Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants

Abstract

The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4-8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

Figure 1.. Generation of replication-competent VSV bearing SARS-CoV-2 spike (rcVSV-CoV2-S).

(A) Representative images of de novo generation of rcVSV-CoV2-S, carrying an EGFP reporter, in transfected 293T-ACE2+TMPRSS2 (F8–2) cells as described in Extended Data Fig. S1. Single GFP+ cells detected at 2–3 days post-transfection (dpt) form a foci of syncytia by 4 dpt. Images are taken by Celigo imaging cytometer (Nexcelom) and are computational composites from the identical number of fields in each well. White bar is equal to 1 millimeter. (B) Entry efficiency of rcVSV-CoV2-S in parental 293T cells, 293T stably expressing ACE2 alone (293T-ACE2) or with TMPRSS2 (293T-ACE2+TMPRSS2). Serial dilutions of virus stocks amplified on Vero-TMPRSS2 cells were used to infect the indicated cell lines in 96-well plates in triplicates. GFP signal was detected and counted by a Celigo imaging cytometer (Nexcelom) 10 hours post-infection. Symbols are individual data points from triplicate infections at the indicated dilutions. Bars represent the average of 3 replicates with error bars indicating standard deviation. A two-way ANOVA was used to compare the differences between cell lines at any given dilution. Adjusted p values from Tukey’s multiple comparisons test are given (ns; not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 2.. Neutralization activity of antibody responses elicited by the Sputnik V vaccine.

(A) Schematic of the Spike substitutions that make up the variants being evaluated in this study. The amino acid positions and corresponding ‘Ancestral’ sequence of the Wuhan isolate is shown. The prevailing WT sequence now has a D614G substitution. All the variants and mutants have D614G. (B) Neutralization activity of individual serum samples against rcVSV-CoV2-S with the WT, variant (B.1.1.7 or B.1.351), or mutant E484K spike proteins. Neutralization is represented by the reciprocal 50% inhibitory dilution factor (1/IC₅₀). Sera samples with no appreciable neutralization against a given virus were assigned a defined 1/IC₅₀ value of 1.0, as values ≤1 are not physiological (Grey shaded area). Dashed line indicates the lowest serum dilution tested (1/IC50 = 20). Geometric mean titers (GMT and 95% CI) for the neutralizing activity of all vaccine sera are indicated below each of the viral spike proteins examined. NS; not significant, *; p<0.05, p < 0.01; ** are adjusted p values from non-parametric one-way ANOVA with Dunn’s multiple comparisons test. (C) For each serum sample, the fold-change in IC₅₀ (reciprocal inhibitory dilution factor) against the indicated variant and mutant spike proteins relative to its IC₅₀ against wild-type (WT) spike (set at 1) is plotted. Adjusted p values were calculated as in (B). Medians are represented by the bars and whiskers demarcate the 95% CI. Neutralization dose-response curves were performed in triplicates, and the mean values from each triplicate experiment are shown as the single data points for each sera sample.

Figure 3.. Dose response curves reveal distinct patterns of neutralizing antibody responses.

Groups (A - D) represent distinct classes of virus neutralizing activity present in the sera samples analyzed. A representative member from each group is shown. Full neutralization curves for all sera tested against all viruses bearing the variant and mutant spike proteins are shown in supplementary Fig. S2. (E) graphs the virus neutralizing titers (VNT = 1/IC₅₀) and 95% CI that can be extrapolated from the nonlinear regression curves. Different colored symbols represent the viruses indicated in the figure key. The open symbols in SP012 (Group D) represent assigned values of 1.0 (for B.1.351 and E484K) when no significant neutralization activity could be detected at the lowest serum dilution used (1:20) or ambiguous fits (for WT) due to very low neutralizing activity. The shaded area represents values that are not physiologically relevant.

Figure 4.. Competitive inhibition of rcVSV-CoV2-S entry by soluble RBD-Fc.

(A) Recombinant RBD-Fc was serially titrated with the infection inoculum containing a fixed amount of rcVSV-CoV2-S bearing WT or the indicated VOC spike proteins. 10 hpi, GFP+ cells were quantified by the Celigo image cytometer. Data points are means of six independent replicates with error bars representing S.D. The number of GFP+ cells in the absence of any RBD-Fc was set to 100% and used to normalize the infection response in the presence of increasing amounts of RBD-Fc. Log[inhibitor] versus normalized response variable slope nonlinear regression curves were generated using GraphPad PRISM (v9.1.0). (B) The IC50 values from each replicate dose response curve generated for a given virus were grouped. The mean (central bar) and SD (whiskers) for each group are indicated. Adjusted p values (*, p<0.05; ****, p<0.0001) from ordinary one-way ANOVA with Dunnett’s multiple comparisons test are indicated. (C) Landscape of slope versus relative IC50 values of all 12 Sputnik sera against the indicated VOC or mutant Spike. Relative IC₅₀ value is defined as the ratio of WT IC₅₀:VOC IC₅₀. Dashed lines indicate quadrants of high/low IC₅₀ and high/low slope.