Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response

A detailed understanding of the molecular features of the neutralizing epitopes developed by viral escape mutants is important for predicting and developing vaccines or therapeutic antibodies against continuously emerging SARS-CoV-2 variants. Here, we report three human monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during first wave of pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, but poorly neutralized Beta and completely failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these three mAbs in complex with trimeric spike protein showed that all three mAbs are involved in bivalent spike binding with two mAbs targeting class-1 and one targeting class-4 Receptor Binding Domain (RBD) epitope. Comparison of immunogenetic makeup, structure, and function of these three mAbs with our recently reported class-3 RBD binding mAb that potently neutralized all SARS-CoV-2 variants revealed precise antibody footprint, specific molecular interactions associated with the most potent multi-variant binding / neutralization efficacy. This knowledge has timely significance for understanding how a combination of certain mutations affect the binding or neutralization of an antibody and thus have implications for predicting structural features of emerging SARS-CoV-2 escape variants and to develop vaccines or therapeutic antibodies against these.

Structural insights for neutralization of BA.1 and BA.2 Omicron variants by a broadly neutralizing SARS-CoV-2 antibody

The SARS-CoV-2 BA.1 and BA.2 (Omicron) variants contain more than 30 mutations within the spike protein and evade therapeutic monoclonal antibodies (mAbs). Here, we report a receptor-binding domain (RBD) targeting human antibody (002-S21F2) that effectively neutralizes live viral isolates of SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Gamma, Delta, and Omicron (BA.1 and BA.2) with IC50 ranging from 0.02 - 0.05 g/ml. This near germline antibody 002-S21F2 has unique genetic features that are distinct from any reported SARS-CoV-2 mAbs. Structural studies of the full-length IgG in complex with spike trimers (Omicron and WA.1) reveal that 002-S21F2 recognizes an epitope on the outer face of RBD (class-3 surface), outside the ACE2 binding motif and its unique molecular features enable it to overcome mutations found in the Omicron variants. The discovery and comprehensive structural analysis of 002-S21F2 provide valuable insight for broad and potent neutralization of SARS-CoV-2 Omicron variants BA.1 and BA.2.

The inactivated NDV-HXP-S COVID-19 vaccine induces a significantly higher ratio of neutralizing to non-neutralizing antibodies in humans as compared to mRNA vaccines

NDV-HXP-S is a recombinant Newcastle disease virus based-vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which expresses an optimized (HexaPro) spike protein on its surface. The vaccine can be produced in embryonated chicken eggs using the same process as that employed for the production of influenza virus vaccines. Here we performed a secondary analysis of the antibody responses after vaccination with inactivated NDV-HXP-S in a Phase I clinical study in Thailand. The SARS-CoV-2 neutralizing and spike binding activity of NDV-HXP-S post-vaccination serum samples was compared to that of matched samples from mRNA BNT162b2 (Pfizer) vaccinees. Neutralizing activity of sera from NDV-HXP-S vaccinees was comparable to that of individuals vaccinated with BNT162b2. Interstingly, the spike binding activity of the NDV-HXP-S vaccinee samples was lower than that of sera obtained from individuals vaccinated with the mRNA vaccine. This let us to calculate ratios between binding and neutralizing antibody titers. Samples from NDV-HXP-S vaccinees had binding to neutralizing activity ratios similar to those of convalescent sera suggesting a very high proportion of neutralizing antibodies and low non-neutralizing antibody titers. Further analysis showed that, in contrast to mRNA vaccination, which induces strong antibody titers to the receptor binding domain (RBD), the N-terminal domain, and the S2 domain, NDV-HXP-S vaccination induces a very RBD focused response with little reactivity to S2. This explains the high proportion of neutralizing antibodies since most neutralizing epitopes are located in the RBD. In conclusion, vaccination with inactivated NDV-HXP-S induces a high proportion of neutralizing antibodies and absolute neutralizing antibody titers comparable to those after mRNA vaccination.

Antibody response to SARS-CoV-2 mRNA vaccine in lung cancer patients: Reactivity to vaccine antigen and variants of concern.

