Human papillomavirus vaccines for cervical cancer.

Cervical cancer is one of the most common causes of cancer-related death in women. As a result of several recent advances in molecular biology, the association between human papillomavirus (HPV) infection and cervical cancer has been firmly established, and the oncogenic potential of certain HPV types has been clearly demonstrated. Several lines of evidence suggest the importance of the host's immune response, especially cellular immune response, in the pathogenesis of HPV-associated cervical lesions. These observations form a compelling rationale for the development of vaccine therapy to combat HPV infection. Both prophylactic and therapeutic HPV vaccine strategies are being developed. Prophylactic strategies currently under investigation focus on the induction of effective humoral immune responses against subsequent HPV infection. In this respect, impressive immunoprophylactic effects have been demonstrated in animals using papillomavirus-like particles (VLPs). VLPs are antigenic and protective, but are devoid of any viral DNA that may be carcinogenic to the host. For treatment of existing HPV infection, techniques to improve cellular immunity by enhancing viral antigen recognition are being studied. For this purpose, the oncogenic proteins E6 and E7 of HPV-16 and -18 are the focus of current clinical trials for cervical cancer patients. The development of successful HPV-specific vaccines may offer an attractive alternative to existing screening and treatment programs for cervical cancer.     

Therapeutic human papillomavirus vaccines: current clinical trials and future directions

Background: Cervical cancer is the second largest cause of cancer deaths in women worldwide. It is now evident that persistent infection with high-risk human papillomavirus (HPV) is necessary for the development and maintenance of cervical cancer. Thus, effective vaccination against HPV represents an opportunity to restrain cervical cancer and other important cancers. The FDA recently approved the HPV vaccine Gardasil for the preventive control of HPV, using HPV virus-like particles (VLP) to generate neutralizing antibodies against major capsid protein, L1. However, prophylactic HPV vaccines do not have therapeutic effects against pre-existing HPV infections and HPV-associated lesions. Furthermore, due to the considerable burden of HPV infections worldwide, it would take decades for preventive vaccines to affect the prevalence of cervical cancer. Thus, in order to speed up the control of cervical cancer and treat current infections, the continued development of therapeutic vaccines against HPV is critical. Therapeutic HPV vaccines can potentially eliminate pre-existing lesions and malignant tumors by generating cellular immunity against HPV-infected cells that express early viral proteins such as E6 and E7. Objective: This review discusses the future directions of therapeutic HPV vaccine approaches for the treatment of established HPV-associated malignancies, with emphasis on current progress of HPV vaccine clinical trials. Methods: Relevant literature is discussed. Results/conclusion: Though their development has been challenging, many therapeutic HPV vaccines have been shown to induce HPV-specific antitumor immune responses in preclinical animal models and several promising strategies have been applied in clinical trials. With continued progress in the field of vaccine development, HPV therapeutic vaccines may provide a potentially promising approach for the control of lethal HPV-associated malignancies.     

A combination of DNA vaccines targeting human papillomavirus type 16 E6 and E7 generates potent antitumor effects

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancers. HPV E6 and E7 are two viral oncoproteins that are consistently expressed in HPV infections and HPV-associated malignancies. We have previously developed DNA vaccines encoding calreticulin (CRT) linked either to HPV type 16 (HPV-16) E6 or to HPV-16 E7, both of which generated significant antitumor effects against E6- and E7-expressing tumors. In this study, we demonstrate that simultaneous vaccination of C57BL/6 mice or HLA-A2 transgenic mice with both CRT/E6 and CRT/E7 DNA vaccines generates significant E6- and E7-specific T-cell immune responses in vaccinated mice. Furthermore, combined vaccination with both CRT/E6 and CRT/E7 DNA generates significantly better therapeutic antitumor effects against HPV E6- and E7-expressing tumors than vaccination with either CRT/E6 DNA or CRT/E7 DNA alone. Our data suggest that it may be desirable to combine DNA vaccines targeting E6 with DNA vaccines targeting E7 to develop effective immunotherapeutic strategies for control of HPV infection and HPV-associated lesions in a clinical setting.     

