Receptor-Binding Domain (RBD) Antibodies Contribute More to SARS-CoV-2 Neutralization When Target Cells Express High Levels of ACE2.

Neutralization assays are experimental surrogates for the effectiveness of infection- or vaccine-elicited polyclonal antibodies and therapeutic monoclonal antibodies targeting SARS-CoV-2. However, the measured neutralization can depend on the details of the experimental assay. Here, we systematically assess how ACE2 expression in target cells affects neutralization by antibodies to different spike epitopes in lentivirus pseudovirus neutralization assays. For high ACE2-expressing target cells, receptor-binding domain (RBD) antibodies account for nearly all neutralizing activity in polyclonal human sera. However, for lower ACE2-expressing target cells, antibodies targeting regions outside the RBD make a larger (although still modest) contribution to serum neutralization. These serum-level results are mirrored for monoclonal antibodies: N-terminal domain (NTD) antibodies and RBD antibodies that do not compete for ACE2 binding incompletely neutralize on high ACE2-expressing target cells, but completely neutralize on cells with lower ACE2 expression. Our results show that the ACE2 expression level in the target cells is an important experimental variable, and that high ACE2 expression emphasizes the role of a subset of RBD-directed antibodies.

Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved variants with substitutions in the spike receptor-binding domain (RBD) that affect its affinity for angiotensin-converting enzyme 2 (ACE2) receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites-a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single-amino acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently Asn501→Tyr (N501Y), cause epistatic shifts in the effects of mutations at other sites. These epistatic shifts shape subsequent evolutionary change-for example, enabling many of the antibody-escape substitutions in the Omicron RBD. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy.

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

Humoral immunogenicity of the seasonal influenza vaccine before and after CAR-T-cell therapy: a prospective observational study.

Recipients of chimeric antigen receptor-modified T (CAR-T) cell therapies for B cell malignancies have profound and prolonged immunodeficiencies and are at risk for serious infections, including respiratory virus infections. Vaccination may be important for infection prevention, but there are limited data on vaccine immunogenicity in this population. We conducted a prospective observational study of the humoral immunogenicity of commercially available 2019-2020 inactivated influenza vaccines in adults immediately prior to or while in durable remission after CD19-, CD20-, or B cell maturation antigen-targeted CAR-T-cell therapy, as well as controls. We tested for antibodies to all four vaccine strains using neutralization and hemagglutination inhibition (HAI) assays. Antibody responses were defined as at least fourfold titer increases from baseline. Seroprotection was defined as a HAI titer ≥40. Enrolled CAR-T-cell recipients were vaccinated 14-29 days prior to (n=5) or 13-57 months following therapy (n=13), and the majority had hypogammaglobulinemia and cellular immunodeficiencies prevaccination. Eight non-immunocompromised adults served as controls. Antibody responses to ≥1 vaccine strain occurred in 2 (40%) individuals before CAR-T-cell therapy and in 4 (31%) individuals vaccinated after CAR-T-cell therapy. An additional 1 (20%) and 6 (46%) individuals had at least twofold increases, respectively. One individual vaccinated prior to CAR-T-cell therapy maintained a response for >3 months following therapy. Across all tested vaccine strains, seroprotection was less frequent in CAR-T-cell recipients than in controls. There was evidence of immunogenicity even among individuals with low immunoglobulin, CD19+ B cell, and CD4+ T-cell counts. These data support consideration for vaccination before and after CAR-T-cell therapy for influenza and other relevant pathogens such as SARS-CoV-2, irrespective of hypogammaglobulinemia or B cell aplasia. However, relatively impaired humoral vaccine immunogenicity indicates the need for additional infection-prevention strategies. Larger studies are needed to refine our understanding of potential correlates of vaccine immunogenicity, and durability of immune responses, in CAR-T-cell therapy recipients.

Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition.

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Antibodies elicited by mRNA-1273 vaccination bind more broadly to the receptor binding domain than do those from SARS-CoV-2 infection.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with mutations in key antibody epitopes has raised concerns that antigenic evolution could erode adaptive immunity elicited by prior infection or vaccination. The susceptibility of immunity to viral evolution is shaped in part by the breadth of epitopes targeted by antibodies elicited by vaccination or natural infection. To investigate how human antibody responses to vaccines are influenced by viral mutations, we used deep mutational scanning to compare the specificity of polyclonal antibodies elicited by either two doses of the mRNA-1273 COVID-19 vaccine or natural infection with SARS-CoV-2. The neutralizing activity of vaccine-elicited antibodies was more targeted to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein compared to antibodies elicited by natural infection. However, within the RBD, binding of vaccine-elicited antibodies was more broadly distributed across epitopes compared to infection-elicited antibodies. This greater binding breadth means that single RBD mutations have less impact on neutralization by vaccine sera compared to convalescent sera. Therefore, antibody immunity acquired by natural infection or different modes of vaccination may have a differing susceptibility to erosion by SARS-CoV-2 evolution.

Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies.

The evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent plasma antibodies are impacted by all mutations to the spike's receptor-binding domain (RBD), the main target of plasma neutralizing activity. Binding by polyclonal plasma antibodies is affected by mutations in three main epitopes in the RBD, but longitudinal samples reveal that the impact of these mutations on antibody binding varies substantially both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBD's receptor-binding motif. The most important site is E484, where neutralization by some plasma is reduced >10-fold by several mutations, including one in the emerging 20H/501Y.V2 and 20J/501Y.V3 SARS-CoV-2 lineages. Going forward, these plasma escape maps can inform surveillance of SARS-CoV-2 evolution.

Attenuated Influenza Virions Expressing the SARS-CoV-2 Receptor-Binding Domain Induce Neutralizing Antibodies in Mice.

An effective vaccine is essential for controlling the spread of the SARS-CoV-2 virus. Here, we describe an influenza virus-based vaccine for SARS-CoV-2. We incorporated a membrane-anchored form of the SARS-CoV-2 spike receptor binding domain (RBD) in place of the neuraminidase (NA) coding sequence in an influenza virus also possessing a mutation that reduces the affinity of hemagglutinin for its sialic acid receptor. The resulting ΔNA(RBD)-Flu virus can be generated by reverse genetics and grown to high titers in cell culture. A single-dose intranasal inoculation of mice with ΔNA(RBD)-Flu elicits serum neutralizing antibody titers against SAR-CoV-2 comparable to those observed in humans following natural infection (~1:200). Furthermore, ΔNA(RBD)-Flu itself causes no apparent disease in mice. It might be possible to produce a vaccine similar to ΔNA(RBD)-Flu at scale by leveraging existing platforms for the production of influenza vaccines.