS-CoV-2 biology from different angles and quickly came to appreciate that it’s unique, that context (cell type, organ, etc.) really matters when doing experiments, and that various techniques used by different researchers can all contribute to our understanding. Here’s some of what we learned.

Lessons from other coronaviruses

If you mention “the coronavirus” these days, people assume you’re talking about SARS-CoV-2. However, there are a number of other coronaviruses. For example, there are four common-cold-causing coronaviruses as well as a couple of other really nasty ones – SARS-CoV (the original) and MERS (Middle Eastern Respiratory Syndrome).

Susanna Lau, University of Hong Kong

Of course, those are just the human-infecting coronaviruses. Additionally, there are lots of coronaviruses hanging out in other animals. We heard a talk by Susanna Lau of the University of Hong Kong on the role bats played in harboring the ancestors of SARS-CoV-2. Often bats don’t experience serious illness from the viruses thanks to their unique immune systems. But other animals are more susceptible, including livestock; Carolyn Machamer of John Hopkins University spoke about Infectious Bronchitis Virus (IBV) which infects birds and sometimes threatens the poultry industry. Researchers have been studying these coronaviruses for years (though with much greater difficulty securing funding) and have found a lot of shared features. In a roundtable discussion led by Susan Weiss of the University of Pennsylvania Perelman School of Medicine, some of the world’s most prominent coronavirus experts (including Machamer, Alexander Gorbalenya of Leiden University, Carolyn Machamer of John Hopkins, Cornelia Bergmann of the Cleveland Clinic, Susan Baker of Loyola University, and Volker Thiel of University of Bern) discussed how our knowledge of other coronaviruses informs current research on SARS-CoV-2.

Much of this previous knowledge has been tremendously advantageous. For example: Since we know things about the largely conserved genetic architecture of coronaviruses, once scientists had the sequence of the viral genome, they could quickly figure out where the different genes were and what each did. This allowed them to do things like isolate the sequence encoding the Spike protein and use it to develop a vaccine. Last meeting we heard from a scientist from Moderna about how this allowed them to so quickly get a vaccine candidate into testing. In this meeting, we heard a talk from Kizzmekia Corbett on how her group at the NIH is developing a platform to be able to take the genetic information from any emerging virus and use it to rapidly create a vaccine. This will hopefully prevent us from getting caught off-guard in the future.

“...quantity isn’t everything, and how well nAbs can bind and block the virus is also an important consideration. In fact, Hatziioannou found that the majority of patients produced some really strong binders, and she was able to isolate some of the strongest to test their therapeutic potential.”

Potential therapeutics

Multiple speakers at the meeting studied how the immune systems of patients reacted in the short term, immediately after infection, to determine which proteins might be targeted as part of potential therapies for COVID-19. Several presenters looked for antibodies, which are proteins produced by special immune cells (B cells) that can bind to specific regions (epitopes) of foreign things (antigens) such as viral proteins. If antibodies bind to a virus in such a way that the virus can no longer infect cells, we call it a “neutralizing antibody” or “nAb.” For SARS-CoV-2, nAbs often bind to the Spike protein and block it from binding to the ACE2 receptor. They have significant value both for the patient (as they can provide some protection against reinfection) as well as potential therapeutic use. Therefore, scientists are really interested in seeing if people make nAbs for SARS-CoV-2: If they do, and if we can isolate the B cells making the nAbs, we can potentially use those cells to make lots more.

Theodora Hatziioannou, The Rockefeller University

One of the scientists leading these efforts is Theodora Hatziioannou, an Associate Professor at The Rockefeller University in NY who’s been looking at the blood plasma (the cell-less part of blood) of recovered patients to test for nAbs. When analyzing data from hundreds of NY donors, what she found was really interesting: About 80% of recovered patients had detectable nAbs, but the distribution of antibody levels within those patients was highly skewed. Most patients had low levels of nAbs, but about 10% were “elite neutralizers,” churning out high levels of nAbs.

Don’t be scared off by her finding that most recovered patients only made small amounts of nAbs – quantity isn’t everything, and how well nAbs can bind and block the virus is also an important consideration. In fact, Hatziioannou found that the majority of patients produced some really strong binders, and she was able to isolate some of the strongest to test their therapeutic potential.