浆细胞样树突状细胞在肝脏疾病中的作用

浆细胞样树突状细胞(pDCs)在外周血中所占比例很小,其选择性地表达Toll样受体TLR7和TLR9。pDCs在接受病原体刺激后快速产生大量Ⅰ型干扰素,产生干扰素之后,pDCs通过激活传统树突状细胞、T细胞、自然杀伤细胞和B细胞形成保护性免疫反应,pDCs分化成为成熟树突状细胞,pDCs抗原提呈能力能够连接天然免疫和获得性免疫反应。已经有报道表明pDCs在病毒感染和癌症中发挥作用。本文中我们综述pDCs在肝脏疾病中的作用。     

Induction of human papilloma virus E6/E7-specific cytotoxic T-lymphocyte activity in immune-tolerant, E6/E7-transgenic mice

Despite promising preclinical results of various therapeutic anticancer immunization strategies, these approaches may not be effective enough to eradicate tumors in cancer patients. While most animal models are based on fast-growing transplantable tumors, malignancies in, for example, cervical cancer patients in general develop much more slowly, which may lead to immune suppression and/or immune tolerance. As a consequence, the immunomodulating signal of any therapeutic immunization regimen should be sufficiently potent to overcome this immunocompromised condition. In previous studies, we demonstrated that an experimental vaccine against human papillomavirus (HPV)-induced cervical cancer, based on Semliki Forest virus (SFV), induces robust HPV-specific cellular immune responses in mice. Now we studied whether this strategy is potent enough to also prime a cellular immune response in immune-tolerant HPV transgenic mice, in which CTL activity cannot be induced using protein or DNA vaccines. We demonstrate that, depending on the route of immunization, SFV-expressing HPV16 E6 and E7 indeed has the capacity to induce HPV16 E7-specific cytotoxic T cells in HPV-transgenic mice.     

TA-CIN, a vaccine incorporating a recombinant HPV fusion protein (HPV16 L2E6E7) for the potential treatment of HPV16-associated genital diseases

Commercially available prophylactic HPV vaccines for cervical cancer prevention have limited use in women with previous viral exposure. Therefore, a therapeutic HPV vaccine would benefit patients with HPV-associated genital diseases. Being developed by Cancer Research Technology Ltd, under license from Xenova Group plc, TA-CIN (Tissue Antigen - Cervical Intraepithelial Neoplasia) is a fusion protein vaccine comprising the HPV16 viral proteins L2, E6 and E7 for the treatment of HPV16-associated genital diseases. In mouse models, TA-CIN induced dose-dependent HPV16-specific CD4 and CD8 T-cell responses, which were enhanced when boosted with the vaccinia-based vector vaccine TA-HPV (Therapeutic Antigen - HPV). A phase I clinical trial of TA-CIN in healthy volunteers reported no serious adverse events and HPV16-specific cellular immune responses. Phase II trials in patients with anogenital and vulval intraepithelial neoplasia investigated heterologous prime/boost strategies with TA-CIN/TA-HPV and TA-HPV/TA-CIN, but neither of the regimens offered advantages over single-agent TA-HPV. A recent phase II trial investigating imiquimod/TA-CIN in patients with vulval intraepithelial neoplasia demonstrated significant infiltration of CD4 and CD8 T-cells in lesion responders and complete lesion regression in 63% of patients. More comprehensive case-controlled trials are needed to define responders to immunotherapy with TA-CIN and verify its prophylactic and therapeutic properties.     

Human papillomavirus 16 E6-specific CD45RA+ CCR7+ high avidity CD8+ T cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer

We defined the nature of the cellular immune response in 5 women with human papillomavirus (HPV) 16+cervical carcinoma at a single time point when surgery was performed for treatment. To monitor the differences in T-cell recognition, 2 approaches of tetramer-guided technology were employed: (i) the in situ localization of major histocompatibility complex class I peptide complexes in the tumor lesions and (ii) the ex vivo sorting of HLA-A*0201-restricted and HPV16 E6-reactive T cells. CD8 T cells from the periphery (peripheral blood lymphocytes), the tumor (tumor-infiltrating lymphocytes), and T cells harvested from draining lymph nodes (T-LN) were analyzed. HPV16 E6 tetramer-sorted lymphocytes from the different anatomic sites recognized an HLA-A*0201-restricted E6 peptide irrespective of the type of antigen-presenting cells used for stimulation as determined by interferon-gamma production: autologous tumor cells, HLA-A*0201 surrogate antigen-presenting cells pulsed with the nominal peptide, and an HLA-A*0201-matched human dendritic cell line transgenic for HPV16 E6. Further analysis showed that the HPV16 E6-reactive CD8 T cells were of high avidity defined by blocking with an anti-CD8-alpha specific monoclonal antibody. We found that HPV16 E6-reactive T cells reside preferentially within the CD45RA+ CCR7+ T-cell subpopulation of tumor-infiltrating lymphocyte, peripheral blood lymphocyte, and T-LN in cervical cancer patients, suggesting that successful immune surveillance of HPV16+ tumor cells in cervical cancer patients is impaired. The CD45RA+/CCR7+ phenotype of HPV antigen-reactive T cells may serve as an indicator of dysfunctional T cells, despite effective interferon-gamma production in response to HPV antigens.     

