Peste des Petits Ruminants Virus-Like Particles Induce a Potent Humoral and Cellular Immune Response in Goats

Abstract

Peste des petits ruminants is a highly contagious acute or subacute disease of small ruminants caused by the peste des petits ruminants virus (PPRV), and it is responsible for significant economic losses in animal husbandry. Vaccination represents the most effective means of controlling this disease, with virus-like particle (VLP) vaccines offering promising vaccine candidates. In this study, a PPRV VLP-based vaccine was developed using a baculovirus expression system, allowing for the simultaneous expression of the PPRV matrix (M), hemagglutinin (H), fusion (F) and nucleocapsid (N) proteins in insect cells. Immunization of mice and goats with PPRV VLPs elicited a robust neutralization response and a potent cellular immune response. Mouse studies demonstrated that VLPs induced a more robust IFN-γ response in CD4⁺ and CD8⁺ T cells than PPRV Nigeria 75/1 and recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. In addition, PPRV VLPs induced a strong Th1 class response in mice, as indicated by a high IgG2a to IgG1 ratio. Goat studies demonstrated that PPRV VLPs can induce the production of antibodies specific for F and H proteins and can also stimulate the production of virus neutralizing antibodies to the same magnitude as the PPRV Nigeria 75/1 vaccine. Higher amounts of IFN-γ in VLP-immunized animal serum suggested that VLPs also elicited a cellular immune response in goats. These results demonstrated that VLPs elicit a potent immune response against PPRV infection in small ruminants, making PPRV VLPs a potential candidate for PPRV vaccine development.

Keywords: baculovirus; cellular response; humoral response; peste des petits ruminants virus; virus-like particles.

Figure 1

Generation and characterization of peste des petits ruminants virus (PPRV) virus-like particles (VLPs). (a) Schematic diagrams are presented for the recombinant plasmids pFastBacDual-2M, pFastBacDual-2F, pFastBacDual-2H, pFastBacDual-2N. (b) Detection of the expression of M, F, H and N. Sf9 cells were mock-infected or infected with recombinant baculoviruses rpFBD-2M, rpFBD-2F, rpFBD-2H, rpFBD-2N or wild-type baculovirus (WBV). Expression was evaluated by IFA using a mouse anti-PPRV M polyclonal antibody, mouse anti-PPRV F polyclonal antibody or sheep polyclonal antibodies against N and F. (c–f) Transmission electron microscopy images of virus and VLPs preparations. Native peste des petits ruminants virus (PPRV) (c), residual baculoviruses (indicated by black triangles) in preparations of VLPs (indicated by white arrow) (d), purified PPRV VLPs (e), and immunogold-labelled PPRV VLPs stained with mouse anti-PPRV H polyclonal antibody followed by gold-labeled goat anti-mouse IgG antibody (f). (g) Western blot depicting VLP protein expression using mouse anti-PPRV M or H polyclonal antibodies or sheep polyclonal antibody sera against N or F.

Figure 2

Immunization induces a humoral immune response and Th1 class immunity in mice. (a) Mice were vaccinated three times via the subcutaneous route at 2-week intervals with VLPs, PPRV Nigeria 75/1 or PBS. Blood sampling and serum isolation were performed 2, 4, and 6 weeks after the first immunization. (b) Virus-neutralizing antibody (VNA) titers were measured by fluorescent antibody viral neutralization (FAVN). (c–i) Serum was diluted by a factor of 5000, and total serum IgG and antibody subtypes were quantified by ELISA. The IgM (c) response were evaluated 1 week after the first vaccination, whereas IgA (d), IgG2a (e), IgG1 (f), IgG3 (g), IgG (h), and IgG2b (i) responses were determined 2 weeks after the second immunization. (j) The IgG1/IgG2a ratio was measured. Dotted lines represent antibody titers greater than ten, indicating positive serum conversion. Significant differences were analyzed by one-way or two-way ANOVA and indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 3

Immunization induces a cell-mediated immune response in mice. Splenocytes from mice were stimulated with inactivated PPRV two weeks after the second immunization. (a) Representative images of IFN-γ- and IL-4-secreting splenocytes. (b,c) ELISpot assay was used to enumerate IFN-γ-secreting (b) or IL-4-secreting (c) splenocytes. Data are depicted as the mean ± SD spot-forming cells (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

Immunization improves IFN-γ-secreting CD4+ and CD8+ T cell responses in mice. Two weeks after the second immunization, splenocytes from three mice in each group were cultured and stimulated with inactivated PPRV Nigeria 75/1. Mouse monoclonal antibodies against CD4, CD8, IFN-γ, and IL-4 were used to identify cells that were double-positive (a) CD4⁺ IFN-γ⁺, (b) CD4⁺ IL-4⁺, (c) CD8⁺ IFN-γ⁺, and (d) CD8⁺ IL-4⁺. Data were analyzed by one-way ANOVA and indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 5

Immunization induces enhanced B cell and dendritic cell recruitment and activation. Two weeks after the second immunization, five mice from each group were euthanized for collection of cells from the inguinal lymph nodes. Mouse monoclonal antibodies against CD19, CD40, CD80, CD86, MCH I, and MCH II were used to identify cells that were double-positive (a) CD19⁺CD40⁺, (b) CD11c⁺CD86⁺, (c) CD11c⁺CD80⁺, (d) CD11c⁺MHC I⁺, and (e) CD11c⁺MHC II⁺. Significant differences were analyzed by one-way ANOVA and indicated as follows: * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 6

Immunization induces a humoral response in goats. (a) Goats were boosted twice every three weeks via the subcutaneous route with VLPs (150 µg or 300 µg), PPRV Nigeria 75/1, PBS, or alum adjuvant after initial immunization. (b) Serum samples were collected 3, 6, 9, 12 and 15 weeks after the first vaccination and virus-neutralizing antibody (VNA) titers were measured by fluorescent antibody viral neutralization (FAVN). Dotted lines represent antibody titers greater than ten, indicating positive serum conversion. (c–d) Total serum IgG (c) and IgM (d) responses were determined at 3 weeks. (e–g) Serum was collected from each goat three weeks after the third immunization for analysis by a PPRV F-, H-, and N-specific ELISA. Significant differences were analyzed by one-way or two-way ANOVA and indicated as follows: *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.

Figure 7

Immunization induces a significant cytokine response in goats. (a–c) Serum samples after the second immunization from animals immunized with PPRV VLPs (150 µg or 300 µg), PPRV Nigeria 75/1, PBS, or alum adjuvant were analyzed for IL-4 (a), IL10 (b) and IFN-γ (c). Significant differences were analyzed by one-way ANOVA and indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.