Antibodies to HPV-16 E6 and E7 proteins as markers for HPV-16-associated invasive cervical cancer.

Transforming proteins E6 and E7 of human papillomaviruses (HPVs) are consistently expressed in HPV-associated cervical cancers. In ELISA with four HPV-16 E6-E7 peptides, patients with HPV-16-associated invasive cervical cancer (group 1) had a greater seroreactivity than all other groups, which included patients with HPV-16-associated cervical intraepithelial neoplasia, invasive cervical cancer patients without HPVs, and unaffected controls. A larger proportion of group 1 sera, as compared to sera of all other groups, was reactive with at least one peptide (49% vs 17-27%), and with two or more peptides (22% vs 0-6%). A clear difference between group 1 and all other groups was also found for high ELISA absorbance values to at least one peptide (22% vs 0-8%). This high seroreactivity of group 1 sera was confirmed by a radioimmunoprecipitation assay with in vitro transcribed and translated HPV-16 E7 protein. Sera from 50% of group 1 but only 3% of controls were reactive in this test. Antibodies to HPV-16 E6 and E7 proteins appear to be virus-specific and disease state-specific markers of HPV-associated cervical cancer.     

Viral recombinant vaccines to the E6 and E7 antigens of HPV-16

Most cancerous lesions of the uterine cervix are linked to persistent infections with human papillomaviruses (HPV), most notably HPV-16 or -18. Vaccine-induced immune responses to the HPV early antigens E6 and E7, which contribute to cell transformation and are thus expressed in these cervical cancers, could potentially eradicate malignant cells. We generated recombinant vaccines based on E1-deleted adenovirus human strain 5 or on vaccinia virus strain Copenhagen expressing either the E6 or E7 oncoproteins of HPV-16. The different vaccines were compared in two experimental mouse tumor models employing Balb/c or C57Bl/6 mice. Data presented here demonstrate that depending on the model either CD4(+) or CD8(+) T cells provide protection to tumor cell challenge, resulting in striking differences in the efficacy of the four vaccines under investigation.     

Nonrandom chromosomal imbalances in human ovarian surface epithelial cells immortalized by HPV16-E6E7 viral oncogenes

We had previously immortalized human ovarian surface epithelial (HOSE) cells using HPV16E6E7 ORFs. In order to identify crucial genetic events involved during cell immortalization, the genomic profile of immortalization of five HOSE cell lines was analyzed by comparative genomic hybridization. Our results showed that chromosomal imbalance was common in HOSE cells after immortalization. The common chromosomal imbalances identified in immortal HOSE cells are: +19q13.1 (5/5 lines), −13q12qter (4/5 lines), +5q15q33 (3/5 lines), +20q11.2q13.2 (3/5 lines) and −22q11.2qter (3/5 lines). Other chromosomal imbalances, which were detected in two of the five immortal HOSE cell lines, included gains on chromosome 1 and 11q12q13, and losses on 2p, 4q, 8p, 10p and 11q14qter. The chromosomal imbalances observed in HOSE cells before immortalization include −8pterp11.2, −11q23qter, −13q12qter and +19 which may represent early genetic events during cell immortalization. The genomic profile was examined in one HOSE cell line (HOSE 6-3) at various stages of immortalization. The genomic profiles of HOSE 6-3 cells after crisis were largely stable. A few additional chromosomal imbalances were detected in the immortalized HOSE cells after an extensive culture period including +11pterq23, −15q23qter, and +17q12qter. Identification of nonrandom chromosomal imbalance in immortalized HOSE cells may facilitate the identification of specific chromosomes harboring genes involved in the immortalization of human ovarian surface epithelial cells.     

