Whole-blood cytokine secretion assay as a high-throughput alternative for assessing the cell-mediated immunity profile after two doses of an adjuvanted SARS-CoV-2 recombinant protein vaccine candidate

Abstract

Objectives: We previously described the Phase I-II evaluation of SARS-CoV-2 recombinant protein candidate vaccine, CoV2-PreS-dTM, with AF03- or AS03-adjuvant systems (ClinicalTrials.gov, NCT04537208). Here, we further characterise the cellular immunogenicity profile of this vaccine candidate using a whole-blood secretion assay in parallel to intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells (PBMCs).

Methods: A randomly allocated subset of 90 healthy, SARS-CoV-2-seronegative adults aged ≥ 18 years who had received (random allocation) one or two separate injections (on study day [D]1 and D22) of saline placebo or CoV2-PreS-dTM formulated with AS03 or AF03 were included. Cytokine secretion was assessed using a TruCulture^® whole-blood stimulation system in combination with multiplex bead array, and intracellular cytokine profiles were evaluated on thawed PBMCs following ex vivo stimulation with recombinant S protein at pre-vaccination (D1), post-dose 1 (D22) and post-dose 2 (D36).

Results: Both methods detected similar vaccine-induced responses after the first and second doses. We observed a Th1 bias (Th1/Th2 ratio > 1.0) for most treatment groups when analysed in whole blood, mainly characterised by increased IFN-γ, IL-2 and TNF-α secretion. Among participants aged ≥ 50 years, the Th1/Th2 ratio was higher for those who received vaccine candidate with AS03 versus AF03 adjuvant. ICS revealed that this higher Th1/Th2 ratio resulted from higher levels of IFN-γ expression and that the vaccine induced polyfunctional CD4⁺ T cells.

Conclusions: The whole-blood cytokine secretion assay is a high-throughput alternative for assessing the quantity and character of vaccine-induced cellular responses.

Keywords: AF03; AS03; CoV2‐PreS‐dTM; SARS‐CoV‐2 recombinant protein candidate vaccine; adjuvant; cell‐mediated immunity; intracellular cytokine staining; multiplex bead array; whole‐blood cytokine secretion assay.

Figure 1

Vaccine‐induced cytokine concentrations in whole‐blood supernatant after S protein stimulation: whole‐blood cytokine secretion assay. Data show background‐subtracted specific cytokine concentrations in pg mL⁻¹ (log scale) in participants receiving the placebo, the non‐adjuvanted SARS‐CoV2 recombinant protein candidate vaccine (None) or SARS‐CoV2 recombinant protein candidate vaccine adjuvanted with AF03 (red dots) or AS03 (blue dots), by age groups (18–49 or ≥ 50 years) at baseline, 21 days after dose 1 (Post 1st) and 14 days after dose 2 (Post 2nd). For the placebo group, data for all three time points are combined. Box and whisker plots represent median (mid‐line of the box) and 25th and 75th percentiles (bottom and top lines, respectively); the whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range or if no value meets this criterion, to the data extremes. Wilcoxon rank P‐values < 0.05 are shown (not adjusted for multiplicity) (exploratory analyses).

Figure 2

Ratios of Th1 (IFN‐γ) to Th2 (IL‐4, IL‐5, IL‐13) responses after one or two doses of SARS‐CoV2 protein adjuvanted with AF03 or AS03 (whole‐blood cytokine secretion assay or intracellular staining of PBMCs). Whole‐blood cell assay: ratios based on background‐adjusted concentrations (values below LLOQ set to LLOQ/2); Intracellular staining (ICS): ratios based on background‐adjusted percentage of CD4⁺ T cells expressing IFN‐γ to those expressing Th2 cytokines. Data points where the response for IFN‐γ was negative are identified as triangles. Wilcoxon rank P‐values < 0.05 are shown (not adjusted for multiplicity) (exploratory analyses).

Figure 3

Frequencies of CD4⁺ T cells expressing different cytokines after stimulation with recombinant full‐length S protein: ICS assay. Data show background‐subtracted CD4⁺ T‐cell frequencies (percentage of CD4⁺ T cells expressing the indicated cytokine; log scale) for participants receiving the placebo, the non‐adjuvanted SARS‐ CoV2 recombinant protein candidate vaccine (None) or SARS‐CoV2 recombinant protein candidate vaccine adjuvanted with AF03 (red dots) or AS03 (blue dots), by age groups (18–49 or ≥ 50 years) at baseline, 21 days after dose 1 (Post 1st) and 14 days after dose 2 (Post 2nd). For the placebo group, data for all three time points are combined. The y‐axis is truncated at 0.001% and any values below this level are censored. The mid‐line of the box denotes the median, and the ends of the box denote the 25th and 75th percentiles. The whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range or if no value meets this criterion, to the data extremes. The Th2 cytokines include co‐expression of CD40L since this reduces the background. Reagents for IL‐5 and IL‐13 were conjugated to the same fluorochrome and were therefore measured as IL‐5 and/or IL‐13. Wilcoxon rank P‐values < 0.05 are shown (not adjusted for multiplicity).

Figure 4

Correlation between cytokine profiles evaluated in the supernatants of whole blood or intracellular staining of PBMCs after the second vaccination (full‐length S protein stimulation). ICS data (frequencies of CD4⁺ T cells producing each cytokine for the recombinant full‐length S protein stimulation) are expressed as percentage of lymphocytes rather than percentage of CD4⁺ T cells in order to be more comparable to the measurement of concentrations in whole blood. Since IL‐5 and IL‐13 were not analysed separately by ICS, the data for those two cytokines were summed for the whole‐blood cytokine secretion assay. Spearman’s rank correlation coefficients (rho) are shown.

Figure 5

Polyfunctionality of CD4⁺ T‐cell responses measured by ICS after stimulation with recombinant full‐length S protein after one or two doses of SARS‐CoV2 protein adjuvanted with AF03 or AS03. Data show background‐subtracted CD4⁺ T‐cell frequencies (percentage of CD4⁺ T cells expressing the indicated cytokine combination; log scale) for participants receiving the SARS‐CoV2 recombinant protein candidate vaccine adjuvanted with AF03 (red dots) or AS03 (blue dots), by age groups (18–49 or ≥ 50 years) at baseline, 21 days after dose 1 (Post 1st) and 14 days after dose 2 (Post 2nd). The y‐axis is truncated at 0.0001%, and any values below this level are censored. Each functional profile is shown in the key on the x‐axis as determined by gating as positive or negative for each marker (see Supplementary figure 5 for the gating strategy and representative scatter plots). Because the focus of this figure was polyfunctional T cells, data for cells expressing only one cytokine are not shown. Also, to limit the size of the graph, cellular profiles for which the frequency of cells was very low are not included. Cells co‐expressing IL‐4 or IL‐17 were either not detected or only present at low frequency; all profiles shown are negative for those cytokines and they are not included in the key on the x‐axis. The mid‐line of the box denotes the median and the ends of the box denote the 25th and 75th percentiles. The whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range or if no value meets this criterion, to the data extremes. Wilcoxon rank P‐values < 0.05 are shown (not adjusted for multiplicity).