Analysis of HLA-DRB1*0901-binding HPV-16 E7 helper T cell epitope

AIM: This study sought to determine the human papillomavirus (HPV)-16 E7 epitopes that would be presented by HLA-DR molecules to CD4-positive T cells in patients with cervical carcinoma. METHODS: HLA-DR binding assays were performed using HPV-16 E7-derived synthetic peptides and, after incubation with these DR-binding peptides, helper T cell frequencies were analyzed in patients whose HLA and HPV genotypes were confirmed. RESULTS: We determined that the E7d peptide, 61CDSTLRLCVQSTHVDIRTL80E, was bound by HLA-DRB1*0901. An increased frequency (0.3-2.4%) of type 2 helper T cell responses was found in HLA-DRB1*0901-positive patients with cervical dysplasia and carcinoma. We found that when IL-12 was combined with E7d-peptide stimulation in vitro, the frequency of type 1 helper T cell responses also increased in patients with carcinoma. CONCLUSION: Thus HPV-16 E7d peptide as an HLA-DRB1*0901-restricted helper T cell epitope might usefully be incorporated into an understanding of the immunological mechanism and immunotherapy for this disease.     

A DNA vaccine constructed with human papillomavirus type 16 (HPV16) E7 and E6 genes induced specific immune responses

OBJECTIVE: Cervical cancer is found highly associated with human papillomaviruses type 16 (HPV16). HPV16 E6 and E7 oncogenes are important transforming genes which have become the main focus of anti-cervical cancer therapy. In this study, a recombinant DNA vaccine candidate, termed HPV16-DNA-E6E7, constructed with HPV16 E7 and E6 genes was generated and used to against HPV16-induced tumors. METHODS: We inserted an E7 DNA fragment into E6 gene to produce a recombinant gene (E6E7-DNA). The E6E7-DNA gene was inserted into a mammalian expression vector, pcDNA 3.1+, to construct the DNA vaccine candidate. Animals (C57BL/6 mice) were immunized with the vaccine candidate with various concentrations (50 microg, 100 microg or 200 microg, respectively), and cytotoxicity measurement and tumor protection assay were carried out to examine the immunological effects of the vaccine candidate. RESULTS: Immunization of with HPV16-E6E7-DNA induced HPV16-specific immune response and also conveyed protection against TC-1 induced tumor in vivo. A survival rate (90%) after 45 days of tumor challenge was observed. The animals injected with a higher dosage of the vaccine (200 microg) exhibited prolonged survival duration of more than 55 days. No transforming activity of the vaccine candidate was detected, as determined by focus formation and degradation of endogenous p53. CONCLUSION: Our results demonstrated that the HPV16-E6E7-DNA compound might become a candidate for HPV16 precautionary and immunotherapy.     

Detection of CD8+ T cell responses to human papillomavirus type 16 antigens in women using imiquimod as a treatment for high-grade vulval intraepithelial neoplasia

OBJECTIVES: To investigate CD8+ T cell reactivity to human papillomavirus (HPV) 16 antigens in patients with high-grade vulval intraepithelial neoplasia (VIN) before, during and after treatment with 5% imiquimod cream. METHODS: CD8-enriched responder cell populations were obtained from 10 patients with high-grade VIN using imiquimod cream as a treatment. Overlapping synthetic peptides covering the entire primary sequences of the HPV16 E6, E7 and E4 proteins were used to screen for CD8+ T cell responses using an ELISPOT assay of interferon (IFN)-gamma release. RESULTS: Reactivity to the proteins was detected in all patients on at least one occasion. With the exception of one patient, CD8+ T cell reactivity generally increased at some stage during treatment. The magnitude and specificities of responses changed over the treatment period. This was particularly noticeable in response to peptides derived from the E4 protein. CD8+ T cell reactivity to HPV16 E7 appeared to be dominant amongst women with high-grade VIN. The magnitude and specificity of response had no correlation with clinical response to imiquimod. CONCLUSIONS: HPV16 specific CD8+ T cell activity was detected in patients with high-grade VIN. Imiquimod use appeared to increase the magnitude of the response and broaden the specificity of response in some patients. Despite the presence of these CD8+ T cells, the disease state persisted; therefore, a role for HPV-specific cytotoxic T cells (CTLs) in VIN resolution remains unproven.     

