Omicron is wildly good at spreading

Omicron is wildly good at spreading

Amongst the plethora countries having different restrictions, the UK news yesterday about further restrictions was no news. But, which immediate future plays out will be a function of a few big unknowns — some already baked into Omicron’s biology and some that can be altered based on how people behave in the coming days and weeks. Further out, the models get fuzzier still. But though they differ in the details, all of them point to SARS-CoV-2 being here to stay. Thus far, data from Gauteng, South Africa, Denmark, the USA and here in the United Kingdom all add to the same picture. Omicron is wildly good at spreading and will infect and re-infect many people who are vaccinated or had prior infections, but is not generating a proportional uptick in hospitalisations or death among populations with high combined vaccination rates and prior outbreaks ie. the seropositives, that is people who had prior infections or vaccination:

As we know there’s lots of data to show it’s more transmissible than Delta. These data suggest it’s 3-5 times more transmissible:

But on the other hand this preprint suggests it’s 35% more transmissible, oh the joy of statistics: “Accounting for under-detection of infection, infection seasonality, nonpharmaceutical interventions, and vaccination, we estimate that the majority of South Africans had been infected by SARS-CoV-2 before the Omicron wave. Based on findings for Gauteng province, Omicron is estimated 100.3% (95% CI: 74.8 - 140.4%) more transmissible than the ancestral SARS-CoV-2 and 36.5% (95% CI: 20.9 - 60.1%) more transmissible than Delta; in addition, Omicron erodes 63.7% (95% CI: 52.9 - 73.9%) of the population immunity, accumulated from prior infections and vaccination, in Gauteng”. The senior author says: “That’s not something that should apply directly to other countries, like the U.S., because it’s very specific to the South African context,” said Shaman. Different strains took off there, leading to an immunological history not as relevant to the Northern Hemisphere. “How much immune erosion we can expect here will be hard to say,” said Shaman. “However, we’re talking about large numbers, so we could imagine it’s going to be pretty potent at running by the immunity of people who’ve already been infected or vaccinated in most places it shows up”:

https://www.medrxiv.org/content/10.1101/2021.12.19.21268073v1

It’s a tricky thing though, to extrapolate the shape of that curve from South African provinces like Gauteng, because the populations there look very different from either Europe or the U.S. People there tend to be younger, and therefore less prone to serious disease than in the U.S., where the population skews older and sicker, with more comorbidities like heart disease and diabetes. Another difference is that in South Africa, Omicron was a standalone surge, whereas here Omicron is hitting on top of Delta surges in the Northeast and Midwest, compounding strains on health care systems. Then there’s the whole ‘HIV thing’.

But, more importantly here’s a fantastic thread explaining intrinsic severity is hard to distinguish from “prior immunity”—our immune system reacts better because we have been vaccinated or previously infected. But there are challenges to this view:

https://twitter.com/roby_bhatt/status/1474339868947980300

And here’s the key point, about the multiple causes of severity: intrinsic and contextual. It's important to separate intrinsic severity (virus itself) from contextual severity (virus in population with lots of acquired immunity):

https://twitter.com/roby_bhatt/status/1475070304716271623

If you are immunologically naive—no prior infection or vaccination—Omicron is still likely more severe than many other variants, but maybe not Delta—which is a beast. And still around. For intrinsic severity, though, we have increasing lines of lab studies pointing to similar results: that it may well also be less intrinsically severe compared with other variants. From the Gupta lab, TMPRSS2 (T2) – one of the receptors for the virus –  is higher in the lower airways and so virus goes the second route in those areas. Omicron spike has reduced ability to use the ACE2+T2 route and doesn't respond to higher T2 levels, unlike Delta in the figure. Also, Omicron entry is not blocked by drugs against T2:

They looked at therapeutics against live Omicron virus versus Delta and find that the REGN2 cocktail (Ronapreve) has almost no in vitro activity against Omicron, though activity of Molnupiravir and Remdesivir are preserved, as shown by other studies:

