Protection induced by virus-like particles containing Toxoplasma gondii microneme protein 8 against highly virulent RH strain of Toxoplasma gondii infection

Abstract

Toxoplasma gondii (T. gondii) microneme protein 8 (MIC8) represents a novel, functional distinct invasion factor. In this study, we generated virus-like particles (VLPs) targeting Toxoplasma gondii MIC8 for the first time, and investigated the protection against highly virulent RH strain of T. gondii in a mouse model. We found that VLP vaccination induced Toxoplasma gondii-specific IgG and IgG1 antibody responses in the sera. Upon challenge infection with RH strain of T. gondii tachyzoites, vaccinated mice showed a significant increase of both IgG antibodies in sera and IgA antibodies in feces compared to those before challenge, and a rapid expansion of both germinal center B cell (B220+, GL7+) and T cell (CD4+, CD8+) populations. Importantly, intranasally immunized mice showed higher neutralizing antibodies and displayed no proinflammatory cytokine IFN-γ in the spleen. Mice were completely protected from a lethal challenge infection with the highly virulent T. gondii (RH) showing no body weight loss (100% survival). Our study shows the effective protection against T. gondii infection provided by VLPs containing microneme protein 8 of T. gondii, thus indicating a potential T. gondii vaccine candidate.

Fig 1. PCR identification of MIC8 and M1 genes and recombinant plasmids pFastBac-MIC8 and pFastBac-M1 digested.

A Prime Script 1^st Strain cDNA Synthesis Kit and total RNA extracted from T. gondii RH strain were used to synthesize cDNA, which was then amplified by PCR to obtain T. gondii MIC8 gene (A). MIC8 plasmid (B) was obtained by cloning T. gondii MIC8 gene into pFastBac vector with EcoRI / XhoI enzymes. Marker: DNA marker; Size of MIC8: 2055bp.

Fig 2. VLPs characterization.

TEM image (A) and Western blot analysis (B). VLPs (5, 10, 30 μg) were loaded for SDS-PAGE. Polyclonal mouse anti-T. gondii antibody and anti-M1 monoclonal antibody were used to probe MIC8 protein and M1 protein, respectively.

Fig 3. Experimental schedule and T. gondii-specific antibody responses upon immunization.

Mice were immunized twice (boost VLP vaccination administered 4 weeks after the prime dose) and challenge infection was performed 4 weeks after boost vaccination. Mice were sacrificed at day 5 post-challenge (A). High levels of T. gondii-specific IgG, IgG1 and IgG2a antibody responses in the sera were determined after boost with ELISA assay (B, C, D, **P < 0.01). Lower levels of IgG2a and IgG2b were determined in IN mice compared to IgG1 (D, E). IM mice showed low levels of isotypes IgG1, IgG2a and IgG2b responses (C, D, E).

Fig 4. T. gondii-specific antibody response profiles in serum and feces following challenge infection.

Immunized mice were challenged orally with T. gondii RH strain 4 weeks after boost vaccination, and T. gondii-specific IgG antibody responses in the sera were detected at day 5 after challenge infection (A; **P < 0.01). IgA antibody responses from feces were also determined after challenge infection (B; **P < 0.01).

Fig 5. Serum neutralizing activities.

Complement-inactivated mouse sera at week 4 after boost were used to react with T. gondii (RH) in vitro, and the mixture of sera (50 μL) and T. gondii (100 tachyzoites) was used to infect mice, including PBS and naïve mouse sera controls. At day 7 after infection, tachyzoites were recovered from mouse abdominal cavity and the replication inhibition of T. gondii was determined (**P < 0.01).

Fig 6. CD4⁺, CD8⁺ T and B cell responses after challenge infection.

The populations of (A) CD4⁺ T cells, (B) CD8⁺ T cells and (C) germinal center B cells were analyzed at days 5 and 16 upon challenge using flow cytometry, and the results are summarized (D). Numbers indicate percentage of cell populations in each quadrant. Higher populations of CD4⁺, CD8⁺ and germinal center B cells were detected in IN+Cha mice (A, B, C, D, *P < 0.05, **P < 0.01).

Fig 7. Proinflammatory cytokine response in the spleen.

To determine the level of inflammatory cytokine IFN-γ and IL-6, mouse spleen cells were cultured in vitro for 5 days. Higher levels of IFN-γ and IL-6 were detected in Naïve+cha and IM+cha compared to the non-infected naïve mice (*P < 0.05). No IFN-γ were detected in IN+Cha mice (A, **P < 0.01). Significantly lower level of IL-6 were detected in IN+Cha mice compared to IM+Cha and Naïve+Cha mice (B, **P < 0.01).

Fig 8. Survival rate and body weight changes upon challenge infection.

Mice that were immunized intranasally or intramusculary with MIC8 VLPs were challenged with T. gondii RH strain. Body weight loss (A) and survival rates of mice (B) were monitored for 16 days. Significant difference was found in body weight loss at 8–12 days post-challenge between IN+cha and Naïve+cha (* P < 0.05).