Every time the virus copies its genome (replicates) it can make little mistakes (mutations). Most mutations are harmless or even detrimental to the virus, but some give the virus beneficial properties under certain selective pressures, like the presence of antibodies. By growing the virus in the presence of different nAbs, Hatziioannou was able to select for viruses that had mutations in the gene for the Spike protein which allowed them to resist binding to the nAbs – a phenomenon called “viral escape.” When she sequenced the mutated Spike genes from those viruses, she found that resistance to different nAbs clustered to mutations in different specific locations on Spike. She then looked at the sequences of the Spike gene from patient samples and found that, although rare, some indeed had mutations in those regions which would likely make them resistant to individual nAbs. So, if you were to try to treat one of those patients with that nAb, they likely wouldn’t respond. However, Hatziioannou also presented evidence that combinations of nAbs targeting different regions (epitopes) of Spike could prevent that viral escape, as it’s unlikely a virus will have a Spike that has multiple resistance-giving mutations. Therefore, she suggested that for treatment/prophylaxis, multiple nAbs be given as an “antibody cocktail.” Indeed, this is the strategy being taken by Regeneron.  

Of course, any nAb still has to be delivered, and this can be difficult and costly because human antibodies aren’t very stable. This has led some scientists to look to alternatives – like llamas! Gerald McInerney of the Karolinska Institute explained how alpacas have unique antibodies called nanobodies which are smaller, more stable, and easier to deliver than human antibodies. His group is working to engineer an alpaca nanobody with strong neutralizing ability and he showed us some of his latest results.

Even more molecular biology

Biochemistry/molecular biology is really my jam, so I was so happy that this meeting was jam-packed with it! I’m really interested in the mechanisms behind molecular marvels and, thankfully, a lot of top scientists are as well. But how do they figure out those processes? A common way to explore how the coronavirus gets into cells and what it does once inside is to test things out in cells in a dish. This is also a great way to screen potential drugs to see if they can prevent the virus from infecting cells and/or causing damage. But different cell types have different levels of various proteins and molecules, and thus might behave differently and give different results in experiments. It’s therefore crucial to do experiments in multiple types of cells and to be cautious with generalizations.

“...by doing experiments in different cell types and different model organisms, and by using different techniques to try to answer the same questions, we are able to study many different aspects of fundamental SARS-CoV-2 viral biology.”

Two cell lines commonly used for coronavirus research are Vero, derived from a monkey’s kidney, and Calu3, human respiratory cells. These lines are chosen in part because they both express the ACE2 receptor, which the coronavirus uses as a cellular docking station. The coronavirus Spike protein sticks out from the viral membrane and latches on to ACE2 to kickstart the viral fusion process. Viral fusion involves the Spike protein undergoing a shape-shift (conformational change) to merge its viral membrane with the cell membrane and dump its contents inside.

To undergo that conformational change, the Spike protein has to be cleaved in a couple of places by protein-cutting enzymes called proteases. As Fang Li from the University of Minnesota explained, there are many proteases capable of doing this, and different cell types express different ones. Further complicating things, some of these proteases, like TMPRSS2, are located at the cell’s plasma membrane (the membrane surrounding the cell) and thus can do the cutting at the cell surface, whereas other proteases are intracellular and can only cleave if the virus gets “swallowed” by the cell in a process called endocytosis. Figure 1 of this publication nicely illustrates this.

Vineet Menachery, The University of Texas Medical Branch

Processing of the virus will thus vary by cell type, as was demonstrated by The University of Texas Medical Branch (UTMB)’s Vineet Menachery. He looked at the importance of the “furin cleavage site,” which is a sequence in the SARS-CoV-2 Spike protein that allows one of the snips to be done by a “furin protein.” Menachery found that the importance of this site was greatly dependent on the cell type – mutating the sequence of the furin cleavage site increased the replication rate in Vero cells but hindered it in in Calu3 cells. Given the conflicting results, he turned to a hamster model, and was was able to share preliminary evidence that disrupting the furin site led to a weaker virus in this model.

