Closing coronavirus spike glycoproteins by structure-guided design

The recent spillover of SARS-CoV-2 in the human population resulted in the ongoing COVID-19 pandemic which has already caused 4.9 million infections and more than 326,000 fatalities. To initiate infection the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface, determining host and tissue tropism, and fusion of the viral and host membranes. Although SARS-CoV- 2 S is the main target of neutralizing antibodies and the focus of vaccine design, its stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a prefusion SARS-CoV-2 S ectodomain trimer construct covalently stabilized in the closed conformation. Structural and antigenicity analysis showed we successfully shut S in the closed state without otherwise altering its architecture. Finally, we show that this engineering strategy is applicable to other {beta}-coronavirus S glycoproteins and might become an important tool for vaccine design, structural biology, serology and immunology studies.

Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

The recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

Two different sarbecoviruses have caused major human outbreaks in the last two decades1,2. Both these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 via the spike receptor-binding domain (RBD)2-6. However, binding to ACE2 orthologs from humans, bats, and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses7-11. Here, we use high-throughput assays12 to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologs. We find that ACE2 binding is an ancestral trait of sarbecovirus RBDs that has subsequently been lost in some clades. Furthermore, we demonstrate for the first time that bat sarbecoviruses from outside Asia can bind ACE2. In addition, ACE2 binding is highly evolvable: for many sarbecovirus RBDs there are single amino-acid mutations that enable binding to new ACE2 orthologs. However, the effects of individual mutations can differ markedly between viruses, as illustrated by the N501Y mutation which enhances human ACE2 binding affinity within several SARS-CoV-2 variants of concern12 but severely dampens it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening consideration of the range of sarbecoviruses with spillover potential.

Deep mutational scans for ACE2 binding, RBD expression, and antibody escape in the SARS-CoV-2 Omicron BA.1 and BA.2 receptor-binding domains

SARS-CoV-2 continues to acquire mutations in the spike receptor-binding domain (RBD) that impact ACE2 receptor binding, folding stability, and antibody recognition. Deep mutational scanning prospectively characterizes the impacts of mutations on these biochemical properties, enabling rapid assessment of new mutations seen during viral surveillance. However, the effects of mutations can change as the virus evolves, requiring updated deep mutational scans. We determined the impacts of all amino acid mutations in the Omicron BA.1 and BA.2 RBDs on ACE2-binding affinity, RBD folding, and escape from binding by the LY-CoV1404 (bebtelovimab) monoclonal antibody. The effects of some mutations in Omicron RBDs differ from those measured in the ancestral Wuhan-Hu-1 background. These epistatic shifts largely resemble those previously seen in the Beta variant due to the convergent epistatically modifying N501Y substitution. However, Omicron variants show additional lineage-specific shifts, including examples of the epistatic phenomenon of entrenchment that causes the Q498R and N501Y substitutions present in Omicron to be more favorable in that background than in earlier viral strains. In contrast, the Omicron substitution Q493R exhibits no sign of entrenchment, with the derived state, R493, being as unfavorable for ACE2 binding in Omicron RBDs as in Wuhan-Hu-1. Likely for this reason, the R493Q reversion has occurred in Omicron sub-variants including BA.4/BA.5 and BA.2.75, where the affinity buffer from R493Q reversion may potentiate concurrent antigenic change. Consistent with prior studies, we find that Omicron RBDs have reduced expression, and identify candidate stabilizing mutations that ameliorate this deficit. Last, our maps highlight a broadening of the sites of escape from LY-CoV1404 antibody binding in BA.1 and BA.2 compared to the ancestral Wuhan-Hu-1 background. These BA.1 and BA.2 deep mutational scanning datasets identify shifts in the RBD mutational landscape and inform ongoing efforts in viral surveillance. Author SummarySARS-CoV-2 evolves in part through mutations in its spike receptor-binding domain. As these mutations accumulate in evolved variants, they shape the future evolutionary potential of the virus through the phenomenon of epistasis. We characterized the functional impacts of mutations in the Omicron BA.1 and BA.2 receptor-binding domains on ACE2 receptor binding, protein folding, and recognition by the clinical LY-CoV1404 antibody. We then compared the measurements to prior data for earlier variants. These comparisons identify patterns of epistasis that may alter future patterns of Omicron evolution, such as turnover in the availability of specific affinity-enhancing mutations and an expansion in the number of paths of antibody escape from a key monoclonal antibody used for therapeutic treatment of COVID-19. This work informs continued efforts in viral surveillance and forecasting.

