Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses

Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape convalescent and vaccine-induced antibody responses has renewed focus on the development of broadly protective T-cell-based vaccines. Here, we apply structure-based network analysis and assessments of HLA class I peptide stability to define mutationally constrained CD8⁺ T cell epitopes across the SARS-CoV-2 proteome. Highly networked residues are conserved temporally among circulating variants and sarbecoviruses and disproportionately impair spike pseudotyped lentivirus infectivity when mutated. Evaluation of HLA class I stabilizing activity for 18 globally prevalent alleles identifies CD8⁺ T cell epitopes within highly networked regions with limited mutational frequencies in circulating SARS-CoV-2 variants and deep-sequenced primary isolates. Moreover, these epitopes elicit demonstrable CD8⁺ T cell reactivity in convalescent individuals but reduced recognition in recipients of mRNA-based vaccines. These data thereby elucidate key mutationally constrained regions and immunogenic epitopes in the SARS-CoV-2 proteome for a global T-cell-based vaccine against emerging variants and SARS-like coronaviruses.

Keywords: CD8(+) T cells; COVID-19; SARS-CoV-2; epitopes; protection; sarbecovirus; vaccine; variants.

Graphical abstract

Figure 1

Structure-based network analysis of the SARS-CoV-2 proteome identifies amino acid residues conserved in lineage B and C coronaviruses (A) Structure-based network analysis schematic for closed spike trimer (PDB: 6VXX) with amino acid residues (nodes) and non-covalent interactions (edges). Edge width indicates interaction strength and node size indicates relative network scores. (B–D) Comparison of SARS-CoV-2 amino acid network scores (binned by network score: <0, 0–2, 2–4, and >4) with sequence entropy for SARS-CoV-2, sarbecoviruses (SARS-CoV-1/bat CoV), and MERS. (E) Alignment of SARS-CoV-2 network scores with sequence entropy values for SARS-CoV-2 in May 2020 and February 2021, sarbecoviruses (SARS-CoV-1/bat CoV) and MERS-CoV. Residues in blue indicate those with network scores >4. Network scores of residues mutated in the B.1.1.7 alpha (red triangles), B.1.351 beta (green triangles), P.1 gamma (yellow triangles), and B.1.617.2 delta variant (purple triangles) are depicted in gray. Yellow boxes indicate new areas of sequence variation in SARS-CoV-2 that emerged between May 2020 and February 2021. Statistical comparisons were made using Mann-Whitney U test. For comparisons of more than two groups, Kruskal-Wallis test with Dunn’s pos hoc analyses was used. Calculated p values were as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S1 and Table S1.

Figure S1

Structure-based network analysis of the SARS-CoV-2 proteome, related to Figure 1 (A) Network diagrams of SARS CoV-2 structural and accessory proteins and (B) NSPs. Node size indicates relative intra-protein network scores.

Figure S2

Correlation of SARS-CoV-2 network scores with SARS-CoV-1 and MERS-CoV network scores, related to Figure 1 Scatterplots comparing SARS-CoV-2 network scores to (A) SARS-CoV-1 network scores and (B) MERS-CoV network scores. Correlations were calculated by Spearman’s rank correlation coefficient.

Figure S3

Spike pseudotyped lentiviral infectivity assay, related to Figure 2 (A) Flow cytometry plots showing %ZsGreen-positive 293T and 293T-ACE2 cells after 60h incubation with ZsGreen backbone lentiviruses pseudotyped with no Spike protein (delta Spike; gray), wild-type (WT) Spike protein (green) or VSV-G (black) envelope protein. Composite pseudotyped lentiviral infectivity data of (B) 293T or (C) 293T-ACE2 cells at five-fold and two-fold dilutions of neat stock virus preparations.

Figure 2

Mutation of highly networked residues in the viral spike protein impairs infectivity and RBD folding (A) Location of networked (blue) and non-networked (red) residues in the closed (PDB: 6VXX) and open (PDB: 6VYB) conformations of the spike protein that were mutated in pseudotyped lentivirus. (B and C) Comparison of network scores and Shannon entropy values between networked residues and non-networked residues selected for mutagenesis. (D) Flow cytometry plots showing the percentage of ZsGreen-positive 293T-ACE2 cells after 60-h incubation with ZsGreen backbone lentiviruses pseudotyped with no spike protein (delta spike; gray), wild-type (WT) spike (green), VSV-G (black), or mutated spike proteins (dark blue, light blue, and red). (E) Comparison of spike pseudotyped lentiviral infectivity of 293T-ACE2 cells after mutation of networked residues with non-conservative mutations (N, dark blue), networked residues with conservative mutations (C, light blue), and non-networked residues with non-conservative mutations (N, red). Data are means of technical triplicates from an experiment performed twice. Statistical analysis by one-way analysis of variance and Mann-Whitney U test. (F) Scatterplot of full spike protein residue network scores and average effect of mutation on monomeric RBD folding. Residues in blue indicate those with high network scores but low effect on monomeric RBD folding (V362, A363, C391, V524, C525). Correlations were calculated by Spearman’s rank correlation coefficient. (G) Location of highly networked residues with low effect on monomeric RBD folding (blue) within the RBD monomer (PDB: 6MOJ) and RBD-distal S1 domain (PDB: 6VXX). (H) Percentage of ZsGreen-positive 293T-ACE2 cells after 60-h incubation with WT spike pseudotyped lentiviruses (green) and non-conservative (blue) or conservative (light blue) mutations to highly networked residues with low effect on monomeric RBD folding. Data are means of technical triplicates from an experiment performed twice. (I) Scatterplot of SARS-CoV-2 RBD residue network scores and average effect of mutation on monomeric RBD folding stability. Residues in blue indicate those that previously had high network scores in the full spike protein but low scores in the RBD monomer. (J) Scatterplot of Shannon entropy values of SARS-CoV-2 RBD residues and average effect of mutation on monomeric RBD folding. Correlations were calculated by Spearman’s rank correlation coefficient. For comparisons of more than two groups, Kruskal-Wallis test with Dunn’s post hoc analyses were used. Calculated p values were as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S2 and Table S2.

