A third COVID-19 vaccine shot markedly boosts neutralizing antibody potency and breadth

COVID-19 (coronavirus disease 2019) vaccines have been rapidly developed and deployed globally as a measure to combat the disease. These vaccines have been demonstrated to confer significant protection, but there have been reports of temporal decay in antibody titer. Furthermore, several variants have been identified with variable degrees of antibody resistance. These two factors suggest that a booster vaccination may be worthy of consideration. While such a booster dose has been studied as a series of three homologous vaccines in healthy individuals, to our knowledge, information on a heterologous regimen remains unreported, despite the practical benefits of such a scheme. Here, in this observational study, we investigated the serological profile of four healthy individuals who received two doses of the BNT162b2 vaccine, followed by a third booster dose with the Ad26.COV2.S vaccine. We found that while all individuals had spike-binding antibodies at each of the timepoints tested, there was an appreciable drop in titer by four months following the second vaccination. The third vaccine dose robustly increased titers beyond that of two vaccinations, and these elicited antibodies had neutralizing capability against all SARS-CoV-2 strains tested in both a recombinant vesicular stomatitis virus-based pseudovirus assay and an authentic SARS-CoV-2 assay, except for one individual against B.1.351 in the latter assay. Thus, a third COVID-19 vaccine dose in healthy individuals promoted not just neutralizing antibody potency, but also induced breadth against dominant SARS-CoV-2 variants. SignificanceCOVID-19 vaccines confer protection from symptomatic disease, but the elicited antibody titer has been found to decrease with time. Furthermore, SARS-CoV-2 variants with relative resistance against antibody neutralization have been identified. To overcome such issues, a third vaccine dose applied as a booster vaccine may be necessary. We studied four healthy individuals who received a heterologous booster dose as a third vaccine. All of these individuals had heightened neutralizing antibody titer following the booster vaccination, and could neutralize nearly all variants tested. Thus, a heterologous third COVID-19 vaccine dose may be a mechanism to both heighten and broaden antibody titers, and could be an additional strategy for controlling the SARS-CoV-2 pandemic.

In vivo kinetics of SARS-CoV-2 infection and its relationship with a person's infectiousness

The within-host viral kinetics of SARS-CoV-2 infection and how they relate to a persons infectiousness are not well understood. This limits our ability to quantify the impact of interventions on viral transmission. Here, we develop data-driven viral dynamic models of SARS-CoV-2 infection and estimate key within-host parameters such as the infected cell half-life and the within-host reproductive number. We then develop a model linking VL to infectiousness, showing that a persons infectiousness increases sub-linearly with VL. We show that the logarithm of the VL in the upper respiratory tract (URT) is a better surrogate of infectiousness than the VL itself. Using data on VL and the predicted infectiousness, we further incorporated data on antigen and reverse transcription polymerase chain reaction (RT-PCR) tests and compared their usefulness in detecting infection and preventing transmission. We found that RT-PCR tests perform better than antigen tests assuming equal testing frequency; however, more frequent antigen testing may perform equally well with RT-PCR tests at a lower cost, but with many more false-negative tests. Overall, our models provide a quantitative framework for inferring the impact of therapeutics and vaccines that lower VL on the infectiousness of individuals and for evaluating rapid testing strategies. SignificanceQuantifying the kinetics of SARS-CoV-2 infection and individual infectiousness is key to quantitatively understanding SARS-CoV-2 transmission and evaluating intervention strategies. Here we developed data-driven within-host models of SARS-CoV-2 infection and by fitting them to clinical data we estimated key within-host viral dynamic parameters. We also developed a mechanistic model for viral transmission and show that the logarithm of the viral load in the upper respiratory tract serves an appropriate surrogate for a persons infectiousness. Using data on how viral load changes during infection, we further evaluated the effectiveness of PCR and antigen-based testing strategies for averting transmission and identifying infected individuals.

Angiotensin converting enzyme 2 is a novel target of the γ-secretase complex

Angiotensin converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system, but also the functional receptor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on structural similarity with other {gamma}-secretase ({gamma}S) targets, we hypothesized that ACE2 may be affected by {gamma}S proteolytic activity. We found that after ectodomain shedding, ACE2 is targeted for intramembrane proteolysis by {gamma}S, releasing a soluble ACE2 C-terminal fragment. Consistently, chemical or genetic inhibition of {gamma}S results in the accumulation of a membrane-bound fragment of ectodomain-deficient ACE2. Although chemical inhibition of {gamma}S does not alter SARS-CoV-2 cell entry, these data point to a novel pathway for cellular ACE2 trafficking.