“These data suggest a tropism shift might have occurred with Omicron, though the next variant may 'revert' with greater clinical severity. In addition we need to understand Omicron infection of other organs.*Vaccines are vital*”

https://twitter.com/GuptaR_lab/status/1474147496825872389

Another group shows in sharp contrast, Omicron is less fusogenic (left panel) and its spike is faintly cleaved (right panel). Our data suggest that the efficacy of spike protein cleavage, fusogenicity and pathogenicity are well correlated each other:

https://twitter.com/SystemsVirology/status/1474759704996122630

And again “at this point there's a consistent clear picture that Omicron has massively decreased fusogenicity when taking the TMPRSS2-dependent path & also that infectivity via that route is correlated w/LRT pathogenicity.”

Once again, none of this means that the ongoing Omicron wave is harmless.  Especially for the elderly or the immunocompromised, the immune defences may not hold as well (and for them, boosters are essential even more so than everyone else). Plus, there are still many, many people around the world who are seronegative, and the high rates of spread of this variant may be a big threat especially in places like that. Less severe than Delta doesn’t mean a cold. If this was the first variant we got back in January 2020 when nobody had prior immunity, it would be much worse than what we faced in that initial wave.

Still, this isn’t the worst case scenario on either counts: there is no substantial severity escape for those who are now seropositive, and it is not as bad as Delta or, yikes, worse, intrinsically. Make sure you’re boosted. Despite the caveat on original antigenic sin: “When a virus infects your body for the first time, your naive memory B cells imprint on specific virus proteins, or antigens, presented to them. These B cells then become either memory B cells or plasma cells. Forever after, they specialise in producing antibodies against those specific antigens. When a slightly mutated form of the virus arrives, these memory B-cells begin pouring forth the antibodies they learned to produce during the first infection. These antibodies bind to multiple epitopes on the virus particles, and in the process they give the slower-moving naive B-cells little chance to learn about any new, mutant virus features”:

https://eugyppius.substack.com/p/more-on-original-antigenic-sin-and

It’s ability to displace Delta seems profound:

There are a few theories for why omicron seemed to spring up suddenly. It may have been spreading all along and was missed by genetic surveillance efforts. But if that’s the case, why didn’t such a transmissible variant change Covid-19 patterns earlier? It’s unclear. Another possibility is that it emerged from a longer-term infection in an immunosuppressed person who didn’t have a strong arsenal of B cells, T cells, and antibodies to rapidly clear out the virus. That would have given it time to acquire mutations that help it elude the immune system.  I think the immunocompromised hypothesis is probably the most likely. However, many might say that usually variants that arise in such cases tend to be less able to infect people with robust immune systems. A third prospect is that omicron may have come from an animal host. Given that SARS-CoV-2 has already shown it can spread to a number of different animals, like minks, bats, tigers, apes, cats, white-tailed deer, it’s not far-fetched that the virus could leap back into people. Some genetic analysis work does hint at this route, but animals are even less studied than humans when it comes to Covid-19. So far, no animal has been found with a virus that looks like it could have spawned omicron.

There’s also the chance that omicron arose from recombination. That’s when an individual is infected with two different varieties of the virus at the same time, allowing the viruses to swap parts, resulting in a new virus that has some traits from each of its parents. If that’s what happened, scientists would expect to see a closer genetic relationship between omicron and the viruses it came from than what is evident in the phylogenetic trees. Figuring out where omicron came from may seem like an academic discussion, but it can reveal potential routes for future changes to the virus and tactics to close them off. In any case, we’re going to learn some interesting virology, but understanding the provenance of this virus can tell us some things about things we might need to do to further stop this pandemic. Here’s its relationships:

With vaccines in mind, there’s also adverts. One of Pfizer’s new vaccine TV commercials never mentions its vaccine brand Comirnaty. In fact, it doesn’t mention vaccines or Covid-19 at all and doesn’t show people wearing masks or social distancing. Yet it’s clear the ad is talking about the pharma’s Covid-19 vaccines. The ad’s voiceover talks about the unremarkable moments and routines that matter, like getting a coffee refill at a diner or Sunday grocery shopping. The images shift from those everyday moments to a scientists and purple lidded glass vials spinning off a production line and being packed into freezers. The sign-off? “At Pfizer, protecting the regular routine in every day, drives us to reach for exceptional. Working to impact hundreds of millions of lives young and old, it’s what we call ‘the pursuit of normal.’”

A second video ad is more overt, featuring NBA Brooklyn Nets player Bruce Brown who talks about how he’ll do “literally anything the team needs me to do out there on the floor. So I really can’t miss any games. When the vaccines became available, there was no doubt I was getting vaccinated. I was super excited because my life is basketball.” One of the final screens shows the words “Don’t miss your shot,” followed by a screen showing the Nets’, Pfizer’s and BioNTech’s logos. Similar to the other ad, the basketball ad is tagged “All rights reserved. December 2021.”

One TV ad first aired last Sunday, and so far has run more than 300 times, according to data from real-time TV ad tracker iSpot.tv. Pfizer expects Comirnaty sales of more than $36 billion in 2021. It forecasted additional sales of $29 billion in 2022 in November, based on 1.7 billion doses sold — although it has the capacity to produce 4 billion. It’s already in the process of signing more deals with more countries for 2022. Some may wonder with sales like that why even bother marketing. It may seem like a waste of resources, and that may be true for now when Pfizer is the only FDA-approved Covid-19 vaccine. However, there are at least seven more emergency use vaccines authorized around the world that are coming for Pfizer’s market share, especially in light of emerging evidence that booster will be needed for some time. Moderna’s Spikevax, J&J’s Janssen vaccine, Novovax’s Nuvaxovid/Covovax, AstraZeneca’s Vaxzevria and Sanofi and GSK’s vaccine will all be competing to become the booster of choice around the world. When and if those vaccines are fully approved in the US, expect more marketing and advertising as the pharma companies jockey for position.

And that means not just consumer awareness or even DTC ads, but also money spent marketing to physicians to become the Covid vaccine of choice with doctor’s offices likely delivering most future booster shots.

Snippets:

The recently emerged Omicron variant encodes 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Now researchers show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for convalescent and vaccinated individuals, but this loss was less pronounced after a third vaccine dose. Most receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only 3 out of 29 mAbs retaining unaltered potency, including the ACE2-mimicking S2K146 mAb1. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs neutralized Omicron through recognition of antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974. The magnitude of Omicron-mediated immune evasion marks a major SARS-CoV-2 antigenic shift. Broadly neutralizing mAbs recognizing RBD epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers:

https://www.nature.com/articles/d41586-021-03825-4

The emergence of Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections. A team investigated whether Omicron escapes antibody neutralisation in South Africans vaccinated with Pfizer BNT162b2. They also investigated if Omicron requires the ACE2 receptor to infect cells. They isolated and sequence confirmed live Omicron virus from an infected person in South Africa and compared plasma neutralisation of Omicron relative to an ancestral SARS-CoV-2 strain, observing that Omicron still required ACE2 to infect. For neutralisation, blood samples were taken soon after vaccination from participants who were vaccinated and previously infected or vaccinated with no evidence of previous infection. Neutralisation of ancestral virus was much higher in infected and vaccinated versus vaccinated only participants but both groups showed a 22-fold escape from vaccine elicited neutralisation by the Omicron variant. However, in the previously infected and vaccinated group, the level of residual neutralisation of Omicron was similar to the level of neutralisation of ancestral virus observed in the vaccination only group. These data support the notion that, provided high neutralisation capacity is elicited by vaccination/boosting approaches, reasonable effectiveness against Omicron may be maintained:

https://www.nature.com/articles/d41586-021-03824-5

Researchers found B.1.1.529 to be markedly resistant to neutralisation by serum not only from convalescent patients, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines. Even serum from persons vaccinated and boosted with mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies to all known epitope clusters on the spike protein, we noted that the activity of 17 of the 19 antibodies tested were either abolished or impaired, including ones currently authorized or approved for use in patients. In addition, we also identified four new spike mutations (S371L, N440K, G446S, and Q493R) that confer greater antibody resistance to B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2:

The above shows resistance to neutralisation by sera. The below to monoclonal antibodies:

https://www.nature.com/articles/d41586-021-03826-3

The rapid development of vaccines has been crucial in battling the ongoing COVID-19 pandemic. However, access challenges remain, breakthrough infections occur, and emerging variants present increased risk. Developing antiviral therapeutics is therefore a high priority for the treatment of COVID-19. Some drug candidates in clinical trials act against the viral RNA-dependent RNA polymerase, but there are other viral enzymes that have been considered good targets for inhibition by drugs. A group report the discovery and characterisation of a drug against the main protease involved in the cleavage of polyproteins involved in viral replication. The drug, PF-07321332, can be administered orally, has good selectivity and safety profiles, and protected against infection in a mouse model. In a phase 1 clinical trial, the drug reached concentrations expected to inhibit the virus based on in vitro studies. It also inhibited other coronaviruses, including severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, and could be in the armoury against future viral threats:

https://www.science.org/doi/10.1126/science.abl4784

The pivotal phase 3 clinical trials of the two-dose messenger RNA (mRNA) vaccines, among the largest ever conducted, led to the notable finding of ∼95% efficacy for prevention of symptomatic COVID-19, 2 months after the second dose. Adenovirus vectored vaccines showed lower protection against infection but achieved >90% protection against severe disease. No vaccines protect against all infections, and very few achieve such a high level of protection as that of the COVID-19 vaccines. In the early months after vaccinations at scale began, around January 2021, post-vaccination infections (or breakthrough infections) were rare, accounting for <1% of COVID-19 cases, and only ∼0.1% resulted in hospitalisation or death in high-income countries. However, by 5 to 6 months after vaccinations began, this pattern changed. Even before vaccine introduction, it was anticipated that a third, booster dose would be necessary to preserve efficacy, but when that would be needed was uncertain.

Variants of SARS-CoV-2 with multiple immune-escape and infectivity-enhancing mutations (particularly in the spike protein, which facilitates infection of human cells) likely arise after chronic infection. Some, such as the Alpha variant, showed increased infectivity and transmissibility, whereas other variants, such as Beta, were less sensitive to neutralisation by vaccine- and infection-induced antibodies. As Delta became prevalent in Israel, the UK, Qatar, and the United States, there were multiple reports of a substantial increase in breakthrough infections after mRNA and adenovirus vectored vaccination. The Israel Ministry of Health reported vaccine effectiveness of 40% against symptomatic infections 4 to 6 months after the second dose, representing a substantial decline (see the figure below). Although initially it was unclear whether this was due to waning immunity over time or the more transmissible Delta variant, it became apparent that time itself was a key driver, with attrition of efficacy seen in the participants of initial clinical trials. Waning immunity occurred, to a variable extent, after all vaccines studied to date, and loss of protection was likely amplified by increased prevalence of Delta.

The Delta variant contains mutations in the spike protein that are divergent from the three prior variants of concern, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). SARS-CoV-2 is an enveloped virus, taking lipid bilayer from infected cells. The infected cell produces the viral spike protein after translation from the viral RNA template, and spike is embedded in the lipid bilayer surrounding the virus core. The spike protein has two main regions, S1 and S2. During transport of spike to the plasma membrane, it is cleaved by cellular furin proteases at the furin cleavage site (FCS) between the S1 and S2 regions. The two cleaved pieces then associate with each other to form semimature spike at the cell surface. For optimal cellular infection of the produced virions, S1S2 also needs processing at the S2′ site adjacent to the FCS; this processing is carried out by transmembrane protease serine 2 (TMPRSS2) in the plasma membrane. Spike is then able to interact with the host cell receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor binding domain (RBD) to drive efficient fusion of the viral membrane with the host cell membrane for entry of the virus.