Figure S4

Shannon entropy values for residues mutated in SARS-CoV-2 spike protein and correlations of spike RBD values with functional mutagenesis data, related to Figure 2 (A) List of matched pairs of networked and non-networked residues in the SARS-CoV-2 Spike proteins targeted for mutagenesis. (B) Comparison of Shannon entropy values between networked residues and non-networked residues in the Sarbecovirus subgenus (SARS-CoV-1/Bat CoV), respectively. (C) Scatterplot of network score of RBD and average effect of mutation on monomeric RBD binding. Statistical comparisons were made using Mann-Whitney U test. (D) Scatterplot of SARS-CoV-1/Bat Shannon entropy values for the RBD and average effect of mutation on monomeric RBD folding. Correlations were calculated by Spearman’s rank correlation coefficient. Calculated P values were as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 3

Stabilization of HLA class I molecules by CD8⁺ T cell epitopes derived from highly networked regions (A) Epitope prioritization pipeline for identification of highly networked CD8⁺ T cell epitopes in SARS-CoV-2. This image was made using BioRender. (B) Representative concentration-based stabilization of surface HLA-A^∗0301 following incubation with no peptide, immunodominant HIV HLA-A^∗0301 epitope RK9 (100 μM), predicted highly networked SARS-CoV-2 epitopes for HLA-A^∗0301 (100 μM), and B^∗08-restricted HIV epitope FL8 (100 μM). (C) Concentration-based HLA class I stabilization of predicted highly networked SARS-CoV-2 CD8⁺ T cell epitopes for HLA-A^∗0301 (0.1-100 μM). The y axis depicts the anti-HLA MFI normalized to the highest value for each HLA class I allele (0-1). HIV HLA-A^∗0301 RK9 epitope is indicated in red. SARS-CoV-2 epitopes with at least 50% relative HLA-A^∗0301 stabilization in comparison to HIV RK9 are indicated in dark blue. The non-HLA-A^∗03-restricted HIV epitope FL8 is depicted in light red. Data are means of technical duplicates from an experiment performed twice. (D) Network-based depiction of A^∗03 RK11 (NSP16; PDB ID: 6W4H, chain A) and A^∗03 KR10 (spike; PDB: 6VXX). (E) Sequence alignments of A^∗03 RK11 and A^∗03 KR10 with the corresponding sequence for SARS-CoV-2, including the emerging variants, bat CoV RaTG13, and all coronaviruses known to infect humans. (F) Fractions of highly networked CD8⁺ T cell epitopes in SARS-CoV-2 with ≤1 amino acid mutation (blue), 2 mutations (green), 3 mutations (red), 4 mutations (orange), and 5 mutations (purple) in SARS-CoV-2 variants, bat CoV RaTG13, and all coronaviruses known to infect humans. (G) Comparison of HLA class I peptide stabilization for SARS-CoV-2 ancestral epitopes and corresponding mutated epitopes in B.1.1.7 alpha (red; A^∗02 VL9) and P.1 gamma (yellow; A^∗01 SY10, A^∗01 NY10, A^∗01 NY11, and B^∗35 SY10) at 100 μM peptide concentration. Statistical comparison was made using Wilcoxon matched-pairs test. (H) Comparison of the fraction of HLA^∗02-restricted highly networked (blue) and non-networked (red) epitope variants (Agerer et al., 2021) that achieve an allelic frequency >0.1 or >0.9. Statistical comparisons of epitope variant frequencies were made using Fisher’s exact test. Calculated p values were as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S3 and Table S3.