Other researchers at the meeting also presented experimental results that differed based on the cell type or method. But this shouldn’t be discouraging or make you not trust science – instead, by doing experiments in different cell types and different model organisms, and by using different techniques to try to answer the same questions, we are able to study many different aspects of fundamental SARS-CoV-2 viral biology. If, like Menachery, scientists get different results in different cell lines, they see the results as the virus trying to tell us something. What is it about one cell type that makes it act differently from another cell type? Menachery, for example, took the conflicting results as evidence that there are potentially as-yet-unknown proteases present in Calu3 cells but not Vero cells that can affect SARS-CoV-2 processing. By looking into these factors, we can better understand the basic biology and potentially find novel therapeutic targets.

So yes, when it comes to SARS-CoV-2, things are complicated, but they’re definitely not hopeless! Years of research on other coronaviruses and the development of a variety of experimental methods have prepared us to face such complications head-on. And hopefully, work on SARS-CoV-2 will help us head off the next pandemic!

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This post is part of a series. Read a summary of the first CSHL COVID meting here, and here for a recap of the third meeting.

The next installment of the COVID/SARS CoV2 Rapid Research Reports series will take place on January 26-27, 2021.

CSHL's COVID/SARS CoV2 Rapid Research Meeting Reports I

Brianna Bibel, CSHL School of Biological Sciences

The COVID/SARS CoV2 Rapid Research Reports is a series of meetings organized by Cold Spring Harbor Laboratory (CSHL) that brings together scientists from around the world to discuss the very latest research on the novel coronavirus SARS-CoV-2 and the disease it causes, COVID-19. The series began in June 2020 and continues with the fifth installment this week.

Brianna “Bri” Bibel is a fifth-year graduate student in the CSHL School of Biological Sciences studying biochemistry and structural biology in Leemor Joshua-Tor’s lab. Bri is the scientist behind the popular blog, The Bumbling Biochemist and Instagram account @thebumblingbiochemist. Her style of explaining basic biochemical experiments is approachable, and appreciated by science teachers and young students alike. Here she guest writes for us, reflecting on the first three installments of the CSHL COVID meeting series.

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Do you remember where you were when you first heard about the novel coronavirus SARS-CoV-2? I do – vividly. I was at the gym and I saw a trending story on Twitter about a strange cluster of pneumonia cases in Wuhan, China. For the next couple of months I’d hear about it sporadically and then, wham! It was here. It was everywhere. And we had a lot of catching up to do.

So I was excited when I learned about a series of virtual meetings being set up by Cold Spring Harbor Laboratory (CSHL). Titled “COVID/SARS CoV2 Rapid Research Reports,” these meetings would feature experts from around the world filling us all in on the very latest research on the virus SARS-CoV-2 and the disease it causes, COVID-19. The first meeting was held June 16-17, 2020, and featured close to 30 talks. I don’t have space to go into every one of them, but I’d like to share some of the highlights.

A short history of coronavirus biology

Susan Weiss, University of Pennsylvania

The meeting was kicked off by a talk I know I needed – a lecture on the history and basic biology of coronaviruses by University of Pennsylvania’s Susan Weiss. Coronaviruses may have been new to many of us, but not to Weiss – SARS-Cov-2 was, of course, but Weiss has years of experience working on other, related, coronaviruses, so was well positioned to get us all up to speed. She told us about how coronaviruses have a single-stranded RNA genome that, although very small compared to our genome, is able to pack in a lot of tools to efficiently hijack our cells and bodies. For example, it makes an RNA-dependent RNA polymerase that it uses to make more copies of its RNA. Some of the copies it makes are full-length copies, which it can package up and ship out to infect more cells, whereas others are shorter “subgenomic” copies that it can use to make proteins from.

Some of these proteins are ones you’ve probably heard a lot about, such as the Spike protein, the protein that juts out from the viral membrane like a crown or halo, giving coronaviruses their name and giving them a way to dock onto our cells by binding to cellular receptors (a membrane protein called ACE2 in the case of SARS-CoV-2). But what fascinated me the most were the “Nsps” (non-structural proteins) she told us about that I’d never heard of. They don’t get that much attention, but many of them have the job of making sure our bodies don’t pay the virus much attention! For example, nsp13, nsp14 & nsp16 help with capping the viral RNAs so that they look like our own RNAs and thus don’t trigger an interferon-mediated immune response.

