Influenza Virus-Like Particles Presenting both Toxoplasma gondii ROP4 and ROP13 Enhance Protection against T. gondii Infection

Abstract

Rhoptry organelle proteins (ROPs) secreted by Toxoplasma gondii (T. gondii) play a critical role during parasite invasion into host cells. In this study, virus-like particles (VLPs) vaccines containing ROP4 and/or ROP13 together with influenza M1 were generated. ROP4+ROP13 VLPs were produced by combining ROP4 VLPs with ROP13 VLPs, and ROP(4 + 13) VLPs by co-infecting insect cells with recombinant baculovirus expressing ROP4 or ROP13. Mice intranasally immunized with ROP(4 + 13) VLPs showed significantly higher levels of IgG, IgG1, IgG2a and IgA antibody responses in sera compared to ROP4+ROP13VLPs. Upon challenge infection by oral route, mice immunized with ROP(4 + 13) VLPs elicited higher levels of IgG and IgA antibody responses in fecal, urine, intestine and vaginal samples as well as CD4⁺ T, CD8⁺ T cells, and germinal center B cell responses compared to other type of vaccines, ROP4 VLPs, ROP13 VLPs, and ROP4+ROP13 VLPs. ROP(4 + 13) VLPs vaccination showed a significant decrease in the size and number of cyst in the brain and less body weight loss compared to combination ROP4+ROP13 VLPs upon challenge infection with T. gondii ME49. These results indicated that the ROP(4 + 13) VLPs vaccination provided enhanced protection against T. gondii infection compared to ROP4+ROP13 VLPs, providing an important insight into vaccine design strategy for T. gondii VLPs vaccines.

Keywords: ROP13; ROP4; Toxoplasma gondii; protection; vaccine; virus-like particle.

Figure 1

Characterization of virus-like particles (VLPs). The 50 and 10 μg of VLPs were loaded for SDS-PAGE and visualized by western blot. As shown in (A), ROP4 VLPs containing ROP4 proteins and M1 (A) were detected. Similarly, ROP13 VLPs containing ROP13 proteins and M1 (B) were also detected. In case of ROP(4 + 13) VLPs, the presence of ROP4, ROP13 proteins and M1 (C) were confirmed. TEM was used to confirm the shape of ROP4 VLPs (A), ROP13 VLPs (B) and ROP(4 + 13) VLPs (C).

Figure 2

High reactivity to T. gondii-specific antibody response in serum. Ten mice per group were immunized with ROP4 VLPs, ROP13 VLPs, ROP4+ROP13 VLPs and ROP(4 + 13) VLPs. Sera were collected 4 weeks after boost immunization and challenge infection with T. gondii ME49. ELISA plate were coated with T.gondii RH (4ug/mL) and sera (1:50 dilution) were incubated in coated plate after serial dilution. T. gondii-specific IgG antibodies response ((A), * p < 0.05, ** p < 0.01, *** p < 0.01). T. gondii-specific IgG1 antibody response ((B), * p < 0.05, ** p < 0.01). T. gondii-specific IgG2a antibody response ((C), * p < 0.05, ** p < 0.01). T. gondii-specific IgG2b antibody response (D). T. gondii-specific IgA antibody response ((E), * p < 0.05, ** p < 0.01).

Figure 3

Intranasal immunization with T. gondii VLPs induced mucosal immunity. Mice (n = 10) were intranasally immunized with ROP4 VLPs, ROP13 VLPs, ROP4+ROP13 VLPs and ROP(4 + 13) VLPs. Mice samples were collected at day 30 after challenge infection with T. gondii ME49. To confirm the activation of mucosal immunity, we identified IgG and IgA antibody responses from vaginal secretions, urine, feces and intestines. IgG and IgA antibody responses from vaginal secretions ((A,B), * p < 0.05, *** p < 0.001). IgG and IgA antibody responses in urine ((C,D), * p < 0.05, *** p < 0.001). IgG and IgA antibody responses in feces ((E,F), * p < 0.05, *** p < 0.001). IgG and IgA antibody response in intestine ((G,H), * p < 0.05, *** p < 0.001).

Figure 4

Cytokine production and antibody secreting cell responses (ASC). Immunized mice (n = 5) were challenge-infected orally with 450 T. gondii ME49 cyst at week 4 after boost immunization. After 4 weeks, isolated splenocytes were collected from infected mice and cells were incubated for 3 days with T. gondii RH. T. gondii VLP is effective in inducing antibody secreting cell responses and cytokines. IgG antibody secreting cells in spleen ((A), * p < 0.05, *** p < 0.001). IgA antibody secreting cells in spleen ((B), * p < 0.05, ** p < 0.01). Cytokine levels of IFN-γ ((C), * p < 0.05, *** p < 0.001) and IL-6 ((D), * p < 0.05, *** p < 0.001) were determined using cytokine ELISA.

Figure 5

The gating strategy of CD4⁺, CD8⁺ T cell and germinal center B cell in spleen and MLN cells. Spleen and MLN cells from mice (n = 5) were collected at day 30 upon challenge infection with T. gondii ME49 and stained with phenotype-specific marker antibodies (CD3, CD4, CD8, B220, GL7). CD4⁺, CD8⁺ T cell gating strategy in spleen cells (A). CD4⁺, CD8⁺ T cell gating strategy in MLN cells (B). Germinal center B cell gating strategy in spleen cells (C). Germinal center B cell gating strategy in MLN cells (D).

Figure 6

Flow cytometric analysis. Collected spleen and MLN cells from mice (n = 5) were stained with specific surface markers (CD3, CD4, CD8, B220, GL7) and analyzed using FACS. CD4⁺ T cell response in spleen (A-a, * p < 0.05, *** p < 0.001). CD4⁺ T cell response in MLN (A-b, * p < 0.05, *** p < 0.001). CD8⁺ T cell response in spleen (B-a, * p < 0.05, ** p < 0.01). CD8⁺ T cell response in MLN (B-b). Germinal center B cell response in spleen (C-a, * p < 0.05, *** p < 0.001). Germinal center B cell response in MLN (C-b, * p < 0.05, *** p < 0.001).

Figure 7

Cyst size and cyst count after T. gondii ME49 challenge. Thirty days after challenge infection with 450 T. gondii ME49 cyst, brain tissues were collected from mice (n = 5). Cyst size ((A), * p < 0.05, *** p < 0.001) and cyst count ((B), * p < 0.05, ** p < 0.01) among VLPs immunization groups were compared. ROP(4 + 13) VLPs of cyst size (27.84um) was significantly reduced compared to naïve challenge (41.04um). Also, 40 cysts were counted from the brains of ROP(4 + 13) VLPs-immunized mice, whereas 660 cysts were counted from naïve challenge mice.

Figure 8

Protective efficacy of immunization with T.gondii VLPs. Naïve or immunized mice (n = 5) were orally infected with 450 T. gondii ME49 cyst after boost immunization. Mice were monitored for 60 days to observe changes in body weight (A) and survival (B) after challenge infection. Upon challenge infection, all immunized mice survived and ROP(4 + 13) VLPs immunization showed the least weight loss, whereas all naïve challenge mice showed rapid weight loss and died on day 35.