Cell-mediated immune responses to different formulations of a live-attenuated tetravalent dengue vaccine candidate in subjects living in dengue endemic and non-endemic regions

Abstract

Three phase II randomized trials evaluated the safety/immunogenicity of two formulations of live-attenuated tetravalent dengue virus (TDEN) vaccine in dengue-endemic (Puerto Rico, Thailand) and non-endemic (US) regions (NCT00350337/NCT00370682/NCT00468858). We describe cell-mediated immune (CMI) responses; safety and humoral responses were reported previously. Participants received two doses of vaccine or control (placebo or the precursor live-attenuated TDEN vaccine) 6 months apart. Selected US participants received a booster 5-12 months post-dose 2. Evaluated subsets of the per-protocol cohorts included 75 primarily dengue virus (DENV)-unprimed US adults, 69 primarily flavivirus-primed Thai adults, and 100 DENV-primed or DENV-unprimed Puerto Rican adults/adolescents/children. T-cell responses were quantified using intracellular cytokine staining (ICS; DENV-infected cell-lysate or DENV-1/DENV-2 peptide-pool stimulation) or IFN-γ ELISPOT (DENV-2 peptide-pool stimulation). Memory B-cell responses were quantified using B-cell ELISPOT. Across populations and age strata, DENV serotype-specific CD4⁺ T-cell responses were slightly to moderately increased (medians ≤0.18% [ICS]), DENV-2-biased, and variable for both formulations. Responses in unprimed subjects were primarily detected post-dose 1. Response magnitudes in primed subjects were similar between doses. Multifunctional CD8⁺ T-cell responses were detected after peptide-pool stimulation. T-cell responses were mostly directed to DENV nonstructural proteins 3 and 5. Memory B-cell responses were tetravalent, of low-to-moderate magnitudes (medians ≤0.25%), and mainly observed post-dose 2 in unprimed subjects and post-dose 1 in primed subjects. A third dose did not boost CMI responses. In conclusion, both formulations of the live-attenuated TDEN vaccine candidate were poorly to moderately immunogenic with respect to B-cell and T-cell responses, irrespective of the priming status of the participants. Abbreviation ATP: according-to-protocol; ICS: Intracellular Cytokine Staining; NS3: Nonstructural protein 3; ELISPOT: Enzyme-Linked ImmunoSpot; JEV: Japanese encephalitis virus; PBMC: peripheral blood mononuclear cells.

Keywords: Cellular-mediated immune responses; Dengue-primed and -unprimed populations; Live-attenuated tetravalent dengue candidate vaccine.

Figure 1.

DENV-specific CD4⁺ T-cell responses in Puerto Rican children. DENV-specific CD4⁺ T-cell responses in Puerto Rican children aged 1–12 years after immunization with the F17 or F19 vaccines are presented for the overall sample subset (a), and stratified by their DENV priming status at baseline (b). Blood samples were obtained prior to each vaccination (Day [D] 0 and month [M] 6), 3 months after the first dose (M3) and 1 month after the second dose (M7). Data are represented in box-and-whiskers plots as percentages of DENV-specific CD4⁺ T cells expressing (after in vitro stimulation) at least IL-2, IFN-γ or TNF-α among all CD4⁺ T cells, with medians, first and third quartiles, and minimum/maximum values presented.

Figure 1.

(Continued).

Figure 2.

DENV-specific CD4⁺ T-cell responses in adults and/or adolescents in the US, Thailand and Puerto Rico. DENV-specific CD4⁺ T-cell responses after immunization with the F17 or F19 vaccines are presented for the overall sample subset of adults in the US and Thailand, and adolescents and adults in Puerto Rico (a), and for US subjects stratified by their DENV priming status at baseline, with numbers of evaluated subjects as indicated (b). Blood samples were obtained prior to each dose (Day [D] 0 and month [M] 6), 1 and 3 months after both the first dose (M1 and M3) and the second dose (M7 and M9). Data are presented in box-and-whiskers plots as percentages of DENV-specific CD4⁺ T cells expressing (after in vitro stimulation) at least two immune markers among IFN-γ, IL-2, TNF-α and CD40L of all CD4⁺ T cells, with medians, first and third quartiles, and minimum/maximum values presented.

