Impact of Previous Common Human Coronavirus Exposure on SARS-CoV-2-Specific T-Cell and Memory B-Cell Response after mRNA-Based Vaccination

Abstract

Objective: T-cell responses against SARS-CoV-2 are observed in unexposed individuals, attributed to previous common human coronavirus (HCoV) infections. We evaluated the evolution of this T-cell cross-reactive response and the specific memory B-cells (MBCs) after the SARS-CoV-2 mRNA-based vaccination and its impact on incident SARS-CoV-2 infections.

Methods: This was a longitudinal study of 149 healthcare workers (HCWs) that included 85 unexposed individuals that were subdivided according to previous T-cell cross-reactivity, who were compared to 64 convalescent HCWs. Changes in specific T-cell response and memory B-cell (MBC) levels were compared at baseline and after two doses of the SARS-CoV-2 mRNA-based vaccine.

Results: A cross-reactive T-cell response was found in 59% of unexposed individuals before vaccination. Antibodies against HKU1 positively correlated with OC43 and 229E antibodies. Spike-specific MBCs was scarce in unexposed HCWs regardless of the presence of baseline T-cell cross-reactivity. After vaccination, 92% and 96% of unexposed HCWs with cross-reactive T-cells had CD4+ and CD8+ T-cell responses to the spike protein, respectively. Similar results to that were found in convalescents (83% and 92%, respectively). Contrarily, higher than that which was observed in unexposed individuals without T-cell cross-reactivity showed lower CD4+ and CD8+ T-cell responses (73% in both cases, p = 0.03). Nevertheless, previous cross-reactive T-cell response was not associated with higher levels of MBCs after vaccination in unexposed HCWs. During a follow-up of 434 days (IQR, 339-495) after vaccination, 49 HCWs (33%) became infected, with a significant positive correlation between spike-specific MBC levels and isotypes IgG+ and IgA+ after vaccination and a longer time to get infected. Interestingly, T-cell cross-reactivity did not reduce the time to vaccine breakthrough infections.

Conclusion: While pre-existing T-cell cross-reactivity enhances the T-cell response after vaccination, it does not increase SARS-CoV-2-specific MBC levels in the absence of previous infection. Overall, the level of specific MBCs determines the time to breakthrough infections, regardless of the presence of T-cell cross-reactivity.

Keywords: SARS-CoV-2; coronavirus; cross-reactive; mRNA vaccine; memory B-cells.

Figure 1

(A) Flow cytometry strategy for the quantification of SARS-CoV-2-specific memory B-cells (MBC). Viable cells (FSC-A/7AAA plot) were plotted with FSC-H/FSC-A parameters to exclude doublets. Single cells were then gated using CD19-APC-Vio770 and CD27-Vio-Bright-FITC to identify memory B-cells. Spike-specific B-cells were then identified with a double staining with the two spike-tetramer on the diagonal of the dot plot. Finally, the use of IgG-VioBlue, IgA-VioGreen, and IgM-APC were used to quantify each specific isotype of spike-specific memory B-cells. The results are recorded as percentage among the total memory B-cells and isotypes among specific memory B-cells. (B) Flow cytometry gating strategy for the quantification of SARS-CoV-2 spike-specific T-cells. After stimulation with the spike peptide, staining of the cells was carried out with the following fluorochrome-conjugated antibodies: CD3-VioBlue, CD4-APC, CD8-FITC, CD14-PerCP, CD20-PerCP, IFN-γ-PE, and FcR blocking reagent. To exclude dead cells, viability 405/520 fixable dye staining was added for the last 10 min of incubation. The samples were measured and analyzed by flow cytometry on a MACSQuant Analyzer 10 using MACSQuantify software. At least 10⁵ cells were analyzed and gated with the following strategy: Single (FSC-A/FSC-H dot plot) and live cells were first selected. Cell debris, monocytes, and B-cells were excluded from the analysis with CD14-and CD20-PerCP antibodies. Then, lymphocytes were selected with an FSC-A/SSC-A dot plot, and CD3 T-cells were gated. IFN-γ expression was finally analyzed separately for CD4+ and CD8+ T-cells. A representative sample of negative control (without stimulation), positive control (stimulated with SEB), and with SARS-CoV-2 spike peptide are shown.

Figure 2

Baseline correlations between antibodies against common human coronavirus (HCoVs), SARS-CoV-2 spike-specific (IgG, IgA, and IgM) antibodies, neutralizing antibodies (NAB), specific memory B-cells (MBCs) and IgG+, IgA+, and IgM+ MBCs isotypes, CD4+ and CD8+ T-cell responses to SARS-CoV-2 spike (S), membrane (M), and nucleocapsid (N) proteins. (A) Spearman test heatmap analysis of the different variables in unexposed subjects with cross-reactivity. (B) Spearman test heatmap of the different variables in convalescent individuals. The grey color represents a positive correlation and the red color a negative correlation. The intensity of the color indicates the R² coefficient. Statistically significant when p < 0.05. * = p < 0.05, and ** = p < 0.01.

Figure 3

Comparison of specific memory B-cells (MBCs, top left) and IgG+ MBC (top right), IgM+ MBCs (bottom left), and IgA+ MBCs (bottom right) isotypes between the different subgroups at baseline (BL) and after two doses of vaccine (PV, post-vaccination). No CR (unexposed without cross-reactivity, green dots and bars), CR (unexposed with cross-reactivity, rose dots and bars), and Conv (convalescent, red dots and bars). Frequencies are indicated at the X axis. Statistically significant when p < 0.05. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Figure 4

Comparison of the magnitude of CD4+ (left) and CD8+ (right) T-cell responses against SARS-CoV-2 S, M, and N proteins between the different subgroups at baseline (BL) and after two doses of vaccine (PV, post-vaccination). No CR (unexposed without cross-reactivity, green dots and bars), CR (unexposed with cross-reactivity, rose dots and bars), and Conv (convalescent, red dots and bars). Frequencies are indicated at the X axis. Statistically significant when p < 0.05. * = p < 0.05, and *** = p < 0.001.

Figure 5

Post-vaccination correlations between antibodies against common human coronavirus (HCoVs), SARS-CoV-2 spike-specific (IgG, IgA, and IgM) antibodies, neutralizing antibodies (NAB), specific memory B-cells (MBCs) and IgG+, IgA+, and IgM+ MBCs isotypes, CD4+ and CD8+ T-cell responses to SARS-CoV-2 spike (S), membrane (M), and nucleocapsid (N) proteins. (A), Spearman test heatmap analysis of the different variables in unexposed subjects with cross-reactivity. (B), Spearman test heatmap of the different variables in convalescent individuals. The grey color represents a positive correlation and the red color a negative correlation. The intensity of the color indicates the R² coefficient. Statistically significant when p < 0.05. * = p < 0.05, and ** = p < 0.01.