Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs

Abstract

Rabies remains an important worldwide public health threat, so safe, effective, and affordable vaccines are still being sought. Virus-like particle-based vaccines targeting various viral pathogens have been successfully produced, licensed, and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs) containing rabies virus (RABV) glycoprotein (G), matrix (M) protein, and membrane-anchored flagellin (EVLP-F) or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L) as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNAs) responses and elicited greater numbers of CD4(+) and CD8(+) T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP) containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin, and heat-labile enterotoxin B subunit possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine candidate for animals.

Keywords: LTB; flagellin; rabies vaccine; rabies virus; virus-like particle.

FIGURE 1

Construction of membrane-anchored flagellin and LTB and identification of cRVLPs. (A) Schematic diagrams for the construction of membrane-anchored flagellin and LTB. (B) Schematic diagrams of the recombinant plasmids pFBD-2MFG and pFBD-2MLG. (C) Western blot analyses of cRVLPs. EVLP-F (a) and EVLP-L (b) were incubated with mouse anti-rabies G monoclonal antibody, rabbit serum against M, mouse anti-flagellin, or mouse anti-LTB monoclonal antibodies. (D) Electron microscopy of cRVLPs. EVLP-F and EVLP-L were stained with 1% sodium phosphotungstate and observed by transmission electron microscopy. (E) Immunoelectron microscopy of cRVLPs. EVLP-F and EVLP-L were stained with anti-flagellin, or anti-LTB monoclonal antibodies followed by a gold-labeled goat anti-mouse IgG antibody.

FIGURE 2

Specific anti-RABV antibody responses in mice. Mice were immunized twice via the i.m. route at 2-week intervals with EVLP, EVLP-F, EVLP-L, or PBS. Serum samples were then collected 1, 2, 4, and 6 weeks after the primary vaccination. (A) VNA titers were measured by FAVN. (B–H) The specific anti-RABV serum IgG and isotype responses were detected by ELISA. The serum dilution factor was 5,000. The serum IgG (B), IgG1 (C), IgG2a (D), IgG2b (E), and IgG3 (F) responses were determined 2 weeks after the second immunization, whereas serum IgM (G) responses were determined 1 week after the first immunization. The IgG1/IgG2a ratio (H) was measured. The data represent the mean and SD from eight mice in each group and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 3

Enzyme-linked immunospot (ELISpot) analyses of IFN-γ and IL-4 secretion in mice. Splenocytes from mice from the four groups were stimulated with inactivated ERA 2 weeks after the second immunization. Splenocytes producing IFN-γ (A) or IL-4 (B) were identified using ELISpot kits. The data represent the mean numbers and SDs of SFCs per million splenocytes from three mice in each group and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 4

Intracellular cytokine staining (ICS) analysis of CD4⁺ and CD8⁺ T cells from mice producing IFN-γ or IL-4. Splenocytes from three mice in each group 2 weeks after the second immunization were cultured and stimulated with inactivated ERA. The splenocytes were then stained with mouse anti-CD4, anti-CD8, anti-IFN-γ, and anti-IL-4 monoclonal antibodies. CD4⁺ T cells producing IFN-γ (A) or IL-4 (B) and CD8⁺ T cells producing IFN-γ (C) or IL-4 (D) are shown. The data represent the means and SDs of double-positive cell percentages and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 5

Flow cytometry assay for recruitment and/or activation of (B) cells and DCs in lymph nodes from mice. Inguinal lymph nodes cells were isolated from 3 mice from each group on days 3, 6, and 9 after the first immunization and were stained with mouse anti-CD19, anti-CD40, anti-CD11C, anti-CD80, anti-CD86, anti-MHC I, and anti-MHC II monoclonal antibodies. The double-positive CD19⁺CD40⁺ (A), CD11c⁺CD86⁺ (B), CD11c⁺CD80⁺ (C), CD11c⁺MHC I⁺ (D), and CD11c⁺MHC II⁺ (E) cells were plotted. The data represent the means and SDs of double-positive cell percentages and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 6

Challenge test in mice. Mice from the four groups were challenged i.m. 4 weeks after the second vaccination with 100 × IMLD₅₀ (n = 8) of the RABV street strain HNPB3 and observed for 21 days. The percentages of surviving mice in the different groups at different time points after challenge were recorded.

FIGURE 7

Specific anti-RABV VNA responses in dogs. Dogs were vaccinated i.m. twice with EVLP, EVLP-F, EVLP-L, or PBS at 2-week intervals. Blood was collected 2 and 4 weeks after the second dose. VNA titers were measured by FAVN. The data represent the means ± SD for five dogs from each group and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).

FIGURE 8

Enzyme-linked immunospot assays of IFN-γ and IL-4 secretion in dogs. PBMCs were isolated from dogs in each group and were stimulated with inactivated ERA. The PMBCs producing IFN-γ (A) or IL-4 (B) were identified using ELISpot kits. The data represent the mean numbers and SDs of SFCs per million PMBCs from 3 dogs in each group and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).