Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination.

Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective.

An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody.

IMPORTANCE: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. Importance: The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Structure of a Vaccine-Induced, Germline-Encoded Human Antibody Defines a Neutralizing Epitope on the SARS-CoV-2 Spike N-Terminal Domain.

Structural characterization of infection- and vaccination-elicited antibodies in complex with antigen provides insight into the evolutionary arms race between the host and the pathogen and informs rational vaccine immunogen design. We isolated a germ line-encoded monoclonal antibody (mAb) from plasmablasts activated upon mRNA vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and determined its structure in complex with the spike glycoprotein by electron cryomicroscopy (cryo-EM). We show that the mAb engages a previously uncharacterized neutralizing epitope on the spike N-terminal domain (NTD). The high-resolution structure reveals details of the intermolecular interactions and shows that the mAb inserts its heavy complementarity-determining region 3 (HCDR3) loop into a hydrophobic NTD cavity previously shown to bind a heme metabolite, biliverdin. We demonstrate direct competition with biliverdin and that, because of the conserved nature of the epitope, the mAb maintains binding to viral variants B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), and B.1.1.529 (omicron). Our study describes a novel conserved epitope on the NTD that is readily targeted by vaccine-induced antibody responses. IMPORTANCE We report the first structure of a vaccine-induced antibody to SARS-CoV-2 spike isolated from plasmablasts 7 days after vaccination. The genetic sequence of the antibody PVI.V6-14 suggests that it is completely unmutated, meaning that this type of B cell did not undergo somatic hypermutation or affinity maturation; this cell was likely already present in the donor and was activated by the vaccine. This is, to our knowledge, also the first structure of an unmutated antibody in complex with its cognate antigen. PVI.V6-14 binds a novel, conserved epitope on the N-terminal domain (NTD) and neutralizes the original viral strain. PVI.V6-14 also binds the newly emerged variants B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), and B.1.1.529 (omicron). Given that this antibody was likely already present in the donor prior to vaccination, we believe that this antibody class could potentially "keep up" with the new variants, should they continue to emerge, by undergoing somatic hypermutation and affinity maturation.

Activity of convalescent and vaccine serum against SARS-CoV-2 Omicron.

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in November 2021 in South Africa and Botswana, as well as in a sample from a traveller from South Africa in Hong Kong1,2. Since then, Omicron has been detected globally. This variant appears to be at least as infectious as Delta (B.1.617.2), has already caused superspreader events3, and has outcompeted Delta within weeks in several countries and metropolitan areas. Omicron hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness2,4-6. Here we investigated the virus-neutralizing and spike protein-binding activity of sera from convalescent, double mRNA-vaccinated, mRNA-boosted, convalescent double-vaccinated and convalescent boosted individuals against wild-type, Beta (B.1.351) and Omicron SARS-CoV-2 isolates and spike proteins. Neutralizing activity of sera from convalescent and double-vaccinated participants was undetectable or very low against Omicron compared with the wild-type virus, whereas neutralizing activity of sera from individuals who had been exposed to spike three or four times through infection and vaccination was maintained, although at significantly reduced levels. Binding to the receptor-binding and N-terminal domains of the Omicron spike protein was reduced compared with binding to the wild type in convalescent unvaccinated individuals, but was mostly retained in vaccinated individuals.

A vaccine-induced public antibody protects against SARS-CoV-2 and emerging variants.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

A public vaccine-induced human antibody protects against SARS-CoV-2 and emerging variants.

The emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility is a public health threat. Some of these variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies, which principally target the receptor binding domain (RBD) on the virus spike glycoprotein. Here, we describe 2C08, a SARS-CoV-2 mRNA vaccine-induced germinal center B cell-derived human monoclonal antibody that binds to the receptor binding motif within the RBD. 2C08 broadly neutralizes SARS-CoV-2 variants with remarkable potency and reduces lung inflammation, viral load, and morbidity in hamsters challenged with either an ancestral SARS-CoV-2 strain or a recent variant of concern. Clonal analysis identified 2C08-like public clonotypes among B cell clones responding to SARS-CoV-2 infection or vaccination in at least 20 out of 78 individuals. Thus, 2C08-like antibodies can be readily induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern. ONE SENTENCE SUMMARY: Protection against SARS-CoV-2 variants by a potently neutralizing vaccine-induced human monoclonal antibody.

The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies and targets both the NTD and the RBD.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.