Detection of T helper responses, but not of human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma

Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T-cell-mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease.     

Molecular biology of human papillomavirus infection and cervical cancer

HPVs (human papillomaviruses) infect epithelial cells and cause a variety of lesions ranging from common warts/verrucas to cervical neoplasia and cancer. Over 100 different HPV types have been identified so far, with a subset of these being classified as high risk. High-risk HPV DNA is found in almost all cervical cancers (>99.7%), with HPV16 being the most prevalent type in both low-grade disease and cervical neoplasia. Productive infection by high-risk HPV types is manifest as cervical flat warts or condyloma that shed infectious virions from their surface. Viral genomes are maintained as episomes in the basal layer, with viral gene expression being tightly controlled as the infected cells move towards the epithelial surface. The pattern of viral gene expression in low-grade cervical lesions resembles that seen in productive warts caused by other HPV types. High-grade neoplasia represents an abortive infection in which viral gene expression becomes deregulated, and the normal life cycle of the virus cannot be completed. Most cervical cancers arise within the cervical transformation zone at the squamous/columnar junction, and it has been suggested that this is a site where productive infection may be inefficiently supported. The high-risk E6 and E7 proteins drive cell proliferation through their association with PDZ domain proteins and Rb (retinoblastoma), and contribute to neoplastic progression, whereas E6-mediated p53 degradation prevents the normal repair of chance mutations in the cellular genome. Cancers usually arise in individuals who fail to resolve their infection and who retain oncogene expression for years or decades. In most individuals, immune regression eventually leads to clearance of the virus, or to its maintenance in a latent or asymptomatic state in the basal cells.     

A cancer cell-specific inducer of apoptosis

Human papillomavirus (HPV) DNA is found in virtually all cervical cancers, strongly suggesting that these viruses are necessary to initiate this disease. The HPV E2 protein is required for viral replication, but E2 expression is usually lost in HPV-transformed cells because of the integration of viral DNA into the host chromosome. Several studies have shown that the reintroduction of E2 into HPV-transformed cells can induce growth arrest and apoptotic cell death. This raises the possibility that E2 could be useful in the treatment of HPV-induced disease. However, the effects of E2 on cell proliferation are not limited to HPV-transformed cells. The E2 protein from HPV type 16 can induce apoptosis via at least two pathways. One pathway involves the binding of E2 to p53 and operates in HPV-transformed cells, many non-HPV-transformed cell lines, and untransformed normal cells. The second pathway requires the binding of E2 to the viral genome and operates only in HPV-transformed cells. A mutation in E2 that significantly reduces the binding of this protein to p53 abrogates the induction of apoptosis in non-HPV-transformed cells and normal cells, but has no effect on the ability of the mutated protein to induce apoptosis in HPV-transformed cells. Here we show that a chimeric protein consisting of this mutant of E2, fused to the herpes simplex virus type 1 VP22 protein, can traffic between cells in a three-dimensional tumor model and induce apoptosis in HPV-transformed cells with high specificity. This cancer cell-specific inducer of apoptosis may be useful in the treatment of cervical cancer and other HPV-induced diseases.     

A virosomal immunization strategy against cervical cancer and pre-malignant cervical disease

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.     