PurposeWe investigated SARS-CoV-2 mRNA vaccine-induced binding and live-virus neutralizing antibody response in NSCLC patients to the SARS-CoV-2 wild type strain and the emerging Delta and Omicron variants. Methods82 NSCLC patients and 53 healthy adult volunteers who received SARS-CoV-2 mRNA vaccines were included in the study. Blood was collected longitudinally, and SARS-CoV-2-specific binding and live-virus neutralization response to 614D (WT), B.1.617.2 (Delta), B.1.351 (Beta) and B.1.1.529 (Omicron) variants were evaluated by Meso Scale Discovery (MSD) assay and Focus Reduction Neutralization Assay (FRNT) respectively. We determined the longevity and persistence of vaccine-induced antibody response in NSCLC patients. The effect of vaccine-type, age, gender, race and cancer therapy on the antibody response was evaluated. ResultsBinding antibody titer to the mRNA vaccines were lower in the NSCLC patients compared to the healthy volunteers (P=<0.0001). More importantly, NSCLC patients had reduced live-virus neutralizing activity compared to the healthy vaccinees (P=<0.0001). Spike and RBD-specific binding IgG titers peaked after a week following the second vaccine dose and declined after six months (P=<0.001). While patients >70 years had lower IgG titers (P=<0.01), patients receiving either PD-1 monotherapy, chemotherapy or a combination of both did not have a significant impact on the antibody response. Binding antibody titers to the Delta and Beta variants were lower compared to the WT strain (P=<0.0001). Importantly, we observed significantly lower FRNT50 titers to Delta (6-fold), and Omicron (79-fold) variants (P=<0.0001) in NSCLC patients. ConclusionsBinding and live-virus neutralizing antibody titers to SARS-CoV-2 mRNA vaccines in NSCLC patients were lower than the healthy vaccinees, with significantly lower live-virus neutralization of B.1.617.2 (Delta), and more importantly, the B.1.1.529 (Omicron) variant compared to the wild-type strain. These data highlight the concern for cancer patients given the rapid spread of SARS-CoV-2 Omicron variant.

mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant

The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from naive vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.

Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

Longitudinal analysis shows durable and broad immune memory after SARS-CoV-2 infection with persisting antibody responses and memory B and T cells

Ending the COVID-19 pandemic will require long-lived immunity to SARS-CoV-2. Here, we evaluate 254 COVID-19 patients longitudinally up to eight months and find durable broad-based immune responses. SARS-CoV-2 spike binding and neutralizing antibodies exhibit a bi-phasic decay with an extended half-life of >200 days suggesting the generation of longer-lived plasma cells. SARS-CoV-2 infection also boosts antibody titers to SARS-CoV-1 and common betacoronaviruses. In addition, spike-specific IgG+ memory B cells persist, which bodes well for a rapid antibody response upon virus re-exposure or vaccination. Virus-specific CD4+ and CD8+ T cells are polyfunctional and maintained with an estimated half-life of 200 days. Interestingly, CD4+ T cell responses equally target several SARS-CoV-2 proteins, whereas the CD8+ T cell responses preferentially target the nucleoprotein, highlighting the potential importance of including the nucleoprotein in future vaccines. Taken together, these results suggest that broad and effective immunity may persist long-term in recovered COVID-19 patients.

Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant

The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies to neutralize these variants. We compared antibody binding and live virus neutralization of sera from naturally infected and spike mRNA vaccinated individuals against a circulating SARS-CoV-2 B.1 variant and the emerging B.1.351 variant. In acutely-infected (5-19 days post-symptom onset), convalescent COVID-19 individuals (through 8 months post-symptom onset) and mRNA-1273 vaccinated individuals (day 14 post-second dose), we observed an average 4.3-fold reduction in antibody titers to the B.1.351-derived receptor binding domain of the spike protein and an average 3.5-fold reduction in neutralizing antibody titers to the SARS-CoV-2 B.1.351 variant as compared to the B.1 variant (spike D614G). However, most acute and convalescent sera from infected and all vaccinated individuals neutralize the SARS-CoV-2 B.1.351 variant, suggesting that protective immunity is retained against COVID-19.

Characterization of neutralizing versus binding antibodies and memory B cells in COVID-19 recovered individuals from India

