Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

Abstract

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8⁺ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

Keywords: Ebola virus; GP and VP40 antigens; SFV replicon vector; immune response; vaccine; virus-like particles.

FIGURE 1

Expression of EBOV GP and VP40 proteins based on SFV DREP vectors. (A) Diagram of constructed recombinant DREP-GP and DREP-VP40 plasmids, nsPs represent the non-structural proteins; (B) Western blot; and (C) Indirect immunofluorescence analysis of GP and VP40 proteins in transfected 293 cells at 48 h post-transfection. β-actin was taken as the internal control in Western blot.

FIGURE 2

TEM analysis of the EBOV VLP assembly in DREP-based GP and VP40 transfected cells. (A–D) TEM analysis of ultrathin sections of cells: (A) 293 cells control; (B) 293 cells transfected with DREP-VP40 for 48 h; (C) 293 cells transfected with DREP-GP for 48 h; (D) 293 cells co-transfected with DREP-GP and DREP-VP40 for 48 h; (E) and (F) the concentrated preparations from the supernatant of cells co-transfected with DREP-GP and DREP-VP40 for 48 h. “N” represents the cell nucleus. The black arrow indicates the assembly of VLPs.

FIGURE 3

Specific antibody responses induced by immunization with DREP-based GP and VP40 vectors. (A) and (B) Determination of specific anti-GP (A) and anti-VP40 (B) antibodies in the sera of BALB/c mice collected at 0, 2, 4, and 6 weeks after the first immunization. The antibody levels were expressed as the log₁₀ IgG titers. (C) and (D) IgG subtype analysis of the specific anti-GP (C) and anti-VP40 (D) antibodies derived from the sera of immunized BALB/c mice. Serum samples from mice immunized with DREP-eGFP were used as the negative control. Results were expressed as the mean of OD450 ± SEM. The results presented are representative of three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

FIGURE 4

ELISA analysis of cytokines secreted by splenocytes from immunized mice upon stimulation with EBOV recombinant GP proteins (rGP). (A–D) Determination of the cytokines IFN-γ (A), IL-2 (B), IL-4 (C), and IL-10 (D) production in the supernatant of splenocytes by ELISA 2 weeks after the final immunization. The results for three independent experiments are shown as the mean ± SD. ^∗p < 0.05.

FIGURE 5

ELISA analysis of cytokines secreted by splenocytes from immunized mice upon stimulation with EBOV recombinant VP40 proteins (rVP40). (A–D) Determination of the cytokines IFN-γ (A), IL-2 (B), IL-4 (C), and IL-10 (D) production in the supernatant of splenocytes by ELISA 2 weeks after the final immunization. The results for three independent experiments are shown as the mean ± SD. ^∗p < 0.05.

FIGURE 6

ELISPOT analysis of IFN-γ secreting CD8⁺ T cells from immunized mice upon stimulation with GP- and VP40-specific epitopes. Splenocytes were isolated from plasmid-immunized mice and were stimulated in vitro with GP- or VP40-specific epitopes. A positive control (PMA/IO) and a negative control (Media) were included. IFN-γ-specific spot forming was observed (A), and the numbers of IFN-γ SFCs/1 × 10⁵ splenocytes were counted (B). The results for three independent experiments are shown as the mean ± SD. ^∗p < 0.05; ^∗∗p < 0.01.