Functional properties of the spike glycoprotein of the emerging SARS-CoV-2 variant B.1.1.529

The recently emerged B.1.1.529 (Omicron) SARS-CoV-2 variant has a highly divergent spike (S) glycoprotein. We compared the functional properties of B.1.1.529 S with those of previous globally prevalent SARS-CoV-2 variants, D614G and B.1.617.2. Relative to these variants, B.1.1.529 S exhibits decreased processing, resulting in less efficient syncytium formation and lower S incorporation into virus particles. Nonetheless, B.1.1.529 S supports virus infection equivalently. B.1.1.529 and B.1.617.2 S glycoproteins bind ACE2 with higher affinity than D614G S. The unliganded B.1.1.529 S trimer is less stable at low temperatures than the other SARS-CoV-2 spikes, a property related to spike conformation. Upon ACE2 binding, the B.1.1.529 S trimer sheds S1 at 37 degrees but not at 0 degrees C. B.1.1.529 pseudoviruses are relatively resistant to neutralization by sera from convalescent COVID-19 patients and vaccinees. These properties of the B.1.1.529 spike glycoprotein likely influence the transmission, cytopathic effects and immune evasion of this emerging variant.

Antibody responses to Omicron BA.4/BA.5 bivalent mRNA vaccine booster shot

The SARS-CoV-2 Omicron variant and its numerous sub-lineages have exhibited a striking ability to evade humoral immune responses induced by prior vaccination or infection. The Food and Drug Administration (FDA) has recently granted Emergency Use Authorizations (EUAs) to new bivalent formulations of the original Moderna and Pfizer mRNA SARS-CoV-2 vaccines that target both the ancestral strain as well as the Omicron BA.4/BA.5 variant. Despite their widespread use as a vaccine boost, little is known about the antibody responses induced in humans. Here, we collected sera from several clinical cohorts: individuals after three or four doses of the original monovalent mRNA vaccines, individuals receiving the new bivalent vaccines as a fourth dose, and individuals with BA.4/BA.5 breakthrough infection following mRNA vaccination. Using pseudovirus neutralization assays, these sera were tested for neutralization against an ancestral SARS-CoV-2 strain, several Omicron sub-lineages, and several related sarbecoviruses. At ~3-5 weeks post booster shot, individuals who received a fourth vaccine dose with a bivalent mRNA vaccine targeting BA.4/BA.5 had similar neutralizing antibody titers as those receiving a fourth monovalent mRNA vaccine against all SARS-CoV-2 variants tested, including BA.4/BA.5. Those who received a fourth monovalent vaccine dose had a slightly higher neutralizing antibody titers than those who received the bivalent vaccine against three related sarbecoviruses: SARS-CoV, GD-Pangolin, and WIV1. When given as a fourth dose, a bivalent mRNA vaccine targeting Omicron BA.4/BA.5 and an ancestral SARS-CoV-2 strain did not induce superior neutralizing antibody responses in humans, at the time period tested, compared to the original monovalent vaccine formulation.

Resistance of SARS-CoV-2 Omicron Subvariant BA.4.6 to Antibody Neutralization

SARS-CoV-2 Omicron subvariants BA.4.6, BA.4.7, and BA.5.9 have recently emerged, and BA.4.6 appears to be expanding even in the presence of BA.5 that is globally dominant. Compared to BA.5, these new subvariants harbor a mutation at R346 residue in the spike glycoprotein, raising concerns for further antibody evasion. We compared the viral receptor binding affinity of the new Omicron subvariants with BA.5 by surface plasmon resonance. We also performed VSV-based pseudovirus neutralization assays to evaluate their antigenic properties using sera from individuals who received three doses of a COVID-19 mRNA vaccine (boosted) and patients with BA.1 or BA.2 breakthrough infection, as well as using a panel of 23 monoclonal antibodies (mAbs). Compared to the BA.5 subvariant, BA.4.6, BA.4.7, and BA.5.9 showed similar binding affinities to hACE2 and exhibited similar resistance profiles to boosted and BA.1 breakthrough sera, but BA.4.6 was slightly but significantly more resistant than BA.5 to BA.2 breakthrough sera. Moreover, BA.4.6, BA.4.7, and BA.5.9 showed heightened resistance over to a class of mAbs due to R346T/S/I mutation. Notably, the authorized combination of tixagevimab and cilgavimab completely lost neutralizing activity against these three subvariants. The loss of activity of tixagevimab and cilgavimab against BA.4.6 leaves us with bebtelovimab as the only therapeutic mAb that has retained potent activity against all circulating forms of SARS-CoV-2. As the virus continues to evolve, our arsenal of authorized mAbs may soon be depleted, thereby jeopardizing the wellbeing of millions of immunocompromised persons who cannot robustly respond to COVID-19 vaccines.

