Background: Toxoplasma gondii (T. gondii) is a significant opportunistic zoonotic protozoan, presenting a substantial risk to human health and livestock. Consequently, the development of an effective vaccine against toxoplasmosis is imperative. This study focuses on the GRA12 protein as a target for developing a recombinant protein vaccine, with its efficacy evaluated through immunization trials in cats. Methods: We expressed recombinant GRA12 protein in E. coli and immunized cats with the purified antigen. The cats were categorized into four groups: G1 (PBS control), G2 (ISA 201 adjuvant alone), G3 (rGRA12 vaccine), and G4 (rGRA12 combined with ISA 201 adjuvant). All cats underwent subcutaneous immunizations on days 0, 14, and 28. Subsequently, serum levels of IgG (including IgG1 and IgG2a subclasses) and cytokines (IFN-γ, IL-2, TNF-α, IL-4, IL-10) were measured by enzyme-linked immunosorbent assay (ELISA). Two weeks after the third immunization (42 DPI), each cat was intraperitoneally infected with 1 × 10⁶T. gondii RH tachyzoites. Oocyst shedding, survival duration, and T. gondii burden were monitored to assess vaccine-induced immunity. Results: The results indicate that immunization with recombinant rGRA12 protein significantly elevated IgG, IgG1, and IgG2a antibody levels in cats. G4 displayed elevated IgG levels post-immunization compared to G1 and G2, with an IgG1/IgG2a ratio > 1, indicating a mixed Th1/Th2 immune response. G4 also showed significantly increased IFN-γ, IL-2, TNF-α, and IL-4 levels compared to G1 (p < 0.05), while IL-10 remained unchanged. After T. gondii infection, total oocyst counts were 4.61 × 10⁶ (G1), 4.49 × 10⁶ (G2), 3.58 × 10⁶ (G3), and 2.59 × 10⁶ (G4), with G3/G4 showing 20.1–27.9% reduction relative to G1 (p < 0.05). Survival analysis revealed that groups G3 and G4 exhibited significantly longer median survival times (38 and 60 days, respectively; G4 with no mortality) compared to G1 and G2 (19 and 26 days, respectively). Additionally, parasite burdens in the brain, heart, lungs, liver, and spleen were significantly reduced in G3/G4 compared to G1/G2 (p < 0.01). Conclusions: In summary, the recombinant GRA12 vaccine significantly enhanced host survival and reduced parasite burden, demonstrating its potential as an effective toxoplasmosis vaccine candidate. These findings provide valuable data for future toxoplasmosis vaccine development.

Background: Toxoplasma gondii, an intracellular protozoan parasite, exists in the host brain as cysts, which can result in Toxoplasmic Encephalitis (TE) and neurological diseases. However, few studies have been conducted on TE, particularly on how to prevent it. Previous proteomics studies have showed that the expression of C3 in rat brains was up-regulated after T. gondii infection. Methods: In this study, we used T. gondii to infect mice and bEnd 3 cells to confirm the relation between T. gondii and the expression of C3. BEnd3 cells membrane proteins which directly interacted with C3a were screened by pull down. Finally, animal behavior experiments were conducted to compare the differences in the inhibitory ability of TE by four chemotherapeutic compounds (SB290157, CVF, NSC23766, and Anxa1). Results: All chemotherapeutic compounds in this study can inhibit TE and cognitive behavior in the host. However, Anxa 1 is the most suitable material to inhibit mice TE. Conclusion: T. gondii infection promotes TE by promoting host C3 production. Anxa1 was selected as the most appropriate material to prevent TE among four chemotherapeutic compounds closely related to C3.