Figure S5

Concentration-based HLA class I-peptide stabilization of predicted SARS-CoV-2 CD8⁺ T cell epitopes, related to Figure 3 Concentration-based HLA class I stabilization of 311 predicted SARS-CoV-2 CD8+ T cell epitopes (0.1-100 μM) across 18 TAP-deficient mono-allelic HLA class I-expressing cell lines. The y axis depicts the anti-HLA MFI normalized to the known immunodominant HIV CD8+ T cell epitope (red) for each HLA class I allele. SARS-CoV-2 epitopes with > 50% relative HLA class I stabilization to the HIV immunodominant epitope indicated in dark blue and those with < 50% relative stabilization are indicated in light blue. (B) HLA class I-peptide stabilization of TAP-deficient B^∗0702, B^∗1402, B^∗3901, B^∗8101 and Cw^∗0701 expressing cell lines following incubation with no peptide (gray), HLA-specific HIV immunodominant HIV epitope (10 μM, red) or Spike ML9 (10 μM, blue). (C) Comparison of normalized anti-HLA MFI for B^∗0702, B^∗1402, B^∗3901, B^∗8101 and Cw^∗0701 following incubation with immunodominant HIV epitope or ML9 peptide at range of concentrations (0.1-100 μM). Statistical comparisons were made using Mann-Whitney U test.

Figure 4

CD8⁺ T cells from convalescent COVID-19 individuals recognize highly networked epitopes derived from structural and accessory proteins (A) Location of highly networked HLA-stabilizing CD8⁺ T cell epitopes in non-structural proteins (NSPs; green) and structural proteins (SPs; purple) across the SARS-CoV-2 proteome. (B) Representative IFN-γ ELISpot data for two pairs of healthy donors (HDs) and COVID-19 patients following incubation with DMSO, anti-CD3/CD28 antibodies, the CEF peptide pool, the highly networked NSP peptide pool (n = 56), the highly networked SP peptide pool (n = 53), and the combined NSP + SP peptide pool (n = 109). (C) Magnitude of IFN-γ⁺ CD8⁺ T cell responses to the CEF peptide pool in HDs (open, n = 20) and COVID-19 patients (filled, n = 30). Mild (filled circles, n = 21) and moderate-to-severe COVID-19 patients (filled diamonds, n = 9) are denoted. (D) Magnitude of IFN-γ⁺ CD8⁺ T cell responses to the highly networked SARS-CoV-2 NSP epitope pool (green), the SP epitope pool (purple), and the combined NSP + SP epitope pool (blue) in HDs (open, n = 20) and COVID-19 patients (filled, n = 30). The number of positive responders relative to the total number of individuals analyzed is indicated. (E) Magnitude of IFN-γ⁺ CD8⁺ T cell responses against the highly networked SARS-CoV-2 SP epitope pool (purple) in mild (n = 21) and moderate-to-severe COVID-19 patients (n = 9). (F) Comparison of the magnitude of IFN-γ⁺ CD8⁺ T cell responses to SP and NSP + SP peptide pools in COVID-19 SP peptide pool responders (n = 15). Statistical comparison was made using a Wilcoxon matched-pairs test. All other statistical comparisons were made using a Mann-Whitney U test. Calculated p values were as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Table 1.

Figure 5

Recipients of mRNA-based vaccines have reduced CD8⁺ T cell reactivity to highly networked spike epitopes (A) Representative IFN-γ ELISpot data for PBMCs from two mRNA-based vaccine recipients with and without CD4⁺ T cells following incubation with DMSO, anti-CD3/CD28 antibodies, the CEF peptide pool, the highly networked SP peptide pool (n = 53), the highly networked spike peptide pool (n = 28), and the full overlapping spike peptide pool. (B) Comparison of the magnitude of IFN-γ⁺ T cell responses against full overlapping spike peptide pool before (open circles) and after CD4⁺ T cell depletion (filled circles) for 23 mRNA vaccine recipients. Blue circles represent individuals with prior SARS-CoV-2 infection. The number of positive responders relative to the total number of vaccinated individuals analyzed is depicted above each dataset. (C) Comparison of the magnitude of IFN-γ⁺ T cell responses against the CEF peptide pool before (open circles) and after (filled circles) CD4⁺ T cell depletion. (D) Comparison of the magnitude of IFN-γ⁺ CD8⁺ T cell responses reactive to full overlapping spike pool (gray) and highly networked SARS-CoV-2 spike epitope pool (red, n = 28 peptides). (E) Representative CD8⁺ T cell responses after 6-day incubation of CFSE-loaded PBMCs with DMSO, anti-CD3/CD28 antibodies, the CEF peptide pool, the highly networked spike peptide pool, and the full overlapping spike peptide pool for three mRNA-based vaccine recipients, which include the two individuals shown in (A) and an additional vaccinated individual with prior COVID-19 infection. (F) Comparison of the magnitude of proliferative CD8⁺ T cell responses (%CD8 CFSE low) following incubation with the CEF peptide pool (orange), the full overlapping spike peptide pool (gray), and the highly networked spike peptide pool (red). A positive response was defined as one with %CD8⁺ CFSE low cells at least 1.5× greater than background wells and greater than 0.2% CD8⁺ CFSE low cells in magnitude following background subtraction. Statistical comparisons were made using a Wilcoxon matched-pairs test. Calculated p values were as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Table S4.