HLA-I表达与维吾尔族妇女宫颈癌前病变及HPV16感染的关系研究

目的：观察人白细胞相关抗原I（human leukocyte antigen class I,HLA-I）表达与维吾尔族妇女宫颈癌前病变进程及高危型HPV16的关系。方法：收集维吾尔族妇女宫颈炎、宫颈内上皮瘤样病变（cervical intraepithelial neoplasia,CIN）和宫颈鳞癌患者的石蜡包埋组织标本共148例,提取组织DNA,应用PCR的方法检测HPV阳性及HPV16型别;同时采用免疫组织化学SP法检测HLA-I蛋白表达水平。结果：（1）在维吾尔族妇女中HLA-I抗原在宫颈炎、CINI-II、CINIII、SCC组中阳性表达逐渐减少,差异有统计学意义（P〈0.001）。（2）HLA-I的阳性表达下降趋势与宫颈癌临床分期、组织分化程度和淋巴结转移密切相关。（3）HPV在宫颈炎、CINI-II、CINIII、宫颈癌中的感染率分别为13%、46%、82%、95%,差异有统计学（P〈0.001）。（4）HPV16在宫颈炎、CINI-II、CINIII、宫颈癌中的感染率分别为4%、30%、68%、85%,差异有统计学（P〈0.001）。（5）在HPV16阳性标本中,存在HLA-I表达缺失的占71%（58/82）,HPV16感染与HLA-I表达呈负相关（r=-0.625,P〈0.001）。结论：（1）HLA-I表达缺陷可能是宫颈病变进展的重要标志,对宫颈癌的预测预警提供依据。（2）HPV16感染在宫颈病变的发展过程中起到了极大的促进作用,是一个很强的致癌因素。（3）HPV16感染与HLA-I表达之间的关系对揭示宫颈癌发病机制提供了客观依据。     

Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intraepithelial lesions (SILs) show quantitative and functional alterations of Langerhans cells (LC). The infiltration of immature LC in the squamous epithelium is mainly controlled by Macrophage Inflammatory Protein 3α/CCL20. After having shown that CCL20 production is altered in HPV-transformed keratinocytes (KC), the possible role of HPV16 E6 and E7 viral oncoproteins in the reduced CCL20 levels observed in SILs was investigated by silencing HPV16 E6 and E7 oncogenes by RNA interference (siRNA). This treatment not only increased CCL20 secretion but also resulted in the modulation of NF-κB p50, p52 and p65 precursor localization. Moreover, silencing of E6 and E7 oncogenes in HPV16-transformed KC induced a significantly higher migratory capacity of LC in a Boyden chamber assay and in an in vitro formed (pre)neoplastic epithelium reminiscent of high-grade SILs. Anti-CCL20 neutralizing antibody experiments showed that the increased migration of LC is due to the re-expression of CCL20 in E6 and E7 siRNA transfected KC. These data suggest that HPV16 E6/E7-induced down-regulation of CCL20 observed during the cervical carcinogenesis may contribute to a diminished capacity of the immune system to control HPV infection. P. Hubert and J. H. Caberg contributed equally to this work.     

Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage

Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G₂ checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G₂ checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.     

