Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T-cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2

Abstract

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but the T-cell response to seasonal coronaviruses remains largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal coronavirus OC43. We identified MHC-bound peptides derived from each of the viral structural proteins (spike, nucleoprotein, hemagglutinin-esterase, membrane, and envelope) as well as non-structural proteins nsp3, nsp5, nsp6, and nsp12. Eighty MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. Fewer and less abundant MHC-I bound OC43-derived peptides were observed, possibly due to MHC-I downregulation induced by OC43 infection. The MHC-II peptides elicited low-abundance recall T-cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T-cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T-cell lines. Among the validated epitopes, spike protein S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. Nucleoprotein N54-68 and hemagglutinin-esterase HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow CD4 T-cell cross-reactivity after infection or vaccination, and to guide selection of epitopes for inclusion in pan-coronavirus vaccines.

Fig 1. Immunopeptidome workflow and HLA-ABC, HLA-DR, and HLA-DP immunopeptidomes in OC43-infected HEK293 cells.

A. Experimental approach: HEK293 cells transduced with CIITA were infected with OC43. After 3 days, cells were collected and pMHC complexes were purified by immunoaffinity. Peptides were eluted from pMHC and analyzed by LC-MS/MS for identification. Identified peptides were used in biochemical and immunological assays. B. MHC expression on the surface of HEK293 cells. Four panels corresponding to the surface expression of HLA-ABC, HLA-DR, HLA-DQ, and HLA-DP are shown. HLA levels on wild-type cells are shown by grey histograms. HLA levels after transduction with CIITA are shown by colored histograms: HLA-ABC (blue), HLA-DR (purple), HLA-DQ (green), and HLA-DP (yellow). Isotype control staining is shown as an open histogram with dotted lines, following the same color scheme. C. Relative protein quantitation of HLA-DR, HLA-DP, and HLA-DQ proteins in CIITA-transfected HEK293 uninfected cells measured by label-free quantitative proteomics. D. Representative dot plots of intracellular staining for OC43 nucleoprotein in uninfected cells (top) and at 3 days after infection (middle); summary of 6 experiments (bottom). E. Representative histograms showing the comparison of surface levels of HLA-ABC, HLA-DR, and HLA-DP on uninfected (dark-shaded histograms) and infected (light-shaded histograms) cells. Graphs show the MFI in uninfected (non) and infected (oc43) cells from 3–6 independent infections. Statistical analysis in D and E by paired t-test, * p<0.05, ** p<0.01, ns: not significant. F. Length distribution of HLA-ABC, HLA-DR, and HLA-DP eluted immunopeptidomes from uninfected (grey histograms) and OC43-infected cells (color histograms). G. Sequence logos of clusters obtained using the Gibbs clustering analysis of HLA-ABC, HLA-DR, and HLA-DP eluted immunopeptidomes from OC43-infected cells; percentage of peptides in each cluster and probable allele are shown.

Fig 2. OC43 peptides in the HEK293-derived HLA-ABC, HLA-DR, and HLA-DP immunopeptidomes.

A. Length distribution of virus-derived peptides within the HLA-ABC, HLA-DR, and HLA-DP immunopeptidomes of OC43-infected cells. B. Ranking of all HLA-ABC, HLA-DR, and HLA-DP eluted peptides according to their precursor ion intensity; viral peptides are shown by colored circles. Sequences are shown for the top five most abundant viral peptides. For each peptide, the two most abundant species in the nested set are indicated. C. HLA-ABC eluted viral peptides. A schematic representation of each source protein and the location of the eluted sequence is shown (first and last residues indicated). D. HLA-DR and HLA-DP eluted viral peptides. A schematic representation as in C; the predicted core epitope in each sequence is underlined. Nested sets of eluted peptides comprising length variants with the same core epitope are shown by lines below the sequence. The peptide sequences highlighted in red were used for biochemical and immunological assays (see Table 1). In C and D, each eluted sequence or nested set was identified by a “P” followed by a number. E. Label-free quantitation of proteins present in infected cells; proteins were ranked from most to least abundant, with viral proteins highlighted in color. F. Relationship between viral protein abundance and eluted peptides abundance. For each source protein, the sum of intensities of all eluted peptides derived from it was used to calculate the eluted peptides abundance.

Fig 3. T-cell recognition of eluted HLA-DR and HLA-DP viral peptides.