Increased Resistance of SARS-CoV-2 Variant P.1 to Antibody Neutralization

The relative resistance of SARS-CoV-2 variants B.1.1.7 and B.1.351 to antibody neutralization has been described recently. We now report that another emergent variant from Brazil, P.1, is not only refractory to multiple neutralizing monoclonal antibodies, but also more resistant to neutralization by convalescent plasma (3.4 fold) and vaccinee sera (3.8-4.8 fold). The cryo-electron microscopy structure of a soluble prefusion-stabilized spike reveals the P.1 trimer to adopt exclusively a conformation in which one of the receptor-binding domains is in the "up" position, with the functional impact of mutations appearing to arise from local changes instead of global conformational alterations. The P.1 variant threatens current antibody therapies but less so the protective efficacy of our vaccines.

Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75

The SARS-CoV-2 Omicron subvariant BA.2.75 emerged recently and appears to be spreading rapidly. It has nine mutations in its spike compared to BA.2, raising concerns it may further evade vaccine-elicited and therapeutic antibodies. Here, we found BA.2.75 to be moderately more neutralization resistant to sera from vaccinated/boosted individuals than BA.2 (1.8-fold), similar to BA.2.12.1 (1.1-fold), but more neutralization sensitive than BA.4/5 (0.6-fold). Relative to BA.2, BA.2.75 showed heightened resistance to class 1 and class 3 monoclonal antibodies to the receptor-binding domain, while gaining sensitivity to class 2 antibodies. The resistance was largely conferred by the G446S and R460K mutations. Of note, BA.2.75 was slightly resistant (3.7-fold) to bebtelovimab, the only therapeutic antibody with potent activity against all Omicron subvariants. BA.2.75 also exhibited higher receptor binding affinity than other Omicron subvariants. BA.2.75 provides yet another example of the ongoing evolution of SARS-CoV-2 as it gains transmissibility while incrementally evading antibody neutralization.

Structural basis for antibody resistance to SARS-CoV-2 omicron variant

The recently reported B.1.1.529 Omicron variant of SARS-CoV-2 includes 34 mutations in the spike protein relative to the Wuhan strain that initiated the COVID-19 pandemic, including 15 mutations in the receptor binding domain (RBD). Functional studies have shown omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here we report a 3.1 [A] resolution cryo-electron microscopy (cryo-EM) structure of the Omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with increased mobility and inter-protomer asymmetry. Many mutations cause steric clashes and/or altered interactions at antibody binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies. HighlightsO_LISARS-CoV-2 omicron spike exclusively adopts 1-RBD-up conformation C_LIO_LIOmicron substitutions alter conformation and mobility of RBD C_LIO_LIA subset of omicron mutations change the local conformation of spike C_LIO_LIThe structure reveals the basis of antibody neutralization escape C_LI

A Novel SARS-CoV-2 Variant of Concern, B.1.526, Identified in New York

Recent months have seen surges of SARS-CoV-2 infection across the globe with considerable viral evolution1-3. Extensive mutations in the spike protein may threaten efficacy of vaccines and therapeutic monoclonal antibodies4. Two signature mutations of concern are E484K, which plays a crucial role in the loss of neutralizing activity of antibodies, and N501Y, a driver of rapid worldwide transmission of the B.1.1.7 lineage. Here, we report the emergence of variant lineage B.1.526 that contains E484K and its alarming rise to dominance in New York City in early 2021. This variant is partially or completely resistant to two therapeutic monoclonal antibodies in clinical use and less susceptible to neutralization by convalescent plasma or vaccinee sera, posing a modest antigenic challenge. The B.1.526 lineage has now been reported from all 50 states in the US and numerous other countries. B.1.526 rapidly replaced earlier lineages in New York upon its emergence, with an estimated transmission advantage of 35%. Such transmission dynamics, together with the relative antibody resistance of its E484K sub-lineage, likely contributed to the sharp rise and rapid spread of B.1.526. Although SARS-CoV-2 B.1.526 initially outpaced B.1.1.7 in the region, its growth subsequently slowed concurrent with the rise of B.1.1.7 and ensuing variants.

