Heterologous sarbecovirus receptor binding domains as scaffolds for SARS-CoV-2 receptor binding motif presentation

Figure 1.. Selection of scaffolds and design. (A) Phylogenetic tree of twenty selected receptor binding domains used in this study; SARS-CoV and SARS-CoV-2 are included for reference. (B) Design schematic of versions 1, 2, and 3 resurfaced constructs, depicting RBM grafts (grey) on heterologous coronavirus scaffolds (indigo). Red dashed boxes indicate differences relative to version 1.

Figure 2.. Resurfaced monomeric immunogen expression. (A) SDS-PAGE analysis of monomers under non-reducing conditions. (B) Representative size exclusion trace with (*) marking the monomeric constructs. Fractions in this peak were pooled for further characterization.

Figure 3.. Resurfaced dimeric immunogen design and expression. (A) RBM-directed Fab B38 was used to confirm conformational integrity of the SARS-2 RBM grafted onto rsYNLF-34C. FAB2G sensors were used with immobilized Fab to measure binding via BLI to rsYNLF-34C at 10 μM, 5 μM, 1 μM, and 0.5 μM. Binding of CR3022 and S309 Fabs to rsYNLF-34C at 10 μM was also assayed, but no binding was detected. (B) SDS-PAGE analysis of dimers under non-reducing conditions. (C) Representative size exclusion trace with (*) marking the dimeric constructs. Fractions in this peak were pooled for use as immunogens. (D) Pairwise comparisons of amino acid sequence identity in the non-RBM portions of selected sarbecovirus RBDs. (E) Pairwise comparisons of amino acid sequence identity in the RBM portions of selected sarbecovirus RBDs.

Figure 4.. Antigenic distance investigation immunizations. Schematic of immunization regimens. Two immunization cohorts (n=5 mice each) were primed with SARS-2 two-proline-stabilized (2P) spike protein on day 0 and then boosted with a cocktail of either the rsSARS-1 and rsWIV1 dimers (“AgDistlow” cohort) or the rsWIV1 and rsYNLF-34C dimers (“AgDisthigh” cohort) at days 21 and 42.

Figure 5.. Antigenic distance investigation immunization serum reactivity. (A) Serum collected at day 35 was assayed in ELISA against different coronavirus antigens. (B) Pseudovirus neutralization was assayed against a panel of sarbecoviruses, ordered here based on spike amino acid sequence similarity to SARS-2. Statistical significance for (A, B) was determined using Kruskal-Wallis test with post-hoc analysis using Dunn’s test corrected for multiple comparisons (* = p < 0.05, ** = p < 0.01); non-significant comparisons not marked.