HPV分型及高危八型定量检测在宫颈病变中的意义

目的分析宫颈病变中的人乳头瘤病毒的亚型分布及病毒载量的情况,以及其与宫颈疾病进展的关系。方法潮州中心医院就诊的220例液基细胞学检查异常的患者行阴道镜检查及组织病理学检查。收集宫颈脱落细胞标本,运用HybriMax导流杂交技术检测HPV的21个亚型,运用实时荧光PCR定量检测八个高危亚型的病毒载量。结果220例样本中,HPV感染率为87.3％,其中HPV16是最主要的基因型（50．91％）,随着宫颈病变程度的增高,总HPV感染率和HPV16感染率逐渐升高。HPV58,HPV33,HPV52,HPV31和HPV18是仅次于HPV16的五种基因亚型,其检出率分别为22．73％,11．82％,11．36％,5．9％和4．54％。高危HPV亚型的病毒载量与宫颈病变的严重程度呈正相关（1=0．373,P〈0．001）。结论尽管HPV52和HPV58是该地区HPV感染的主导亚型,但在宫颈病变患者中HPV感染以HPV16为主,而与宫颈癌最相关的是HPV16和HPV18。病毒载量是一个潜在的确定子宫颈癌及癌前病变的辅助指标。     

Intrabody strategies for the treatment of human papillomavirus-associated disease

Human papillomaviruses (HPVs) are associated with a variety of epithelial lesions, including benign genital warts and cervical intraepithelial neoplasia. Both cause significant morbidity in the general population, with cervical intraepithelial neoplasia progressing to cervical cancer in a subset of women who cannot resolve their infection. At present, there are no antiviral agents for the treatment of genital HPV infections, with many lesions requiring surgical intervention. Although other approaches are available for the treatment of genital warts, HPV infection cannot usually be cured and lesion recurrence is often a problem. A growing understanding of the molecular biology of HPV infection has identified several viral protein functions that may serve as drug targets. Among these are the HPV E1 and E2 proteins, which are necessary for viral genome replication and partitioning, and the E6 and E7 proteins, which are necessary for cell proliferation and apoptotic inhibition. With the exception of E1, these proteins lack enzymatic activity and achieve their effects by interacting with cellular proteins. Protein-protein interactions are in general quite difficult to inhibit using conventional small molecule drugs, but are amenable to inhibition using intracellular antibodies or intrabodies, which bind the viral proteins and sterically inhibit their association with cellular partners. The lack of homology between viral and cellular proteins, and the fact that HPV infections can be treated topically, makes them particularly well suited to the intrabody approach. This review covers the various strategies that are being considered for the treatment of HPV infections and the different intrabody formats that have been used to inhibit HPV function in model systems. The clinical utility of the approach is considered alongside the general difficulties of using protein molecules as intracellular therapeutics.     

Induction of cytotoxic T lymphocytes with dendritic cells transfected with human papillomavirus E6 and E7 RNA: implications for cervical cancer immunotherapy

Human papillomavirus (HPV) infection is associated with cervical cancer. The high-risk HPV E6 and E7 oncoproteins are constitutively expressed in most cervical carcinoma cells, and are, therefore, attractive antigens for cytotoxic T-lymphocyte (CTL)-mediated immunotherapy. The objective of this study was to evaluate the use of dendritic cells (DCs) transfected with RNA encoding the E6 and E7 protein for cervical cancer immunotherapy. The authors have shown that DCs transfected with RNA-encoding antigen stimulate potent antigen-specific CTL responses in vitro and in vivo. In this study, they tried to determine whether DCs transfected with E6 and E7 RNA stimulate primary, antigen-specific CTL responses in vitro. The results show that DCs pulsed with E6 or E7 RNA stimulate antigen-specific CTL responses that recognize and lyse DCs transfected with E6 and E7 RNA and human cervical carcinoma cells expressing the E6 and E7 products, and the lysis was comparable to that achieved with E6 and E7 peptide-pulsed DCs. Dendritic cells cotransfected with both E6 and E7 RNA stimulate CTLs that are more effective at lysing human cervical cancer cells. This study provides a rationale for the development of cervical carcinoma immunotherapy using DCs transfected with HPV E6 and E7 RNA.     

Human papillomavirus and cervical cancer

Human papillomaviruses (HPVs) are double stranded DNA viruses. To date, over 100 genotypes have been identified. Oncogenic types of HPV, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, are commonly associated with cervical cancer, with HPV16 being the most frequently detected genotype in women with cervical cancer worldwide. Genital HPV infections are common among young sexually active women and usually transient. Only a small fraction of infected women develop cervical cancer, implying the involvement of environmental and genetic cofactors in cervical carcinogenesis. The epidemiology of HPV infections and current advances on HPV vaccines will be reviewed here.