Promotion of Cellular and Humoral Immunity against Foot-and-Mouth Disease Virus by Immunization with Virus-Like Particles Encapsulated in Monophosphoryl Lipid A and Liposomes

Abstract

Virus-like particles (VLPs) have emerged as promising vaccine candidates against foot-and-mouth disease (FMD). However, such vaccines provide a relatively low level of protection against FMD virus (FMDV) because of their poor immunogenicity. Therefore, it is necessary to design effective vaccine strategies that induce more potent immunogenicity. In order to investigate the means to improve FMD VLP vaccine (VLP_FMDV) immunogenicity, we encapsulated VLPs (MPL/DDA-VLP_FMDV) with cationic liposomes based on dimethyldioctadecylammonium bromide (DDA) and/or monophosphoryl lipid A (MPL, TLR4 agonist) as adjuvants. Unlike inactivated whole-cell vaccines, VLP_FMDV were successfully encapsulated in this MPL/DDA system. We found that MPL/DDA-VLP_FMDV could induce strong cell-mediated immune responses by inducing not only VLP-specific IFN-γ⁺CD4⁺ (Th1), IL-17A⁺CD4⁺ (Th17), and IFN-γ⁺CD8⁺ (activated CD8 response) T cells, but also the development of VLP-specific multifunctional CD4⁺ and CD8⁺ memory T cells co-expressing IFN-γ, TNF-α, and IL-2. In addition, the MPL/DDA-VLP_FMDV vaccine markedly induced VLP-specific antibody titers; in particular, the vaccine induced greater Th1-predominant IgG responses than VLP_FMDV only and DDA-VLP_FMDV. These results are expected to provide important clues for the development of an effective VLP_FMDV that can induce cellular and humoral immune responses, and address the limitations seen in current VLP vaccines for various diseases.

Keywords: TLR4 agonist; foot-and-mouth disease; immunogenicity; liposome; vaccine; virus-like particles.

Figure 1

Physical characterization of virus-like particle (VLP) vaccines formulated with dimethyldioctadecylammonium bromide (DDA) or/and monophosphoryl lipid A (MPL). Particle size distributions of VLP_FMDV (A), DDA (B), MPL/DDA (C), DDA-VLP_FMDV (D), and MPL/DDA-VLP_FMDV (E) were measured by DLS analysis. VLP_FMDV: recombinant food-and-mouth disease VLP vaccine; MPL/DDA: MPL and DDA formulation; DDA-VLP_FMDV: VLP_FMDV formulated with DDA; MPL/DDA-VLP_FMDV: VLP_FMDV formulated with DDA and MPL. (F) Bar graph is expressed as the mean ± SD of 3 samples per group. ** p < 0.01.

Figure 2

Analysis of VLP-specific CD4⁺ and CD8⁺ T cell responses. Mice (n = 5 animals/group) were immunized twice with PBS (G1), DDA (G2), MPL/DDA (G3), VLP_FMDV (G4), DDA-VLP_FMDV (G5), or MPL/DDA/VLP_FMDV (G6). (A,B) Single-cell suspensions were re-stimulated with 10 μg/mL VLP for 12 h and analyzed for VLP-specific Th1 (IFN-γ-producing CD4⁺ T cells), Th2 (IL-5-producing CD4⁺ T cells), Th17 (IL-17A-producing CD4⁺ T cells) and activated CD8⁺ T cells (IFN-γ-producing CD8⁺ T cells) by intracellular cytokine (IFN-γ, IL-5, IL-17A) staining based on T cell-specific makers (CD3, CD4, CD8). (B) The percentages of VLP-specific Th1, Th2 and Th17, and activated CD8⁺ T cells in spleens from immunized mice were analyzed by flow cytometry. (C) Single-cell suspensions of splenocytes were treated with 10 μg/mL VLP for 24 h, and supernatants were collected and evaluated for VLP-specific cytokines (IFN-γ, IL-5 and IL-17A) by ELISA. The means ± SD shown are representative of two independent experiments. * p < 0.05, *** p < 0.001. n.s.: not significant.

Figure 3

Analysis of antigen-specific multifunctional CD4⁺ T cells. (A) Gating strategy for multifunctional CD4⁺ memory T cells. Memory CD4+ T cells (determined by increased CD44 expression) gated to show CD3⁺CD4⁺CD44⁺ T cells. For multifunctional CD4⁺ T cells analysis, cells were identified by intracellular cytokine (IFN-γ, TNF-α, and IL-2) staining based on memory CD4⁺ T cell gating. (B) The percentage of VLP-specific total cytokine (IFN-γ, TNF-α, and/or IL-2)-producing cells among splenic CD4⁺CD44⁺ memory T cells. (C) Pie charts representing the mean frequencies of cells co-expressing IFN-γ, TNF-α, and/or IL-2. The relative amounts of single-, double- and triple-functional CD4⁺CD44⁺ memory T cells are indicated as pie arcs. Means ± SD (n = 5 mice/group) shown are representative of two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s.: not significant.

Figure 4

Analysis of antigen-specific multifunctional CD8⁺ T cells. (A) Gating strategy for multifunctional CD8⁺ memory T cells. (B) Percentage of VLP-specific total cytokine (IFN-γ, TNF-α, and/or IL-2)-producing cells among splenic CD8⁺CD44⁺ memory T cells. (C) Pie charts represent the mean frequencies of cells co-expressing IFN-γ, TNF-α, and/or IL-2. The relative amounts of single-, double- and triple-functional CD4⁺CD44⁺ memory T cells are indicated as pie arcs. The means ± SD (n = 5 mice/group) shown are representative of two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s.: not significant.

Figure 5

Analysis of VLP-specific serum antibody isotype. Mice were immunized twice with VLP_FMDV in different adjuvant combinations. Two weeks after final immunization, serum from five mice in each group was obtained, and the VLP-specific IgG, IgM, IgG1 and IgG2a were analyzed using ELISA. Data from one of two independent experiments are shown. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s.: not significant.