HPV16-specific cytotoxic T lymphocyte responses are detected in all HPV16-positive cervical cancer patients

OBJECTIVES: The specific CTL response against human papillomavirus (HPV) antigens in women with cervical cancer has been poorly studied. Immunological monitoring of this response is central for understanding the principles that underlie successful immunotherapeutic strategies. The aim of the study was to investigate the HPV16 E6/E7-specific CTL immune response in a group of untreated HPV16-positive cervical cancer patients. METHODS: Peripheral blood mononuclear cells from 21 untreated cervical cancer patients and 4 healthy controls were isolated prior to any therapy. Autologous monocyte-derived dendritic cells (MDDCs) were transiently transfected with HPV16 E6 or E7 expression vectors and used for one round of in vitro restimulation and as target cells in chromium release assays with restimulated peripheral blood lymphocytes. RESULTS: Transfected monocyte-derived dendritic cells were differentiated to exhibit a fully mature phenotype. HPV16 E6 and E7 transgenes were expressed and translated as measured by RT-PCR and intracellular flow cytometry, respectively. All HPV16-associated cervical cancer patients showed evidence of specific CTLs. Lytic activity for HPV16 E6 (11/12) and/or E7 (8/9) was above 30% at the 100:1 effector to target ratio. None of the HPV16-negative cervical cancer patients or healthy controls were above 15% of lysis. CONCLUSIONS: These data suggest that HPV-specific cytolytic immune responses can be detected in all untreated cervical cancer patients. Our approach, using dendritic cells for restimulation and as target cells, may enhance immunomonitoring of cervical cancer patients.     

Heat shock fusion protein-based immunotherapy for treatment of cervical intraepithelial neoplasia III

OBJECTIVES: SGN-00101 (HspE7, Nventa, San Diego, CA) is a novel therapeutic vaccine consisting of a fusion protein containing an M. bovis BCG heat shock protein (Hsp65) covalently linked to the entire sequence of HPV 16 E7. This trial was designed to evaluate the efficacy and toxicities of HspE7 in women with CIN III. METHODS: HIV (-) women with biopsy-proven CIN III were eligible. Two cohorts were accrued; one cohort to establish efficacy and a second cohort with a longer follow-up period to improve the precision of the trial to estimate response rates. Each patient underwent 3 monthly subcutaneous vaccinations with 500 microg of HspE7 followed by monthly colposcopic follow-up for 1 month in cohort 1 and an extended observation period (2 months) in cohort 2. All patients then underwent a LEEP or cone biopsy of the cervix. A complete pathologic response (pCR) was defined as no evidence of CIN or CIN I (only HPV changes). A partial response (PR) was defined as colposcopic lesion regression of >50% in size. Cervicovaginal lavage samples were obtained at each visit for HPV typing using MY09/ MY11 HPV PCR. RESULTS: Seventy-two patients were registered and screened, of whom 64 were eligible. Fifty-eight patients completed the trial and were evaluable (31 in cohort 1, 27 in cohort 2). There were no significant epidemiologic or HPV type differences between the 2 cohorts so responses were combined for analysis. Of the 58 evaluable patients, 13 (22.5%) had a pCR; 32 (55%) had a PR and 11 (19%) had stable disease. Two (3.5%) patients in cohort 2 had microinvasive disease and were defined as progressive disease. Thirty-three of 58 (57%) of the patients were infected with HPV 16 prior to vaccination or in subsequent visits. There was no significant difference in regression in women infected with HPV 16 compared to those without HPV 16 infection (88% vs. 70%; p=0.12). Women who had a previous LEEP or ablation for CIN were 2.7 times more likely to have a complete response compared to patients without previous treatment, although the difference was not statistically significant (95% CI for rate ratio: 0.95-6.19, p=0.10). At a cellular level, there was a significant association between local inflammation and response; lower grade of lesional inflammation correlated with a response to HspE7 (p=0.04 using Wilcoxon rank sum test). CONCLUSIONS: HspE7 appeared to demonstrate activity in women with CIN III and met a priori assumptions for efficacy; however, it is unclear whether this response was due to natural regression rather than treatment effect. HspE7, which targets the HPV 16 E7 oncoprotein, had efficacy in patients infected with HPV types other than 16, suggesting cross-reactivity. A larger randomized, controlled trial is needed to better define efficacy and to identify subsets of women most likely to benefit from immunotherapy.     

