| Toxoplasma gondii is an intracellular opportunistic protozoon, tachyzoites of parasite may convert to bradyzoites in the presence of cysts when the proliferation was inhibited in immunocompetent individuals. Cysts existed in eye and brain could last a few years or even a lifetime which causing central nervous system damage and visual impairment to the host. However, bradyzoites in the cysts might proliferate fast in patients suffering from immunodeficiency or low immunity (e.g. cancer or organ transplant recipients) that leading to severe clinical symptoms and consequences. For instance, the activation of bradyziotes to tachyzoites might be the main reason for the death of AIDS patients. Therefore, prevention or control of chronic infections caused by bradyzoites has an extremely important significance.Currently, there was no effective chemical drugs to treat toxoplasmosis. An effective vaccine is considered to be the best strategy to control the infection. Antigen study of T. gondii is mainly focusing on tachyzoite stage, but fewer on bradyzoite stage. However, damage caused by bradyzoite in cysts is more serious. Vaccine investigation that consisted of bradyzoite-specific antigen as a target antigen is very important to prevent T. gondii chronic infection. SAG2C/2D/2X which belong to T. gondii surface antigen SAG2superfamily, are specifically expressed on bradyzoites stage, SAG2C and/or SAG2D has(have) been demonstrated to be expressed before the conversion of tachyzoites to bradyzoites by immunofluorescence analysis, and SAG2C/2D/2X are further proved to play an important role for cysts in the mechanism of presence in the brain tissue. Otherwise, potential capacity of SAG2C/2D/2X as vaccine candidates against toxoplasmosis has not been yet studied and evaluated.In this study, we constructed the recombinant eukaryotic plasmid DNA vaccines pVAXl-SAG2C (pSAG2C), pVAXl-SAG2D (pSAG2D), pVAXl-SAG2X (pSAG2X), and examined their immunogenicity and protective effects in BALB/c mice challenged with the T. gondii type ⅡPRU strain. Mice vaccinated with pSAG2C, pSAG2D, or pSAG2X showed higher levels of serum IgG antibodies when compared with empty plasmid or PBS groups(d35, p<0.05), especially pSAG2C, pSAG2X and co-immunized groups, IgG antibody titers increased4weeks after the last immunization, and the difference was statistically significant(p<0.01). ELISA assays showed a significant difference in IL-2production by mice immunized with pSAG2C, pSAG2D, or pSAG2X(p<0.05), and the co-immunized group was detected the. highest secretion (486.3±79.1)pg/m, obviously higher than single gene treated and control groups(p<0.01); however, there was no difference in IL-10production between tested and control groups (p>0.05). Additionally, splenocyte proliferation in vitro indicated the strong immunogenicity of the recombinant plasmids. The immune response was characterized by a strong Thl response and increased expression of IFN-y secreted by CD8+T lymphocytes. Mice were intragastrically with T. gondii PRU strain cysts to evaluate the protective effects of DNA vaccines. A significant cyst burden reduction rate was calculated in the mice immunized with pSAG2C, pSAG2D, or pSAG2X,72%,23%and70%respectively when compared with PBS group(p<0.05). In particular, the cyst number of combination immunized group was263±50(reduction rate77%), which were fewer than any of the single plasmid vaccinated mice group(p<0.01).Besides, we analyzed the structure and homology of protein SAG2C/2D/2X by using bioinformatics softwares. Alignment result of the three protein sequences is98%, which indicating that SAG2C/2D/2X might be homologous proteins. The domain of SAG2C/2D/2X protein was characterized with the plurality of (3sheet, that may be associated with the predicted protein structure function. We also predicted and screened6CD8+T cell epitopes, which have high affinity score (Percent Rank<10%) to MHC (H-2)molecules. Immunogenicity of the epitopes was evaluated by immunizing BALB/c mice. Finally, immunized mice were challenged with the cyst of T. gondii type ⅡPRU strain to define the protective efficacy of the epitopes.Immune response induced by epitope vaccinated mice was much efficient and vigorous than control groups; the cyst burden of epitopes immunized mice reduced to some extent. Furthermore, peptides TSTTKSVTF and QALVPNSSL identified from SAG2C, SAG2D, and SAG2X in vitro lymphocyte proliferation essay displayed better immunogenicity which might be excellent candidates for epitope vaccine and targeted therapy study in the future.In brief, DNA vaccines encoding bradyzoite-specific antigen SAG2C/2D/2X of T. gondii could induce BALB/c mice to produce a comprehensive humoral and cellular immune responses, and generate efficient protection for the immunized mice by reduction of the cysts number in the mice brains. In addition, the screened antigen epitopes immune mice can effectively stimulate mouse memory T lymphocyte proliferation, which can be used as the epitope vaccines and targeted therapeutic candidate compositions, provides a candidate component effective for prevention and treatment chronic infection of T. gondii. |
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