人乳头瘤病毒16型E＿6E＿7基因反义质粒对人宫颈癌细胞恶性的逆转作用

构建可为地塞米松诱导表达的人乳头瘤病毒１６型（ＨＰＶ－１６）Ｅ＿６Ｅ＿７基因反义质粒（ｐ１６ａｓＥ＿６Ｅ＿７Ｎｅｏ），利用磷酸钙沉淀法将其分别转染到ＨＰＶ－１６阳性的人宫颈癌细胞株Ｃａｓｋｉ和ＨＰＶ阴性的人宫颈癌细胞株Ｃ－３３Ａ中。地塞米松诱导反义质粒表达后，ＣａｓＫｉ细胞失去其恶性表型，而Ｃ－３３Ａ细胞的生长特性及恶性行为未发生变化。说明反义质粒能够改变Ｃａｓｋｉ细胞的恶性表型，且这种改变是通过特异性抑制Ｅ＿６Ｅ＿７基因表达实现的。     

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.     

Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model

HPV L1-based virus-like particles vaccines (VLPs) efficiently induce temporary prophylactic activity through the induction of neutralizing antibodies; however, VLPs that can provide prophylactic as well as therapeutic properties for longer periods of time are needed. For this purpose, we generated a novel HPV 16 L1-based chimeric virus-like particle (cVLP) produced in plants that contains a string of T-cell epitopes from HPV 16 E6 and E7 fused to its C-terminus. In the present study, we analyzed the persistence of specific IgG antibodies with neutralizing activity induced by immunization with these cVLPs, as well as their therapeutic potential in a tumor model of C57BL/6 mice. We observed that these cVLPs induced persistent IgG antibodies for over 12 months, with reactivity and neutralizing activity for VLPs composed of only the HPV-16 L1 protein. Efficient protection for long periods of time and inhibition of tumor growth induced by TC-1 tumor cells expressing HPV-16 E6/E7 oncoproteins, as well as significant tumor reduction (57 %), were observed in mice immunized with these cVLPs. Finally, we discuss the possibility that chimeric particles of the type described in this work may be the basis for developing HPV prophylactic and therapeutic vaccines with high efficacy.     

The HPV-16 E7 oncogene sensitizes malignant cells to IFN-alpha-induced apoptosis.

Interferons (IFNs) exert antitumor effects in several human malignancies, but their mechanism of action is unclear. There is a great variability in sensitivity to IFN treatment depending on both tumor type and the individual patient. The reason for this variable sensitivity is not known. The fact that several IFN-induced anticellular effects are exerted through modulation of proto-oncogenes and tumor suppressor genes may indicate that the malignant genotype may be decisive in the cell's sensitivity to IFN. To determine if a deregulated oncogene could alter the cellular response to IFN, a mouse lymphoma cell line (J3D) was stably transfected with the viral human papillomavirus-16 (HPV-16) E7 oncogene. The E7-transfected cells and their respective mock-transfected sister clones were treated with IFN-alpha and examined for possible IFN-induced anticellular effects. We found that the E7-transfected clones were greatly sensitized to IFN-alpha-induced apoptosis compared with their mock-transfected counterparts. Induction of apoptosis in the transfected cells correlated with the ability of IFN to activate parts of the proapoptotic machinery specifically in these cells, including activation of caspases and the proapoptotic protein Bak. In summary, our data suggest that transfection of malignant cells with the E7 oncogene can sensitize them to IFN-alpha-induced apoptosis. This demonstrates that an oncogenic event may alter the cellular sensitivity to IFN and might also have implications for treatment of HPV-related diseases with IFN.     

Differences in HPV 16- and HPV 18 E6/E7 oncogene expression between in situ and invasive adenocarcinomas of the cervix uteri