Structural basis for broad coronavirus neutralization

Three highly pathogenic {beta}-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARS-CoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight {beta}-coronavirus spike glycoproteins, including all five human-infecting {beta}-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryo-electron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with {beta}-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.

Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor, and is a major determinant of host range and a dominant target of neutralizing antibodies. Here we experimentally measure how all amino-acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBDs surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail

The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-CoV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the cilgavimab (AZD1061) mAb. Here, we show that this residue substitution remodels the ACE2-binding site allosterically, thereby dampening receptor recognition severely and altering the epitopes recognized by these three mAbs. Although vaccine-elicited neutralizing antibody titers are decreased similarly against the E406 mutant and the Delta or Epsilon variants, broadly neutralizing sarbecovirus mAbs, including a clinical mAb, inhibit the E406W spike mutant.

Delta breakthrough infections elicit potent, broad and durable neutralizing antibody responses

The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

Structure, receptor recognition and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein

The recent isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. Here, we determined cryo-electron microscopy structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and identified that it binds canine, feline and porcine aminopeptidase N (APN encoded by ANPEP) orthologs which serve as entry receptors. Introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single nucleotide polymorphisms could account for the detection of this virus in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 S-mediated entry, indicating elicitation of cross-neutralizing activity among -coronaviruses. These data provide a blueprint of the CCoV-HuPn-2018 infection machinery, unveil the viral entry receptor and pave the way for vaccine and therapeutic development targeting this zoonotic pathogen.

Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020

Following its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic resulting in unprecedented efforts to reduce transmission and develop therapies and vaccines (WHO Emergency Committee, 2020; Zhu et al., 2020). Rapidly generated viral genome sequences have allowed the spread of the virus to be tracked via phylogenetic analysis (Worobey et al., 2020; Hadfield et al., 2018; Pybus et al., 2020). While the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced, allowing continent-specific variants to emerge. However, within Europe travel resumed in the summer of 2020, and the impact of this travel on the epidemic is not well understood. Here we report on a novel SARS-CoV-2 variant, 20E (EU1), that emerged in Spain in early summer, and subsequently spread to multiple locations in Europe. We find no evidence of increased transmissibility of this variant, but instead demonstrate how rising incidence in Spain, resumption of travel across Europe, and lack of effective screening and containment may explain the variants success. Despite travel restrictions and quarantine requirements, we estimate 20E (EU1) was introduced hundreds of times to countries across Europe by summertime travellers, likely undermining local efforts to keep SARS-CoV-2 cases low. Our results demonstrate how a variant can rapidly become dominant even in absence of a substantial transmission advantage in favorable epidemiological settings. Genomic surveillance is critical to understanding how travel can impact SARS-CoV-2 transmission, and thus for informing future containment strategies as travel resumes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the first pandemic where the spread of a viral pathogen has been globally tracked in near real-time using phylogenetic analysis of viral genome sequences (Worobey et al., 2020; Hadfield et al., 2018; Pybus et al., 2020). SARS-CoV-2 genomes continue to be generated at a rate far greater than for any other pathogen and more than 500,000 full genomes are available on GISAID as of February 2020 (Shu and McCauley, 2017). In addition to tracking the viral spread, these genome sequences have been used to monitor mutations which might change the transmission, pathogenesis, or anti-genic properties of the virus. One mutation in particular, D614G in the spike protein, has received much attention. This variant (Nextstrain clade 20A) seeded large outbreaks in Europe in early 2020 and subsequently dominated the outbreaks in the Americas, thereby largely replacing previously circulating lineages. This rapid rise led to the suggestion that this variant is more transmissible, which has since been corroborated by phylogenetic (Korber et al., 2020; Volz et al., 2020) and experimental evidence (Plante et al., 2020; Yurkovetskiy et al., 2020). Following the global dissemination of SARS-CoV-2 in early 2020 (Worobey et al., 2020), intercontinental travel dropped dramatically. Within Europe, however, travel and in particular holiday travel resumed in summer (though at lower levels than in previous years) with largely uncharacterized effects on the pandemic. Here we report on a novel SARS-CoV-2 variant 20E (EU1) (S:A222V) that emerged in early summer 2020, presumably in Spain, and subsequently spread to multiple locations in Europe. Over the summer, it rose in frequency in parallel in multiple countries. As we report here, this variant, 20E (EU1), and a second variant 20A.EU2 with mutation S477N in the spike protein accounted for the majority of sequences in Europe in the autumn of 2020.

