全酿酒酵母HPV16-E7疫苗的构建及免疫原性研究

目的构建人乳头状瘤病毒16型（HPV16）E7重组全酿酒酵母疫苗,评价疫苗的免疫效应。方法将HPV16 E7 cDNA亚克隆人酵母表达载体pYES2／NT,重组质粒转化酿酒酵母,经诱导表达、热灭活制备全酵母HPV16-E7疫苗,免疫C57BL／6小鼠。ELISA、MTT、LDH法分别检测疫苗诱导的细胞因子、脾淋巴细胞增殖和细胞毒性T淋巴细胞（CTL）应答水平。结果全酵母疫苗能够有效表达HPV16-E7蛋白;疫苗小鼠免疫,能够刺激脾淋巴细胞增殖,并诱导出Th1型细胞因子和特异性CTL杀伤效应;免疫效果优于单纯HPV16-E7蛋白免疫（P〈0．01）。结论全酵母疫苗既能表达HPV16-E7蛋白,又能有效诱导特异性CTL免疫应答。研究结果为进一步进行疫苗抗肿瘤疗效研究,提供了实验基础。     

Human papillomavirus type 16 E7 peptide(38-61) linked with an immunoglobulin G fragment provides protective immunity in mice

OBJECTIVE: To explore whether the recombinant protein (Human papillomavirus (HPV) type16 E7 peptide(38-61) linked with an immunoglobulin G fragment) will generate protective immunity in mouse model. METHODS: In our study, we combined the HPV16 E7 peptide(38-61) with a murine IgG heavy chain constant region to construct a chimeric protein compound, which was highly expressed as inclusion bodies in a bacterial expression system with Escherichia coli. The purified chimeric protein was injected into C57BL/6 mice and the efficiency of the chimeric vaccine candidate was evaluated by antibody response assay, T cell proliferation assay, CTL assay, tumor challenge assay and therapeutic experiment. RESULTS: The chimeric vaccine candidate was able to induce anti-HPV antibodies as well as to elicit HPV16 E7-specific CTLs and T cell proliferation in a pre-clinical mouse model. It was also able to effectively protect mice against the challenge of HPV16-positive tumor cells, and to eradicate HPV16-expressing tumors in mice. CONCLUSIONS: The chimeric protein vaccine can induce E7-specific immune responses and protect mice against challenge of HPV16-positive tumor, even eradicate developed tumor. The results indicated a possibility to use the chimeric protein vaccine to protect human against HPV infection.     

Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA

ObjectiveTo generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine.Materials and methodsWe have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA.ResultsThe cGM–CSF–E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM–CSF–E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ.ConclusionThrough in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.     

腺病毒伴随病毒表达载体表达人乳头状瘤病毒16型E6/E7基因的构建及应用

目的　为了了解人乳头状瘤病毒 (Humanpapillomavirus ,HPV) 1 6型的E6 /E7基因在细胞恶性转化中所起的作用 ,利用腺病毒伴随病毒载体 (AAVHelper -FreeSystem)构建和表达人乳头状瘤病毒 1 6型E6 /E7基因。方法　在pLXSN1 6E6E7质粒中经PCR扩增回收HPV 1 6E6E7基因片段 ,连接于T载体上进行测序 ,将正确的HPV 1 6E6E7插入pAAV -IRES -hrGFP质粒 ,协同pAAV -RC质粒和pHelper质粒共转染HEK 2 93细胞 ,包装表达HPV 1 6E6E7基因的重组腺病毒伴随病毒 ,收获病毒 ,并检测病毒的感染效率。结果　在包装细胞系HEK 2 93细胞中能形成较高感染效率的腺病毒伴随病毒 ,激光共聚焦检测可发现HEK 2 93细胞内有绿色荧光蛋白表达 ,HEK 2 93细胞经PCR可扩增出特异性的HPV 1 6E6E7基因片段 ,经流式细胞仪检测重组病毒的感染效率为71 3%。结论　携带人乳头状瘤病毒 1 6型E6E7基因的腺病毒伴随病毒可感染细胞 ,并在细胞内表达 ,可望用于宫颈癌病因学的研究     

Modified HPV16 E7/HSP70 DNA vaccine with high safety and enhanced cellular immunity represses murine lung metastatic tumors with downregulated expression of MHC class I molecules

