Immunogenicity and Protective Efficacy of a Recombinant Toxoplasma gondii GRA12 Vaccine in Domestic Cats

<b>Background:</b><i>Toxoplasma gondii</i> (<i>T. gondii</i>) 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. <b>Methods:</b> We expressed recombinant GRA12 protein in <i>E. coli</i> 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 × 10⁶<i>T. gondii</i> RH tachyzoites. Oocyst shedding, survival duration, and <i>T. gondii</i> burden were monitored to assess vaccine-induced immunity. <b>Results:</b> 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 (<i>p</i> < 0.05), while IL-10 remained unchanged. After <i>T. gondii</i> 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 (<i>p</i> < 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 (<i>p</i> < 0.01). <b>Conclusions:</b> 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.