Structural Basis for Accommodation of Emerging B.1.351 and B.1.1.7 Variants by Two Potent SARS-CoV-2 Neutralizing Antibodies

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented a response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for both ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies like 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.

Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class

Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilized VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486RBD. Despite these similarities, class members bound both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N343 on a neighboring RBD - a quaternary interaction accommodated by an increase in RBD separation of up to 12 [A]. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions. HighlightsO_LIDetermine structures of VH1-2-derived antibodies 2-43, 2-15, and H4 in complex with SARS-CoV-2 spike C_LIO_LIDefine a multi-donor VH1-2-antibody class with modular components for RBD and quaternary recognition C_LIO_LIReveal structural basis of RBD-up and RBD-down recognition within the class C_LIO_LIShow somatic hypermutations and avidity to be critical for potency C_LIO_LIDelineate changes in spike conformation induced by CDRH3-mediated quaternary recognition C_LI

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.

Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors

We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.

The Functional Landscape of SARS-CoV-2 3CL Protease

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CLpro), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CLpro, and validate our results both in yeast and in authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CLpro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CLpro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.

SARS-CoV-2 Neutralizing Antibody Responses Are More Robust in Patients with Severe Disease

We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ~5-fold and ~7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.

Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants

The SARS-CoV-2 Omicron variant continues to evolve, with new BQ and XBB subvariants now rapidly expanding in Europe/US and Asia, respectively. As these new subvariants have additional spike mutations, they may possess altered antibody evasion properties. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals who were boosted with a WA1/BA.5 bivalent mRNA vaccine. Compared to the ancestral strain D614G, serum neutralizing titers against BQ and XBB subvariants were lower by 13-81-fold and 66-155-fold, respectively, far beyond what had been observed to date. A panel of monoclonal antibodies capable of neutralizing the original Omicron variant, including those with Emergency Use Authorization, were largely inactive against these new subvariants. The spike mutations that conferred antibody resistance were individually studied and structurally explained. Finally, the ACE2-binding affinities of the spike proteins of these novel subvariants were found to be similar to those of their predecessors. Taken together, our findings indicate that BQ and XBB subvariants present serious threats to the efficacy of current COVID-19 vaccines, render inactive all authorized monoclonal antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.

Antibody Evasion Properties of SARS-CoV-2 Omicron Sublineages

The identification of the Omicron variant (B.1.1.529.1 or BA.1) of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in Botswana in November 20211 immediately raised alarms due to the sheer number of mutations in the spike glycoprotein that could lead to striking antibody evasion. We2 and others3-6 recently reported results in this Journal confirming such a concern. Continuing surveillance of Omicron evolution has since revealed the rise in prevalence of two sublineages, BA.1 with an R346K mutation (BA.1+R346K) and B.1.1.529.2 (BA.2), with the latter containing 8 unique spike mutations while lacking 13 spike mutations found in BA.1. We therefore extended our studies to include antigenic characterization of these new sublineages. Polyclonal sera from patients infected by wild-type SARS-CoV-2 or recipients of current mRNA vaccines showed a substantial loss in neutralizing activity against both BA.1+R346K and BA.2, with drops comparable to that already reported for BA.12,3,5,6. These findings indicate that these three sublineages of Omicron are antigenically equidistant from the wild-type SARS-CoV-2 and thus similarly threaten the efficacies of current vaccines. BA.2 also exhibited marked resistance to 17 of 19 neutralizing monoclonal antibodies tested, including S309 (sotrovimab)7, which had retained appreciable activity against BA.1 and BA.1+R346K2-4,6. This new finding shows that no presently approved or authorized monoclonal antibody therapy could adequately cover all sublineages of the Omicron variant.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.