OBJECTIVE: To explore whether the modified E7-HSP70, which has been introduced mutations in two zinc-binding motifs of E7, will eliminate its transformation potential and enhance the immunogenicity of fusion protein and repress E7 containing tumors with a low level of MHC-I molecules to lung metastatic in murine model. METHODS: In this study, we examined the transforming properties of mutant E7 oncoprotein by the soft agar colony-formation assays, explored the immunogenicity of modified E7-HSP70 gene by various cellular and humor immune responses and evaluated the effect of treating lung metastatic tumor with a low expressing MHC-I molecules by tumor challenge assay and therapeutic experiment. RESULTS: The mutant E7 oncoprotein has completely lost its transforming properties as measured in the soft agar colony-formation assays. Modified E7-HSP70 gene inducted stronger E7-specific cellular immune response than that induced by unmodified E7-HSP70. More importantly, the new construct significantly reduced the number of B16-HPV16E7 lung metastases. CONCLUSION: The modified E7-HSP70 gene may be as a powerful and safe DNA vaccine in controlling the hematogenous spread of HPV16E7-associated tumors with low expression of MHC-I molecules. In addition, the B16-HPV16E7 lung metastasis model can be used to test the efficacy of various E7-specific vaccines and immunotherapeutic strategies in settings more relevant to clinical requirements.     

Absence of human papillomavirus E6-E7 transforming genes from HPV 16 and 18 in malignant ovarian carcinoma

Ovarian carcinoma is one of the frequent causes of death from malignancies in the United States. A report excited the scientific community when human papillomavirus were identified in advanced epithelial ovarian carcinoma tissues in 10 of 12 patients. A few studies also identified HPV DNA in ovarian carcinoma tissues. However, several researchers employing polymerase chain reaction techniques and using different oligonucleotide probes did not detect HPV DNA in ovarian carcinoma tissues. The objective was to determine the presence of the E6-E7 genes of HPV types 16 and 18 in archived paraffin-embedded malignant ovarian carcinoma using primers targeting. Archived human malignant ovarian cancer tissues (N = 20 cases) embedded in paraffin blocks were processed, and DNA was extracted and the presence of DNA verified by p53 amplifications. PCR analyses were performed on the extracted DNA together with appropriate controls. The results showed an absence of E6-E7 genes of HPV types 16 and 18 in ovarian carcinoma. However, the presence of other HPV types or gene regions is not ruled out and more studies are needed to resolve the question of HPV involvement in ovarian carcinogenesis.     

反义HPV16 E6/E7-EGFP重组质粒的构建及其对宫颈癌SiHa细胞的促凋亡作用

目的:构建HPV16早期基因E6/E7的反义重组质粒,探讨其对SiHa细胞的促凋亡作用。方法:将HPV16E6/E7基因片段反向克隆于真核表达载体pEGFP-C1并转染SiHa细胞,用RT-PCR方法检测转染后SiHa细胞E6、E7基因mRNA的表达,West-ernblot方法检测转染后E6/E7蛋白的表达,流式细胞仪检测转染后细胞的凋亡率。结果:成功构建携带HPV16E6/E7基因反义片段的真核表达载体,转染该质粒后,SiHa细胞E6、E7基因的mRNA和蛋白均明显下调;转染后细胞凋亡率为(59.3±11.3)%,明显高于转染空载体组[(9.4±1.8)%]和未转染组[(2.1±0.4)%](P<0.05)。结论:反义HPV16E6/E7基因可下调宫颈癌细胞中E6/E7癌基因的表达,诱导宫颈癌细胞凋亡,为宫颈癌的基因治疗提供了实验依据。     

HPV16/18 E7-pulsed dendritic cell vaccination in cervical cancer patients with recurrent disease refractory to standard treatment modalities

OBJECTIVE: To evaluate the potential of human papillomavirus (HPV) type 16 and 18 E7 antigen-loaded autologous dendritic cells (DC) as a therapeutic cellular vaccine in a case series of cervical cancer patients harboring recurrent/metastatic disease refractory to standard treatment modalities. METHODS: Autologous monocyte-derived DC were pulsed with recombinant HPV16 E7 or HPV18 E7 oncoproteins and administered to 4 cervical cancer patients. Vaccinations were followed by subcutaneous administration twice daily of low doses of human recombinant interleukin-2 (1 x 10(6) IU/m2) from day 3 to day 7. Safety, toxicity, delayed type hypersensitivity reactions (DTH), clinical responses, and induction of serological and cellular immunity against HPV16/18 E7 were monitored. RESULTS: The vaccine was well-tolerated in all patients and no local or systemic side effects or toxicity were recorded. Three out of four patients were found to be significantly immunocompromised before starting the vaccination treatment, as assessed by DTH with a panel of recall antigens. Specific humoral and cellular CD4+ T cell responses to the E7 vaccine were detected in 2 patients, as detected by ELISA and by IFN-gamma ELISpot assays, respectively. Increased numbers of E7-specific IFN-gamma secreting CD8+ T cells were detected in all patients after vaccination. Swelling and induration (i.e., a positive DTH response) to the intradermal injection of HPV E7 oncoprotein and/or irradiated autologous tumor cells were detected in two patients after six vaccinations. No objective clinical responses were observed. However, both patients who developed a positive DTH to the vaccine experienced a slow tumor progression (i.e., 13 months survival) while DTH unresponsive patients died within 5 months from the beginning of therapy. CONCLUSIONS: Autologous DC pulsed with HPV16/18 E7 proteins can induce systemic B and T cell responses in patients unresponsive to standard treatment modalities. However, treatment-induced immunosuppression may impose severe limitations on the efficacy of active vaccination strategies in late stage cervical cancer patients. DC-based vaccination trials are warranted in immunocompetent cervical cancer patients with early stage disease and/or limited tumor burden, and at significant risk for tumor recurrence or disease progression.     