Seroprevalence of SARS-CoV-2 among children visiting a hospital during the initial Seattle outbreak

Children are strikingly underrepresented in COVID-19 case counts1-3. In the United States, children represent 22% of the population but only 1.7% of confirmed SARS-CoV-2 cases1. One possibility is that symptom-based viral testing is less likely to identify infected children, since they often experience milder disease than adults1,4-7. To better assess the frequency of pediatric SARS-CoV-2 infection, we serologically screened 1,775 residual samples from Seattle Children's Hospital collected from 1,076 children seeking medical care during March and April of 2020. Only one child was seropositive in March, but seven were seropositive in April for a period seroprevalence of {approx} 1%. Most seropositive children (6/8) were not suspected of having had COVID-19. The sera of seropositive children had neutralizing activity, including one that neutralized at a dilution >1:18,000. Therefore, an increasing number of children seeking medical care were infected by SARS-CoV-2 during the early Seattle outbreak despite few positive viral tests.

Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

Investigating the mechanisms of SARS-CoV-2 cellular infection is key to better understand COVID-19 immunity and pathogenesis. Infection, which involves both cell attachment and membrane fusion, relies on the ACE2 receptor that is paradoxically found at low levels in the respiratory tract, suggesting that additional mechanisms facilitating infection may exist. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding Ig-like lectin 1 (SIGLEC1) function as auxiliary receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the N-terminal domain (NTD) or to the conserved proteoglycan site at the base of the Receptor Binding Domain (RBD), while poorly neutralizing infection of ACE2 over-expressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the Receptor Binding Motif (RBM), while potently neutralizing infection of ACE2 over-expressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.

Single-dose replicating RNA vaccine induces neutralizing antibodies against SARS-CoV-2 in nonhuman primates

The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.

Structural and functional analysis of a potent sarbecovirus neutralizing antibody

SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than one million infections and 73,000 deaths1,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of a SARS survivor infected in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

Omicron BA.1 and BA.2 neutralizing activity elicited by a comprehensive panel of human vaccines

The SARS-CoV-2 Omicron variant of concern comprises three sublineages designated BA.1, BA.2, and BA.3, with BA.2 steadily replacing the globally dominant BA.1. We show that the large number of BA.1 and BA.2 spike mutations severely dampen plasma neutralizing activity elicited by infection or seven clinical vaccines, with cross-neutralization of BA.2 being consistently more potent than that of BA.1, independent of the vaccine platform and number of doses. Although mRNA vaccines induced the greatest magnitude of Omicron BA.1 and BA.2 plasma neutralizing activity, administration of a booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1 and BA.2 across all vaccines evaluated. Our data suggest that although BA.1 and BA.2 evade polyclonal neutralizing antibody responses, current vaccine boosting regimens may provide sufficient protection against Omicron-induced disease.

SARS-CoV-2 spike conformation determines plasma neutralizing activity

Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV-2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.

Molecular basis of immune evasion by the delta and kappa SARS-CoV-2 variants

Worldwide SARS-CoV-2 transmission leads to the recurrent emergence of variants, such as the recently described B.1.617.1 (kappa), B.1.617.2 (delta) and B.1.617.2+ (delta+). The B.1.617.2 (delta) variant of concern is causing a new wave of infections in many countries, mostly affecting unvaccinated individuals, and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing the impact of individual mutations on immune evasion. Mutations in the B.1.617.1 (kappa) and B.1.617.2 (delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including an unexpected remodeling of the B.1.617.2 (delta) N-terminal domain. The binding affinity of the B.1.617.1 (kappa) and B.1.617.2 (delta) receptor-binding domain for ACE2 is comparable to the ancestral virus whereas B.1.617.2+ (delta+) exhibits markedly reduced affinity. We describe a previously uncharacterized class of N-terminal domain-directed human neutralizing monoclonal antibodies cross-reacting with several variants of concern, revealing a possible target for vaccine development.

