Humoral responses to the SARS-CoV-2 spike and receptor binding domain in context of pre-existing immunity confer broad sarbecovirus neutralization

Figures

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  Figure 1. Immunization regimens. Two immunization cohorts were used for this study (n=5 mice per cohort). Mice were primed with SARS-2 2-proline- stabilized spike protein on day -21 and then boosted with either the same spike protein (“Spike Boost” cohort) or a trimeric SARS-2 RBD (“RBD Boost” cohort) at days 0 and 21.

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  Figure 2. Serum response to immunization regimens. Serum collected at day 35 from the Spike Boost cohort (A) and the RBD boost cohort (B) was assayed in ELISA different coronavirus antigens. (C, D) Additional ELISAs were performed using day 35 serum samples against RaTG13 and SHC014 RBDs. (E, F) Percent of day 35 serum antibody binding lost in competition ELISAs compared to a no IgG control. SARS- 2, SARS-1, and WIV1 RBDs were used as coating antigens. Statistical significance across all panels was determined using Kruskal-Wallis test with post-hoc analysis using Dunn’s test corrected for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). All pairwise comparisons were performed, and only statistically significant comparisons are indicated on the plots.

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  Figure 3. Flow cytometry gating scheme and results. (A) Gating strategy to select for CD3-/CD19+/IgM-/IgG+ cells to isolate the memory B cell population. (B) Antigen specific memory IgG responses were identified using a combination of SARS-2 spike, SARS-2 RBD, and SARS-2 RBM hg RBD flow hooks. SARS-2 spike-directed B cell responses were further separated by reactivity to into the RBM, RBD remainder (excluding RBM epitopes), and spike remainder (excluding RBM and RBD epitopes). (C) Spike-directed responses were binned into RBM, RBD remainder (excluding RBM epitopes), and spike remainder (excluding RBD and RBM epitopes) populations using relevant probes in flow cytometry. Data is shown as a percentage of total spike-specific IgG+ B-cells. Statistical significance was determined using Kruskal-Wallis test with posthoc analysis using Dunn’s test corrected for multiple comparisons (*p < 0.05, **p < 0.01); ns, not significant.

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  Figure 4. Sarbecovirus neutralization. (A) Neutralization was assayed across a range of sarbecoviruses, ordered here from left to right based on genetic similarity of the spike gene to SARS-2. (B) Neutralization potency was computed by dividing the NT50 value for each sample by the adjusted serum ELISA endpoint titer for the corresponding sarbecovirus RBD. Comparisons across both panels were performed using the Mann-Whitney U test (*p < 0.05, **p < 0.01); ns, not significant.