目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预...目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。展开更多
Globally,about 70% of cervical cancers are associated with human papillomavirus (HPV)-16 or HPV-18 infection.A meta-analysis of epidemiologic studies in China showed that HPV was present in 98% of cervical cancer samp...Globally,about 70% of cervical cancers are associated with human papillomavirus (HPV)-16 or HPV-18 infection.A meta-analysis of epidemiologic studies in China showed that HPV was present in 98% of cervical cancer samples.The HPV-16/18 AS04-adjuvanted vaccine Cervarix has shown a high level of protection against HPV-16/18 infections and associated cervical lesions.This phase Ⅰ trial (NCT00549900) assessed the safety,tolerability,and immunogenicity of the vaccine in Chinese.Thirty healthy Chinese females,aged 15 to 45 years with a median age of 29.5 years,received three doses of Cervarix in Months 0,1,and 6.Safety was assessed via recording solicited local and systemic symptoms within 7 days and unsolicited symptoms within 30 days after each vaccination.Serious adverse events,new onset of chronic diseases,and other medically significant conditions were recorded throughout this trial.As an exploratory objective,HPV-16/18 antibody titers were determined by enzyme-linked immunosorbent assay in serum samples collected in Months 0 and 7.Pain at the injection site was the most frequently reported local symptom.Two subjects reported medically significant adverse events.Both cases were assessed as unrelated to vaccination by the investigator.In Month 7,100% seroconversion was observed for both anti-HPV-16 and anti-HPV-18 with high geometric mean antibody titers.HPV-16/18 AS04-adjuvanted vaccine,evaluated for the first time in Chinese females,was generally well tolerated and immunogenic,as previously shown in global studies.展开更多
Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by...Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.展开更多
In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used ...In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-induced 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β in lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1 （-）/E6 group and the LPS control group or the pcDNA3.1 （-） control group （P 〈 0.01）, but no significant difference existed between pcDNA3.1 （-） control group and LPS control group （ P 〉 0.05）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.展开更多
文摘目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。
文摘Globally,about 70% of cervical cancers are associated with human papillomavirus (HPV)-16 or HPV-18 infection.A meta-analysis of epidemiologic studies in China showed that HPV was present in 98% of cervical cancer samples.The HPV-16/18 AS04-adjuvanted vaccine Cervarix has shown a high level of protection against HPV-16/18 infections and associated cervical lesions.This phase Ⅰ trial (NCT00549900) assessed the safety,tolerability,and immunogenicity of the vaccine in Chinese.Thirty healthy Chinese females,aged 15 to 45 years with a median age of 29.5 years,received three doses of Cervarix in Months 0,1,and 6.Safety was assessed via recording solicited local and systemic symptoms within 7 days and unsolicited symptoms within 30 days after each vaccination.Serious adverse events,new onset of chronic diseases,and other medically significant conditions were recorded throughout this trial.As an exploratory objective,HPV-16/18 antibody titers were determined by enzyme-linked immunosorbent assay in serum samples collected in Months 0 and 7.Pain at the injection site was the most frequently reported local symptom.Two subjects reported medically significant adverse events.Both cases were assessed as unrelated to vaccination by the investigator.In Month 7,100% seroconversion was observed for both anti-HPV-16 and anti-HPV-18 with high geometric mean antibody titers.HPV-16/18 AS04-adjuvanted vaccine,evaluated for the first time in Chinese females,was generally well tolerated and immunogenic,as previously shown in global studies.
文摘Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.
文摘In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-induced 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β in lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1 （-）/E6 group and the LPS control group or the pcDNA3.1 （-） control group （P 〈 0.01）, but no significant difference existed between pcDNA3.1 （-） control group and LPS control group （ P 〉 0.05）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.