India is one of the countries most affected by the recent COVID-19 pandemic. Characterization of humoral responses to SARS-CoV-2 infection, including immunoglobulin isotype usage, neutralizing activity and memory B cell generation, is necessary to provide critical insights on the formation of immune memory in Indian subjects. In this study, we evaluated SARS-CoV-2 receptor-binding domain (RBD)-specific IgG, IgM, and IgA antibody responses, neutralization of live virus, and RBD-specific memory B cell responses in pre-pandemic healthy versus convalescent COVID-19 individuals from India. We observed substantial heterogeneity in the formation of humoral and B cell memory post COVID-19 recovery. While a vast majority (38/42, 90.47%) of COVID-19 recovered individuals developed SARS-CoV-2 RBD-specific IgG responses, only half of them had appreciable neutralizing antibody titers. RBD-specific IgG titers correlated with these neutralizing antibody titers as well as with RBD-specific memory B cell frequencies. In contrast, IgG titers measured against SARS-CoV-2 whole virus preparation, which includes responses to additional viral proteins besides RBD, did not show robust correlation. Our results suggest that assessing RBD-specific IgG titers can serve as a surrogate assay to determine the neutralizing antibody response. These observations have timely implications for identifying potential plasma therapy donors based on RBD-specific IgG in resource-limited settings where routine performance of neutralization assays remains a challenge. ImportanceOur study provides an understanding of SARS-CoV-2-specific neutralizing antibodies, binding antibodies and memory B cells in COVID-19 convalescent subjects from India. Our study highlights that PCR-confirmed convalescent COVID-19 individuals develop SARS-CoV-2 RBD-specific IgG antibodies, which correlate strongly with their neutralizing antibody titers. RBD-specific IgG titers, thus, can serve as a valuable surrogate measurement for neutralizing antibody responses. These finding have timely significance for selection of appropriate individuals as donors for plasma intervention strategies, as well as determining vaccine efficacy.

Serology in Children with Multisystem Inflammatory Syndrome (MIS-C) associated with COVID-19

ObjectivesWe aimed to measure SARS-CoV-2 serologic responses in children hospitalized with multisystem inflammatory syndrome (MIS-C) compared to COVID-19, Kawasaki Disease (KD) and other hospitalized pediatric controls. MethodsFrom March 17, 2020 - May 26, 2020, we prospectively identified hospitalized children at Childrens Healthcare of Atlanta with MIS-C (n=10), symptomatic PCR-confirmed COVID-19 (n=10), KD (n=5), and hospitalized controls (n=4). With IRB approval, we obtained prospective and residual blood samples from these children and measured SARS-CoV-2 spike (S) receptor binding domain (RBD) IgM and IgG binding antibodies by quantitative ELISA and SARS-CoV-2 neutralizing antibodies by live-virus focus reduction neutralization assay. We statistically compared the log-transformed antibody titers among groups and performed correlation analyses using linear regression. ResultsAll children with MIS-C had high titers of SARS-CoV-2 RBD IgG antibodies, which correlated strongly with neutralizing antibodies (R2=0.667, P<0.001). Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG antibody titers (geometric mean titer [GMT] 6800, 95%CI 3495-13231) than children with COVID-19 (GMT 626, 95%CI 251-1563, P<0.001), children with KD (GMT 124, 95%CI 91-170, P<0.001) and other hospitalized pediatric controls (GMT 85 [all below assay limit of detection], P<0.001). All children with MIS-C also had detectable RBD IgM antibodies, indicating recent SARS-CoV-2 infection. RBD IgG titers correlated with erythrocyte sedimentation rate (ESR) (R2=0.512, P<0.046) and with hospital and ICU lengths of stay (R2=0.590, P=0.010). ConclusionQuantitative SARS-CoV-2 RBD antibody titers may have a role in establishing the diagnosis of MIS-C, distinguishing it from other similar clinical entities, and stratifying risk for adverse outcomes. Table of Contents SummaryChildren with MIS-C have high antibody titers to the SARS-CoV-2 spike protein receptor binding domain, which correlate with neutralization, systemic inflammation, and clinical outcomes. Whats Known on This SubjectAlthough the clinical features of a multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19 have been recently described, the serologic features of MIS-C are unknown. What This Study AddsIn this case series, all hospitalized children with MIS-C had significantly higher SARS-CoV-2 binding and neutralizing antibodies than children with COVID-19 or Kawasaki Disease. SARS-CoV-2 antibodies correlated with metrics of systemic inflammation and clinical outcomes, suggesting diagnostic and prognostic value.

Rapid generation of neutralizing antibody responses in COVID-19 patients

SARS-CoV-2 is currently causing a devastating pandemic and there is a pressing need to understand the dynamics, specificity, and neutralizing potency of the humoral immune response during acute infection. Herein, we report the dynamics of antibody responses to the receptor-binding domain (RBD) of the spike protein and virus neutralization activity in 44 COVID-19 patients. RBD-specific IgG responses were detectable in all patients 6 days after PCR confirmation. Using a clinical isolate of SARS-CoV-2, neutralizing antibody titers were also detectable in all patients 6 days after PCR confirmation. The magnitude of RBD-specific IgG binding titers correlated strongly with viral neutralization. In a clinical setting, the initial analysis of the dynamics of RBD-specific IgG titers was corroborated in a larger cohort of PCR-confirmed patients (n=231). These findings have important implications for our understanding of protective immunity against SARS-CoV-2, the use of immune plasma as a therapy, and the development of much-needed vaccines.