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.     

Aptima法人乳头瘤病毒E6/E7 mRNA检测在子宫颈癌筛查中的流行病学分析及其诊断价值

目的 通过Aptima法检测人乳头瘤病毒(HPV)E6/E7 mRNA,研究其分型和定量检测结果在不同年龄和子宫颈活检中的分布情况,探讨其在子宫颈病变中的诊断价值.方法 选取2019年1月至2020年1月于郑州大学第三附属医院行HPV E6/E7 mRNA检测的患者为研究对象,收集HPVE6/E7 mRNA阳性患者的年...     

Immunization with adenoviral vectors carrying recombinant IL-12 and E7 enhanced the antitumor immunity to human papillomavirus 16-associated tumor

OBJECTIVE: Human papillomavirus (HPV) infection play a significant role in cervical carcinogenesis, and HPV oncoprotein E7 has important functions in the formation and maintenance of cervical cancers. Interleukin-12 (IL-12) has been reported to induce cellular immune responses, and has also been demonstrated to suppress the growth of tumors and the expression of E7. Here, we investigate the utility of adenovirus E7 (AdE7) and adenovirus IL-12 (AdIL-12) for protection against TC-1 tumor using an animal model. METHODS: The antitumor effects induced by AdIL-12 and/or E7 were assessed by measurements of tumor size. E7-specific antibody and INF-gamma production in sera were measured, as were T-helper cell proliferative responses. Cytotoxic T-lymphocytes (CTL) and T cell subset depletion studies were also performed. RESULTS: Infection of tumor sites with a combination of AdIL-12 and AdE7 resulted in an antitumor effect which was significantly more profound than that which resulted from singular infections with either AdIL-12 or AdE7. Combined infection resulted in regression of 9-mm-sized tumors in approximately 80% of our experimental animals as compared to the PBS group. Serum levels of E7-specific antibody and INF-gamma production, as well as T-helper cell proliferative responses, were found to be significantly higher in coinfected with AdIL-12 and AdE7 group than in single infection with either AdIL-12 or AdE7 group. CTL responses only exhibited by the AdIL-12 and AdE7 coinjected group suggested that these tumor suppression effects were mediated primarily by CD8+ and, to a lesser degree, by CD4+ T cells. CONCLUSION: Combined injection with adenovirus carrying IL-12 and E7 induced significant antitumor immunity against TC-1 tumors. They may prove useful in clinical applications for the treatment of HPV-associated tumors.     

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.     

Investigation of uterine arterial chemoembolization and uterine arterial infusion chemotherapy for advanced cervical cancer before radical radiotherapy: a long-term follow-up study

Purpose

To evaluate the immunotherapeutic potentials for human dendritic cells (DCs) loaded with different HPV16-associated antigens, including HPV16E7 (E) protein, HPV16E7 polypeptide (P), as well as CpG-oligodeoxynucleotide (ODN) 2006 as a promising immune adjuvant for vaccination against cervical carcinoma.

Methods

DCs derived from human peripheral blood and cord blood were isolated and loaded with HPV-derived protein or peptides, in combination with CpG-ODN2006 as a potential adjuvant. The IL-12 level, the allogeneic T cell-stimulatory capacity and the cytotoxicity of cytotoxic T lymphocytes (CTLs) were evaluated in vitro. Furthermore, an immune reconstitution model of human cervical carcinoma in SCID mice was used to assess the anti-tumor effects in vivo. The tumor sizes, the expression of IgG and IFN-γ, and the presence of the human CD3⁺, CD4⁺ and CD8⁺ T cells were measured in the mice inoculated with different DCs.

