Huaiyu Zhou

Evaluation of the immune response induced by multiantigenic DNA vaccine encoding SAG1 and ROP2 of Toxoplasma gondii and the adjuvant properties of murine interleukin-12 plasmid in BALB/c mice

The heavy incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. In the present study, we constructed a multiantigenic DNA vaccine, eukaryotic plasmid pcDNA3.1-SAG1-ROP2, expressing surface protein SAG1 and rhoptry protein ROP2 of Toxoplasma gondii, and examined the expression ability of the DNA vaccine in HeLa cells by Western blot. Afterwards, we investigated the efficacy of pcDNA3.1-SAG1-ROP2 with or without co-administration of a plasmid encoding murine interleukin-12 (pIL-12) as a genetic adjuvant to protect Bagg albino/c mice against toxoplasmosis. After T. gondii RH strain challenge, mice immunized with pcDNA3.1-SAG1-ROP2 displayed significant high survival rates. Moreover, the protection was markedly enhanced by pIL-12 co-administration. The results of lymphocyte proliferation assay, cytokine, and antibody determinations show that mice immunized with pcDNA3.1-SAG1-ROP2 elicited stronger humoral and Th1-type cellular immune responses than those immunized with single-gene plasmids, empty plasmid, or phosphate-buffered saline. Furthermore, co-immunization with IL-12 genes resulted in a dramatic enhancement of these responses. Our study indicates that the introduction of multiantigenic DNA vaccine is more powerful and efficient than single-gene vaccine, and the co-delivery of pIL-12 further enhanced the potency of multiantigenic DNA vaccine.

Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice

BackgroundToxoplasmosis, 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.MethodsIn 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 1×104 tachyzoites of T. gondii and the survival time of mice was observed and recorded every day.ResultsMice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) 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 1×104 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).ConclusionsThe 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.

Vaccines against Toxoplasma gondii: Status, challenges and future directions

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect a wide range of animals including humans. This single known species in the genus Toxoplasma is considered as one of the most successful eukaryotic pathogens which is of major medical and veterinary importance. Effective vaccines may contribute toward preventing and controlling the spread of toxoplasmosis. The present communication addresses the current status of development of vaccines against T. gondii. Further discussion is made on the difficulties along with challenges, such as vaccine construct, mode of vaccine administration and standardization of immunization evaluation. Finally suggestions are made on possible directions for future research on the development of vaccines against T. gondii.

Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis

BackgroundToxoplasma gondii is an obligate intracellular parasite that can pose a serious threat to human health by causing toxoplasmosis. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance. Aspartic proteases play essential roles in the T. gondii lifecycle. The parasite has four aspartic protease encoding genes, which are called toxomepsin 1, 2, 3 and 5 (TgASP1, 2, 3 and 5, respectively).MethodsBioinformatics approaches have enabled us to identify several promising linear-B cell epitopes and potential Th-cell epitopes on TgASP1, thus supporting its potential as a DNA vaccine against toxoplasmosis. We expressed TgASP1 in Escherichia coli and used the purified protein to immunize BALB/c mice. The antibodies obtained were used to determine where TgASP1 was localized in the parasite. We also made a TgASP1 DNA vaccine construct and evaluated it for the level of protection conferred to mice against infection with the virulent RH strain of T. gondii.ResultsTgASP1 appears to be a membrane protein located primarily at the tip of the T. gondii tachyzoite. Investigation of its potential as a DNA vaccine showed that it elicited strong humoral and cellular immune responses in mice, and that these responses were mediated by Th-1 cells. Mice immunized with the vaccine had greater levels of protection against mortality following challenge with T. gondii RH tachyzoites than did those immunized with PBS or the empty vector control.ConclusionsTgASP1 is a novel candidate DNA vaccine that merits further investigation.

