Dynamics of SARS-CoV-2 VOC neutralization and novel mAb reveal protection against Omicron

To evaluate SARS-CoV-2 variants we isolated SARS-CoV-2 temporally during the pandemic starting with first appearance of virus in the Western hemisphere near Seattle, WA, USA, and isolated each known major variant class, revealing the dynamics of emergence and complete take-over of all new cases by current Omicron variants. We assessed virus neutralization in a first-ever full comparison across variants and evaluated a novel monoclonal antibody (Mab). We found that convalescence greater than 5-months provides little-to-no protection against SARS-CoV-2 variants, vaccination enhances immunity against variants with the exception of Omicron BA.1, and paired testing of vaccine sera against ancestral virus compared to Omicron BA.1 shows that 3-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a 2-dose regimen. We also reveal a novel Mab that effectively neutralizes Omicron BA.1 and BA.2 variants over clinically-approved Mabs. Our observations underscore the need for continued vaccination efforts, with innovation for vaccine and Mab improvement, for protection against variants of SARS-CoV-2. SummaryWe isolated SARS-CoV-2 temporally starting with emergence of virus in the Western hemisphere. Neutralization analyses across all variant lineages show that vaccine-boost regimen provides protection against Omicron BA.1. We reveal a Mab that protects against Omicron BA.1 and BA.2 variants.

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.

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.

Divergence of delta and beta variants and SARS-CoV-2 evolved in prolonged infection into distinct serological phenotypes

Characterizing SARS-CoV-2 evolution in specific geographies may help predict the properties of variants coming from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from the ancestral virus in a person with advanced HIV disease. Infection was before the emergence of the Beta variant first identified in South Africa, and the Delta variant. We compared early and late evolved virus to the ancestral, Beta, Alpha, and Delta viruses and tested against convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, whereas late virus was similar to Beta, exhibiting vaccine escape and, despite pre-dating Delta, strong escape of Delta-elicited neutralization. This example is consistent with the notion that variants arising in immune-compromised hosts, including those with advanced HIV disease, may evolve immune escape of vaccines and enhanced escape of Delta immunity, with implications for vaccine breakthrough and reinfections. HighlightsO_LIA prolonged ancestral SARS-CoV-2 infection pre-dating the emergence of Beta and Delta resulted in evolution of a Beta-like serological phenotype C_LIO_LISerological phenotype includes strong escape from Delta infection elicited immunity, intermediate escape from ancestral virus immunity, and weak escape from Beta immunity C_LIO_LIEvolved virus showed substantial but incomplete escape from antibodies elicited by BNT162b2 vaccination C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/21263564v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1194bfdorg.highwire.dtl.DTLVardef@1cbe318org.highwire.dtl.DTLVardef@aa74f8org.highwire.dtl.DTLVardef@e57969_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

The continuous evolution of SARS-CoV-2 in South Africa: a new lineage with rapid accumulation of mutations of concern and global detection

Global genomic surveillance of SARS-CoV-2 has identified variants associated with increased transmissibility, neutralization resistance and disease severity. Here we report the emergence of the PANGO lineage C.1.2, detected at low prevalence in South Africa and eleven other countries. The emergence of C.1.2, associated with a high substitution rate, includes changes within the spike protein that have been associated with increased transmissibility or reduced neutralization sensitivity in SARS-CoV-2 VOC/VOIs. Like Beta and Delta, C.1.2 shows significantly reduced neutralization sensitivity to plasma from vaccinees and individuals infected with the ancestral D614G virus. In contrast, convalescent donors infected with either Beta or Delta showed high plasma neutralization against C.1.2. These functional data suggest that vaccine efficacy against C.1.2 will be equivalent to Beta and Delta, and that prior infection with either Beta or Delta will likely offer protection against C.1.2.

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.

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 M range in vitro. Furthermore, Exebryl-1, a {beta}-amyloid anti-aggregation molecule for Alzheimers therapy, was shown to have antiviral activity between 10 to 66 M, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral. Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).