Lentinan improved the efficacy of vaccine against Trichinella spiralis in an NLRP3 dependent manner

Abstract

There is an urgent need for the development of new, improved vaccine adjuvants against T. spiralis infection. Polysaccharides are effective, safe, and biodegradable as adjuvant. In our study, we first observed the protective efficacy of lentinan as adjuvant against helminth T. spiralis infection. Recombinant T. spiralis Serpin (rTs-Serpin) immunoscreened from a cDNA library of T. spiralis, as a vaccine, protect host against Trichinella infection. The reduction rate of helminth burden of rTs-Serpin+lentinan-immunized mice was significantly increased compared with rTs-Serpin+FCA -immunized mice. rTs-Serpin+lentinan induced IgG1-dominant immune response and higher levels of IFN-γ and IL-4. rTs-Serpin+lentinan displayed a lower reduction rate of parasite burden in NLRP3-/- mice than that in WT mice and lower level of IgG1 than that in WT mice. The level of IL-4, but not IFN-γ, from NLRP3-/- mice immunized by rTs-Serpin+lentinan was significantly lower than that from WT mice, suggesting that NLRP3 is associated with rTs-Serpin+lentinan -triggering Th2 protective immunity against T. spiralis infection. In summary, we revealed that lentinan was a novel adjuvant against T. spiralis infection via NLRP3. NLRP3 therefore represents an important target for adjuvant discovery and the control of T. spiralis infection.

Fig 1. Protective immunity induced by immunizing mice with lentinan against T. spiralis.

The number of adults recovered from intestines (A) and muscle larvae (ML) per gram (LPG) in skeletal muscles (B) from mouse groups immunized subcutaneously with PBS (PBS), rTs-Serpin (rTs-Serpin), rTs-Serpin emulsified with Freund’s adjuvants (rTs-Serpin+FCA, and rTs-Serpin emulsified with lentinan (rTs-Serpin+lentinan), after challenge with 500 ML of T. spiralis. The adult worms (C) and muscle larvae (D) reduction rates were analyzed based on the the mean number of adult worms or the recovered muscle larvae per gram (LPG) of muscle from vaccinated groups compared with PBS group. Results are expressed as the mean ± SD of 10 mice per group. The data shown are representative of three independent experiments. *p<0.05, ** p < 0.01, vs the as indicated by the line (Tukey multiple comparison following ANOVA).

Fig 2. Analysis of humoral immune responses.

(A) The levels of anti-rTs-serpin IgG in the sera of immunized mice or control mice were measured by ELISA. (B) The levels of specific IgE rTs-serpin in the sera of immunized mice were measured. (C) The IgG1 subclass responses against rTs-serpin were detected at different time points. (D) The IgG2a subclass responses against rTs-serpin were detected at different time points. The values shown for each group are the mean + SD of the antibody levels (n = 10) from three individual experiments * p < 0.05 as indicated by the line (one-way ANOVA with Tukey’s post-test).

Fig 3. Analysis of cytokine production from CD4⁺ T cells.

CD4⁺ T cells secreting IFN-γ (A) and IL-4 (B) production was measured by ELISA one week after the final immunization. The data are the mean ± SD of each group (n = 10) from three independent experiments. * p < 0.05 as indicated by the lines (one-way ANOVA with Tukey’s post-test).

Fig 4. Expression of NLRP3 in CD4⁺ T cells.

Results are relative to the expression at day 0. Results are the mean ± SD (n = 10) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 as indicated by line (one-way ANOVA with Tukey’s post-test).

Fig 5. Protective immunity induced by immunizing WT and NLRP3^-/- mice with lentinan against T. spiralis.

The adult worms (A) and muscle larvae (B) reduction rates were analyzed based on the the mean number of adult worms or the recovered muscle larvae per gram (LPG) of muscle from vaccinated groups compared with PBS group from immunized WT and NLRP3^-/- mice after challenge with 500 ML of T. spiralis. Results are expressed as the mean ± SD of 10 mice per group. The data shown are representative of three independent experiments. *p<0.05 vs the as indicated by the line (Tukey multiple comparison following ANOVA).

Fig 6. Analysis of humoral immune responses in WT and NLRP3^-/- mice.

(A) The levels of anti-rTs-serpin IgG in the sera of WT and NLRP3^-/- mice were measured by ELISA. (B) The levels of specific IgE rTs-serpin in the sera of immunized mice were measured by ELISA. (C) The IgG1 subclass responses against rTs-serpin in WT and NLRP3^-/- mice were detected at different time points. (D) The IgG2a subclass responses against rTs-serpin in WT and NLRP3^-/- mice were detected at different time points. The values shown for each group are the mean + SD of the antibody levels (n = 10) from three individual experiments * p < 0.05 as indicated by the line (one-way ANOVA with Tukey’s post-test).

Fig 7. Analysis of cytokine production from WT and NLRP3^-/- CD4⁺ T cells.

WT and NLRP3^-/- CD4⁺ T cells secreting IFN-γ (A) and IL-4 (B) production was measured by ELISA one week after the final immunization. The data are the mean ± SD of each group (n = 10) from three independent experiments. * p < 0.05 as indicated by the lines.