Detection of antibodies neutralizing historical and emerging SARS-CoV-2 strains using a thermodynamically coupled de novo biosensor system

With global vaccination efforts against SARS-CoV-2 underway, there is a need for rapid quantification methods for neutralizing antibodies elicited by vaccination and characterization of their strain dependence. Here, we describe a designed protein biosensor that enables sensitive and rapid detection of neutralizing antibodies against wild type and variant SARS-CoV-2 in serum samples. More generally, our thermodynamic coupling approach can better distinguish sample to sample differences in analyte binding affinity and abundance than traditional competition based assays.

Safety and immunogenicity of a SARS-CoV-2 recombinant protein nanoparticle vaccine (GBP510) adjuvanted with AS03: a phase 1/2, randomized, placebo-controlled, observer-blinded trial

BackgroundVaccination has helped to mitigate the COVID-19 pandemic. Ten traditional and novel vaccines have been listed by the World Health Organization for emergency use. Additional alternative approaches may better address ongoing vaccination globally, where there remains an inequity in vaccine distribution. GBP510 is a recombinant protein vaccine, which consists of self-assembling, two-component nanoparticles displaying the receptor-binding domain (RBD) in a highly immunogenic array. MethodsWe conducted a randomized, placebo-controlled, observer-blinded, phase 1/2 trial to evaluate the safety and immunogenicity of GBP510 (2-doses at a 28-day interval) adjuvanted with or without AS03 in adults aged 19-85 years. The main outcomes included solicited and unsolicited adverse events; anti-SARS-CoV-2 RBD IgG antibody and neutralizing antibody responses; T-cell immune responses. FindingsOf 328 participants who underwent randomization, 327 participants received at least 1 dose of vaccine. Each received either 10 g GBP510 adjuvanted with AS03 (n = 101), 10 g unadjuvanted GBP510 (n = 10), 25 g GBP510 adjuvanted with AS03 (n = 104), 25 g unadjuvanted GBP510 (n = 51), or placebo (n = 61). Most solicited adverse events were mild-to-moderate in severity and transient. Higher reactogenicity was observed in the GBP510 adjuvanted with AS03 groups compared to the non-adjuvanted and placebo groups. Reactogenicity was higher post-dose 2 compared to post-dose 1, particularly for systemic adverse events. The geometric mean concentrations of anti-SARS-CoV-2-RBD IgG antibody reached 2163.6/2599.2 BAU/mL in GBP510 adjuvanted with AS03 recipients (10 g/25 g) by 14 days after the second dose. Two-dose vaccination with 10 g or 25 g GBP510 adjuvanted with AS03 induced high titers of neutralizing antibody via pseudovirus (1369.0/1431.5 IU/mL) and wild-type virus (949.8/861.0 IU/mL) assays. InterpretationGBP510 adjuvanted with AS03 was well tolerated and highly immunogenic. These results support further development of the vaccine candidate, which is currently being evaluated in Phase 3. FundingCoalition for Epidemic Preparedness Innovations RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for research articles published by December 31, 2021, using the terms "COVID-19" or "SARS-CoV-2," "vaccine," and "clinical trial." In previously reported randomized clinical trials, we found that mRNA vaccines were more immunogenic than adenovirus-vectored vaccines. Solicited adverse events were more frequent and more severe in intensity after the first dose compared to the second dose for adenovirus-vectored vaccines, whereas they increased after the second dose of mRNA or recombinant spike-protein nanoparticle vaccines. Added value of this studyThis is the first human study evaluating the immunogenicity and safety of recombinant SARS-CoV-2 protein nanoparticle with and without adjuvant AS03, designed to elicit functional cross-protective responses via receptor-binding domain (RBD). Both 10 and 25 g of GBP510 with AS03 formulations were well tolerated with an acceptable safety profile. Potent humoral immune responses against the SARS-CoV-2 RBD were induced and peaked by day 42 (14 days after the second dose). In addition, GBP510 adjuvanted with AS03 elicited a noticeable Th1 response, with production of IFN-{gamma}, TNF-, and IL-2. IL-4 was inconsistent and IL-5 nearly inexistent response across all groups. Implications of the available evidenceThe results from this phase 1/2 trial indicate that GBP510 adjuvanted with AS03 has an acceptable safety profile with no vaccine-related serious adverse events. Two-dose immunization with GBP510 adjuvanted with AS03 induced potent humoral and cellular immune responses against SARS-CoV-2.

SARS-CoV-2 immune evasion by variant B.1.427/B.1.429

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.