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之间有很强的联系。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PV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感染性乳头瘤病毒颗粒，这些颗粒可以转导封装的标记质粒，下文称为假病毒粒子。这种方法代表了乳头瘤病毒研究的技术突破。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。假病毒生产技术正被用于探索乳头瘤病毒感染的基本特征和抗体介导的中和机制。我们已经确定，细胞蛋白转化酶furin对L2末端的切割是乳头瘤病毒感染所必需的。这种切割可能暴露了一个保守细胞受体的结合位点，因为在其他研究中，我们已经确定，广泛交叉型中和L2抗体结合了furin切割位点下游的肽。在溶液中，这个裂解位点不暴露在成熟的病毒粒子上。相反，只有在细胞表面和/或细胞外基质接触并导致衣壳构象改变后，它才会暴露出来。我们已经确定，令人惊讶的是，由许可的L1 VLP疫苗诱导的主要类中和抗体不能阻止细胞表面结合。相反，这些抗体阻止了L2蛋白切割所需的病毒粒子构象的改变，导致病毒粒子保留在细胞表面。相反，我们广泛交叉中和的L2疫苗诱导的抗体诱导病毒粒子从细胞表面分离，可能是通过阻止次级细胞表面受体的接合。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。我们发现，雌性小鼠生殖道的感染，即使是单层宫颈内细胞，也需要将基底膜暴露于病毒。衣壳与基底膜紧密结合，但不与完整柱状上皮或层状鳞状上皮的顶端表面结合。利用表达假病毒粒子的红色荧光蛋白（RFP）感染后基于全组织荧光成像的定量分析，我们进一步确定nonoxynol-9 （N-9）和Conceptrol（一种含有N-9的非对称杀精剂）在体内显著增强乳头瘤病毒感染，可能是由于它能够穿透上皮层，从而使基底膜暴露于病毒。有趣的是，如果在卡拉胶中配制壬氧醇醚-9而不是正常的胶凝剂，则没有检测到感染。卡拉胶是一种藻类多糖，广泛应用于加工食品和化妆品中，也是一些非处方润滑剂的主要胶凝剂。我们之前已经证明，卡拉胶和含有它的产品，在体外是非常有效的HPV感染抑制剂。结果表明，使用N-9杀精剂的妇女可能会增加感染生殖器HPV的风险，这种风险可能通过在卡拉胶凝胶中重新配制N-9来消除。我们目前正在与NCI校外分部DCP合作，启动一项卡拉胶作为杀微生物剂预防年轻女性生殖器HPV感染的临床试验。我们在小鼠宫颈阴道刺激模型中的研究表明，损害生殖上皮完整性的其他干预措施也可能增强HPV感染。获取宫颈外和宫颈内细胞用于细胞学（Pap）筛查，会有意破坏上皮。因此，我们试图确定细胞学标本（巴氏涂片）收集程序是否使恒河猴模型的子宫颈更容易感染HPV。在未发表的研究中，我们发现巴氏涂片收集程序极大地增强了表达HPV16假病毒的RFP的感染。然而，在标本采集后，使用卡拉胶凝胶而不是Surgilube作为内部指检的润滑剂，在很大程度上消除了感染增强效果。这些发现表明，女性细胞学筛查可能会导致HPV感染易感性的短暂增强，而在检查期间使用卡拉胶凝胶可能会减轻这种增强。我们开发了一种方法，在N-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的阴道内疫苗接种策略的关键。在正在申请专利的研究中，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。全身反应可与肌肉内注射优化Ad5载体引起的反应相媲美。阴道内接种疫苗可以将免疫反应集中到女性生殖道，从而提高针对HSV和HIV感染的疫苗的有效性，以及对HPV诱导的肿瘤进行免疫治疗的有效性。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上皮，无论是简单的、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，而破坏的上皮则易受感染。相比之下，NC1-60组中几乎所有的人上皮肿瘤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表明它们可能在肿瘤诊断或细胞毒性基因治疗应用中有用。在概念验证研究中，我们记录了在腹腔内注入表达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。

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 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 PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. This methodology represents a technical breakthrough in papillomavirus research. We have exploited this technology in our basic virologic and translational research efforts. Pseudovirus production technology is being used to explore the basic features of papillomavirus infection and mechanisms of antibody-mediated neutralization. We have determined that a cleavage of the terminus of L2 by the cellular proprotein convertase furin is required for papillomavirus infection. This cleavage presumably exposes a binding site for a conserved cellular receptor, because in other studies we have determined that broadly cross-type neutralizing L2 antibodies bind the peptide immediately downstream of the furin cleavage site. This cleavage site is not exposed on muture virions in solution. Rather it becomes exposed only after engagement of the cell surface and/or extracellular matrix and a resulting conformation change in the capsid. We have determined that, surprisingly, the major class of neutralizing antibodies induced by the licensed L1 VLP vaccines does not prevent cell surface binding. Rather these antibodies prevent the change in virion conformation required for furin cleavage of L2, resulting in retention of the virions on the cell surface. In contrast, the antibodies induced by our broadly cross-neutralizing L2 vaccines induce the disassociation of the virions from the cell surface, presumably by preventing engagement of a secondary cell surface receptor. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs. We have found that the infection of the female mouse genital tract, even of monolayer endocervical cells, requires exposure the basement membrane to the virus. The capsids bind avidly to the basement membrane but not to the apical surfaces of intact columnar or stratified squamous epithelia. Using a quantitative assay based on whole tissue fluorecence imaging after infection with red fluorescent protein (RFP) expressing pseudovirions, we have further determined that nonoxynol-9 (N-9) and Conceptrol, an over the counter spermacide that containing N-9, dramatically potentiate in vivo papillomavirus infection, presumably due to it's ability to permeablize the epithelial layers and thereby expose the basement membrane to the virus. Interesting, no infection was detected if nonoxynol-9 was formulated in carrageenan rather than its normal gelling agent. Carrageenan is an algal polysaccharide widely used in processed food and cosmetics and is the main gelling agent in some over-the-counter lubricants. We had previously shown that carragenen, and products that contain it, are extremely potent inhibitors of HPV infection in vitro. The results suggest that women using N-9 spermacides may be at increased risk of acquiring genital HPV infection and that this risk might be eliminated by reformulation of N-9 in a carrageenan based gel. We are currently working with the NCI extramural division DCP to initiate a clinical trial of carrageenan as a microbicide to prevent genital HPV infection in young women. Our studies in the mouse cervicovaginal challenge model suggested that other interventions that compromise the integrity of the genital epithelium might also potentiate HPV infection. Acquisition of ecto- and endocervical cells for cytology (Pap) screening disrupts the epithelium by design. Therefore we sought to determine whether the cytology specimen (Pap smear) collection procedure renders the cervix more susceptible to HPV infection in a rhesus macaque model. In unpublished studies, we found that the Pap smear collection procedure greatly potentiated infection by RFP expressing HPV16 pseudovirus. However, use of a carrageenan gel rather than Surgilube as the lubricant used for the internal digital exam after specimen collection largely abrogated the infection enhancing effect. These findings suggest that cytology screening in women might lead to a transient enhancement of susceptibility to HPV infection and that use of a carrageenan-based gel during the examine might mitigate this enhancement. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with N-9 has proven to be the key to our recent development of an effective intravaginal vaccination strategy. In patent pending studies, 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 intramuscular injection of optimized Ad5 vectors. Intravaginal vaccination could serve to focus immune responses to the female genital tract and so increase the effectiveness of vaccines directed against HSV and HIV infections and for immunotheraphy of HPV induced neoplasia. 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 test, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human epithelial tumor lines in the NC1-60 panel were highly susceptible to infection in vitro. The remarkable specificity of HPV pseudovirus binding and infection suggests that they may be useful in tumor diagnostic or cytotoxic gene 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 instillation of RFP-expressing pseudovirus.