Human papillomavirus type 16 E6 and E7 gene variations in Indian cervical cancer

OBJECTIVES: Human papillomavirus type 16 is a causative factor for development of cervical cancer. The E6 and E7 genes of HPV 16 are critical to the process of immortalization and transformation of host cells. Recent reports suggest that variants of these two genes may contribute to the risk of malignant progression of cancer in the uterine cervix. However, no data exist on sequence variations of HPV 16 E6 and E7 genes that may exist in India. Therefore, we examined intratype variations in the E6 and E7 viral genes in DNA isolated from HPV 16-positive cervical scrapes and biopsies. METHODS: The open reading frames of the E6 and E7 genes were amplified by PCR and then directly sequenced by the fluorescent dye dideoxy termination method.Results. In addition to the prototype E6 gene sequence, five sets of mutations of the E6 gene were identified. The European prototype (350T) was detected in 9.1% of the study group while the European variant (350G) was seen in 28% of patients. The remaining variants (a combination of the 350G mutation with 335T, 145T, or 419G) were significantly associated with cases compared to controls. The 350G + 145T variant was found at much higher incidence in cases in younger women, suggesting that this variant may be associated with aggressive tumor behavior. Interestingly the 350G + 419G combination was found only in controls. There was no significant association between the four genotypes of E7 and any stage of tumor progression or age. CONCLUSIONS: The results indicate that specific mutations in the E6 gene are found in young Indian women with high-grade squamous intraepithelial lesions and invasive cancer, suggesting that these mutations represent more oncogenically active HPV 16. Whether this increased oncogenecity is due to differences in p53 inactivation, ineffective keratinocyte differentiation, and/or altered response to the immune system by these oncogenic E6 mutants remains to be clarified.     

含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1-E7AS的构建及鉴定

目的:构建含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1- E7AS并鉴定,探讨腺相关病毒介导的反义E7基因技术用于治疗早期宫颈癌的可能性。方法:使用RT -PCR法扩增全长HPV16型E7基因,利用基因重组法将目的片段反向插入腺相关病毒骨架质粒pUF1并酶切鉴定。结果:RT PCR法扩增全长315bp的HPV16型E7基因,装入pGEM -Teasy载体进行测序,经NCBI数据库blast检索为100%的符合,经基因重组获得含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS,AccⅠ和KpnⅠ双酶切鉴定pUF1 E7AS。结论:含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS可成功构建。     

人乳头状瘤病毒16型E7基因重组腺相关病毒载体的构建及E7 mRNA和蛋白在真核细胞中的表达

目的构建含人乳头状瘤病毒16型E7(HPV16E7)基因的重组腺相关病毒(rAAV)载体,并验证HPV16E7mRNA和蛋白在真核细胞中的表达。方法将含腺相关病毒(AAV)末端反向重复序列及HPV16E7基因的重组质粒pAAVMCSE7、含rep/cap基因的质粒pAAVRC及辅助质粒pAAVhelper共同转染胚胎肾细胞系HEK293细胞,回收、纯化病毒颗粒并鉴定,斑点杂交法测定病毒滴度,RTPCR技术、蛋白印迹法验证HPV16E7mRNA和蛋白在真核细胞中的表达。结果电镜鉴定结果显示真核细胞中有病毒颗粒存在,斑点杂交法测定病毒滴度为1×1011/ml。含HPV16E7基因的rAAV载体转染真核细胞后,在细胞内可检测到HPV16E7mRNA和蛋白的表达。结论本实验成功构建了含HPV16E7基因的rAAV载体,经验证HPV16E7mRNA和蛋白在真核细胞内有表达。     