To evaluate the importance of high-risk human papillomavirus (HPV) types in in situ and invasive adeno- and adenosquamous carcinomas (ACISs/ACs, and ASCISs/ASCs) of the cervix uteri, we analyzed HPV infection and HPV 16- and HPV 18 E6/E7 oncogene expression in different histologic subtypes. Using the polymerase chain reaction (PCR) technique, 29 of 33 (88%) ACISs, 2 of 2 (100%) ASCISs, 46 of 54 (85%) ACs, and 8 of 10 (80%) ASCs were found to be HPV 16- and/or HPV 18-positive. In 25 of 35 (71%), 10 of 35 (29%), and 4 of 35 (11%) ACISs/ASCISs, HPV 16, HPV 18, and HPV 16 and HPV 18 were detected, respectively. Invasive ACs/ASCs were more frequently infected with HPV 18 (36 of 64, 56%) than with HPV 16 (28 of 64, 44%). Ten (16%) of these cases were positive for HPV 16 and HPV 18. In ACISs/ASCISs, HPV 16 oncogene expression predominated (62%) relative to HPV 18 (25%) expression, whereas in invasive ACs/ASCs, only 21% of the cases expressed HPV 16, but 48% of the cases expressed HPV 18 oncogenes. Thus, detection of HPV 18 in ACISs/ASCISs might be associated with an increased risk of progression. HPV oncogene expression was not dependent on histologic subtype of in situ or invasive AC. Normal glandular epithelia and glandular dysplasias (GDs, n = 4) were always negative concerning HPV oncogene expression. In HPV 16- and HPV 18-double-infected cases, HPV 18 oncogene expression was most frequently detected, and we did not find a coexpression of HPV 16- and HPV 18-specific oncogenes in purely glandular lesions or in cases with an additional CIN (cervical intraepithelial neoplasia) II or CIN III. HPV E6/E7 expression of the same HPV type in both in situ or invasive ACs and associated CIN II/III suggest that these lesions might be histogenetically related.     

Identification of genital tract papillomaviruses HPV-6 and HPV-16 in warts of the oral cavity

Warty lesions of the oral cavity were examined for etiologic association with genital tract papillomaviruses HPV-6, HPV-11, and HPV-16. DNAs extracted from ten oral biopsies were screened for HPV genomic sequences by Southern transfer hybridization with 32P-labeled viral DNA probes. Nonstringent hybridization with an HPV-6 probe revealed papillomavirus DNA sequences in four of seven tissues with histologic evidence of papillomatosis, in none of two tissues without histologic evidence of papillomatosis, and in one tissue that was not examined by histology. Stringent hybridization tests with HPV-6 and HPV-16 probes identified the genome in one tissue as being HPV-16, in a second tissue as being HPV-6 subtype a, and in a third tissue as HPV-6 (subtype unidentified); papillomavirus DNA sequences in two tissues are as yet not identified. An additional case of HPV-6 or HPV-11 related oral cavity lesion was diagnosed by in situ hybridization of paraffin sections with a 35S-labeled, mixed HPV-6 + HPV-11 probe. The hybridization in the positive section was extensive and confined to epithelial nuclei. The oral lesions associated with genital tract papillomaviruses were asymptomatic, multiple or single, and were located in different parts of the oral cavity, for example, on the gingivae, on the tongue, on the lip, on the tonsillar pillar, and on the floor of the mouth.     

Adenovirus transformation revertant resistant to retransformation by E1 but not by SV40-T and HPV16-E7 oncogenes.

We have previously described a revertant cell line which expresses a dominant tumor suppressor phenotype to E1 but not to heterologous oncogenes such as c-myc, N-ras, or polyoma middle t (Sircar et al. (1988) Oncogene 3, 725-728). DNA tumor virus oncogenes have been suggested to transform cells via the common mechanism of sequestering the Rb-105 antioncoprotein. This paradigm would predict that our revertant cell line, which is resistant to retransformation by E1a, should also be resistant to the other members of the Rb-105 binding family of oncogenes. To test this hypothesis we transfected the revertant cell line with plasmids bearing SV40-T or the HPV16-E7 oncogenes. Because transformation was obtained by both oncogenes at efficiencies similar to the transformation of a related revertant cell line, the results suggest that the resistance phenotype is specific to E1a. This specificity was further confirmed by cell fusion experiments.     