Results

The antigen-loaded DCs displayed obvious anti-tumor activities in vitro and in vivo, and showed no toxicity to normal cells. The level of IL-12, an important cytokine for immune response, was up-regulated in all mice inoculated with antigen-loaded DCs. Stimulation and activity of CTLs were increased after treatment with antigen-loaded DCs. Significantly, DCs loaded with HPV16E7 polypeptide (P) showed the most distinguished immunotherapeutic activities, and such effect was further enhanced when HPV16E7 polypeptide (P) was used in combination with CpG-ODN2006. Interestingly, the same results were obtained in vivo: the tumor size was decreased, and IgG and IFN-γ levels were increased after the SCID mice were inoculated with the loaded DCs.

Conclusions

HPV16E7 polypeptide combined with the immune adjuvant CpG-ODN2006 could be a suitable HPV16-associated tumor antigen. The research provides a new strategy for generating DCs vaccines for immunotherapy of cervical cancer.     

宫颈癌及癌前病变组织中Notch1及HPV16 E6/E7表达的研究

目的 探讨Notch1受体和人乳头瘤病毒16感染在宫颈癌前病变和宫颈癌发生发展中的作用。方法 采用免疫组化SP法检测18例宫颈上皮内瘤变（cervical intraepithelial neoplasia,CIN）和35例宫颈癌标本中Notch1受体及HPV16E6/E7蛋白的表达,以34例正常宫颈组织及慢性宫颈炎组织作为对照。比较各组间Notch1及HPV16E6/E7表达的差异,并分析Notch1与HPV16E6/E7表达的关系。结果 Notch1蛋白在细胞胞浆、胞核及胞膜中表达,HPV16E6/E7在细胞核中表达。从对照组到CIN组到宫颈癌组,Notch1及HPV16E6/E7的表达均逐渐增强（P〈0.01）。Notch1的阳性表达与宫颈癌不同分期、分化程度、淋巴结是否转移无关（P〉0.05）。在宫颈癌组中Notch1与HPV16E6/E7的表达均呈正相关性（P〈0.01）。结论 Notch1表达与HPV16E6/E7感染可能与CIN及宫颈癌的发生密切相关,两者在宫颈癌的发病机制中可能协同发挥作用。     

HPV16 E6E7癌蛋白通过TWEAK/Fn14信号途径对细胞增殖和凋亡的影响

目的:探讨HPV16 E6E7对TWEAK/Fn14信号途径及细胞增殖和凋亡的影响。方法:制备表达HPV16 E6E7的逆转录病毒,感染原代角质形成细胞。用流式细胞仪分析可诱导纤维母细胞生长因子14(Fn14)表达,Real-time RT-PCR和Western blot法检测调解活化正常T细胞表达和分泌的趋化因子(RANTES)、单核细胞趋化蛋白-1(MCP-1)、干扰素-γ诱导蛋白10水平(IP10)和肿瘤坏死因子(TNF)受体相关因子(TRAF2)表达,生物化学法检测Ras GTpase活性,TUNEL检测细胞的凋亡,MTT法检测细胞增殖。结果:表达HPV16 E6E7的逆转录病毒感染角质形成细胞后,Fn14高表达,RANTES、MCP-1、IP10和TRAF2表达增加,Ras GTpase活性升高。与未感染表达HPV16 E6E7逆转录病毒的细胞比较,表达E6E7感染的角质形成细胞对TNF-α刺激不敏感,细胞增殖显著增加(P0.05)。结论:表达HPV16 E6E7细胞激活TWEAK/Fn14信号途径,从而上调RANTES和TRAF2表达,导致细胞增殖,这可能是高危型HPV的一个致病机制。     

Extended effects of human papillomavirus 16 E6-specific short hairpin RNA on cervical carcinoma cells

Most cervical carcinomas express high-risk human papillomavirus (HPV) E6 and E7 oncogenes. Small interfering RNA can mediate sequence-specific inhibition of gene expression in mammalian cells. To find a most effective short hairpin RNA (shRNA) for HPV16 E6 messenger RNA (mRNA) and investigate the extended effects of the HPV16 E6 shRNA on cervical carcinoma cells, we stably transfected SiHa cells with four shRNA expression vectors (E6A-D). HPV16 E6A shRNA was found to be the most efficient in our study, which caused the reduction of HPV16 E6 mRNA to 10% in SiHa cells but did not reduce HPV18 E6 mRNA expression in HeLa cells. We subsequently demonstrated that E6A could stably express shRNA and effectively reduce HPV16 E6 and E7 viral genes expression in SiHa cells for more than 4 months. After E6 and E7 repression, there was a dramatic accumulation of p53, p21, and hypophosphorylated pRb proteins in cells. Furthermore, cell proliferation, colony formation ability, tumorigenicity, and in vitro cell invasive capability were suppressed substantially in E6A-transfected cells. These results suggest that the use of shRNA expression vector may be a potential approach for the treatment of persistent HPV infection and HPV-positive cervical carcinoma.     

