Vaccination With a Single Consensus Envelope Protein Ectodomain Sequence Administered in a Heterologous Regimen Induces Tetravalent Immune Responses and Protection Against Dengue Viruses in Mice

Abstract

The development of a safe and effective tetravalent dengue vaccine that elicits protection against all dengue virus (DENV) serotypes is urgently needed. The consensus sequence of the ectodomain of envelope (E) protein of DENV (cE80) has been examined as an immunogen previously. In the current study, a cE80 DNA (D) vaccine was constructed and evaluated in conjunction with the cE80 protein (P) vaccine to examine whether both vaccines used together can further improve the immune responses. The cE80 DNA vaccine was administrated using either a homologous (DNA alone, DDD) or heterologous (DNA prime-protein boost: DDP or DPP) regimen, and evaluated for immunogenicity and protective efficacy in mice. Among the three DNA-based immunization regimens tested, DDP immunization is the optimal immunization regimen that elicited the greatest systemic immune response and conferred protection against all four DENV serotypes. This work provides innovative ideas for the development of consensus E-based dengue vaccines and the testing of optimal immunization regimens.

Keywords: E80; consensus sequence; dengue virus; envelope; prime-boost; tetravalent vaccine.

FIGURE 1

Immunization schedule and humoral immune response to DENV2 induced by different immunization regimens. (A) Immunization, sampling, and challenge timeline. (B) Anti-DENV2 IgG antibody titers. (C) Anti-DENV2 nAb titers in serum samples (n = 8) and data represent the GMT + SD. (D–G) Distribution of serum DENV2-specific IgG subclass responses, (D) IgG1; (E) IgG2a; (F) IgG2b; and (G) IgG3. The levels of IgG subclass were expressed as 450 nm OD. Data were expressed as means + SD (n = 8). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 2

CD8⁺ T cell responses against DENV2 elicited by different immunization regimens. (A) Representative expression of CD44^High T cells (gate on CD3⁺ CD8⁺ T cells). Quantification of the frequency of (B) CD44^High CD62L^High and (C) CD44^High CD62L^Low cells among CD8⁺ T cells. Data were expressed as mean + SD (n = 8). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 3

DENV2-specific cytokine-secreting splenocytes in different immunization groups. Cytokine-secreting splenocytes were determined after stimulation with purified DENV2 particles. The positive splenocytes were counted and expressed as the mean SFU/3 × 10⁵ cells. (A) IL-4-positve and (B) IFN-γ-positive splenocytes with representative ELISPOT images, respectively. Data were expressed as mean + SD (n = 8). ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 4

Protective efficacy against DENV2 challenge in different immunization regimens. Mice immunized with different regimens were challenged i.c. with DENV2 and monitored daily for body weight and survival rate for 12 consecutive days (n = 8). (A–C) Percentage changes of body weight from day 0 were determined as 100 × (weight post-challenge)/(weight pre-challenge). Data were expressed as mean ± SD, (D–F) The survival rate shown as the percentage of survivors. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 5

DENV1–4-specific cytokine-secreting splenocytes elicited by the DDP immunization regimen. Cytokine-secreting splenocytes were determined after stimulation with purified DENV1–4 particles, individually. The positive splenocytes were counted and expressed as the mean SFU/3 × 10⁵ cells. (A) IL-4-positve and (B) IFN-γ-positive splenocytes with representative ELISPOT images, respectively. Data were expressed as mean + SD (n = 8). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 6

Protective efficacy against DENV1, DENV3, and DENV4 induced by the DDP immunization regimen. Mice immunized with the DDP regimen were challenged i.c. with DENV1, DENV3, and DENV4 and monitored daily for 12 days (n = 8). Percentage changes of body weight from day 0 after challenge with (A) DENV1, (B) DENV3, and (C) DENV4. The percentage was determined as 100 × (weight post-challenge)/(weight pre-challenge). Data were expressed as mean ± SD. (D) The survival rate post challenge with DENV4 was shown as the percentage of survivors. ^∗P < 0.05, ^∗∗P < 0.01.