One mutation, P681R (Pro681 → Arg), is located in the FCS and is specific to Delta. The mutation is associated with increased cleavage of spike into S1 and S2 fragments. The role of P681R might be related to other spike mutations—for example, in the amino-terminal domain (NTD) of S1, where Delta bears a deletion of amino acids 157–158—as well as T19R, G142D, and R156G mutations. These NTD mutations lead to substantial rearrangement of the NTD that may have allosteric effects on the RBD and/or promote binding with additional cellular receptors to increase infectivity. Spike mutations that increase infectivity could enable virus to rapidly attach and infect epithelial respiratory cells, avoiding the relatively sparse neutralizing antibodies in the mucosa. Furthermore, Delta’s spike protein can achieve membrane fusion far more efficiently than can other variants. This ability to fuse cells to generate syncitia (multinucleated cells) might enable virus to propagate from one cell to another without needing to exit the cell, avoiding exposure to neutralizing antibodies.

Delta has also demonstrated moderate evasion from neutralizing antibodies, which appears to be partly related to the RBD mutation L452R with a less clear contribution from T478R in the RBD. Mutations in the Delta NTD have also been shown to reduce recognition by NTD-specific neutralizing antibodies. Together, immune evasion and increased replication likely underpin Delta’s ability to cause reinfection and vaccine breakthrough.

A consistent finding across studies has been the high viral load associated with Delta infections, no matter whether they occur among unvaccinated or vaccinated individuals. Recent transmission studies of the Delta variant have also revealed some distinct features, particularly a faster onset of illness and clearance. Faster clearance of the virus and a shorter duration of infectivity was noted in a study of vaccinated compared with unvaccinated people. The magnitude of transmissibility by individuals with Delta breakthrough infections appears to be approximately half that compared with unvaccinated individuals, which is supported by reduced culture-positive virus in some vaccinated individuals with high viral loads [as assessed by <25 cycle threshold (Ct), the number of cycles required for a positive result in real-time polymerase chain reaction (RT-PCR) tests]. A recent report of nearly 140,000 people who were contacts of individuals with RT-PCR–confirmed COVID-19 showed that both the AstraZeneca and Pfizer/BioNTech vaccines suppressed transmission, but their capacity to do so was markedly lower for Delta compared with Alpha and lower in people vaccinated with AstraZeneca compared with the Pfizer/BioNTech vaccine.

Furthermore, two independent reports have confirmed that high amounts of viral RNA (Ct <25) occur in asymptomatic Delta breakthrough infections, and that these individuals could be transmitting SARS-CoV-2 to others. However, distinction should be made between infectious virus and Ct value, and the relationship between the two in vaccinated versus unvaccinated individuals needs further evaluation. Overall, transmission from vaccinated individuals is increased by the Delta variant, compared with previous strains, according to the setting and length of time elapsed from initial vaccination.

