Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒病毒颗粒蛋白和疫苗

基本信息

批准号：
8937668
负责人：
JOHN T. SCHILLER
金额：
$ 206.07万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8937668
关键词：
Adenovirus Vector Adenoviruses Animal Model Animals Antibodies Antibody Formation Antigen Targeting Antigens Anus Auras B-Lymphocytes Back Basement membrane Benign Binding Biological Assay CD4 Positive T Lymphocytes CD8B1 gene Capsid Cavia Cell Culture Techniques Cell Line Cell surface Cervical Clinical Clinical Trials Collaborations Communicable Diseases Cooperative Research and Development Agreement Costa Rica Coupled Cutaneous Dependence Development Diagnostic Disease Division of Cancer Epidemiology and Genetics Dose Dyes Epithelial Cells Epithelium Female Ganciclovir Gene Transfer Genes Genital system HIV Infections Heparitin Sulfate Human Human Herpesvirus 2 Human Papilloma Virus Vaccine Human Papillomavirus Human papilloma virus infection Human papillomavirus 16 Image Immune Immune response Immunocompetent Immunologics Implant In Vitro Infection Infection prevention Intervention Intraperitoneal Injections Legal patent Lesion Life Cycle Stages Macaca mulatta Malignant - descriptor Malignant Neoplasms Manuscripts Measures Mediating Memory Metastatic Malignant Neoplasm to the Ovary Methods Modeling Modification Molecular Mouse Strains Mus Neoplasms Nodule Nude Mice Oropharyngeal Oryctolagus cuniculus Papilloma Papillomavirus Papillomavirus Infections Peritoneum Pharmaceutical Preparations Pharmacotherapy Plasmids Prevention Process Production Proteins Proteoglycan Publications Reagent Reporting Research Resistance SIV Simplexvirus Site Specificity Spermatocidal Agents Staining method Stains Surveys T cell response T-Lymphocyte Tail Technology Testing Therapeutic Agents Tissues Translational Research Translations Tropism Uveal Melanoma Vaccination Vaccine Clinical Trial Vaccines Vagina Virion Woman base cervicovaginal cytotoxic gene therapy gene transfer vector genetic vaccine in vivo interest intraepithelial killings man mouse model neoplastic cell neutralizing antibody novel oral infection ovarian neoplasm particle pathogen prevent prophylactic research clinical testing response tumor vaccination strategy vaccine development vaccine effectiveness vaccine evaluation vector vector-induced

项目摘要

Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital, anal and oropharyngeal lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is primarily concerned with development of vaccines and other infection inhibition strategies against HPV and the elucidation of the HPV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited this technology in our basic virologic and translational research efforts. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs. We have used this assay to define the molecular mechanism used by HPV to infect its target tissue and to determine how the antibodies induced L1- and L2-based prophylactic vaccines prevent infection. Key to the process is an obligatory binding to the basement membrane of a disrupted epithelium. This binding induces a conformational change required for subsequent steps in infection. The in vitro neutralizing assays we previously developed for L1 VLP vaccine analysis have proven to be relatively insensitive measures of protective L2 antibodies. Based on our understanding of the in vivo infectious process, we have recently developed a novel in vitro neutralizing assay that is 1000-fold more sensitive measure of L2 antibody activity. This assay will be critical in the further clinical development of L2-based vaccines, which we previously discovered to induce antibodies that, unlike L1 VLP vaccines, broadly cross-neutralize divergent mucosal and cutaneous HPV types. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with the over-the-counter spermicide nonoxonol-9 has proven to be the key to our recent development of an effective, and we believe practical, intravaginal vaccination strategy. We have found that intravaginal pseudovirus vaccination of N-9 treated mice induces strong systemic and mucosal T and B cell responses to target antigens transduced by the pseudovirions. Systemic responses rival those induced by previously optimized Ad5 vectors. Intravaginal responses are remarkably strong, with up to 80% of all intravaginal CD8 T cells staining tetramer positive for the targeted antigen. Most of the induced T appear to be intraepithelial, and high level of effector memory CD8 T cells are maintained in the vaginal tract 100 days after vaccination. Critically, CD8 IEL's were not induced after systemic vaccination with Ad5 vectors. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract and so might increase the effectiveness of vaccines directed against HSV and HIV infections and against HPV induced neoplasia. This concept was tested in an SIV/rhesus macaque intravaginal challenge model in collaboration with Dr. Franchini. In collaboration with Dr. Jeff Cohen, vectors expressing HSV antigens are being tested in mouse and guinea pig HSV-2 challenge models. We have determined that intravaginal delivery of Adenovirus 5 vectors induce similar locals T cell responses as intravaginal delivery of HPV pseudovirions and are superior at inducing systemic T cell responses. In collaborated with Crucell/Johnson & Johnson, we have determine that Ad26 and Ad35 vectors behave similarly. Crucell has extensive expertise in GMP production of their adenovirus vectors, which should greatly facilitate translation of our findings into a clinical trial. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we have conducted a broad infection tropism survey. In patent pending studies, we demonstrated that intact murine epithelium at all sites, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human-derived epithelial cell lines in the NC1-60 panel were highly susceptible to infection in vitro. The remarkable specificity of HPV pseudovirus binding and infection appears to be mediated by specific heparan sulfate modifications of proteoglycan on the tumor cell surfaces that mimic those normally found on the basement membrane. The results suggest that HPV pseudovirions may be useful in tumor diagnostic or cytotoxic gene or drug therapy applications. In proof of concept studies, we documented highly specific binding and infection, and dramatic imaging, of human ovarian tumor nodules implanted in nude mouse peritoneum after intraperitoneal injection of RFP-expressing pseudovirus. In a preliminarly study using a mouse model of ovarian metastases, intraperitoneal injection of Herpes TK-expressing HPV psuedovirions followed by ganciclovir treatment, increased survival of tumor bearing mice. A manuscript reporting these findings is being prepared. A CRADA with Aura Biosciences was initiated to facilitate further development and clinical testing of this approach to tumor therapy. With CCR's Peter Choyke and Aura, we are investigating tumor imaging and killing by capsids coupled to an infrared dye, IR700. Tumor therapy studies in several mouse models and particularly in rabbit zenograft model of human uveal melanoma have produced exceptionally encouraging results. We have begun a virologic and immunologic characterization of a recently discovered papillomavirus for the domestic mouse. Large papillomas are induced on the muzzles and tails (but interesting not the backs) in immunodeficient strains but not in any of the many immunocompetent strains that we have tested. Interesting, strains vary in their dependence on CD8 and CD4 T cells for control of papillomavirus formation, requiring either both or just one or the other. A manuscript reporting these results was recently accepted in PLoS Pathogen. We can now take advantage of the large set of immunological reagents and genetically modified mouse strains to provide the first in depth understanding of the immunological control of papillomavirus infection. A long standing collaboration with DCEG colleagues have resulted in several publications related to the NCI-sponsored prophylactic HPV vaccine clinical trial in Costa Rica. Highlights include demonstration of strong vaccine-type specific protection for four years in women receiving only one or two doses of the vaccine, remarkable persistence of the antibody responses after only one dose, and protection from oral infection by the vaccine targeted types.

乳头瘤病毒（PVS）感染动物和人的上皮，它们通常在感染部位诱导良性增殖。然而，人类生殖器，肛门和口咽病变的恶性进展与某些人乳头瘤病毒（HPV）类型之间存在很强的联系，最常见于HPV 16。我们的研究主要关注疫苗和其他感染抑制HPV的发展以及HPV生命周期的启示。我们已经开发了一种简单有效的策略，以产生高滴度的传染性乳头瘤病毒颗粒，这些乳头病毒颗粒会导致封装的标记质粒，即假病。我们已经在基本的病毒和翻译研究工作中利用了这项技术。我们已经使用伪病毒技术来开发HPV的首个宫颈阴道挑战模型。我们已经使用该测定法来定义HPV感染其靶组织的分子机制，并确定抗体如何诱导基于L1和L2的预防性疫苗预防感染。该过程的关键是在上皮中断的地下膜的强制性结合。这种结合引起了后续感染步骤所需的构象变化。我们先前为L1 VLP疫苗分析开发的体外中和测定已被证明是保护性L2抗体的相对不敏感的测量。根据我们对体内感染过程的理解，我们最近开发了一种新型的体外中和测定法，该测定法对L2抗体活性的敏感度更高。该测定在基于L2的疫苗的进一步临床开发中至关重要，我们以前发现，该疫苗诱导抗体，与L1 VLP疫苗不同，该抗体将跨上和不中和的粘膜和皮肤HPV类型广泛地中和。我们开发了一种诱导女性生殖道有效的HPV假病毒感染的方法，并在瞬时中断使用非处方药物杀精子剂nonoxonol-9诱导了人们，这已被证明是我们最近开发有效发展的关键，并且我们认为实用的，静脉内疫苗接种策略。我们发现，在N-9治疗的小鼠的阴道内假病毒疫苗接种会诱导强烈的全身性和粘膜T和B细胞反应对伪病毒转导的靶抗原的反应。系统性响应与先前优化的AD5矢量引起的响应相媲美。阴道内反应非常强，最多80％的所有阴道内CD8 T细胞染色四聚体的靶向抗原呈阳性。大多数诱导的t似乎是上皮内的，并且在疫苗接种后100天，在阴道中维持高水平的效应记忆CD8 T细胞。至关重要的是，与AD5载体进行全身疫苗接种后，CD8 IEL未被诱导。腔内假病毒疫苗接种是一种有前途的方法，是将免疫反应集中在女性生殖道上，因此可能会提高针对HSV和HIV感染的疫苗的有效性以及针对HPV诱导的肿瘤的疫苗。该概念在与Franchini博士合作的SIV/Rhesus Macaque内部丢进挑战模型中进行了测试。与杰夫·科恩（Jeff Cohen）博士合作，正在在小鼠和豚鼠HSV-2挑战模型中测试表达HSV抗原的载体。我们已经确定，腺病毒5个载体的阴道内输送诱导类似的当地T细胞反应，与HPV伪病毒的阴道递送，并且在诱导全身T细胞反应方面表现出色。在与Crucell/Johnson＆Johnson合作的情况下，我们确定AD26和AD35矢量的行为类似。 Crucell在GMP生产其腺病毒载体方面具有广泛的专业知识，这应该大大促进我们的发现转化为临床试验。为了更普遍地评估HPV伪病毒作为基因转移车的潜力，我们进行了广泛的感染Tropism调查。在申请专利研究中，我们证明了所有部位的完整鼠上皮，无论是简单，柱状还是鳞状，都对病毒粒子结合和感染都具有高度抗性，而易感的上皮易感性。相反，NC1-60面板中几乎所有人类衍生的上皮细胞系都非常容易在体外感染。 HPV假病毒结合和感染的显着特异性似乎是由特异性硫酸乙酰肝素对蛋白聚糖的修饰在肿瘤细胞表面上模仿了通常在基底膜上发现的肿瘤细胞表面上的介导的。结果表明，HPV假病毒可能在肿瘤诊断或细胞毒性基因或药物治疗应用中有用。在概念研究证明中，我们记录了高度特异性的结合和感染以及戏剧性的成像，这些人卵巢肿瘤结节在腹膜内注射表达RFP的假病毒后植入裸鼠腹膜中的人卵巢肿瘤结节。在使用卵巢转移小鼠模型的初步研究中，腹膜内注射疱疹表达TK TK的HPV伪动物，然后进行Ganciclovir治疗，增加了肿瘤轴承小鼠的存活率。正在准备报告这些发现的手稿。发起了具有Aura生物科学的CRADA，以促进对这种肿瘤治疗方法的进一步发展和临床测试。使用CCR的Peter Choyke和Aura，我们正在调查肿瘤成像和杀死的衣壳与红外染料IR700相连。在几种小鼠模型，特别是人类紫veal瘤模型中的肿瘤疗法研究中产生了极大的令人鼓舞的结果。我们已经开始了最近发现的家用小鼠乳头瘤病毒的病毒学和免疫学表征。在免疫缺陷菌株中诱导了大乳头瘤，但在我们已经测试过的许多免疫能力菌株中没有诱发。有趣的是，菌株对CD8和CD4 T细胞的依赖性有所不同，以控制乳头瘤病毒形成，这两者都需要或仅一个或另一个。最近在PLOS病原体中接受了报告这些结果的手稿。现在，我们可以利用大量的免疫试剂和转基因的小鼠菌株，以深入了解乳头瘤病毒感染的免疫控制。与DCEG同事进行了长期的合作，导致了与NCI赞助的预防性HPV疫苗临床试验有关的几个出版物。亮点包括在仅接受一两剂疫苗的女性中表现出强疫苗型特异性保护，仅接受一剂疫苗的持续存在明显的持久性，以及仅接受一剂剂量的抗体反应，并通过疫苗靶向类型的口服感染。