高危型HPV 感染对宫颈病变组织IFN-r、IL-10表达的影响

目的：探讨高危型人乳头瘤病毒（HPV）感染对宫颈病变组织干扰素-r(IFN-r)、白细胞介素-10(IL-10)的影响。方法：收集我 院2013 年1 月到2015 年1 月妇科门诊行宫颈活检患者150 例,根据病理检查结果,将患者分为观察组（宫颈癌33 例、CINⅠ期 26 例、CINⅡ期28 例、CINⅢ期23 例）110 例,对照组（慢性宫颈炎患者）40 例。提取两组患者宫颈组织标本,检测观察组 HPV-DNA、HPV 分型,及两组IFN-r、IL-10 蛋白及mRNA 表达,分析高危型HPV感染与宫颈病变组织IFN-r、IL-10 表达的关系。 结果：观察组HPV-DNA阳性87 例（79.1%）,其中HPV16 型47 例（42.7%）、HPV18 型20 例（18.2%）感染率最高;且HPV16、18 感 染率在CINⅠ期、CINⅡ期、CINⅢ期以及宫颈癌组中的感染率差异均有统计学意义（P<0.05）,Spearman相关性分析显示,HPV16、 18 与宫颈病变程度均呈现正相关关系（r=0.896,0.786;均P<0.05）。IFN-r、IL-10 表达水平在CINⅠ期、CINⅡ期、CINⅢ期、宫颈 癌、对照组表达水平差异均具有统计学意义（P<0.05）,Spearman 相关性分析显示,IFN-r与宫颈病变程度呈现负相关关系（r=-0. 567,P<0.05）,IL-10 呈现正相关关系（r=0.678,P<0.05）。且HPV16、HPV18 感染率与IFN-r表达呈现负相关关系（rHPV16/18=-0. 678,-0.675,均P<0.05）,与IL-10 呈现正相关关系（rHPV16/18=0.582,0.778,均P<0.05）。结论：IFN-r表达随着宫颈病变加重或者 HPV16、HPV18 感染率升高逐渐降低,IL-10 则逐渐升高。     

A novel kinase-based ATP assay using molecular beacon

Quantitation of E6 oncogene sequences of the human papillomavirus type 16 by real-time or quantitative PCR (qPCR) is used to determine the viral load, which correlates with the degree of the cervical neoplastic lesions. In the presence of EvaGreen, a new DNA intercalating fluorochrome, we obtained consistent and reproducible qPCR amplification curves and thermal denaturation profiles identical to those of the authentic E6–HPV16 (human papillomavirus 16) genome from the amplification products derived from a construct carrying the E6–HPV16 oncogene. E6–HPV16 quantitation in the presence of EvaGreen, therefore, is reproducible and specific and may be used to determine HPV16 viral load.     

人乳头瘤病毒16型早期基因E6、E7的克隆及表达

高危人乳头瘤病毒16型的感染与宫颈癌的发病密切相关。HPV16E6和E7蛋白在大多数HPV16相关宫颈癌及其癌前病变中持续表达,因此E6和E7蛋白可作为制备HPV16相关肿瘤及其癌前病变治疗性疫苗的靶抗原〔2〕。采用套式PCR方法,从宫颈癌患者组织中扩增出E6、E7基因并成功构建了包含E6、E7的表达质粒PET-E6、PET-E7。SDS聚丙烯酰胺凝胶电泳和Western-blotting分析结果表明,外源基因E6,E7在T7启动子控制下可获得稳定表达。     

Fusion of CTLA-4 with HPV16 E7 and E6 Enhanced the Potency of Therapeutic HPV DNA Vaccine

Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4) with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6). pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6.     

Neutralizing antibodies against oncogenic human papillomavirus as a possible determinant of the fate of low-grade cervical intraepithelial neoplasia

To determine whether neutralizing antibodies (NAs) against HPV16 is responsible for a higher regression rate of low-grade cervical intraepithelial neoplasia (CIN1), we investigated an association between the presence of the NAs and the fate of the HPV16-related CIN1. All the women examined in this study had HPV16 positive cervix. The women were allocated into four groups by their cervical pathology, i.e., non-pathological (n:7), CIN1 (n:37), CIN2/3 (n:19), and cervical cancer (n:13). Their sera were tested for the presence of NAs against HPV16 by an in vitro assay using HPV16-pseudovirions. As for the CIN1 cases, clinical regression of the lesions were compared between NA-positive and NA-negative groups. Copy number of HPV16-DNA in smear samples was measured by quantitative PCR. The incidence of the presence of the NAs in the women with a non-pathological cervix (85.7%) was significantly higher than in the CIN1 cases (21.5%), the CIN2/3 cases (15.7%), and the cervical cancer cases (0%) (p<0.0001). The regression of the CIN1 lesion was closely associated with the presence of the N As (p=0.0002). The presence of the NAs was associated with low-level copy number of the viral DNA relative to the NA-negative group (p=0.05). The presence of the NAs against HPV16 was associated with a higher regression rate of HPV-related CIN1 lesions. The NAs seem to have a role in deterring HPV-related cervical lesions from progressing to CIN2/3 by inhibiting the infection with de novo replicated HPV. This study further suggests that HPV vaccine to induce the NAs may be effective in eliminating CIN lesions, especially in the NA-negative cases.     

Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region

Cervical cancer is caused by infection by high-risk human papillomavirus (HPV), especially HPV16. Limitations in current treatments of cervical cancers call for the development of new and improved immunotherapies. This study aims at investigating the efficacy of a novel vaccine consisting of modified HPV 16E7 fused with human cytotoxic T-lymphocyte antigen 4 (CTLA4). The regions in HPV16 E7 gene associated with its transformation and CTL-enhanced response were modified; the resultant HPV16mE7 was fused with extracellular region of CTLA4 to generate HPVml6E7-eCTLA4 fusion protein. Binding of this fusion protein to B7 molecules expressed on antigen presenting-cells (APCs) was demonstrated. C57BL/6 (H-2^b) mice immunized with low dose of the fusion protein (10 μg) produced higher titer antibody and stronger specific CTL response, and expressed higher levels of IFN-γ and IL-12, compared with those immunized with HPVml6E7 only or admixture of HPVml6E7 and CTLA4, or PBS; and were protected from lethal dose tumor challenge. Tumor growth was retarded and survival prolonged in mouse models with the fusion protein treatment. Our results demonstrate that fusion of HPV16 E7 with eCTLA4 targeting APCs resulted in enhanced immunity, and that this fusion protein may be useful for improving the efficacy of immunotherapeutic treatments of cervical cancer and other HPV16 infection-associated tumors.     

Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4⁺ and CD8⁺ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4⁺ and CD8⁺ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8⁺ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4⁺ and CD8⁺ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical cancer. Although more than 100 distinct HPV genotypes have been described, and at least 20 are associated with cervical cancer, HPV type 16 (HPV-16) is by far the most frequently detected in cervical neoplasia regardless of the geographical origin of the patients (4). In the last few years significant advances have been made in the development of candidate prophylactic vaccine against cervical cancer and HPV-related infections. In several large prospective randomized studies, virus-like particles consisting of the HPV-16 and HPV-18 major capsid protein L1 (L1-VLPs) have shown promise in protecting young healthy females against persistent infection with HPV-16 and HPV-18 and their associated cervical intraepithelial neoplasia (reviewed in reference 12). These data strongly suggest that the implementation of large-scale L1-VLP-based prophylactic vaccinations have the potential to dramatically reduce worldwide cervical cancer rates in the years to come.Unfortunately, because HPV infection is endemic in humans and there is a long latency from HPV infection to the development of invasive cervical cancer in women, even if prophylactic L1-based vaccinations are implemented on a worldwide scale today it would take decades to perceive any significant benefit. Consistent with this view, an estimated 5 million cervical cancer deaths will occur in the next 20 years due to existing HPV infections (4, 12). Thus, the current development of therapeutic vaccines for protection against persistent HPV infections, cervical cancer, and its precursor lesions remains an area of great interest.Although the interactions between the host immune system and HPV-infected cells are still not completely understood, several lines of evidence suggest that protection against HPV-related infections by L1-VLP-based vaccines is likely conferred by the generation of high levels of neutralizing antibodies (12, 38). Nevertheless, a potential crucial role of L1-specific T-cell responses and the involvement of T cells in mediating the production of neutralizing antibodies and antiviral effect in infected hosts has been previously hypothesized (8, 24). This point may be particularly noteworthy in patients harboring HPV-infected cervical lesions because several studies have demonstrated the critical importance of both cytotoxic (CD8⁺) and helper (CD4⁺) T cells in achieving clinical responses (1, 5, 16-18, 20, 23). However, limited information is currently available to evaluate whether cell-mediated immune responses to L1-VLP may have any significant therapeutic effect in cervical cancer patients harboring HPV-16 positive tumors. Furthermore, to our knowledge, no direct comparison of the therapeutic efficacy of L1 and E7-specific immune responses against naturally HPV-16-infected cervical cancer have been yet reported in human patients.In the present study we have analyzed and quantified by highly sensitive real-time PCR (RT-PCR) the RNA levels of E6, E7, and L1 in flash-frozen biopsy specimens obtained from HPV-16-infected cervical carcinomas and in short- and long-term primary cultures of HPV-16-positive cervical tumors. In addition, we have studied the kinetics of expression of these genes and proteins during the establishment of HPV-16-positive primary tumors in vitro. Finally, using completely autologous systems of naturally infected HPV-16-positive human tumors, we have carefully studied the phenotype and function of L1-specific CD4⁺ and CD8⁺ T-lymphocyte responses generated by VLP-loaded dendritic cells (DCs) and compared their therapeutic potential to those elicited by DC loaded with the E7 oncoprotein.     