“When confronted with something “foreign,” an animal’s immune system has to decide what to do about it and how “paranoid” to be. Instead of focusing on host defense...bats err on the side of tolerance.”

Bats as reservoirs

Speaking of immune response, next up we heard from Linfa Wang from the University of Singapore about how bats are able to serve as reservoirs for viruses like SARS-CoV-2 because they have better balanced immune responses than us. When confronted with something “foreign,” an animal’s immune system has to decide what to do about it and how “paranoid” to be. Instead of focusing on host defense, which risks potentially over-reacting and causing autoimmune-related disorders and/or excessive inflammation, bats err on the side of tolerance. Wang explained his work showing that bats actually have a different way to control inflammation than other mammals, so they’re able to harbor viruses and allow those viruses to replicate within their bodies (and potentially go on to infect others) without getting sick themselves. Consistent with Wang’s findings, the University of Hong Kong’s Leo Poon told us that, since 2005 when he discovered the first bat coronavirus, he, and other labs around the world, have found many, many more (including ones closely related to SARS-CoV-2), stressing the importance of continuous surveillance in case they develop the capacity to infect other species.

Spike proteins

Figure 5 from Model for structural rearrangements of SARS-Cov-2 S protein. Image source.

As a graduate student in a structural biology lab, I know I’m biased, but some of my favorite talks came during the “molecular biology” session, where we got to hear about early work solving the structure of the coronavirus Spike protein. This protein is a bit like a molecular Transformer; after docking to ACE2 receptors on cell surfaces and getting cleaved by protease(s), it’s able to undergo a dramatic conformational change that enables it to fuse the viral membrane with the cell membrane, thereby releasing the viral genome into our cells.

I remember how surprised I was when the first SARS-CoV-2 Spike structure was published so quickly back in March, and at the meeting I got to learn more about how that was possible. Similar to Weiss, Jason McLellan from the University of Texas had a history of work on other coronaviruses – he had already solved the structures of the Spike proteins from SARS-CoV and MERS. So as soon as he received the sequence for the Spike protein, he cloned it and stuck it into cells to have them make Spike protein he could purify and take “molecular pictures” of. Thanks to his prior work on the “original” SARS-CoV & MERS Spike proteins, he knew that this Spike likely wouldn’t want to sit still for its photo-op. But he also knew a couple of changes he could make to the protein to help stabilize it in the pre-fusion conformation. By switching out a couple of amino acids (protein letters) in part of the protein, he was able to “freeze” it and then literally freeze it in a thin sea of vitreous ice (glasslike water) and use cryo-electron microscopy to solve its structure.

Overall, McLellan found that it looked very similar to the SARS-CoV Spike protein, but he was able to identify key differences in the part of the Spike responsible for binding the ACE2 receptor (the receptor binding domain). The structure was great, but McClellan and his lab weren’t satisfied – there were missing regions in their model and they were getting poor expression of their original construct, so McClellan told us about ongoing work in his lab trying to engineer a more stable, better-expressing version.

Neutralizing antibodies

David Veesler, University of Washington

One of the reasons it’s so valuable to know the structure of the SARS-CoV-2 Spike protein is because pre-fusion Spike can be used to look for neutralizing antibodies, as we got to hear about in a talk by David Veesler of the University of Washington. Antibodies are small proteins made by the immune system that bind specifically to foreign molecules (aka antigens) such as viral proteins; neutralizing antibodies are a subclass of antibodies that bind to a virus in such a way that they prevent the virus from infecting cells, thus “neutralizing” their threat. The search for neutralizing antibodies for SARS-CoV-2 has been focused on the Spike protein because, if antibodies bind to the Spike protein in the right places, they can prevent it from binding ACE2 and/or undergoing fusion, and thus prevent the virus from getting in.

Veesler developed a pseudovirus assay he could use to test for neutralizing antibodies. In these experiments, he used a different virus, MLV, to make “fake” SARS-CoV-2 viral particles that express the SARS-CoV-2 Spike protein and a fluorescence reporter, and then added this pseudovirus to cells. If the virus can infect cells, those cells will start glowing. But if the virus can’t get in because neutralizing antibodies are present, the cells won’t glow. Veesler and his collaborators used this assay to look for antibodies in the blood of SARS patients, hoping to find cross-reactive neutralizing antibodies (thanks to the high similarity between the Spike proteins of SARS and SARS-CoV-2). And they found some – Veesler used cryo-EM to solve the structure of one of them bound to SARS-CoV-2, and is now taking it into clinical development for use as a therapeutic.

