S Protein-Reactive IgG and Memory B Cell Production after Human SARS-CoV-2 Infection Includes Broad Reactivity to the S2 Subunit

Abstract

The high susceptibility of humans to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the cause of coronavirus disease 2019 (COVID-19), reflects the novelty of the virus and limited preexisting B cell immunity. IgG against the SARS-CoV-2 spike (S) protein, which carries the novel receptor binding domain (RBD), is absent or at low levels in unexposed individuals. To better understand the B cell response to SARS-CoV-2 infection, we asked whether virus-reactive memory B cells (MBCs) were present in unexposed subjects and whether MBC generation accompanied virus-specific IgG production in infected subjects. We analyzed sera and peripheral blood mononuclear cells (PBMCs) from non-SARS-CoV-2-exposed healthy donors and COVID-19 convalescent subjects. Serum IgG levels specific for SARS-CoV-2 proteins (S, including the RBD and S2 subunit, and nucleocapsid [N]) and non-SARS-CoV-2 proteins were related to measurements of circulating IgG MBC levels. Anti-RBD IgG was absent in unexposed subjects. Most unexposed subjects had anti-S2 IgG, and a minority had anti-N IgG, but IgG MBCs with these specificities were not detected, perhaps reflecting low frequencies. Convalescent subjects had high levels of IgG against the RBD, S2, and N, together with large populations of RBD- and S2-reactive IgG MBCs. Notably, IgG titers against the S protein of the human coronavirus OC43 were higher in convalescent subjects than in unexposed subjects and correlated strongly with anti-S2 titers. Our findings indicate cross-reactive B cell responses against the S2 subunit that might enhance broad coronavirus protection. Importantly, our demonstration of MBC induction by SARS-CoV-2 infection suggests that a durable form of B cell immunity is maintained even if circulating antibody levels wane.IMPORTANCE The recent rapid worldwide spread of SARS-CoV-2 has established a pandemic of potentially serious disease in the highly susceptible human population. Key issues are whether humans have preexisting immune memory that provides some protection against SARS-CoV-2 and whether SARS-CoV-2 infection generates lasting immune protection against reinfection. Our analysis focused on pre- and postinfection IgG and IgG memory B cells (MBCs) reactive to SARS-CoV-2 proteins. Most importantly, we demonstrate that infection generates both IgG and IgG MBCs against the novel receptor binding domain and the conserved S2 subunit of the SARS-CoV-2 spike protein. Thus, even if antibody levels wane, long-lived MBCs remain to mediate rapid antibody production. Our study results also suggest that SARS-CoV-2 infection strengthens preexisting broad coronavirus protection through S2-reactive antibody and MBC formation.

Keywords: COVID-19; IgG antibodies; SARS-CoV-2; memory B cells; spike protein.

FIG 1

Serum IgG levels against SARS-CoV-2 and non-SARS-CoV-2 proteins in non-SARS-CoV-2-exposed and COVID-19 convalescent subjects. Sera were collected from (i) 21 healthy donors sampled from 2011 to 2014 (HD), (ii) 20 SARS-CoV-2-negative health care workers sampled in 2020 (HCW), and (iii) 26 COVID-19 convalescent subjects sampled 4 to 9 weeks after symptom onset (Conv). (A) Serum IgG concentrations measured by ELISA against the SARS-CoV-2 spike (S), receptor binding domain (RBD), S2 subunit, and nucleocapsid (N). Columns represent individual HD and convalescent subjects in order of ascending titers against S. The assigned cutoff for positivity is shown by the horizontal gray bar. (B) Proportions of serum IgG against SARS-CoV-2 RBD, S2, and N for individual convalescent subjects. (C) Serum IgG concentrations against the S protein of the HCoV OC43 in Conv, HD, and HCW subjects. (D to F) Serum IgG concentrations against the S protein of HCoV 229E (D), influenza virus H1 hemagglutinin (E), and TTd (F) in Conv and HD subjects. (G) Correlation between serum IgG concentrations against the S2 subunit of SARS-CoV-2 and the S protein of the HCoV OC43. Significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant) for comparisons of serum IgG concentrations between subject groups was determined by the Wilcoxon rank sum test. Correlations were tested by Spearman correlation analysis with corresponding robust regression models.

FIG 2

Analysis of IgG memory B cells (MBCs) reactive to SARS-CoV-2 and non-SARS-CoV-2 proteins in non-SARS-CoV-2-exposed and COVID-19 convalescent subjects. PBMCs for MBC analysis were collected from (i) 21 healthy donors sampled from 2011 to 2014 (HD) and (ii) 26 COVID-19 convalescent subjects sampled 4 to 9 weeks after symptom onset (Conv). PBMCs were stimulated in vitro to induce MBC differentiation into Ab-secreting cells. Antigen-specific quantitation of MBC-derived Ab (IgG)-secreting cells (MASCs) by ELISpot assay or of MBC-derived polyclonal (IgG) Abs (MPAbs) by ELISA provided a measure of the abundance of specific IgG MBCs. (A) IgG MBCs reactive to the SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N) in convalescent subjects. The assigned cutoff for positivity is shown by the horizontal gray bar. (B) IgG MBCs reactive to the influenza virus H1 hemagglutinin and TTd in convalescent subjects. (C) Proportions of IgG MBCs reactive to the SARS-CoV-2 RBD, S2, and N for individual convalescent subjects. A bar representing the mean value for the HD cohort is included for comparison. In all HD samples, MPAb IgG levels against RBD, S2, and N were below the cutoff for assay positivity. (D) Comparison of serum IgG concentrations (upper panels) and numbers of IgG MBCs (lower panels) reactive to the SARS-CoV-2 S (left-hand side) and N (right-hand side) proteins. Serum IgG was measured by ELISA; IgG MBC numbers were based on ELISA of MPAbs. Dilution curves are shown for individual convalescent subjects; curves for 4 subjects are shown in different colors to identify particular response patterns. (E to H) IgG MBCs reactive to the S proteins of HCoVs OC43 (E) and 229E (F), the H1 hemagglutinin (G), and TTd (H) in convalescent and HD subjects. Significance (*, P < 0.05; ns, not significant) for comparisons of IgG MBC numbers between subject groups was determined by the Wilcoxon rank sum test.