DNA vaccines encoding IL-2 linked to HPV-16 E7 antigen generate enhanced E7-specific CTL responses and antitumor activity

DNA vaccination has emerged as a promising strategy for cancer immunotherapy. However, since DNA vaccines have low immunogenicity, various strategies have been developed to enhance the potency of DNA vaccines. In the current study, we aim to determine whether the potency of the DNA vaccine encoding human papillomavirus type 16 (HPV-16) E7 antigen can be enhanced by IL-2. We have generated a DNA vaccine encoding IL-2 linked to HPV-16 E7 antigen. Our results indicate that the DNA vaccine encoding a fusion of IL-2 and E7 proteins generated the highest frequency of E7-specific CD8(+) T cells. We also found that the DNA vaccine encoding a fusion of IL-2 and E7 proteins generated the strongest protective as well as therapeutic anti-tumor effect against E7-expressing tumors. In addition, it was observed that CD8(+) T cells were mainly responsible for the antitumor effect generated by the DNA vaccine encoding a fusion of IL-2 and E7 proteins. Thus, we conclude that the linkage of IL-2 to HPV-16 E7 antigen significantly enhances the DNA vaccine potency against E7-expressing tumors. Our strategy may potentially be used in other antigenic systems to control infectious diseases and/or cancer.     

Identification in humans of HPV-16 E6 and E7 protein epitopes recognized by cytolytic T lymphocytes in association with HLA-B18 and determination of the HLA-B18-specific binding motif

Human papilloma virus type 16 (HPV-16) is the HPV most frequently associated with cervical carcinoma in humans. For the prevention or treatment of cervical carcinoma, the E6 and E7 oncoproteins appear to be good targets for vaccine-induced cytotoxic T lymphocytes (CTL). Lipopeptide vaccination is an efficient way of stimulating cellular responses. However, to synthesize effective lipopeptides, it is necessary to define which epitopes are immunogenic. In this study we first determined that peptide 80 - 88 of the E6 protein was recognized by CTL from a healthy donor in association with the HLA-B18 molecule. We then defined the HLA-B18 anchoring peptide motif by testing the binding of various short peptides with the HLA-B18 molecule and showed that it was related to the HLA-A1-specific peptide motif. Furthermore, in analyzing the potential E7 epitopes susceptible to associating with HLA-B18, we demonstrated that peptide E7 44 - 52 gave the strongest binding. It could also be recognized by CTL from peripheral blood mononuclear cells (PBMC) of the same healthy donor. Finally, with PBMC from a patient with a cervical intraepithelial neoplasia grade 3, we found CTL which recognized the E6 80 - 88 epitope. We have hence identified two peptides encoded by the E6 and E7 proteins which are presented by the HLA-B18 molecule and could be included in a vaccine against HPV-16.     

The seroprevalence of IgG antibodies to human papillomavirus (HPV) types HPV-16, HPV-18, and HPV-11 capsid-antigens in mothers and their children

Human papillomavirus (HPV) types causing anogenital lesions and cancer are accepted as being sexually transmitted. The methods whereby children acquire these anogenital type HPV infections are unclear. The present study determined the prevalence of anti-HPV-16, HPV-11 and HPV-18 IgG antibodies in mothers and their children in an attempt to identify evidence of HPV transmission from mother to child. HPV virus-like particles (VLP) VLP-16, VLP-11 and VLP-18 were used in enzyme-linked immunosorbent assay to identify IgG antibodies in serum from 100 mothers and their 111 children. Antibodies to VLP-16, VLP-11 and VLP-18 were found in serum from 17%, 21% and 16% of mothers, respectively and seroprevalences were 9%, 11.7% and 9.9%, respectively amongst the children. Of the 111 children, 23 (20.7%) showed antibodies to one or more of the three HPV types tested. Seven of these (30.4%) HPV IgG positive children had the same antibodies to one or more HPV types as their mothers. The prevalence of HPV-11 was similar in children of seropositive compared with seronegative mothers (14% and 11%, respectively). The prevalence of HPV-16 and HPV-18 was higher in children of seropositive mothers compared with seronegative mothers (for HPV-16, 18% and 7%, respectively, P = 0.1, for HPV-18, 19% and 8%, respectively, P = 0.2). None of these differences were statistically significant indicating a lack of correlation between antibodies in mothers and children and no evidence to support vertical or horizontal mother to child transmission of HPV infection. Indications were of multiple sources of HPV infection in the children.     

Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results

The ATHENA (Addressing THE Need for Advanced HPV Diagnostics) HPV study evaluated the clinical usefulness of the cobas HPV Test (Roche Molecular Systems, Pleasanton, CA) for high-risk human papillomavirus (HR-HPV) testing (14 HR types) and individual HPV-16/HPV-18 genotyping in women undergoing routine cervical cytology screening in the United States. For the study, 47,208 women were recruited, including 32,260 women 30 years or older with negative cytology. All women with positive results for HR-HPV (n = 4,219) plus a subset of HR-HPV- women (n = 886) were referred for colposcopy and biopsy. The overall prevalence of HR-HPV was 6.7% and of HPV-16/HPV-18 was 1.5%. Cervical intraepithelial neoplasia grade 2 (CIN 2) or worse was found in 1.2% of women examined. The estimated absolute risk of CIN 2 or worse in HPV-16+ and/or HPV-18+ women was 11.4% (95% confidence interval [CI], 8.4%-14.8%) compared with 6.1% (95% CI, 4.9%-7.2%) in HR-HPV+ and 0.8% (95% CI, 0.3%-1.5%) in HR-HPV- women. These analyses validate the 2006 American Society of Colposcopy and Cervical Pathology guidelines for HPV-16/HPV-18 genotyping, which recommend referral to colposcopy of HPV-16/HPV-18+ women with negative cytology.     

Tissue engineered cartilage using human articular chondrocytes immortalized by HPV-16 E6 and E7 genes

Chondrocytes are useful as a cell culture system for studying arthritic degeneration in tissue engineered cartilage. However, primary chondrocytes have short in vitro lifespan and rapid shift of collagen phenotype. In this study, we used a high dosage of retroviral vector LXSN16E6E7 to transduce human primary chondrocytes and obtained an actively proliferating cell line, designated hPi, which expresses HPV-16 E6/E7 mRNA in early passages. Parental primary chondrocytes cease to grow after five passages, whereas hPi could be propagated beyond 100 passages without requiring additional cell elements in defined medium. After 48 passages, hPi can also give many profiles similar to those of parental primary chondrocyte, including type II collagen in mRNA and protein level, aggrecan in mRNA level, lacunae in type I collagen matrices, and morphology with GAG-specific Alcian blue staining. hPi has shown neoplastic transformation, as examined by NOD-SCID mice tumorigenicity assays for 3 months. Our results indicated that human primary chondrocytes could be immortalized by transduction with HPV-16 E6/E7, preserving stable cartilage-specific differentiation markers. The established chondrocyte cell line could provide a novel model to engineer cartilage in vitro and in vivo for cartilage repair research and clinical application.     

HPV16,18,58-mE67重组腺病毒的构建及克隆表达

目的 构建能同时表达HPV16,18,58三亚型E6-E7优化融合基因的重组腺病毒,为下一步获得适合国情的宫颈癌HPV三价治疗性候选疫苗打基础.方法 常规扩增、构建HPV16,18,58的E6-E7融合基因,PCR定点突变消除其转化活性后以IRES相连并重组入pAdeno-X腺病毒载体系统;体外细胞、软琼脂培养及裸鼠成瘤实验检测突变基因转化活性去除情况;重组腺病毒电击转染293包装细胞,Western blot分析外源基因表达情况.结果 三型重组融合基因序列与设计完全一致,其中突变基因的转化活性显著降低,Western blot等显示所构建重组腺病毒在293细胞中高效表达外源基因.结论 成功构建表达HPV16,18,58三型E6-E7融合基因的重组腺病毒,并有效消除了其转化活性,为下一步疫苗研发打下了基础.     

Cervical intraepithelial neoplasia 3, coinfected with HPV-16 and -18--case report.

Recently, detection of human papillomavirus (HPV)mRNA expression was made possible by in situ hybridization. We described a patient with cervical intraepithelial neoplasia (CIN) 3, showing a distinctive and rare form of co-infection with HPV type 16 and 18. HPV-16 was detected in high grade squamous intraepithelial neoplastic lesion (CIN 3) and HPV-18 was in low grade lesion just adjacent to the HPV-16 infected area. This case suggests that HPV infection may be one of the most responsible causative agents producing malignant transformation and two distinctive HPV types can also simultaneously infect the squamous epithelium of the uterine cervix.