Figure 2.

(Continued).

Figure 3.

DENV-specific T-cell responses in US adults (ICS with peptide pool stimulation). (a) Representative flow plots of IFN-γ vs TNF-α production by CD4⁺ T cells or CD8⁺ T cells (left-hand and right-hand panels, respectively) after stimulation of peripheral blood mononuclear cells with negative control or the DENV-2 nonstructural (NS) 3 peptide pool are shown. Cells co-expressing other functions (CD107a, IL-2, CD40L or MIP-1β) are highlighted in color. Data are from one representative subject at 1 month after the second dose (M7). (b) Magnitudes of responses of CD4⁺ T cells and CD8⁺ T cells (left-hand and right-hand panels, respectively) producing at least IFN-γ after stimulation with DENV-1 envelope (E), DENV-2 E, DENV-1 NS3, or DENV-2 NS3 peptide pools are presented for 13 vaccinated subjects. PBMC were obtained pre-vaccination (D0) and 1 month after the second dose (M7). Data are background (negative control value)-subtracted. For the subjects with a D0 value available, a response was considered positive if it was ≥0.05%, and the background-subtracted value at M7 was at least 3 times higher than the subject-matched value at day 0.

Figure 4.

Specificity of T-cell responses in DENV-unprimed subjects. The magnitudes and specificity of background (medium)-subtracted T-cell responses to different DENV antigens measured at 1 month after the second dose (Month 7) by IFN-γ ELISPOT are presented. Samples were collected from a subset of US subjects (with subject numbers indicated) who were DENV-unprimed at baseline. Each bar represents the response of one subject. A response was considered positive if it was ≥55 spot-forming units (SFU) per 10⁶ PBMC, and exceeded response to the negative control by four-fold. (a) Magnitudes of T-cell responses to individual DENV-2 antigens measured after stimulation with peptide pools covering DENV-2 nonstructural (NS) 1, NS2, NS3, NS4, NS5, capsid (C), premembrane (PrM) and envelope (E) proteins are presented. (b) Relative proportions of the T-cell response to DENV-1, DENV-2, DENV-3 and DENV-4 NS3 peptide pools were determined for the subjects with DENV-2 NS3 specific response in the analysis shown in (a).

Figure 5.

DENV-specific B-cell responses to tetravalent live-attenuated DENV vaccines in different study populations. (a) Frequencies of DENV-specific B-cells for the overall sample subsets of study populations in the US and Thailand (adults) and Puerto Rico (adults and adolescents) after immunization with F17 or F19 vaccine are presented. Blood samples were obtained prior to each dose (Day [D] 0 and month [M] 6), at 1 and 3 months after both the first dose (M1 and M3) and the second dose (M7 and M9). Box-and-whiskers plots represent the percentages of responding B cells with medians, first and third quartiles, and the minimum/maximum values measured. (b) B-cell responses in the Puerto Rican subjects after subtraction of the pre-vaccination responses by subject are presented. (c) B-cell responses in the US subjects stratified by their DENV priming status at baseline are presented, with numbers of evaluated subjects indicated (numbers may vary by time-point and serotype).

Figure 5.

(Continued).

Figure 6.

Correlations between memory B-cell responses and neutralizing antibody responses in Thai adults. Memory B-cell responses and DENV-specific neutralizing antibody responses observed in Thai adults are presented at pre-vaccination (pre), by the subjects’ baseline Japanese encephalitis virus (JEV) priming status (a), and at pre-vaccination, 1 month after the first (P1(M1)) and the second (PII(M7)) dose, by DENV serotype (b).

Figure 7.

Focus on the patient section.