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Evaluation of Immunogenicity of Cocktail DNA Vaccine Containing Plasmids Encoding Complete GRA5, SAG1, and ROP2 Antigens of Toxoplasma gondii in BALB/C Mice.

ABSTRACT: BACKGROUND:Severe and fatal complications of toxoplasmosis urge development of effective vaccines against the disease. The current study was performed to evaluate cocktail DNA vaccine containing plasmids encoding GRA5, SAG1, and ROP2 genes of Toxoplasma gondii in BALB/c mice in Tarbiat Modares University in 2012. METHODS:The plasmids containing complete GRA5, SAG1, and ROP2 genes were mass extracted and then the recombinant plasmids were administered via intramuscular injections according to immunized mice three times with three-week intervals. Then splenocytes were cultured, and proliferation as well as cytokine assays were carried out. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis. RESULTS:The results of cytokine assay for INF-? were higher in the mice that received the cocktail DNA containing recombinant plasmids. Evaluation of proliferation of splenocytes using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay indicated induction of cellular response. Measurement of total IgG and the isotypes of IgG1 and IgG2a showed that the cocktail DNA stimulated IgG and IgG2a production in comparison with the control groups (P<0.05). Furthermore, the survival rate of mice in the groups that received the cocktail DNA was significantly higher than that in the control groups (P<0.05). CONCLUSION:Administration of the cocktail DNA vaccine led to production of higher levels of IFN-?, confirmed by secretion of IgG2a, and the immune response was shifted toward Th1. Thus, the cocktail DNA containing the recombinant plasmids can be an appropriate candidate for immunization against toxoplasmosis.

SUBMITTER: Naserifar R

PROVIDER: S-EPMC4724836 | biostudies-literature | 2015 Oct-Dec

REPOSITORIES: biostudies-literature

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Evaluation of Immunogenicity of Cocktail DNA Vaccine Containing Plasmids Encoding Complete GRA5, SAG1, and ROP2 Antigens of Toxoplasma gondii in BALB/C Mice.

Naserifar Razi R Ghaffarifar Fatemeh F Dalimi Abdolhosein A Sharifi Zohreh Z Solhjoo Kavous K Hosseinian Khosroshahi Kami K

Iranian journal of parasitology 20151001 4

<h4>Background</h4>Severe and fatal complications of toxoplasmosis urge development of effective vaccines against the disease. The current study was performed to evaluate cocktail DNA vaccine containing plasmids encoding GRA5, SAG1, and ROP2 genes of Toxoplasma gondii in BALB/c mice in Tarbiat Modares University in 2012.<h4>Methods</h4>The plasmids containing complete GRA5, SAG1, and ROP2 genes were mass extracted and then the recombinant plasmids were administered via intramuscular injections a ...[more]

PMID: 26811726

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Project description:BACKGROUND:Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available. RESULTS:We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1's N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen. CONCLUSIONS:Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

Project description:Toxoplasmosis is a cosmopolitan zoonotic infection, caused by a unicellular protozoan parasite known as Toxoplasma gondii that belongs to the phylum Apicomplexa. It is estimated that over one-third of the world's population has been exposed and are latently infected with the parasite. In humans, toxoplasmosis is predominantly asymptomatic in immunocompetent persons, while among immunocompromised individuals may be cause severe and progressive complications with poor prognosis. Moreover, seronegative pregnant mothers are other risk groups for acquiring the infection. The life cycle of T. gondii is very complex, indicating the presence of a plurality of antigenic epitopes. Despite of great advances, recognize and construct novel vaccines for prevent and control of toxoplasmosis in both humans and animals is still remains a great challenge for researchers to select potential protein sequences as the ideal antigens. Notably, in several past years, constant efforts of researchers have made considerable advances to elucidate the different aspects of the cell and molecular biology of T. gondii mainly on microneme antigens, dense granule antigens, surface antigens, and rhoptry proteins (ROP). These attempts thereby provided great impetus to the present focus on vaccine development, according to the defined subcellular components of the parasite. Although, currently there is no commercial vaccine for use in humans. Among the main identified T. gondii antigens, ROPs appear as a putative vaccine candidate that are vital for invasion procedure as well as survival within host cells. Overall, it is estimated that they occupy about 1%-30% of the total parasite cell volume. In this review, we have summarized the recent progress of ROP-based vaccine development through various strategies from DNA vaccines, epitope or multi epitope-based vaccines, recombinant protein vaccines to vaccines based on live-attenuated vectors and prime-boost strategies in different mouse models.

Project description:BACKGROUND: Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. METHODS: In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10(4) tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. RESULTS: Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-?) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×10(4) tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05). CONCLUSIONS: The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.

Dataset Information

Evaluation of Immunogenicity of Cocktail DNA Vaccine Containing Plasmids Encoding Complete GRA5, SAG1, and ROP2 Antigens of Toxoplasma gondii in BALB/C Mice.

Publications

Evaluation of Immunogenicity of Cocktail DNA Vaccine Containing Plasmids Encoding Complete GRA5, SAG1, and ROP2 Antigens of Toxoplasma gondii in BALB/C Mice.

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