AIM： To analyze the biological role of the surface antigen of Toxoplasma gondii（Tgondii） in development of vaccine. METHODS： The surface antigen of Tgondii （SAG1） was expressed in vitro. The immune response of t...AIM： To analyze the biological role of the surface antigen of Toxoplasma gondii（Tgondii） in development of vaccine. METHODS： The surface antigen of Tgondii （SAG1） was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of Tgondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on Tgondii tachyzoites under electromicroscope. RESULTS： The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-y, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-α were undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION： SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against Tgondii may be regulated by both hormone- and cell-mediated immune response.展开更多
OBJECTIVE: To characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis. METHODS: Mice wer...OBJECTIVE: To characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis. METHODS: Mice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally. RESULTS: Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival. CONCLUSION: Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.展开更多
Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ aga...Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ against Toxoplasma gondii (T gondii) infection in mice Methods A fragment of the IFN γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100?μg, and a booster vaccination was given at the same dosage after two weeks Control groups were injected with pcDNA3 blank plasmid or normal saline At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC ELISA for the determination of IFN γ, IL 2 and IL 10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera Results The recombinant plasmid, pcIFN γ was constructed The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN γ, IL 2 and NO in mice injected with pcROP1 and pcIFN γ were higher than in those injected with pcROP1 alone There was no difference in IgG antibody levels between the two groups Conclusion The genetic adjuvant, pcIFN γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection展开更多
To characterize the immune response induced by SAG1 encoding plasmid combined with IL 2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis Methods Mice were co inje...To characterize the immune response induced by SAG1 encoding plasmid combined with IL 2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis Methods Mice were co injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL 2 expression vector at a dose of 100 μg Booster immunizations were employed 2 more times at 3 week interval As controls, mice were inoculated with PBS or empty plasmid pcDNA3 Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN γ, as well as IL 4 To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally Results Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid With respect to the IgG isotype, co inoculation of IL 2 expression plasmid enhanced the level of IgG2a and the production of IFN γ Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival Conclusion Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co inoculation with IL 2 expression plasmid The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T gondii infection warrants further investigation展开更多
The dense granule protein 4 （GRA4） is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3, 1-GRA4 （pGRA4）, encoding for the GRA4 a...The dense granule protein 4 （GRA4） is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3, 1-GRA4 （pGRA4）, encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine （PC）, 8 mmol/L dioleoyl phosphatidylethanolamine （DOPE）, and 4 mmol/L 1,2-diodeoyl-3-（trimethylammonium） propane （DOTAP）. C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay （ELISA） of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon （IFN）-7 and interleukin （IL）-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th 1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.展开更多
基金Supported by China Ministry of Human Affairs and Department of Science and Technology of Shandong Province, No. 031050115
文摘AIM： To analyze the biological role of the surface antigen of Toxoplasma gondii（Tgondii） in development of vaccine. METHODS： The surface antigen of Tgondii （SAG1） was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of Tgondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on Tgondii tachyzoites under electromicroscope. RESULTS： The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-y, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-α were undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION： SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against Tgondii may be regulated by both hormone- and cell-mediated immune response.
文摘OBJECTIVE: To characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis. METHODS: Mice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally. RESULTS: Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival. CONCLUSION: Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.
文摘Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ against Toxoplasma gondii (T gondii) infection in mice Methods A fragment of the IFN γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100?μg, and a booster vaccination was given at the same dosage after two weeks Control groups were injected with pcDNA3 blank plasmid or normal saline At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC ELISA for the determination of IFN γ, IL 2 and IL 10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera Results The recombinant plasmid, pcIFN γ was constructed The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN γ, IL 2 and NO in mice injected with pcROP1 and pcIFN γ were higher than in those injected with pcROP1 alone There was no difference in IgG antibody levels between the two groups Conclusion The genetic adjuvant, pcIFN γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection
文摘To characterize the immune response induced by SAG1 encoding plasmid combined with IL 2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis Methods Mice were co injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL 2 expression vector at a dose of 100 μg Booster immunizations were employed 2 more times at 3 week interval As controls, mice were inoculated with PBS or empty plasmid pcDNA3 Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN γ, as well as IL 4 To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally Results Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid With respect to the IgG isotype, co inoculation of IL 2 expression plasmid enhanced the level of IgG2a and the production of IFN γ Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival Conclusion Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co inoculation with IL 2 expression plasmid The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T gondii infection warrants further investigation
基金supported by the Science Foundation of the Health Bureau of Zhejiang Province, China (Nos. 2003QN003 and 2005A001)the Science Foundation of the Science and Technology Department of Zhejiang Province, China (No. 2006C13022)
文摘The dense granule protein 4 （GRA4） is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3, 1-GRA4 （pGRA4）, encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine （PC）, 8 mmol/L dioleoyl phosphatidylethanolamine （DOPE）, and 4 mmol/L 1,2-diodeoyl-3-（trimethylammonium） propane （DOTAP）. C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay （ELISA） of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon （IFN）-7 and interleukin （IL）-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th 1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.
