Despite recent major advances in developing effective vaccines against toxoplasmosis, finding new protective vaccination strategies remains a challenging and elusive goal as it is critical to prevent the disease. Over the past few years, various experimental approaches have shown that developing an effective vaccine against T. gondii is achievable. However, more remains unknown due to its complicated life cycle, difficulties in clinical translation, and lack of a standardized platform. This minireview summarizes the recent advances in the development of T. gondii vaccines and the main obstacles to developing a safe, effective and durable T. gondii vaccine. The successes and failures in developing and testing vaccine candidates for the T. gondii vaccine are also discussed, which may facilitate the future development of T. gondii vaccines.

The immune system may aberrantly silence when against “altered self”, which consequently may develop into malignancies. With the development of tumor immunology and molecular biology, the deepened understanding of the relationship between parasites and tumors shifts the attitude towards parasitic pathogens from elimination to utilization. In recent years, the antitumor impact implemented by protozoan parasites and the derived products has been confirmed. The immune system is activated and enhanced by some protozoan parasites, thereby inhibiting tumor growth, angiogenesis, and metastasis in many animal models. In this work, we reviewed the available information on the antitumor effect of parasitic infection or induced by parasitic antigen, as well as the involved immune mechanisms that modulate cancer progression. Despite the fact that clinical trials of the protozoan parasites against tumors are limited and the specific mechanisms of the effect on tumors are not totally clear, the use of genetically modified protozoan parasites and derived molecules combined with chemotherapy could be an important element for promoting antitumor treatment in the future.

Toxoplasma gondii (T. gondii) can infect almost all warm-blooded animals and is a major threat to global public health. Currently, there is no effective drug or vaccine for T. gondii. In this study, bioinformatics analysis on B and T cell epitopes revealed that TGGT1_316290 (TG290) had superior effects compared with the surface antigen 1 (SAG1). TG290 mRNA-LNP was constructed through the Lipid Nanoparticle (LNP) technology and intramuscularly injected into the BALB/c mice, and its immunogenicity and efficacy were explored. Analysis of antibodies, cytokines (IFN-γ, IL-12, IL-4, and IL-10), lymphocytes proliferation, cytotoxic T lymphocyte activity, dendritic cell (DC) maturation, as well as CD4⁺ and CD8⁺ T lymphocytes revealed that TG290 mRNA-LNP induced humoral and cellular immune responses in vaccinated mice. Furthermore, T-Box 21 (T-bet), nuclear factor kappa B (NF-kB) p65, and interferon regulatory factor 8 (IRF8) subunit were over-expressed in the TG290 mRNA-LNP-immunized group. The survival time of mice injected with TG290 mRNA-LNP was significantly longer (18.7 ± 3 days) compared with the survival of mice of the control groups (p < 0.0001). In addition, adoptive immunization using 300 μl serum and lymphocytes (5*10⁷) of mice immunized with TG290 mRNA-LNP significantly prolonged the survival time of these mice. This study demonstrates that TG290 mRNA-LNP induces specific immune response against T. gondii and may be a potential toxoplasmosis vaccine candidate for this infection.

Toxoplasma gondii , which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii . The lowest limit of detection of the assay was 31 copies/μL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii , showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii . Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

Background: Toxoplasma gondiiis an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondiivaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.Methods: First, theompdcanduprtgenes ofT. gondiiwere deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel–Cox) test, Student’sttest and one-way ANOVA.Results: The RHΔompdcΔuprtwere constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8⁺T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%).Conclusions: The avirulent RHΔompdcΔuprtstrain can provide strong anti-T. gondiiimmune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.Graphical