Immunogenicity of peptide-based vaccine composed of epitopes from Echinococcus granulosus rEg.P29

Abstract

Echinococcus granulosus is one of the main causes of economic loss in the livestock industry because of its food-borne transmission. Cutting off the transmission route is a valid prevention method, and vaccines are the most effective means of controlling and eliminating infectious diseases. However, no human-related vaccine has been yet marketed. As a genetic engineering vaccine, recombinant protein P29 of E. granulosus (rEg.P29) could provide protection against deadly challenges. In this study, we generated peptide vaccines (rEg.P29_T , rEg.P29_B , and rEg.P29_T+B ) based on rEg.P29 and an immunized model was established by subcutaneous immunization. Further evaluation showed that peptide vaccine immunization in mice induced T helper type 1 (Th1)-mediated cellular immune responses, leading to high levels of rEg.P29 or rEg.P29_B -specific antibodies. In addition, rEg.P29_T+B immunization can induce a higher antibody and cytokine production level than single-epitope vaccines, and immune memory is also longer. Collectively, these results suggest that rEg.P29_T+B has the potential to be developed as an efficient subunit vaccine for use in areas where E. granulosus is endemic.

Keywords: E. granulosus; Th1 cells; antibody; immune memory; immunogenicity; peptide vaccine.

FIGURE 1

Multi‐epitope peptide design and scheme of immunization. (A) Schematic representation and amino acid sequences of multi‐epitope peptide, rEg.P29_T, rEg.P29_B, rEg.P29_T+B. (B) C57BL/6 mice (n = 5) were subcutaneously immunized following the prime‐boost protocol, we performed two boosts (−3 and 0). Spleen sample and serum sample were collected and tested at indicated time points.

FIGURE 2

Antibody responses of splenic lymphocytes in multi‐epitope vaccinated mice. At 1, 2, 4, 8 and 16 weeks post‐immunization, serum samples were collected to determine the antibody level. (A and B) rEg.P29 and rEg.P29_B ‐specific antibodies were detected using ELISA. (C and D) Titers of rEg.P29 and rEg.P29_B ‐specific IgG antibodies in serum from C57BL/6 mice. The splenocytes were collected from mice immunized with peptides, protein, PBS, or CpG at 2 weeks after immunization, and the secretion of rEg.P29 and rEg.P29_B‐specific IgM and IgG in splenocytes were measured using ELISPOT. (E) rEg.P29‐specific IgM and IgG spot‐forming cells. (F) rEg.P29_B‐specific IgM and IgG spot‐forming cells. (****p < .001; ***p < .005; **p < .01; *p < .05; p > .05, ns).

FIGURE 3

Antigen‐specific IgG1 and IgG2a subtype responses. At 1, 2, 4, 8, and 16 weeks post‐immunization, sera samples were subjected to ELISA for IgG1/IgG2a ratio specific to rEg.P29 or rEg.P29_B in mice. (A) rEg.P29‐specific IgG1/IgG2a ratio. (B) rEg.P29_B‐specific IgG1/IgG2a ratio.

FIGURE 4

ELISA analysis of antigen‐specific T‐cell‐mediated immune responses in mice. At 1, 2, 4, 8, and 16 weeks post‐immunization, splenocytes were collected to determine the cytokine level. (A–E) At indicated time interval, mice were killed and splenocytes were isolated and stimulated with different antigen. The supernatants were tested for cytokine productions by ELISA. (****p < .001; ***p < .005; **p < .01; *p < .05; p > .05, ns).

FIGURE 5

ELISPOT analysis of antigen‐specific T‐cell‐mediated immune responses in mice. The splenocytes were collected from mice immunized with peptides, protein,PBS or CpG at 2 weeks after immunization, and the secretion of antigen‐specific IFN‐γ and IL‐4 in splenocytes was measured using ELISPOT. (A and B) antigen‐specific IFN‐γ spot‐forming cells. (C and D) antigen‐specific IL‐4 spot‐forming cells. Representative images of IFN‐γ or IL‐4 spot‐forming cells are shown as A and C. (****p < .001; ***p < .005; **p < .01; *p < .05; p > .05, ns).

FIGURE 6

Flow cytometry analysis of antigen‐specific CD4⁺ and CD8⁺ T‐cell secretion of IFN‐γ and IL‐4 in splenocytes after immunization. The splenocytes were collected from mice immunized with peptides, protein, PBS, or CpG at 2 weeks after immunization. Antigen‐specific CD4⁺/CD8⁺ T‐cell secretion of IFN‐γ/IL‐4 was determined using and flow cytometry assay. (A) The gating strategies for analyzing the secretion of IFN‐γ and IL‐4 in T cells. (B) Representative flow cytometric plots for measuring CD4⁺ T‐cells secreting cytokines. Analyses for (C and D) antigen‐specific CD4⁺ T‐cell secretion of IFN‐γ and IL‐4 from the spleen.(E) Representative flow cytometric plots for measuring CD8⁺ T‐cells secreting cytokines. Analyses for (F and G) antigen‐specific CD8⁺ T‐cell secretion of IFN‐γ and IL‐4 from the spleen. (****p < .001; ***p < .005; **p < .01; *p < .05; p > .05, ns).

FIGURE 7

Memory, plasma cells, and plasmablasts phenotype of B cells. At 1, 2, 4, 8, and 16 weeks post‐immunization, splenocytes were collected to determine the memory, plasma cells, and plasmablasts phenotype of B cells. (A) The gating strategies for analyzing the secretion of plasma cells and plasmablasts. Analyses for (B) CD19⁺CD138⁻B cells from the spleen. Analyses for (C) CD19⁺CD138⁺ plasmablasts from the spleen. Analyses for (D) CD19⁻CD138⁺ plasma cells from the spleen (E) Representative flow cytometry plots for measuring memory B cells. Analyses for (F) CD19⁺IgD⁻IgG⁺memory B cells. Analyses for (G) CD19⁺IgD⁻IgA⁺memory B cells. (****p < .001; ***p < .005; **p < .01; *p < .05; p > .05, ns).

FIGURE 8

Memory phenotype of CD4⁺T and CD8⁺ T cells. At 1, 2, 4,8, and 16 weeks post‐immunization, splenocytes were collected to determine the memory phenotype of CD4⁺T and CD8⁺T cells. (A) Representative contour plot analysis of gated CD4⁺T and CD8⁺T cells expressing CD44 and CD62L in spleen. Numbers of CD4⁺T cells presenting the cell surface phenotype (B) CD44⁺CD62⁻T cells and (C) CD44⁺CD62⁺T cells. Numbers of CD8⁺T cells presenting the cell surface phenotype (D) CD44⁺CD62⁻T cells and (E) CD44⁺CD62⁺T cells.(****p < .001; ***p < .005; **p < .01; *p < .05; p > .05, ns).