Time appears to be the key driver of the post-vaccination reduction in effectiveness, as demonstrated from a study of 3.4 million members of the Kaiser Permanente health care organisation that found a similar pattern of decline in immunity against multiple variants from 2 months after the second dose. Although many studies have confirmed a reduction in serum concentrations of neutralizing antibodies from 4 to 6 weeks after vaccination, the picture is less clear for CD4+ and CD8+ T cell responses, with studies showing small changes consistent with the development of immune memory. The clinical waning of immunity after the first 2 months is particularly notable in people over 60 years of age, in whom susceptibility increased for both symptomatic infections and hospitalisations, as first noted in Israel and later confirmed in multiple US Centers for Disease Control and Prevention (CDC) reports. Circulating and tissue-neutralizing antibodies are expected to wane in a few months despite maintenance of specific memory B cell populations in the circulation. T cells, normally mobilized in response to infection, are thought to protect individuals from severe disease. That hospitalisations were increasingly noted in people of advanced age with breakthrough infections is consistent with poorer B cell and T cell responses to vaccination in older people, as shown in a study examining responses to the Pfizer/BioNTech mRNA vaccine. More studies exploring the trajectory of vaccine-induced cellular responses over time and according to age are needed.

Although the clinical trials of mRNA vaccines used a short time interval between two doses, 3 to 4 weeks for Pfizer/BioNTech and Moderna, shortage of the vaccines in many countries led to adoption of 8- to 16-week spacing. Scotland and Canada found that extended spacing of mRNA vaccines led to >80% effectiveness against symptomatic infection in the first few weeks after vaccination. Moreover, the most substantial drop-off in vaccine effectiveness (before the Delta variant became dominant) was observed using a 3- to 4-week dosing interval, such as in Israel, the United States, and Qatar. A direct comparison of short and long dose spacing for the Pfizer/BioNTech vaccine demonstrated that a 16-week spacing between doses resulted in optimal humoral immune responses. Administration of two mRNA vaccine doses, closely spaced by 3 to 4 weeks, may have acted as a primary immunisation—maximally inducing neutralizing antibodies but compromising durable immunity. That compromise may take the form of both humoral and cellular immunity waning in high-risk individuals, such as the elderly or immunocompromised as early as 2 months after the second dose.

Immunologic studies of responses to boosters, given 6 months after the last vaccine dose, have uniformly shown the induction of very high amounts of neutralizing antibodies, which correlates with protection from breakthrough infection. In Israel, where more than 1.1 million people over 60 years of age received an mRNA vaccine booster dose 6 months after the second dose, restoration of more than 90% effectiveness against severe COVID-19 was achieved. The restoration of vaccine effectiveness against hospitalisations and deaths with a booster dose was subsequently demonstrated for adults aged 40 years and older. A large, placebo-controlled randomized trial of the Pfizer/BioNTech booster indicated 95% efficacy, with reduction of symptomatic infections across all adults. Data are lacking for other vaccines and the durability of this effect. With continued circulating virus over time, it is likely that improved efficacy of a booster dose will be further demonstrated, in addition to reduced transmission and fewer cases of Long Covid (which can probably occur after vaccine breakthrough infection).

The high transmission rates observed in North America and Europe, where vaccine coverage is greatest, portends selection of vaccine escape variants of SARS-CoV-2 that could overcome some of the protection against severe disease. These variants are likely to arise during chronic infections in those with suboptimal vaccine responses, such as people who are immune-compromised, or where vaccine waning has occurred. New variants may evolve from Delta or may be radically different and could even be recombinants of variants due to mixed infections within individual hosts. Recent identification of B.1.1.529 (Omicron) with multiple spike mutations in southern Africa is a reminder of the ongoing threat posed by SARS-CoV-2. Continued transmission in highly vaccinated populations underscores the need for expansion of vaccination across age groups while maintaining nonpharmacological interventions, such as mask wearing. Investigation of intranasal vaccine preparations as a means of preventing breakthrough infection, development of pan-sarbecovirus vaccines, and exploration of the potential for antiviral medications should also be explored to limit transmission:

