Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

Figures

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  Fig 1. Overview of SARS-CoV-2 epitope selection, protein engineering, and immunization. (A) SARS-CoV-2 Spike protein (yellow) interacting with ACE2 (blue), PDB ID: 6M0J. A zoomed view shows SARS-CoV-2:ACE2 interacting residues. (B) Spike protein RBM (436–507) sequence. Purple residues indicate ACE2 binding, dots above residues indicate B38 or CC12.1 antibody binding. Immunogens models 1–3 span the putative B cell epitope FNCY (486–489). (C) VH62 model with CDR1 (yellow), CDR2 (blue), CDR3 (red). (D) Timeline of priming (day 0), and booster shot (day 21), with blood draws (days 0, 21, 30, 45). Figure adapted from images created with BioRender.com.

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  Fig 2. ELISA binding of immune sera of mice after prime-boost. Binding to RBD (A) and Spike protein (B) of sera at the timepoints indicated. Closed symbols refer to individual mice. Open symbols refer to binding of pre-immune sera. Day 30 statistical significance analysis. (RBD): group 1 vs. 4, p = 0.086; group 2 vs. 4, p = 0.054; group 3 vs. 4,—value; group 1 vs 3, p = 0.008; group 2 vs 3, p = 0.003. (Spike) group 1 vs. 4, p = 0.003; group 2 vs. 4, p = 0.009; group 3 vs. 4, p = 0.532; group 1 vs 3, p = 6.4E-06; group 2 vs 3, p = 1.69E-07. Results from one experiment representative of three independent runs each performed at different times. Sera tested in duplicate. Color scheme: red = group 1; orange = group 2; green = group 3; blue = group 4.

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  Fig 3. Polyclonal antibodies in immune sera share paratope specificity with Class 1 human neutralizing antibodies B38 and CC12.1. Immune sera were tested in a cross-competition RBD binding assay by ELISA. (A) Heatmap of neutralizing antibody contact residues (purple) with RBM region (positions 437–508). Black dots indicate ACE2 contact residues. (B) Titration of binding of HRP-B38 to RBD in ELISA. Inset: Slope of inhibition of HRP-B38 binding by unlabeled antibody B38. Effective 50% binding concentrations were (~1.5 ng/well). (C) Binding of HRP-B38 in the presence of 1:50 dilution of individual mouse serum (day 45). (D) Titration of binding of HRP-CC12.1 to RBD in ELISA. Inset: Slope of inhibition of HRP-CC12.1 binding by unlabeled antibody CC12.1. (E) Binding of HRP-CC12.1 in the presence of 1:50 dilution of individual mouse serum (day 45). Empty columns; pre-immune sera. Black columns indicate maximal binding of HRP-labeled antibody in ELISA buffer. Color scheme: red = group 1; orange = group 2; green = group 3; blue = group 4.

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  Fig 4. Neutralization of authentic SARS-CoV-2 WA1 isolate and VOCs B.1.351/Beta. Neutralization of authentic SARS-CoV-2 isolate WA1 and VOCs B.1.351 and B.1.617.2 by immune sera was measured by focus reduction neutralization test (FRNT) on TMPRSS2-Vero cells. 3-fold serial dilutions started at 1:50. Percent neutralization and percent infection are relative to media-only control. Data shown are the mean and SD of 2 biological replicates. Dotted lines mark 50% neutralization. Color scheme: red = group 1; orange = group 2; green = group 3; blue = group 4. NMS = normal mouse serum.

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  Fig 5. Neutralization of authentic SARS-CoV-2 VOC B.1.617.2/Delta isolate. Neutralization of authentic SARS-CoV-2 isolate B.1.617.2 by immune sera was measured by focus reduction neutralization test (FRNT) on TMPRSS2-Vero cells. 3-fold serial dilutions started at 1:50. Percent neutralization and percent infection are relative to media-only controls. Data shown are the mean and SD of 2–4 biological replicates. Dotted lines mark 50% neutralization. Color scheme: red = group 1; orange = group 2; green = group 3; blue = group 4. NMS = normal mouse serum.

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  Fig 6. Neutralization of authentic SARS-CoV-2 VOC BA.1/Omicron isolate. Neutralization of authentic SARS-CoV-2 isolate BA.1 by immune sera was measured by focus reduction neutralization test (FRNT) on TMPRSS2-Vero cells. 3-fold serial dilutions started at 1:50. Percent neutralization and percent infection are relative to media-only controls. Data shown are the mean and SD of 4 biological replicates. Dotted lines mark 50% neutralization. Color scheme: red = group 1; orange = group 2; green = group 3; blue = group 4. NMS = normal mouse serum.

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  Fig 7. Comparison of IC50s from neutralization of authentic SARS-CoV-2 by immune sera from groups 1–4. IC50s from focus reduction neutralization tests (FRNTs) using the indicated variants of SARS-CoV-2 (Figs 4–6). Each point is 1 mouse. Significance determined by ordinary one-way ANOVA with comparison to reference group 4. * = p<0.033.

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  Fig 8. Conformational analysis of SARS2-CoV-2 RBM structure and predicted conformation of VH Model 2. In the Spike S1 protein (A) the tip of the RBM ridgeline is shown in dark green and the FNCY patch in dark blue. In Model 2 (ribbon B) and (space filling C) the modeled CDR2 loop is shown in dark green, while the grafted RBM epitope is in dark blue. Panels on the right show the interface between ACE2 and SARS-CoV-2 RBM (D), and ACE2 and the CDR2 loop of Model 2 (E). Color scheme: A: gray—RBD; green—RBM; blue–ACE2 contact residues; B and C: yellow -CDR1; green—CDR2; orange—CDR3; blue—SARS-Cov-2 RBM residues grafted in CDR2; gray—tetanus toxoid epitope; D: pink—ACE2; green—SARS-Cov-2 RBM residues; E: pink—ACE2; blue—SARS-Cov-2 RBM residues grafted in CDR2.

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  Fig 9. Overview of key amino acid residues in the RBM in or around the 486Phe-Asn-Cys-Tyr489 “FNCY” patch. The heatmap shows neutralizing antibodies clustered by contact residues (dark red squares), and their relation to ACE2 binding residues (red dots). The middle barplot shows the percentage of all MHC-II alleles that can now not effectively present a mutated residue compared to wildtype with a threshold of a NetMHCIIpan rank of 10. The top plot indicates which residues are mutated in the parental/WA1, Beta, Delta, and Omicron Variants of Concern. For discussion on antibodies A23-58.1 [40], B1.182.1 [40], SE12 [21, 41], and S2K146 [42] see text.