BACKGROUND

Toxoplasma gondii, an intracellular parasitic protozoan, which infects almost all warm-blooded animals, including humans, causes toxoplasmosis. However, we lack effective drugs and vaccines to control toxoplasmosis, representing a clinical challenge. Therefore, safe and effective vaccines are urgently needed. In this study, a self-replicating mRNA vaccine comprising four T. gondii antigens: ROP18, TGME49_237490, TGME49_268230, and MIC13, named 4x-mRNA-LNP (lipid nanoparticle), was developed, and its protective efficacy was evaluated in mice.

METHODS

The expression of this vaccine in eukaryotic Human embryonic kidney 293 T (HEK-293 T) cells and mouse myoblast (C2C12) cells were analyzed, followed by enzyme-linked immunosorbent assay (ELISA) evaluation of the elicited humoral immune response. Subsequently, the vaccine-triggered immune responses in mice were detected, including antibody titers, T lymphocyte subsets, and cytokine levels. Finally, its immunoprotective effects were evaluated after challenging mice with T. gondii PRU oocysts or tachyzoites of different strains and analyzing the pathological changes, parasite loads, and mouse survival time. Western blotting and ELISA confirmed the successful eukaryotic expression and immunogenicity of 4x-mRNA, respectively. Statistical analyses, including the log-rank (Mantel-Cox) test, Student's t-test, and one-way ANOVA, were performed using GraphPad Prism software.

RESULTS

Mice vaccinated with 4x-mRNA-LNP generated higher levels of IgG1 and IgG2a antibodies (P < 0.05) and cytokines (IL-2, IL-4, IL-10, IL-12, IFN-γ) (P < 0.05) compared with the control group. The high specific IgG titer was maintained for at least 10 weeks after the last vaccination. The proportion of CD3CD4 T cells and CD3CD8 T cells also increased significantly (P < 0.05), along with increased spleen cell proliferation in 4x-mRNA-LNP-vaccinated mice. Notably, limited pathological changes and < 10 fg of parasites/mg were found in the immunized mice tissues post-pathogen challenge. During observation for 30 days, 4x-mRNA-LNP-immunized mice survived significantly longer under challenge with lethal doses of RH, ME49, or WH6 tachyzoites (survival rates = 60%, 80%, and 60%, respectively). Following PRU oocyst challenge, vaccinated mice had notably decreased cyst burdens (72.5%, P < 0.05) compared with control mice.

CONCLUSIONS

The 4x-mRNA-LNP vaccine triggered effective long-term antibody levels in mice, thus representing a promising candidate to further develop anti-toxoplasmosis vaccines.