子宫颈癌组织中人乳头状瘤病毒16 E6 mRNA表达与survivin蛋白表达的相关性

目的探讨宫颈癌组织中人乳头状瘤病毒(HPV)16E6mRNA表达与survivin蛋白表达的相关性。方法采用半定量PCR技术和免疫组化链霉菌抗生物素蛋白-过氧化物酶连接法,检测慢性宫颈炎、宫颈上皮内瘤变(CIN)及宫颈癌共148例患者宫颈组织中HPV16E6mRNA及survivin蛋白的表达。结果148例患者中,HPV16阳性共37例,其中慢性宫颈炎5例、CINⅠ6例、CINⅡ～Ⅲ11例及宫颈癌15例。慢性宫颈炎、CINⅠ、CINⅡ～Ⅲ及宫颈癌组织中,HPV16E6mRNA的表达水平分别为0.3±0.4、0.6±0.4、1.8±0.6及2.4±0.6;survivin蛋白阳性表达率分别为7%、31%、63%及84%。CINⅡ～Ⅲ及宫颈癌组织中,HPV16E6mRNA的表达水平及survivin蛋白阳性表达率均显著高于慢性宫颈炎及CINⅠ组织,两者比较,差异均有统计学意义(P<0.01)。宫颈癌组织中,HPV16E6mRNA的表达水平与survivin蛋白阳性表达率呈显著正相关关系(γs=0.62,P<0.05)。结论HPV16E6mRNA的表达水平与宫颈病变的进展有关,survivin蛋白阳性表达率升高可能与宫颈癌的发生、发展密切相关。     

FHIT基因、HPV16E6的表达与宫颈鳞癌发病的相关性研究

目的：探讨抑癌基因脆性组氨酸三联（fragile histine triad,FHIT）基因、人乳头瘤病毒16E6（HPV16E6）蛋白在宫颈鳞癌中的表达及其相互关系。方法：应用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连接（SP）法观察40例宫颈鳞癌、30例宫颈上皮内瘤样病变（CIN）和30例正常宫颈组织中FHIT基因、HPV16E6的表达。结果：FHIT基因在正常宫颈组织、CIN和宫颈鳞癌组织中的阳性表达率分别为96.7%,66.7%和30.0%,各组间阳性等级表达比较差异有统计学意义（χ2=43.595,P〈0.001）。FHIT基因阳性表达在宫颈鳞癌病理分级、临床分期中的比较,差异无统计学意义（χ2分别为3.378和3.315,均P〉0.05）。HPV16E6在正常宫颈组织、CIN和宫颈鳞癌组织中的阳性表达率分别为13.3%,53.3%和82.5%,各组间阳性等级表达比较差异有统计学意义（χ2=32.538,P〈0.001）。HPV16E6蛋白阳性表达在宫颈鳞癌病理分级、临床分期中的比较,差异无统计学意义（χ2分别为0.231和1.399,均P〉0.05）。结论：宫颈鳞癌中FHIT基因表达减少或缺失,以及HPV16E6的高检出率,提示二者在宫颈鳞癌的发生发展中起重要作用,可能是宫颈鳞癌发病机制之一。通过对FHIT基因和HPV16E6蛋白的致病机制以及相互影响的研究,有助于揭示宫颈癌发病机制,二者有可能用于临床宫颈癌诊断、预防和基因治疗。     

Efficacy of human papillomavirus-16 vaccine to prevent cervical intraepithelial neoplasia: a randomized controlled trial

OBJECTIVE: Human papillomavirus (HPV) virus-like particle (VLP) vaccines have demonstrated effectiveness in preventing persistent HPV infections. Whether protection lasts longer than 18 months and, thus, impacts rates of cervical intraepithelial neoplasia (CIN) 2-3 has not yet been established. We present results from an HPV16 L1 VLP vaccine trial through 48 months. METHODS: A total of 2,391 women, aged 16-23 years, participated in a randomized, double-blind, placebo-controlled trial. Either 40 mug HPV16 L1 VLP vaccine or placebo was given intramuscularly at day 1, month 2, and month 6. Genital samples for HPV16 DNA and Pap tests were obtained at day 1, month 7, and then 6-monthly through month 48. Colposcopy and cervical biopsies were performed if clinically indicated and at study exit. Serum HPV16 antibody titer was measured by radioimmunoassay. RESULTS: Among 750 placebo recipients in the per protocol population, 12 women developed HPV16-related CIN2-3 (6 CIN2 and 6 CIN3). Among 755 vaccine recipients, there were no cases (vaccine efficacy 100%, 95% confidence interval [CI] 65-100%). There were 111 cases of persistent HPV16 infection in placebo recipients and 7 cases in vaccine recipients (vaccine efficacy 94%, 95% CI 88-98%). After immunization, HPV16 serum antibody geometric mean titers peaked at month 7 (1,519 milli-Merck units [mMU]/mL), declined through month 18 (202 mMU/mL), and remained relatively stable between month 30 and month 48 (128-150 mMU/mL). CONCLUSION: The vaccine HPV16 L1 VLP provides high-level protection against persistent HPV16 infection and HPV16-related CIN2-3 for at least 3.5 years after immunization. Administration of L1 VLP vaccines targeting HPV16 is likely to reduce risk for cervical cancer. LEVEL OF EVIDENCE: I.