In late 2020, the Delta and Kappa variants were detected, and the Delta variant became globally dominant by June 2021. A team show that vaccine-elicited serum-neutralizing activity is reduced against these variants. Based on biochemistry and structural studies, the authors show that mutations in the domain that binds the ACE2 receptor abrogate binding to some monoclonal antibodies but do not improve ACE2 binding, suggesting that they emerged to escape immune recognition. Remodelling of the N-terminal domain allows the variants to escape recognition by most neutralizing antibodies that target it. The work could guide the development of next-generation vaccines and antibody therapies:

https://www.science.org/doi/10.1126/science.abl8506

To develop therapies against emerging variants, it is important to understand the viral biology and the effect of mutations. However, this is challenging because live virus can only be studied in a few laboratories that meet stringent safety standards. Now a team describe a virus-like particle (VLP) that comprises the four SARS-CoV-2 structural proteins, but instead of packaging viral RNA, it packages messenger RNA (mRNA) that expresses a reporter protein. The amount of reporter expressed in receiver cells depends on the efficiency of packaging and assembly in the producer cells and the efficiency of entry into receiver cells. Mutations in the nucleocapsid protein that are found in more transmissible variants increase mRNA packaging and expression. The VLPs provide a platform for studying the effect of mutations in the structural proteins and for screening therapeutics:

https://www.science.org/doi/10.1126/science.abl6184

Although efforts have been made to understand the biology of SARS-CoV-2, a major focus has been on investigating genetic variation in the virus. However, progress is hampered by the need to perform experiments involving SARS-CoV-2 in biosafety level 3 (BSL3) laboratories, which require substantial training for safe operation. Now a team offer an alternative to using live virus, introducing a new SARS-CoV-2 virus-like particle (VLP) system. The authors innovate on previous VLP systems by incorporating a reporter construct to study infection. Illustrating the system’s utility, they use VLPs to characterize mutations in SARS-CoV-2 variants of concern.

SARS-CoV-2 VLPs are created by expressing the four structural proteins, spike, membrane, envelope, and nucleocapsid, in a packaging cell line. Upon expression, VLPs consisting of these four proteins and a lipid membrane self-assemble and are released from the cell. Despite resembling SARS-CoV-2 morphologically, traditional VLPs cannot be used to study the effect of a mutation on fitness because they lack genetic material to deliver to target cells. They introduced a key innovation. They first identified the SARS-CoV-2 packaging signal, a genetic marker used to identify full-length genomes for packaging into the virion. This packaging signal was incorporated into the 3’ untranslated region of a luciferase reporter plasmid, causing the resulting transcripts to be packaged within VLPs. They show that VLPs deliver these luciferase reporters to target cells, allowing the resulting signal to be used as a proxy for SARS-CoV-2 infection. Thus, the effects of particular mutations on the strength of the luciferase signal can be used to determine modulation of SARS-CoV-2 infection (see the figure).

In the broader context of studying SARS-CoV-2 genetic variation, VLPs represent a middle ground between two commonly used methodologies: infectious clones and pseudovirus vectors. SARS-CoV-2 infectious clones are the gold standard because they create recombinant virus, incorporating mutations anywhere in the genome. However, using SARS-CoV-2 infectious clones is technically challenging and creates live SARS-CoV-2 that requires BSL3 laboratories for study. This limits the use of SARS-CoV-2 infectious clones to laboratories with access to such facilities and willingness to invest in developing a specialized skill set.

Pseudovirus systems are the leading alternative to using SARS-CoV-2 infectious clones. In these systems, SARS-CoV-2 spike protein is expressed in cells along with a noncoronavirus packaging system and a reporter gene, with the most common being lentivirus-based. Like the VLPs developed here, pseudoviruses self-assemble, incorporating spike proteins on their surface and packaging reporter messenger RNA. The primary advantage of pseudovirus systems is their ease of use, allowing rapid analysis of spike mutations. Pseudoviruses can be generated in the widely available 293T cell line by simply expressing a small number of proteins. Additionally, because pseudoviruses replace replication genes, they do not undergo continued amplification in target cell lines. This makes them safe to use in BSL2 laboratories, which are available to most researchers. However, the only SARS-CoV-2 protein incorporated into pseudoviruses is spike. Because substantial genetic variation occurs outside of spike, the pseduovirus systems have limited applicability to study SARS-CoV-2 variants.

