Toxoplasma gondii (T. gondii) causes considerable financial losses in the livestock industry and can present serious
threats to pregnant women, as well as immunocompromised patients. Therefore, it is required to design and
produce an efficient vaccine for controlling toxoplasmosis. The present study aimed to evaluate the protective
immunity induced by RMS protein (ROP18, MIC4, and SAG1) with Freund adjuvant, calcium phosphate nanoparticles (CaPNs), and chitosan nanoparticles (CNs) in BALB/c mice. The RMS protein was expressed in
Escherichia coli (E. coli) and purified using a HisTrap HP column. Thereafter, cellular and humoral immunity was
assessed by injecting RMS protein on days 0, 21, and 35 into four groups [RMS, RMS-chitosan nanoparticles
(RMS-CNs), RMS-calcium phosphate nanoparticles (RMS-CaPNs), and RMS-Freund]. Phosphate buffered saline
(PBS), CNs, CaPNs, and Freund served as the four control groups. The results displayed that vaccination with
RMS protein and adjuvants significantly elicited the levels of specific IgG antibodies and cytokines against
toxoplasmosis. There were high levels of total IgG, IgG2a, and IFN-γ in vaccinated mice, compared to those in the
control groups, especially in the RMS-Freund, indicating a Th-1 type response. The vaccinated and control mice
were challenged intraperitoneally with 1 × 103 tachyzoites of the T. gondii RH strain four weeks after the last
injection, and in RMS-Freund and RMS-CaPNs groups, the highest increase in survival time was observed (15
days). The RMS can significantly increase Th1 and Th2 responses; moreover, multi-epitope vaccines with adjuvants can be a promising strategy for the production of a vaccine against toxoplasmosis