Toxoplasma gondii: expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris

Toxoplasma gondii is an obligate intracellular protozoan which infects most species of warm-blooded animals and causes toxoplasmosis. Previous immunological and immunization studies have demonstrated the potential role of T. gondii antigens SAG1 and GRA2 as a vaccine candidate. In the present study, we have cloned, expressed, and purified a recombinant protein SAG1–GRA2 in Pichia pastoris. Results showed that P. pastoris was a robust system producing a large amount of highly purified and biological activity protein. BALB/c mice immunized with SAG1–GRA2 elicited stronger humoral and cellular responses in comparison to control groups. This immunization resulted in an enhanced Th1 immune response as measured by IgG2a antibody production and increased splenocyte IFN-γ production, whereas no IL-4 was detected. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in SAG1–GRA2-immunized mice was observed in comparison to control groups. Our data demonstrate that SAG1–GRA2 triggered a protective response against toxoplasmosis. Therefore, SAG1–GRA2 protein might be a good candidate for the further development of a multiantigenic vaccine.

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA− and aroD− attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG1‐2/CTA2/B expressing a multi‐antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti‐T. gondii IgG values increased markedly in the BRD509/pSAG1‐2‐CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0·008). With CTA2/B genetic adjuvant, the T. gondii‐specific response was predominantly Th1, indicating that the CTA2/B genetic adjuvant was able to overcome the strong Th2‐bias of the antigen (IgG2a >> IgG1). Antigen‐specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0·009; P = 0·006). Culture supernatants from antigen‐stimulated splenocytes from mice in these groups were also examined by ELISA for Th1‐ and Th2‐type cytokines; mean IFN‐γ levels produced after oral immunization with BRD509/pSAG1‐2‐CTA2/B were about nine‐fold higher than after immunization with BRD509/pSAG1‐2 (P = 0·007). On the other hand, the levels of IL‐4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG1‐2‐CTA2/B was markedly longer than other groups (P = 0·003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.

Protective immune response against Toxoplasma gondii elicited by a recombinant DNA vaccine with a novel genetic adjuvant.

Previous immunological studies from our laboratory have demonstrated the potential role of Toxoplasma gondii antigens SAG1 and GRA2 as vaccine candidates. To further evaluate the vaccines effects, a series of recombinant DNA vaccines pVAX1-SAG1, pVAX1-GRA2 and pVAX1-SAG1-GRA2, termed pSAG1, pGRA2 and pSAG1-GRA2, respectively, were constructed. A plasmid pVAX1-S/PreS2, termed pSPreS2 encoding hepatitis B virus (HBV) surface antigen (HBsAg) S and PreS2 as a novel genetic adjuvant, was also constructed. The expression abilities of those DNA plasmids were examined in HFF cells by Western blotting. Then BALB/c mice were intramuscularly immunized with DNA plasmids and followed by challenging with the highly virulent T. gondii RH strain. The results demonstrated that the recombinant DNA vaccine pSAG1-GRA2 was capable of eliciting high levels of antibodies, a Th1 type of immune response with significant production of IFN-γ and low levels of IL-4 or IL-10 in BALB/c mice, and partial protection against the acute phase of toxoplasmosis as compared to pSAG1, pGRA2 and controls. In addition, the adjuvant pSPreS2 formulated with DNA vaccine induced a Th1 type of immune response and therefore might be a novel genetic adjuvant to DNA vaccine for further investigation.

Enhancement of protective immune responses induced by Toxoplasma gondii dense granule antigen 7 (GRA7) against toxoplasmosis in mice using a prime-boost vaccination strategy

Effective vaccines against Toxoplasma gondii may contribute to preventing and controlling the spread of toxoplasmosis, which is important for improving outcomes of infections in humans and livestock animals. The dense granule antigen 7 (GRA7) of T. gondii might be an immunodominant antigen for a vaccine candidate. In the present study, a further exploration of its vaccine effect, a heterologous prime-boost vaccination strategy with a recombinant eukaryotic plasmid pEGFP-GRA7 and a recombinant protein GRA7 expressed from a prokaryotic plasmid pET30-GRA7, was performed in BALB/c mice. The data reveal that a DNA prime-protein boost vaccination induces both humoral and cellular immune responses against T. gondii associated with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ). Challenge experiments further show that the DNA prime-protein boost vaccination significantly increases survival rate (60%), compared with controls in which all died within 8 days of challenge. Therefore, the DNA prime-protein boost vaccination based on GRA7 might be a promising regimen for further development of an effective vaccine against T. gondii.

