Cellular interferon-gamma and interleukin-2 responses to SARS-CoV-2 structural proteins are broader and higher in those vaccinated after SARS-CoV-2 infection compared to vaccinees without prior SARS-CoV-2 infection

Abstract

Class I- and Class II-restricted epitopes have been identified across the SARS-CoV-2 structural proteome. Vaccine-induced and post-infection SARS-CoV-2 T-cell responses are associated with COVID-19 recovery and protection, but the precise role of T-cell responses remains unclear, and how post-infection vaccination ('hybrid immunity') further augments this immunity To accomplish these goals, we studied healthy adult healthcare workers who were (a) uninfected and unvaccinated (n = 12), (b) uninfected and vaccinated with Pfizer-BioNTech BNT162b2 vaccine (2 doses n = 177, one dose n = 1) or Moderna mRNA-1273 vaccine (one dose, n = 1), and (c) previously infected with SARS-CoV-2 and vaccinated (BNT162b2, two doses, n = 6, one dose n = 1; mRNA-1273 two doses, n = 1). Infection status was determined by repeated PCR testing of participants. We used FluoroSpot Interferon-gamma (IFN-γ) and Interleukin-2 (IL-2) assays, using subpools of 15-mer peptides covering the S (10 subpools), N (4 subpools) and M (2 subpools) proteins. Responses were expressed as frequencies (percent positive responders) and magnitudes (spot forming cells/106 cytokine-producing peripheral blood mononuclear cells [PBMCs]). Almost all vaccinated participants with no prior infection exhibited IFN-γ, IL-2 and IFN-γ+IL2 responses to S glycoprotein subpools (89%, 93% and 27%, respectively) mainly directed to the S2 subunit and were more robust than responses to the N or M subpools. However, in previously infected and vaccinated participants IFN-γ, IL-2 and IFN-γ+IL2 responses to S subpools (100%, 100%, 88%) were substantially higher than vaccinated participants with no prior infection and were broader and directed against nine of the 10 S glycoprotein subpools spanning the S1 and S2 subunits, and all the N and M subpools. 50% of uninfected and unvaccinated individuals had IFN-γ but not IL2 or IFN-γ+IL2 responses against one S and one M subpools that were not increased after vaccination of uninfected or SARS-CoV-2-infected participants. Summed IFN-γ, IL-2, and IFN-γ+IL2 responses to S correlated with IgG responses to the S glycoprotein. These studies demonstrated that vaccinations with BNT162b2 or mRNA-1273 results in T cell-specific responses primarily against epitopes in the S2 subunit of the S glycoprotein, and that individuals that are vaccinated after SARS-CoV-2 infection develop broader and greater T cell responses to S1 and S2 subunits as well as the N and M proteins.

Fig 1. S, N, and M peptide subpools and their relationship to each protein structure.

The structures of the S glycoprotein, and N and M proteins and their individual domains are aligned with the peptide subpools for each protein. Since constituent 15mer peptides partially overlap, each subpool also partially overlaps adjoining subpools. Consensus amino acid positions are shown in each protein. Some differences are reported, for example the S heptad repeat is variously reported to end at amino acid 1211 (reference #19), and 1213 (reference #20). Spike S glycoprotein (reference #20): Signal peptide (S); N-terminal domain (NTD); RBD (receptor-binding domain); SD1/SD2 (subdomain 1 and 2); 1 (fusion peptide); 2 (HR1, heptad repeat); 3 (central helix); CD (connector domain); 4 (HR2, heptad repeat); 5 (transmembrane domain); 6 (cytoplasmic tail); the S1/S2 and S2’ cleavage sites (reference #23). S protein subpools predominantly aligned with S domains: Sp1, Sp2 NTD; Sp3 NTD/RBD; Sp4 RBD; Sp5 SD1/SD2; Sp6 SD1/SD2 including S1/S2 cleavage site; Sp7 fusion peptide; Sp8 heptad repeat; Sp9 central helix/connector domain; Sp10 heptad repeat/transmembrane domain/cytoplasmic tail. Nucleocapsid protein N (reference #24): domain N1; domain N2 (N-terminal domain [NTD] or RNA binding domain); domain N3 (contains a serine-arginine rich region); domain N4 (C-terminal domain [CTD]; domain N5. N protein subpools predominantly aligned with N domains: Np1 domains N1 and part of N2; Np2 domains N2 and N3; Np3 domains N3 and N4; Np4 domains N4 and N5. Membrane protein M (reference #26): N (NTD, N-terminal domain); I, II and III (transmembrane domains); C (CTD, C-terminal domain). M protein subpools predominantly aligned with M domains: Mp1 NTD, 1, II and III; Mp2 CTD.