553 例女性宫颈高危型HPV 感染与宫颈病变的相关性研究

目的:研究女性生殖道高危型人乳头瘤病毒(high risk human papilloma virus,hrHPV)感染现状及子宫颈上皮内瘤样病变(cervical intraepithelial neoplasia,CI N)的现患率,并分析该人群年龄与HPV感染及宫颈癌前病变的相关性以及hrHPV病毒载量与宫颈病变之间剂量-效应关系。方法:对553例30岁-78岁有性生活史的女性进行以人群为基础的横断面调查。对所有接受筛查的妇女均行宫颈HPV检测及电子阴道镜下病理活检,并以病理结果作为诊断子宫颈病变的金标准。资料采用EXCEL整理,利用x2检验和非条件Logistic回归分析危险因素和CI N的关系。结果:553例受检对象总体阳性检出率为18.0%。hrHPV感染情况在不同年龄段分布有显著差异,hrHPV病毒载量越高宫颈病变的程度越重。结论:不同年龄段hrHPV感染情况不同,hrHPV病毒载量与宫颈病变之间存在剂量-效应关系。     

Design,Immune Responses and Anti-Tumor Potential of an HPV16 E6E7 Multi-Epitope Vaccine

Cervical cancer is a common type of cancer among women worldwide and infection with high-risk human papillomavirus (HPVs) types represents the major risk factor for the etiopathogenesis of the disease. HPV-16 is the most frequently identified HPV type in cervical lesions and expression of E6 and E7 oncoproteins is required for the uncontrolled cellular proliferation. In the present study we report the design and experimental testing of a recombinant multi-epitope protein containing immunogenic epitopes of HPV-16 E6 and E7. Tumor preventive assays, based on the engraftment of TC-1 cells in mice, showed that the E6E7 multi-epitope protein induced a full preventive anti-tumor protection in wild-type mice, as well as in mice deficient in expression of CD4⁺ T cells and TLR4 receptor. Nonetheless, no anti-tumor protection was observed in mice deficient in CD8⁺ T cells. Also, the vaccine promoted high activation of E6/E7-specific T cells and in a therapeutic-approach, E6E7 protein conferred full anti-tumor protection in mice. These results show a potential use of this E6E7 multi-epitope antigen as a new and promising antigen for the development of a therapeutic vaccine against tumors induced by HPV.     

不同地区HPV16 E7基因的克隆及序列差异分析

采用PCR扩增、pGEM T载体克隆和核苷酸序列分析的方法对一例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与野生型 (德国标准株 )及已发表的HPV16湖北株 (HPVHB)进行了比较。结果发现武汉地区HPV16型E7基因仅第 5 4位出现一个同义突变 ,而高发区HPV16型E7基因存在差异 ,第 77位氨基酸由精氨酸 (Arg)变为半胱氨酸 (Cys) ,第 96位由谷氨酰氨酸 (Gln)变为精氨酸 (Arg) ,E7蛋白的二级结构及亲、疏水性也相应改变 ,与野生型有较大差异     

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach

The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells.