Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（pv）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类乳头瘤病毒（HPV）类型，最常见的是HPV 16之间存在很强的关联。我们的研究主要涉及针对HPV的疫苗和其他感染抑制策略的开发以及HPV生命周期的阐明。我们已经开发了一种简单而有效的策略来产生高滴度的传染性乳头瘤病毒颗粒，这些颗粒可以转导封装的标记质粒，即假病毒粒子。我们已经在基本病毒学和转化研究工作中利用了这项技术。我们已经使用我们的假病毒技术开发了hpv的第一个宫颈阴道攻击模型。我们已经使用该试验来确定HPV感染其靶组织的分子机制，并确定抗体诱导的基于L1和l2的预防性疫苗如何预防感染。这个过程的关键是与被破坏的上皮基底膜的强制性结合。这种结合诱导了感染后续步骤所需的构象变化。我们之前开发的用于L1 VLP疫苗分析的体外中和试验已被证明对保护性L2抗体相对不敏感。基于我们对体内感染过程的理解，我们最近开发了一种新的体外中和试验，对L2抗体活性的测量灵敏度提高了1000倍。该试验将对进一步临床开发l2基疫苗至关重要，我们之前发现这种疫苗可以诱导抗体，与L1 VLP疫苗不同，它可以广泛交叉中和不同的粘膜和皮肤HPV类型。我们开发了一种方法，在非处方杀精剂壬诺醇-9短暂破坏后诱导有效的HPV假病毒感染女性生殖道，这已被证明是我们最近开发有效且我们认为实用的阴道内疫苗接种策略的关键。我们发现，经N-9处理的小鼠阴道内假病毒疫苗可诱导对假病毒粒子转导的靶抗原产生强烈的全身和粘膜T细胞和B细胞反应。系统反应可与先前优化的Ad5载体诱导的反应相媲美。阴道内反应非常强烈，高达80%的阴道内CD8 T细胞对靶抗原染色为四聚体阳性。大多数诱导的T细胞出现在上皮内，接种后100天阴道内仍保持高水平的效应记忆CD8 T细胞。关键的是，在系统接种Ad5载体后，CD8 IEL没有被诱导。阴道内假病毒疫苗接种是一种很有前途的方法，可以将免疫反应集中到女性生殖道，因此可以提高针对HSV和HIV感染以及HPV诱导的肿瘤的疫苗的有效性。与Franchini博士合作，在SIV/恒河猴阴道内挑战模型中对这一概念进行了测试。与Jeff Cohen博士合作，正在小鼠和豚鼠HSV-2攻击模型中测试表达HSV抗原的载体。我们已经确定阴道内递送腺病毒5载体诱导了与阴道内递送HPV假病毒粒子相似的局部T细胞反应，并且在诱导全身T细胞反应方面优于阴道内递送。在与Crucell/Johnson & Johnson的合作中，我们确定了Ad26和Ad35载体的行为相似。Crucell在腺病毒载体的GMP生产方面拥有丰富的专业知识，这将极大地促进我们的研究结果转化为临床试验。为了更广泛地评估HPV假病毒作为基因转移载体的潜力，我们进行了广泛的感染倾向调查。在正在申请专利的研究中，我们证明了完整的小鼠所有部位的上皮，无论是单纯的、柱状的还是鳞状的，都对病毒粒子结合和感染具有高度的抵抗力，而破坏的上皮则很敏感。相比之下，NC1-60组中几乎所有人源性上皮细胞系都对体外感染高度敏感。HPV假病毒结合和感染的显著特异性似乎是由肿瘤细胞表面的蛋白聚糖的特异性硫酸肝素修饰介导的，这些修饰类似于通常在基底膜上发现的修饰。结果表明，HPV假病毒粒子可能在肿瘤诊断或细胞毒性基因或药物治疗应用中有用。在概念验证研究中，我们记录了在腹腔注射表达rfp的假病毒后，植入裸鼠腹膜的人卵巢肿瘤结节的高度特异性结合和感染，以及戏剧性的成像。在一项使用卵巢转移小鼠模型的初步研究中，腹腔注射表达疱疹tk的HPV假病毒体，然后用更昔洛韦治疗，增加了荷瘤小鼠的生存。报告这些发现的手稿正在准备中。与Aura Biosciences合作的CRADA旨在促进这种肿瘤治疗方法的进一步开发和临床试验。与CCR的Peter Choyke和Aura一起，我们正在研究衣壳与红外染料IR700耦合的肿瘤成像和杀伤。肿瘤治疗研究在几种小鼠模型，特别是兔移植瘤模型的人葡萄膜黑色素瘤已经产生了非常令人鼓舞的结果。我们已经开始对最近发现的一种家鼠乳头瘤病毒进行病毒学和免疫学鉴定。大的乳头状瘤是在免疫缺陷菌株的口鼻和尾巴上诱导的（但有趣的是不是在背部），而不是在我们测试过的许多免疫能力菌株中。有趣的是，菌株在控制乳头瘤病毒形成时对CD8和CD4 T细胞的依赖性不同，要么需要两者，要么只需要其中一种。报告这些结果的手稿最近被《公共科学图书馆·病原体》接受。我们现在可以利用大量的免疫试剂和转基因小鼠菌株，首次深入了解乳头瘤病毒感染的免疫控制。与DCEG同事的长期合作已导致与哥斯达黎加nci赞助的预防性HPV疫苗临床试验相关的若干出版物。重点包括证明仅接种一剂或两剂疫苗的妇女在四年中具有很强的疫苗类型特异性保护，仅接种一剂疫苗后抗体反应的显著持久性，以及疫苗靶向类型对口腔感染的保护。

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.