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。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。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。两项由nci赞助的卡拉胶作为杀微生物剂预防年轻女性生殖器HPV感染的临床试验即将开始。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可能会增强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。在一项提交发表的研究中，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。基于假病毒的小鼠宫颈阴道攻击模型也被用于探索乳头瘤病毒感染的基本特征以及抗体介导的病毒在女性生殖道中的中和机制。在最近发表在《美国国家科学院院刊》上的一项研究中，我们发现，在乳头瘤病毒特有的过程中，感染的初始步骤发生在与基底膜上的硫酸庚聚糖蛋白聚糖（HSPG）初始结合之后，这是细胞表面结合之前。具体来说，病毒粒子经历构象变化，暴露L2次要衣壳蛋白的n端被furin（一种我们在体内感染位点大量检测到的细胞蛋白酶）切割。这种切割反过来导致高度保守的L2交叉中和表位暴露，这些表位位于切割位点的下游。阻断HSPG相互作用或furin切割可预防宫颈感染。在一个需要几个小时的过程中，病毒粒子从基底膜转移到角质形成细胞的表面，侵入创伤部位，然后病毒粒子被内化。在《细胞宿主和微生物》杂志接受发表的一项研究中，我们确定L1型VLP疫苗（其类型是市售的）和L2型疫苗（我们开发的是一种更广泛的交叉型中和替代疫苗）诱导的抗体通过不同的机制阻断体内感染。L1 VLP抗体阻断与基底膜的结合，但允许与角质形成细胞结合。然而，L2交叉中和表位不暴露，病毒粒子不内化。广泛交叉中和HPV类型的L2抗体允许初始病毒粒子与基底膜HSPG结合。然而，在稍后的时间点组织中没有检测到病毒粒子，这表明L2抗体阻止了向角化细胞的稳定转移。这些研究很可能提供最详细的机制，了解病毒如何在活的哺乳动物宿主中感染其相关组织，以及疫苗诱导的中和抗体如何在体内预防感染。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。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细胞似乎在上皮内，接种后100天阴道内仍保持高水平的效应记忆CD8 T细胞。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。这一概念目前正在与Franchini博士合作，在SIV/恒河猴阴道内挑战模型中进行测试。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。

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. 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. 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. Two NCI-sponsored clinical trial of carrageenan as a microbicide to prevent genital HPV infection in young women will soon commence. Our studies in the mouse cervicovaginal challenge model suggested that other interventions that compromise the integrity of the genital epithelium might 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 a study submitted for publication, 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. The pseudovirus-based mouse cerviovaginal challenge model is also being used to explore the basic features of papillomavirus infection and the mechanisms of antibody-mediated neutralization of the virus in the female reproductive tract. In a study recently published in PNAS, we found that, in a process unique to papillomaviruses, the initial steps in infection occur after initial binding to heperan sulfate proteoglycans (HSPG) on the basement membrane, prior cell surface binding. Specifically the virions undergo a conformational change that exposes the N-terminus of the L2 minor capsid protein to cleavage by furin, a cellular protease that we detect in abundance at the sites of infection in vivo. This cleavage in turns leads to exposure of highly conserved L2 cross-neutralizing epitopes that are immediately downstream of the cleavage site. Blocking HSPG interactions or furin cleavage prevented cerviovaginal infection. In a process that takes several hours, the virions transfer from the basement membrane to the surface of keratinocytes invading the site of trauma, and the virions are then internalized. In a study accepted for publication in Cell Host and Microbes, we determined that antibodies induced by L1 VLP vaccines (of type that are how commercially available) and L2 vaccines (which we developing as a more broadly cross type-neutralizing alternative) block in vivo infection by distinct mechanisms. L1 VLP antibodies block binding to the basement membrane, but allow binding to the keratinocytes. However, the L2 cross-neutralizing epitopes are not exposed and the virions are not internalized. L2 antibodies that broadly cross-neutralize HPV types permit the initial virion binding to the basement membrane HSPG. However, the virions are not detected in the tissue at later time points, suggesting that the L2 antibodies prevent stable transfer to the keratinocytes. These studies may well provide the most detailed mechanistic understanding of how a virus, in a living mammalian host, infects its relevant tissue and how vaccine induced neutralizing antibodies prevent infection in vivo. 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 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. 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 is now being testing in an SIV/rhesus macaque intravaginal challenge model in collaboration with Dr. Franchini. 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 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 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.