A. Ex vivo T-cell responses to OC43 eluted peptides (pooled by HLA allele) in pre-pandemic PBMC samples from donors with a partial HLA match to HEK293 cells. The plot shows IFN-γ production measured by ELISpot (SFU/10⁶ cells); pie graphs show the percentage of donors responding to the pool. B-C. T cells from partially HLA-matched pre-pandemic donors were expanded in vitro by stimulation with each of the eluted peptides. IFN-γ responses by expanded T-cell populations to the same peptide presented by a single allele antigen-presenting cells (APC) are shown in (B) for the HLA-DP peptides presented by DPA1*03:01/DPB1*04:01 (DP4.1) and in (C) for the DR peptides presented by DRB1*15:01 (DR2b) or DRB5*0101 (DR2a); pie graphs show the percentage of donors responding to the peptide. In each panel, donors are represented by a different color. D. Summary of responses of single-peptide in vitro expanded T cells to the eluted peptides, grouped by allele. E-F. Lowest peptide dose (10–10⁻⁷ μg/mL) able to elicit a positive response to each eluted peptide, in experiments where the single-peptide in vitro expanded T cells were tested for IFN-γ response to different doses of HLA-DP (E) or HLA-DR (F) eluted peptides presented by single allele APC (as in B-C). Each symbol represents a different donor. G. Response of single-peptide in-vitro expanded T cells to peptide stimulation followed in IFN-γ intracellular cytokine secretion (ICS) assay. Dot plots show CD4 expression (x-axis) and IFN-γ production (y-axis). DMSO was used as a negative control. Responses >3-fold background (red boxes) were considered positive. The gating strategy and other controls are presented in S5A and S5B Fig. H-I. Summary of IFN-γ producing cell percentages in ICS assays for multiple donors for HLA-DP (H) and HLA-DR (I) peptides; only positive responses are shown. In A-C, statistical analysis to determine positive ELISpot responses was done by distribution-free resampling (DFR) method [98]; the size of the filled symbols indicates positive responses by DFR2x or DFR1x, while negative responses are shown as empty symbols. In A and D, statistical analysis was done by unpaired t-test (ns: not significant).

Fig 4. Epitope-specific T-cell cross-reactivity between OC43 and other human coronaviruses.

A. Screening of cross-reactive T-cell responses to eluted peptides in partially HLA-matched pre-pandemic donors. T-cell lines were expanded in vitro by stimulation with the eluted OC43 peptides, and IFN-γ responses (SFU/10⁶ cells) to OC43 (green) or SARS-CoV-2 (blue) peptides presented by single allele APC were measured. Pies show the fraction of responding donors to each peptide. B. For the two cross-reactive peptides (P4, P11), the screening was extended to more donors. C. Responses ex vivo and after in vitro expansion of T cells with P4 or P11 peptides from OC43 (green lines), or after heterologous expansion with P4 or P11 peptides from SARS-CoV-2 (blue lines), in pre-pandemic donors; responses to OC43 peptides are shown as green symbols and responses to SARS-CoV-2 peptides are shown as blue symbols. D. Dose-response assay for P4 and P11 in pre-pandemic donors. T cells were expanded in vitro with the OC43 peptide (OC43-expanded, top row) or SARS-CoV-2 peptide (CoV2-expanded, bottom row) and IFN-γ responses of each expanded line to the OC43 peptide (green) or SARS-CoV-2 peptide (blue) were tested using single allele APC as before. E. Same as D but for COVID-19 convalescent donors. F. Lowest observed dose for a positive response for the cross-reactive peptides (tested in panels D and E). Pre-pandemic donors are shown as circles and COVID-19 donors as triangles. G. Experimental binding of OC43 peptides (green) and the SARS-CoV-2 homologs (blue) to the relevant alleles. Half-maximal inhibitory concentration (IC₅₀) values are shown. H. Sequence alignment of OC43 peptides and their SARS-CoV-2 homologs. OC43 sequences shown on top, with predicted core epitope shown in magenta and flanking regions in green; SARS-CoV-2 sequences on bottom, with residues different from OC43 shown and dots indicating identical residues. Predicted SARS-CoV-2 core epitope highlighted in turquoise with flanking regions shown in blue. Positions within the 9-mer core epitope are indicated by numbers shown below the sequences; major T-cell contacts are enclosed in circles. Arrowheads indicated gaps in the aligned sequences. If OC43 and SARS-CoV-2 epitopes are different both are shown. Grey bars show positions of identical residues at T-cell contacts positions. I. Experimental binding of P4 and P11 OC43 peptides and their homologs in other coronaviruses to the relevant alleles. J. IFN-γ responses of T-cell lines expanded in vitro with OC43 peptides (OC43 expanded, top row) or with SARS-CoV-2 peptides (CoV2 expanded, bottom row), to P4 and P11 peptides from OC43, SARS-CoV-2, and the other seasonal coronaviruses, presented by relevant single allele APC. In A-E and J, ELISpot statistical analysis by DFR method [98]; positive responses shown as filled symbols and negative responses as empty symbols. In B and E, statistical analysis was done by unpaired t-test. * p<0.05).