Persistence of Th1/Tc1 responses one year after tetravalent dengue vaccination in adults and adolescents in Singapore

Abstract

To characterize the cell mediated immunity (CMI) induced by the investigational CYD tetravalent dengue vaccine (TDV), we developed a whole-blood, intracellular cytokine staining (ICS) assay and a multiplex assay, each requiring 3 mL of blood. We assessed CMI before and 28 d after a first and third injection of CYD-TDV and one year after the third injection in a subset of 80 adolescents and adults enrolled in a phase II trial in Singapore (ClinicalTrial.gov NCT NCT00880893). CD4/IFNγ/TNFα responses specific to dengue NS3 were detected before vaccination. Vaccination induced YF-17D-NS3-specific CD8/IFNγ responses, without significant TNFα, and a CYD-specific Th1/Tc1 cellular response in all participants, which was characterized by predominant IFNγ secretion compared with TNFα, associated with low level IL-13 secretion in multiplex analysis of peripheral blood mononuclear cells (PBMC) supernatants after restimulation with each the CYD vaccine viruses. Responses were directed mainly against CYD-4 after the first vaccination, and were more balanced against all four serotypes after the third vaccination. The same qualitative profile was observed one year after the third vaccination, with approximately 2-fold lower NS3-specific responses, and 3-fold lower serotype-specific cellular responses. These findings confirm previous observations regarding both the nature and specificity of cellular responses induced by CYD-TDV, and for the first time demonstrate the persistence of cellular responses after one year. We also established the feasibility of analyzing CMI with small blood samples, allowing such analysis to be considered for pediatric trials.

Keywords: cell mediated immunity; cytokines; intracellular cytokine staining; tetravalent dengue vaccine; whole blood.

Figures

Figure 1. DEN NS3-specific CD4 responses pre-exist in adults, and DENV NS3-specific CD8 responses can be recalled by vaccination Dengue virus NS3-specific CD4+ (left) and CD8+ (right) T cell responses in CYD-TDV vaccinees (top) and placebo controls (bottom). Geometric mean (GM) interferon-gamma (IFNγ) and tumor-necrosis-factor-α (TNFα) production in CD4 (CD3+CD8-) (left) and CD8 T cells (right) following ex vivo stimulation of whole blood with DENV NS3 peptides in dengue vaccine group (top panel) and placebo group (bottom) before and 28 d after the first, before and 28 d after the third vaccination, and 1 y after the last vaccination as measured by ICS. For each subject, first the background cytokine levels in the negative control (medium+DMSOs) were subtracted from DENV NS3 peptide pool A and B stimulated cytokine levels. Second, the responses from pool A and pool B were combined by taking the higher percentage of the two pools for each subject. Responses shown are the geometric mean (GM) cytokine percentages of these combined results. (A) Percentages of IFNγ+ or TNFα+ CD4 cells in adult and adolescent vaccinees (B). Percentages of IFNγ+ or TNFα+ CD4 cells in adult and adolescent placebo recipients (C) Percentages of IFNγ+ and TNFα+ CD8 cells in adult and adolescent vaccinees. (D) Percentages of IFNγ+ and TNFα+ CD8 cells in adult and adolescent placebo recipients

Figure 2. Tetravalent CYD vaccination induces mostly CD8+ YF-17D NS3-specific responses YF-17D NS3-specific CD4+ (left) and CD8+ (right) T cell responses in CYD-TDV vaccinees (top) and placebo controls (bottom). Geometric mean (GM) interferon-gamma (IFNγ) and tumor-necrosis-factor-α (TNFα) production in CD4 (CD3+CD8-) (left) and CD8 T cells (right) following ex vivo stimulation of whole blood with YF-17D NS3 peptides in dengue vaccine group (top) and placebo group (bottom panel) before and 28 d after the first, before and 28 d after the third vaccination, and 1 y after the last vaccination (V07) as measured by ICS. For each subject, first the background cytokine levels in the negative control (medium+DMSOs) were subtracted from YF-17D NS3 peptide pool A and B stimulated cytokine levels. Second, the responses from pool A and pool B were combined by taking the higher percentage of the two pools for each subject. Responses shown are the geometric mean cytokine percentages of these combined results.(A) Percentages of IFNγ+ or TNFα+ CD4 cells in adult and adolescent vaccinees (B). Percentages of IFNγ+ or TNFα+ CD4 cells in adult and adolescent placebo recipients (C) Percentages of IFNγ+ and TNFα+ CD8 cells in adult and adolescent vaccinees. (D) Percentages of IFNγ+ and TNFα+ CD8 cells in adult and adolescent placebo recipients

Figure 3. Tetravalent CYD vaccination induces serotype-specific responses dominated by IFNγ. IFNγ was produced following ex vivo stimulation of PBMCs with CYD1–4 monovalent vaccine with a predominant response against serotype 4 after primary CYD-TDV vaccination and a different balance after booster vaccination. Geometric mean (GM) interferon-gamma (IFNγ) production following ex vivo stimulation of freshly purified PBMCs with CYD-1, CYD-2, CYD-3, and CYD-4 monovalent vaccine in dengue vaccine group (top) and placebo group (bottom) in adults (left) and adolescents (right) before and 28 d after the first, before and 28 d after the third vaccination and 1 y after the last vaccination as measured by Luminex. Responses shown are geometric mean IFNγ levels in pg/mL in the CYD-TDV group and placebo group in adults and adolescents from CYD1–4 stimulated PBMCs with the background IFNγ level in the negative control (CY110 stimulated PBMCs) subtracted. (A) IFNγ secretion in adult vaccinees (B) IFNγ secretion in adult placebo recipients (C) IFNγ secretion in adolescent vaccines (D) IFNγ secretion in adolescent placebo recipients

Figure 4. Tetravalent CYD vaccination induces serotype-specific TNFα and IL-13 responses lower than for IFNγ, but with a good pattern correlation in both age groups. Geometric mean TNFα and IL-13 production following ex vivo stimulation of freshly purified PBMCs with CYD-1, CYD-2, CYD-3, and CYD-4 monovalent vaccine in adults (top) and adolescents (bottom) before and 28 d after the first, before and 28 d after the third vaccination, and 1 y after the last vaccination as measured by Luminex. Responses shown are geometric mean TNFα (left) and IL-13 levels (right) in pg/mL in the dengue vaccine (CYD-TDV) group in adults and adolescents from CYD1–4 stimulated PBMCs with the background cytokine level in the negative control (CY110 stimulated PBMCs) subtracted. (A) TNFα secretion in adult vaccinees (B) TNFα secretion in adolescent vaccinees (C) IL-13 secretion in adult vaccinees (D) IL-13 secretion in adolescent vaccines.