Fig 2. Group 1, Group 2, and Group 3: Median IFN-γ, IL2, and IFN-γ+IL2 responses to S, N, and M subpools.

Medians of the magnitudes of responses of Group 1 (uninfected, unvaccinated), Group 2 (uninfected, vaccinated), and Group 3 (infected, vaccinated) IFN-γ, IL2, and IFN-γ+IL2 responses (spot forming cells/million PBMCs, sfc/m) to S glycoprotein (Sp1 –Sp10), N protein (Np1 –Np4), and M protein (Mp1, Mp2) peptide subpools. *Significant differences between Group 2 and Group 3 indicate that prior SARS-CoV-2 infection significantly increased responses to each subpool as indicated.

Fig 3. Correlations between summed IFN-γ, IL2, and IFN-γ+IL2 responses to S sub-pools and serum IgG responses to Spike glycoprotein.

Summed IFN-γ, IL2, and IFN-γ+IL-2 responses (spot forming cells/million PBMCs, sfc/m) to S glycoprotein correlated with serum IgG responses to Spike glycoprotein. Total n per graph = 198: Uninfected unvaccinated n = 11, Uninfected vaccinated n = 179, Infected vaccinated n = 8. Of note, 8 individuals in the uninfected unvaccinated group and 2 individuals in the uninfected vaccinated group had values of zero for frequencies of cells producing both IFN-γ and IL-2 in response to S peptides. While the zero values were used for statistical analyses, they were plotted as values of 1 to enable visualization on the log axis. Correlations were assessed using the non-parametric Spearman’s correlation analysis.

Fig 4. Hierarchical distribution of IFN-γ, IL2, and IFN-γ+IL2 responses to S, N, and M peptide subpools.

Responses to each stimulant were used, including to all 10 S subpools, all 4 N subpools and each M subpool. In addition, responses to megapools each containing all S, or N, or M peptides combined into one pool, Smp, Nnp, and Mmp respectively, were also used. IFN-γ responses (g), IL2 responses (2), IFN-γ+IL2 responses (g/2). When Components 1 and 2 are compared, responses to Medium only (M4, M4 2 and M4 3) fell outside this correlation. However, S and M peptides fall within distinct aggregate areas defined by purple or blue circles respectively, although responses to N protein (gold circle) overlap the S and M protein areas. S glycoprotein peptide subpool Sp6 falls within each aggregated area (green arrows): IFN-γ (gSp6), IL2 (2Sp6), IFN-γ+IL2 (g/2Sp6).

Fig 5. Scatter plot comparison of all subjects.

Component 1 and Component 2 values appear to differentiate participants based on prior infection and vaccination. Participants who were infected and vaccinated had higher Component 2 values reflecting responses to the N and M proteins; however, there are two outlier subjects (blue arrows). In contrast, uninfected and vaccinated participants had lower Component 2 values reflecting the responses to the S protein. Participants who were uninfected and unvaccinated are distinct and cluster around 0.

Fig 6. Heat map profiles of all IFN-γ, IL2, and IFN-γ+IL2 responses to each S, N, and M peptide subpool.

Group 1 = uninfected, unvaccinated; Group 2: uninfected, vaccinated; Group 3: infected, vaccinated. All IFN-γ (γ), IL2 (2), and IFN-γ+IL2 (γ2) responses of individual subjects with each S, N and M peptide subpool were compared with each other using the Pearson correlation coefficient (r), a value of “1” is shown in bright red and represents 100% positive correlation, and a value of -1 in bright blue represents 100% negative correlation. Intersection of each parameter is boxed. Sp6 and Mp1: γSp6 and γMp1, 2Sp6 and 2Mp1, and γ2Sp6 and γ2Mp1, were mapped on Groups 1, 2, and 3. Sp6 and Mp2: γSp6 and γMp2, 2Sp6 and 2Mp2, and γ2Sp6 and γ2Mp2, were mapped on Group 3.