Toxoplasma gondii: bioinformatics analysis, cloning and expression of a novel protein TgIMP1.

Toxoplasma gondii is an obligate intracellular protozoan parasite, infecting a large variety of animals and human beings. In recent years, the study of DNA vaccine against T. gondii has made a great progress; however, few vaccines have completely controlled toxoplasmosis. Thus people started to look for more effective antigenic proteins. Here we report a novel T. gondii protein termed immune mapped protein 1 (TgIMP1). We used multiple bioinformatics approaches to predict the physical and chemical characters, signal peptide, transmembrane domain, epitope, topological structure and function of the protein, and we theoretically determined that the TgIMP1 has multiple epitopes, and with immunogenicity, suggesting that the TgIMP1 may be a vaccine candidate against toxoplasmosis. Then the gene coding TgIMP1 was obtained by PCR and connected with cloning vector. Recombinant plasmid was identified by PCR, double digestion and sequencing analysis. Then the TgIMP1 gene was directly inserted into the eukaryotic expression vector pBudCE4.1, so that the recombinant eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed. After identification by PCR and restriction enzyme digestion, the recombinant plasmid pBudCE4.1-TgIMP1 was transfected into cells of HFF, and then identified by RT-PCR. The results showed that the eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed and was transfected to the HFF cells successfully.

Compound DNA vaccine encoding SAG1/ SAG3 with A2/B subunit of cholera toxin as a genetic adjuvant protects BALB/c mice against Toxoplasma gondii

BackgroundIntracellular parasites, such as T. gondii, present a plurality of antigens because of the complexity of its life cycle. Compound DNA vaccines bring a new approach and hope for the treatment of toxoplasmosis. In this study, a DNA vaccine encoding two major surface antigens SAG1, SAG3 from T. gondii, with A2/B subunit of cholera toxin as a genetic adjuvant was constructed.MethodsBALB/c mice were immunized intramuscularly with PBS, pcDNA3.1, pSAG1, pSAG1/SAG3 and pSAG1/SAG3-CTXA2/B three times separately. Immunized mice were tested for IgG antibody and IFN-γ and IL-4 production by ELISA. The proliferation of T cells was measured by DNA synthesis assay and the lymphocyte subsets of spleen cells by flow cytometry. All the immunized mice were challenged with 103 highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally and the survival times were recorded.ResultsAn enhanced production of IgG antibodies, antigen-specific lymphocyte proliferation and IFN-γ production from splenic cells were induced in mice immunized with pSAG1/SAG3 compared to mice immunized with pSAG1 (P<0.05). Introduction of CTXA2/B further enhanced the Th1 cell-mediated immunity with higher levels of IFN-γ, lymphocyte proliferation activity and percentage of CD8+ T-cells. When challenged with lethal doses of T. gondii (1×103), all control mice (PBS and empty plasmid group) died within 6 days. Mice immunized with pSAG1 died within 8 days. While 20% and 40% survival rate were achieved from mice immunized with pSAG1/SAG3 and pSAG1/SAG3-CTXA2/B.ConclusionsThis study indicates the compound DNA vaccine encoding T. gondii antigens SAG1, SAG3 with CTXA2/B gene was a promising DNA vaccine candidate against toxoplasmosis, which could effectively enhance the humoral and cellular immune response and prolong survival time in vaccinated mice.