SARS-CoV-2 spike conformation determines plasma neutralizing activity

Abstract

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 S ₁ 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.

Figure 1.. Prefusion-stabilization of SARS-CoV-2 S enhances S₁ subunit antibody titers.

(A) Antibody binding titers elicited by SARS-CoV-2 infection or vaccination to the prefusion S (S), the N-terminal domain (NTD), the receptor-binding domain (RBD), and the S₂ subunit in the prefusion (S2(Pre)) and postfusion (S2(Post)) conformations, as measured by ELISA. (B-D) Antibody binding titers in matched cohorts of individuals previously infected with SARS-CoV-2 before and after vaccination with BNT162b2 (B), Ad26.COV2.S (C), or AZD1222 (D). Each point represents a single patient plasma sample, bars represent geometric means, and error bars represent geometric standard deviations. Protruding colored bars (B-D) mark the geometric mean of individuals that were not previously infected with SARS-CoV-2. Fit curves are shown Figure S1 and S2.

Figure 2.. SARS-CoV-2 neutralization is determined by S₁ subunit targeting antibodies.

(A) SARS-CoV-2 S pseudotyped VSV neutralization titers elicited by natural infection or vaccination. The dotted line is the limit of detection, the colored bars are GMTs and the black error bars are geometric standard deviations. Fit curves are shown in Fig. S3. (B-F) Correlation between plasma neutralizing activity and prefusion S (B), N-terminal domain (C), receptor-binding domain (D), prefusion S₂ (E), and postfusion S₂ (F) binding titers shown with a linear regression fit to the log of neutralization and binding titers. The black shaded regions represent 95% confidence intervals. (G-H) Binding (G) and neutralization (H) titers resulting from depletion of polyclonal antibodies targeting prefusion S, prefusion S₂, and postfusion S₂. Fit curves are shown in Fig. S5 and S6. Statistical significance between groups of paired data were determined by Wilcoxon rank test and *P < 0.05.

Figure 3.. Broad neutralization of SARS-CoV-2 variants is mediated by RBD-specific antibodies.

(A-B) Plasma binding titers resulting from mock, Wuhan-Hu-1 RBD (left) and NTD (right) depletion of polyclonal antibodies. (B-D) Plasma neutralizing activity against G614 S VSV (B), Delta S VSV (C) and Omicron S VSV (D) after mock, Wuhan-Hu-1 RBD or NTD depletion of polyclonal antibodies. Mock consists of depletion carried out with beads without immobilized antigen. Statistical significance between groups of paired data were determined by Wilcoxon rank test and *P < 0.05, ***P < 0.001, ****P < 0.0001.