“...if you use multiple antibodies you can prevent “viral escape,” a scenario whereby rare mutations in the Spike protein prevent the antibody from binding and therefore allow the virus in. ”

Later in the meeting we heard from Christos Kyratsous from Regeneron Pharmaceuticals about their development of neutralizing monoclonal antibody cocktails. Kyratsous explained that if you use multiple antibodies you can prevent “viral escape,” a scenario whereby rare mutations in the Spike protein prevent the antibody from binding and therefore allow the virus in. While some versions of the virus might have one of those mutations, it’s much less likely that a virus would have mutations that prevent binding of both antibodies. To find the antibodies to put in their cocktail, Kyratsous and his team immunized mice which had been genetically altered to produce human antibodies, and screened the mice’s blood along with blood from patients who had recovered from COVID-19 for the presence of neutralizing antibodies, choosing the most promising ones to take into clinical development. Kyratsous’ talk came just days after Regeneron had announced their first clinical trial, so it was especially exciting to hear about.

Antivirals and vaccines

Last but definitely not least was the session on “Antivirals and Vaccines,” where talks featured experts from Moderna and Gilead (among others), companies I’d heard so much about in the news but mostly in soundbytes and short pieces aimed at a more general audience. Here I got to hear about the science of Moderna’s mRNA vaccine and Gilead’s RNA dependent RNA polymerase (RdRp) inhibitor remdesivir, at a deeper, geekier, level.

Remdesivir is a nucleotide analog – essentially a “fake” RNA letter that the viral RdRp gets tricked into using, resulting in RdRp getting stuck and being unable to successfully copy the viral RNA. As we heard from Gilead’s Danielle Porter, it’s actually a repurposed drug. It was initially found by Gilead in a screen for hepatitis C drugs, and was later provided to the CDC and U.S. Army Medical Research Institute of Infectious Diseases as part of a library of molecules the government agencies could screen for effectiveness against various infectious diseases. The government scientists found it potentially useful against Ebola, but it was put aside after a different treatment (monoclonal antibodies) worked better. Since remdesivir had the potential to target viruses that depend on viral RdRps, scientists tested it against SARS-CoV-2 and got some promising early results that led to larger clinical trials. Because Gilead had already taken remdesivir fairly far along in the testing process for the Ebola trials, scientists knew a lot about its safety profile - and they knew how to make it – enabling it to come to market quickly.

Matthew Hall, National Center for Advancing Translational Sciences

The story of remdesivir highlights the potential of drug repurposing and, if there are other existing drugs that could be repurposed to treat COVID-19, Matthew Hall wants to find them! He told us how he and his team at the NIH are running high-throughput screens of various SARS-CoV-2-related assays against all approved drugs, and sharing the data freely through their OpenData Portal, which they also use to host data from similar assays run around the world, allowing for the most people possible to benefit.   

This meeting was an amazing opportunity, especially as a graduate student. I got to hear directly from the scientists whose names graced the papers I’d been poring over as I tried to make sense of what was going on. If I had to sum up the spirit of the first meeting in one word, it’d be “unity.” Despite the fact that we were coming from all over the world, despite the fact that attendees were of all training levels and most of us were completely outside of the virology field, there was a strong sense that we were all in this together. That those with the knowledge were willing to share it freely, with unprecedented speed and scale. That labs from different institutions and even different countries were teaming up in the fight. Coming away from the meeting, not only did I have hope about tackling the disease, but I also had hope that the future of science – all science – will be much more collaborative and open.

“Despite the fact that we were coming from all over the world, despite the fact that attendees were of all training levels and most of us were completely outside of the virology field, there was a strong sense that we were all in this together.”

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This post is part of a series. Go here for a recap of the second COVID meeting, and here for the third meeting.

The next installment of the COVID/SARS CoV2 Rapid Research Reports series will take place on January 26-27, 2021.