| Context: Toxoplasma gondii is a ubiquitous intracellular protozoan parasite which causes zoonotic disease known as Toxoplasmosis. It can infect all warm-blooded vertebrates including human beings and consequently has serious impact on public health and livestock sectors. So far, there is no effective treatment for this disease. Developing an effective vaccine against T. gondii infection is in urgent need.Objectives:In this study, we constructed a multiantigenic DNA vaccine expressing ROP16and GRA7of T. gondii and a recombinant plasmid encoding murine costimulatory molecule B7-2as a genetic adjuvant. After intramuscular immunization in Kunming mice, we assessed the protective efficacy of these two fragments with or without the genetic adjuvant B7-2. In this regard, it is supposed to provide a promising immunization strategy and contribute to the development of T. gondii vaccines.Methods:The ROP16gene and GRA7gene fragments were amplified by PCR from genomic DNA of T. gondii RH strain as well as the B7-2gene fragment was amplified by PCR from cDNA of murine spleens using their respective primers. PCR products were cloned into pEASY-T1vector and identified by sequencing in both directions. Then, these target genes were subcloned into the eukaryotic expression plasmid pEGFP-C1respectively to produce pEGFP-ROP16, pEGFP-GRA7, pEGFP-ROP16-GRA7and pEGFP-B7-2. After identification by restriction enzymes digestion, PCR and sequencing, these recombinant plasmids were transfected into HEK293T cells. Fluorescence microscopy, SDS-PAGE and Western blotting were performed to detect the target protein expressions. Large-scale plasmid preparation and purification were performed for the preparation of DNA vaccines. Mice were inoculated with DNA vaccine with or without the genetic adjuvant B7-2in each thigh skeletal muscle. After that, the immune responses were evaluated using cytokine and antibody determinations, T lymphocyte subsets analysis and the survival times of mice post acute T. gondii RH strain challenge.Results: The recombinant plasmids pEGFP-ROP16, pEGFP-GRA7, pEGFP-ROP16-GRA7and pEGFP-B7-2with the correct insert orientation were verified by PCR, restriction enzyme digestion and DNA sequencing. The sizes of ROP16, GRA7, ROP16-GRA7and B7-2were2124bp,708bp,2832bp and930bp, respectively. The green fluorescence was observed in HEK293T cells transfected with pEGFP-ROP16, pEGFP-GRA7, pEGFP-ROP16-GRA7, pEGFP-B7-2or pEGFP-C1, whereas no fluorescence was observed in the untransfected cells. Specific proteins at the size of76kDa,30kDa,106kDa and34kDa were recognized by Western blotting using anti-STAg mouse sera or anti-B7-2antibody. Higher levels of IgG titers and IgG2a subclass titers, IFN-y secretion and percentage of CD8+T cells were elicited in mice immunized with pEGFP-ROP16-GRA7compared to mice immunized with pEGFP-ROP16or pEGFP-GRA7(P<0.05). Introduction of B7-2further enhanced these humoral and cellular (Thl, CD8+T cell) immune responses as well as prolonged survival times were observed in these treatment groups after acute T. gondii challenge.Conclusions:Vaccination with T. gondii ROP16/GRA7elicited specific humoral, Th1, and CD8+T cell immune responses. Combining ROP16with GRA7provided a greater level of protective immunity than the single antigen. A genetic adjuvant B7-2was capable of improving the efficacy of T. gondii ROP16/GRA7DNA vaccines. Thus, it might be a promising genetic adjuvant to DNA vaccine against T. gondii for further investigation. |
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