Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒病毒颗粒蛋白和疫苗

• 批准号: 8157217
• 负责人: JOHN T. SCHILLER
• 金额: $ 170.27万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8157217
• 关键词: Papillomavirus; Proteins; Vaccines; Virion

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.

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。但是，人类生殖器病变的恶性进展与某些人类乳头瘤病毒（HPV）类型之间存在很强的联系，最常见于HPV 16。我们的研究主要涉及疫苗的发展和针对HPV的其他感染抑制策略以及PV生命周期的阐明。 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，这些乳头瘤病毒颗粒会导致封装的标记质粒，以下称为伪文化。这种方法代表了乳头瘤病毒研究中的技术突破。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。我们发现，雌性小鼠生殖道的感染，即使是单层宫颈细胞的感染，都需要将基底膜暴露于病毒。衣壳与地下膜平均结合，但不与完整柱状或分层鳞状上皮的顶端表面结合。 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上皮层，从而将基底膜暴露于病毒。有趣的是，如果在角叉菜胶中而不是其正常胶凝剂中配制了非氧合诺酚-9。角叉菜胶是一种广泛用于加工食品和化妆品的藻类多糖，是某些非处方润滑剂的主要胶凝剂。结果表明，使用N-9精子剂的妇女可能会增加获得生殖器HPV感染的风险，并且在基于角叉菜胶的凝胶中，N-9可以消除这种风险。 Carrageenan作为菌心的两项NCI赞助的临床试验，以防止年轻女性的生殖器HPV感染。我们在小鼠宫颈阴道挑战模型中的研究表明，损害生殖器上皮完整性的其他干预措施可能会增强HPV感染。通过设计筛选细胞学（PAP）的外核细胞和遗传细胞的获取会通过设计破坏上皮。因此，我们试图确定细胞学标本（PAP涂片）收集程序是否使子宫颈在恒河猴模型中更容易受到HPV感染的影响。在提交出版的一项研究中，我们发现PAP涂片收集程序大大增强了RFP表达HPV16假病毒的感染。但是，在标本收集大部分大量消除了感染增强效果之后，将其使用胶胶凝胶而不是手术室用作内部数字检查的润滑剂。这些发现表明，女性的细胞学筛查可能会导致对HPV感染的敏感性的短暂增强，并且在检查期间使用基于角叉菜胶的凝胶可能会减轻这种增强。基于假病毒的小鼠子宫颈挑战模型也被用来探索乳头瘤病毒感染的基本特征，以及在女性生殖道中抗体介导的中和该病毒中和的机制。在最近在PNA中发表的一项研究中，我们发现，在乳头瘤病毒独有的过程中，感染的初始步骤在初始结合与硫酸葡萄糖蛋白聚糖蛋白聚糖（HSPG）的基底膜上，即先前的细胞表面结合。具体而言，病毒体经历了构象变化，该构象变化暴露了L2小帽蛋白的N末端，以纤维蛋白裂解，这是一种细胞蛋白酶，我们在体内感染部位发现了丰富的细胞蛋白酶。这种裂解在弯曲中导致高度保守的L2跨中和表位，这些表位立即在裂解位点的下游。 阻断HSPG相互作用或脂肪蛋白裂解阻止了宫颈阴道感染。在一个需要几个小时的过程中，病毒体从地下膜转移到侵入创伤部位的角质形成细胞表面，然后将病毒体内化。在一项在细胞宿主和微生物中发表的研究中，我们确定了L1 VLP疫苗（类型（类型为商业可用）和L2疫苗（我们作为一种更广泛的交叉类型中和替代方案）在体内通过不同机制在体内感染中阻断的抗体。 L1 VLP抗体阻断与基底膜结合，但允许与角质形成细胞结合。但是，L2交叉中和表位未暴露，病毒体并未内化。 L2抗体广泛中和HPV类型允许初始病毒粒子结合与基底膜HSPG的结合。然而，在以后的时间点没有在组织中检测到病毒体，这表明L2抗体可阻止稳定转移到角质形成细胞上。这些研究很可能提供了对活的哺乳动物宿主中如何感染其相关组织以及疫苗诱导中和抗体阻止体内感染的最详细的机械理解。 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，这证明是我们最近开发有效的阴道疫苗接种策略的关键。在申请专利的研究中，我们发现N-9治疗的小鼠的内部伪病毒疫苗接种可诱导对伪病毒转导的靶抗原的强大的全身性和粘膜T和B细胞反应。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，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。 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。现在，这个概念正在与Franchini博士合作进行SIV/Rhesus猕猴的浸润爆发挑战模型进行测试。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。