The SARS-CoV-2 VLPs used here offer researchers several advantages over pseudoviruses. Rather than relying on the packaging machinery of another virus, VLPs use SARS-CoV-2 proteins and recapitulate packaging, assembly, and release, as occurs in genuine virus infection. In principle, this allows the effects of variant mutations on these processes to be studied. Similarly, because all four structural proteins are incorporated into SARS-CoV-2 VLPs, additional genetic variation can be captured. Like pseudoviruses, VLPs do not undergo subsequent rounds of replication, allowing them to be used safely in BSL2 laboratories.

Illustrating the utility of SARS-CoV-2 VLPs, they characterized several nucleocapsid mutations. SARS-CoV-2 nucleocapsid is a hotspot for coding mutations, particularly within its serine-rich (SR) motif. Although its exact function is unclear, the SR motif has many phosphorylated amino acids and is located within a region of intrinsic structural disorder. Using their SARS-CoV-2 VLP system, they analyzed the effects of several common nucleocapsid mutations and found that several enhanced infection, including those present in the Alpha, Gamma, and Delta variants. These data are consistent with findings using SARS-CoV-2 infectious clones.

The finding that nucleocapsid mutations enhance SARS-CoV-2 infection has important implications. To date, most studies of SARS-CoV-2 genetic variation have focused on spike. This is understandable, because spike binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2), and is thus the primary determinant of infection. Additionally, because spike is the target of available vaccines, determining if mutations affect protection is a pressing question. However, recent studies suggest that nucleocapsid mutations lead to enhanced virulence and fitness, highlighting the need to characterize genetic variation elsewhere in the viral genome. Because SARS-CoV-2 VLPs recapitulate enhancement of infection by these nucleocapsid mutations, they can be used to characterize mutations in emerging variants, such as deletion of amino acids 31 to 33 in the nucleocapsid protein of the Omicron variant.

Although a promising platform, there are limitations of this SARS-CoV-2 VLP system. Only the four structural proteins are present. Thus, like pseudoviruses, the scope of variation that can be captured is limited. For example, variant mutations in the viral replication machinery cannot be examined with VLPs. Additionally, while allowing for safe use in BSL2 laboratories, the inability of VLPs to undergo continued replication makes them unsuitable to study virulence or transmission. Furthermore, although data presented here by Syed et al. suggest that enhancement of infection by VLPs and live SARS-CoV-2 are correlated, additional work is needed to determine how closely VLPs model infection. As SARS-CoV-2 evolves, it is critical that the effects of new mutations are characterized:

A really fascinating paper now – I don’t know any other virus that does this. A group provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralisation by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis:

https://www.pnas.org/content/119/1/e2111400119

Finally today, I note “Young progressives have constructed a fantasy world where they are protagonists in the most catastrophic, consequential moment in history, and they’re baffled why the actual world keeps going”:

https://www.vox.com/2021/12/16/22837830/covid-pandemic-climate-change-great-resignation-2021

Graphs:

Time series of primary series/booster vaccinations (top), new COVID cases (middle) and current hospitalisations due to COVID (bottom) in the US:

Emerging hot spots in the US. The x-axis is growth rate of new cases compared to last week, the y-axis is the new case per hundred, and z is the latitude. Each state is colour coded by vaccination rate:

Back to Work Chart and Upcoming Vaccine Catalysts:

 

Percentage of hospital bed utilisation by US State:

US vaccinations:

Daily vaccinations in key regionns:

Vaccinations vs Deaths in various countries:

Vaccine orders by country. The number on top of each bar shows the % of the population that the orders in place can cover. Note that countries whose orders cover <25% of their population were excluded:

Google search interest of three terms regarding COVID vaccines over the past 3 months:

Daily vaccinations in the US and Google trends of keywords around vaccination:

Justin Stebbing
Managing Director

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