NVX-CoV2373 vaccination induces functional SARS-CoV-2-specific CD4+ and CD8+ T cell responses

Abstract

NVX-CoV2373 is an adjuvanted recombinant full-length SARS-CoV-2 spike trimer protein vaccine demonstrated to be protective against COVID-19 in efficacy trials. Here we demonstrate that vaccinated individuals made CD4+ T cell responses after 1 and 2 doses of NVX-CoV2373, and a subset of individuals made CD8+ T cell responses. Characterization of the vaccine-elicited CD8+ T cells demonstrated IFN-γ production. Characterization of the vaccine-elicited CD4+ T cells revealed both circulating T follicular helper (cTfh) cells and Th1 cells (IFN-γ+, TNF-α+, and IL-2+) were detectable within 7 days of the primary immunization. Spike-specific CD4+ T cells were correlated with the magnitude of the later SARS-CoV-2-neutralizing antibody titers, indicating that robust generation of CD4+ T cells, capable of supporting humoral immune responses, may be a key characteristic of NVX-CoV2373 that utilizes Matrix-M adjuvant.

Keywords: Adaptive immunity; Cellular immune response; Immunology; T cells.

Figure 1. Spike-specific CD4⁺ T cells following NVX-CoV2373 vaccination.

(A) Representative FACS plots of AIM⁺ (sCD40L⁺OX40⁺) CD4⁺ T cells on day 0 (D0), D7, and D28 after vaccination. (B and C) Spike-specific AIM⁺ CD4⁺ T cell responses in vaccinees (black dots) and placebo controls (gray dots). The same data are graphed as grouped (B) and paired comparisons (C). (D) Representative FACS plots of AIM⁺CXCR5⁺ CD4⁺ cTfh cells (blue dots are AIM⁺ CD4⁺ T cells overlaid on total CD4⁺ T cells in black). (E and F) Spike-specific AIM⁺ cTfh cell responses in vaccinees and placebo controls. The same data are graphed as grouped (E) and paired comparisons (F). Dotted line indicates limit of quantitation (LOQ) for the assay, and was calculated as the geometric mean of all sample DMSO wells multiplied by the geometric SD. Percentage responders are calculated as responses ≥ LOQ divided by the total samples in the group. Paired data were analyzed by Wilcoxon’s signed-rank test. Data shown as geometric mean ± geometric SD. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. Cytokine-producing spike-specific CD4⁺ T cell responses following NVX-CoV2373 vaccination.

Proportion of (A) IFN-γ⁺, (B) TNF-α⁺, and (C) IL-2⁺ spike-specific CD4⁺ T cells detected following peptide stimulation. (D) Representative FACS plots and (E) proportion of IFN-γ⁺ intracellular CD40L⁺ (iCD40L⁺) responses in spike-specific CD4⁺ T cells on days 0, 7, and 28 after vaccination. (F) Proportion of spike-specific CD4⁺ T cells expressing iCD40L and producing IFN-γ, TNF-α, IL-2, or GzmB (“secreted-effector⁺”). Predominant multifunctional profiles of spike-specific CD4⁺ T cells with 1, 2, 3, 4, or 5 functions were analyzed on (G) day 0 (D0), (H) D7, and (I) D28 after vaccination. (J) Pie charts depicting the proportion of spike-specific CD4⁺ T cells exhibiting 2, 3, 4, or 5 functions on day 7 and day 28 after immunization. Functionality is defined as a cell expressing iCD40L and any combination of IFN-γ, TNF-α, IL-2, or GzmB. Dotted line indicates LOQ for the assay, and was calculated as the geometric mean of all sample DMSO wells multiplied by the geometric SD. Percentage responders was calculated as responses ≥ LOQ divided by the total samples in the group. Paired data were analyzed by Wilcoxon’s signed-rank test. Data shown as geometric mean ± geometric SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. Spike-specific CD8⁺ T cells are induced following NVX-CoV2373 vaccination.

(A) Representative FACS plots of AIM⁺ (CD69⁺4-1BB⁺) CD8⁺ T cells on day 0 (D0), D7, and D28 after vaccination. (B and C) Spike-specific AIM⁺ CD8⁺ T cells responses in vaccinees (black dots), placebo controls (gray dots), and convalescent COVID-19 donors (blue dots). (D) Representative FACS plots of IFN-γ responses in total spike-specific CD8⁺ T cells. IFN-γ⁺ (E), TNF-α⁺ (F), and IL-2⁺ (G) spike-specific CD8⁺ T cell responses in vaccinees, placebo controls, and convalescent COVID-19 controls. (H) Secreted-effector⁺ spike-specific CD8⁺ T cells (sum of CD8⁺ T cells expressing any combination of IFN-γ, TNF-α, IL-2, or GzmB, excluding GzmB single positives). (I) Pie charts depicting the proportion of spike-specific CD8⁺ T cells exhibiting 1, 2, 3, or 4 functions on day 7 and day 28 after immunization. Functionality was defined as a cell expressing any combination of IFN-γ, TNF-α, IL-2, or GzmB, excluding GzmB single positives. Dotted line indicates limit of sensitivity for the assay, and was calculated as the geometric mean of all sample DMSO wells multiplied by the geometric SD factor. Percentage responders was calculated as responses ≥ LOQ divided by the total samples in the group. Paired data were analyzed by Wilcoxon’s signed-rank test. Data shown as geometric mean ± geometric SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Association between T cell and antibody responses following NVX-CoV2373 vaccination.

Correlation between day 7 SARS-CoV-2 spike–specific AIM⁺ CD4⁺ T cells and (A) day 21 microneutralization titers or (B) D28 SARS-CoV-2 spike–specific AIM⁺ CD4⁺ T cells and day 35 neutralization titers. Correlation between day 28 SARS-CoV-2 spike–specific AIM⁺ CD4⁺ T cells and (C) day 35 hACE2 binding inhibition or (D) day 35 spike IgG. Correlation between spike-specific AIM⁺ CD4⁺ cells and AIM⁺ CD8⁺ cells on (E) day 7 and (F) day 28. Data were analyzed by Spearman’s correlation.

Figure 5. Preexisting T cell immunity does not impact NVX-CoV2373 vaccine responses.

(A) Spike-specific AIM⁺ CD4⁺ T cells at baseline and after vaccination grouped according to donors with detectable AIM⁺ CD4⁺ T cell responses on day 0 (blue dots) or no detectable AIM⁺ CD4⁺ T cell responses on day 0 (black dots). Effect of preexisting AIM⁺ CD4⁺ T cells on (B) spike-specific AIM⁺ cTfh cells, (C) spike-specific AIM⁺ CD8⁺ T cells, (D) total spike-specific CD4⁺ T cells and (E) spike-specific CD8⁺ T cell cytokine production. Dotted line indicates limit of sensitivity for the assay, and was calculated as the geometric mean of all sample DMSO wells multiplied by the geometric SD. Data were analyzed by Mann-Whitney test. Data shown as geometric mean ± geometric SD. *P < 0.05; ***P < 0.001; ****P < 0.0001.