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.     

人乳头状瘤病毒16型E6、E7基因诱导的人子宫颈永生化上皮细胞系的建立及其生物学特性的鉴定

目的 建立人宫颈上皮的永生化细胞系,并进行生物学特性进行鉴定。方法 用含人乳头状瘤病毒(HPV)16型E6、E7基因的腺病毒伴随病毒感染人胎儿宫颈上皮细胞,培养、传代。采用激光共聚焦免疫荧光和PCR技术检测HPV16型E6、E7基因的表达情况;取20代细胞,采用软琼脂培养、染色体核型分析和Scid小鼠皮下接种等方法检测细胞系的生物学特性;采用电子显微镜观察细胞的形态。结果 20代细胞的表型仍保留原代上皮细胞的特征,表现为单层生长和锚锭依赖性生长。激光共聚焦免疫荧光和PCR技术检测显示,细胞内有HPV16型E6、E7基因的表达,其基因片断长度为829bp。20代细胞进行软琼脂培养不形成克隆;scid小鼠皮下接种未成瘤;染色体核型分析：勾二倍体和多倍体,11号染色体可能为HPV16型E6、E7基因整合的部位。电子显微镜观察可见张力原纤维,证实细胞为鳞状上皮来源。结论 成功建立了HPV16型E6、E7基因诱导的宫颈上皮永生化细胞系,且其生物学特性稳定,可进一步用以宫颈癌病因和发病相关机制的研究。     

Influence of HPV16 E6/7 on the expression of FGF2 and FGFR type B in cervical carcinogenesis

This study investigates the influence of fibroblast growth factor receptor type B (FGFRb) and fibroblast growth factor on cervical carcinogenesis associated with HPV16 E6/7 infection, using primary cancer cells isolated from Taiwanese patients with cervical cancer. Functional interaction between FGFRb in Cx cells and HPV16 E6/7 transfected Cx cells (CxWJ cells) following treatment with FGF-7, according to cell growth, invasive ability, and tumor growth in SCID mice. Our results indicate that the downregulation of FGFRb gene expression in CxWJ cells partially represses proliferation and the invasive ability provided by FGF-7 stimulation. In SCID mice, the FGF2 and FGFR1 gene expression ascend in CxWJ tumor nodule. These data provide evidence of a functional interaction between HPV16 E6/7 in FGFRb and FGF2, suggesting that cooperative stimulation of HPV E6/7 in inactivated FGFRb and the upregulation of FGF2 may be necessary to completely overcome the oncogenic function associated with the progression of cervical carcinogenesis.     

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     

Human papillomavirus type 16 E2 and E6/E7 variants

OBJECTIVES: Polymorphisms in human papillomavirus (HPV) type 16 have been shown to be related to geographic areas and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Certain variants have been reported as being more likely to cause cervical disease; our objectives were to identify new HPV16 polymorphisms, to determine the linkage of the E2 and E6/E7 regions and to determine the minimum sequence necessary to classify variants. METHODS: We sequenced the complete E2, E6, and E7 regions in all HPV16-positive cervical samples identified in a case-control study of pre-invasive cervical disease. RESULTS: In the 100 samples analyzed, only one new polymorphism was identified, a synonymous change, T3205A, in region E2. The frequency distribution of variants in the sample set was 37 European prototypes and 27 E-G350, 16 AA, 5 Af1, 2 Af2, 8 E-C109G, 3 E-G131G, and 2 As. As shown by others, region E7 varied much less than E6 and E2. CONCLUSIONS: In each case, E2 changes were linked to the expected E6/E7 changes, and there was no evidence for recombination. The linkage between E2 and E6/E7 allows variant classification to be based on a short E6 sequence (nt 109-350).