Understanding 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疫苗和其他药物的开发，以预防和治疗HPV感染及其诱导的瘤变，阐明HPV生命周期，并利用这些研究中获得的见解开发针对其他癌症和慢性疾病的治疗方法。我们已经开发了一种简单而有效的策略来产生乳头瘤病毒样颗粒（vlp）和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。我们在基本病毒学和转化研究工作中利用了这些技术。我们利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，并利用该试验确定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。该过程的关键是与破坏上皮基底膜上的硫酸肝素蛋白聚糖（HSPGs）的强制性结合。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。大多数诱导T细胞是长寿命的上皮组织内驻留效应记忆CD8 T细胞（Trms）。关键的是，在用多肽或病毒载体全身接种后，CD8 IEL不会被诱导。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感染和HPV诱导瘤变的疫苗的有效性。与NIAID的Jeff Cohen博士合作，在小鼠和豚鼠HSV-2攻击模型中对表达HSV抗原的载体进行了测试。阴道内接种这些载体减少了阴道内HSV接种引起的局部病理，但标准的肠外接种没有。在CRADA与Crucell/Janssen合作的一项研究中，我们已经确定Ad26和Ad35载体诱导的局部T细胞反应与阴道内递送的HPV假病毒粒子相似，并且在诱导全身T细胞反应方面更优越。Crucell在腺病毒载体的GMP生产方面拥有广泛的专业知识，该公司正在将我们的发现推向临床试验。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癌和黑色素瘤细胞系在体外对VLP/伪病毒结合/感染高度敏感。HPV VLP/假病毒结合和感染的显著特异性是由肿瘤细胞表面的特异性HSPG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 vlp /假病毒粒子可能在肿瘤诊断或肿瘤导向的细胞毒性基因或药物偶联应用中有用。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研究中，在更昔洛韦治疗后，腹腔注射表达疱疹tk的HPV假病毒，增加了荷瘤小鼠的生存。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。基于CCR的Peter Choyke早期基于抗体的研究，我们正在研究衣壳与红外染料IR700偶联的肿瘤成像和杀伤。染料偶联VLPs在几种小鼠模型中的肿瘤治疗研究，特别是在人葡萄膜黑色素瘤的兔移植物模型中，已经产生了非常令人鼓舞的结果。这些研究已经导致FDA批准了眼部黑色素瘤治疗的1期和2期试验。剂量和频率递增试验的治疗阶段已经完成，我们现在正在收集随访安全性和有效性数据。在其他临床前研究中，我们获得了NCI主任创新奖，我们正在测试假设，即先前存在的对巨细胞病毒的免疫反应可以通过假病毒转导在肿瘤中特异性表达病毒抗原，从而有效地直接作用于肿瘤。为了进一步研究HPV病毒粒子组装的分子机制，并开发适合工业规模GMP生产的假病毒生产系统，我们设计并改进了在无细胞系统中产生DNA质粒转导假病毒粒子的方法。最初的基本机制发现被首次发表。最近，我们发表了一些方法，在化学定义的无细胞反应中产生假病毒粒子的滴度，与我们优化的细胞内组装反应产生的滴度相媲美。在未发表的研究中，我们发现了有效包裹RNA分子的方法。这些进展应该有助于将我们的临床前疫苗和肿瘤治疗研究转化为更多的人体试验。与DCEG同事的长期合作导致了许多与nci在哥斯达黎加赞助的预防性HPV疫苗临床试验（CVT）相关的出版物，在过去的一年里，重点是进一步完善疗效和安全性分析。根据最近发表的来自CVT的特别分析，即使是单剂Cervarix也能产生长期保护（现在是11年），我们在去年与我们的DCEG同事一起设计，获得NCI和盖茨的资助，并开始在哥斯达黎加女孩中进行一剂和两剂Cervarix和Gardasil-9的疗效试验。此外，一项比较一剂Cervarix和三剂Gardasil的免疫原性非劣效性试验正在进行中，并已完全入组。与麦吉尔大学的同事合作，我们发表了第一个随机对照临床研究的结果，表明局部应用卡拉胶凝胶可以保护年轻女性免受生殖器HPV感染。

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 agent to prevent and treat HPV infections and the neoplasia they induce, the elucidation of the HPV life cycle, and using the insights obtained in these studies to develop treatments against other cancers and chronic diseases. We have developed a simple and efficient strategy for generating papillomavirus-like particles (VLPs) and high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited these technologies in our basic virologic and translational research efforts. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs and 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 heparan sulfate proteoglycans (HSPGs) on the basement membrane of a disrupted epithelium. 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. Most of the induced T are long lived intraepithelial tissue resident effector memory CD8 T cells (Trms). Critically, CD8 IEL's were not induced after systemic vaccination with peptides or viral vectors. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract and so should increase the effectiveness of vaccines directed against HSV infections and against HPV induced neoplasia. In collaboration with Dr. Jeff Cohen, NIAID, vectors expressing HSV antigens are were tested in mouse and guinea pig HSV-2 challenge models. Intravaginal vaccination with these vectors reduced local pathology induced by intravaginal HSV inoculation, but standard parenteral vaccination did not. In a CRADA collaborated with Crucell/Janssen, we have determined that Ad26 and Ad35 vectors induce similar locals T cell responses as intravaginal delivery of HPV pseudovirions and are superior at inducing systemic T cell responses. Crucell has extensive expertise in GMP production of their adenovirus vectors, and the company is moving our findings toward a clinical trial. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we 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 carcinoma and melanoma cell lines in the NC1-60 panel were highly susceptible to VLP/psuedovirus binding/infection in vitro. The remarkable specificity of HPV VLP/pseudovirus binding and infection is mediated by specific HSPG modifications on the tumor cell surfaces that mimic those normally found on the basement membrane. The results suggest that HPV VLPs/pseudovirions may be useful in tumor diagnostic or tumor-directed cytotoxic gene or drug conjugate 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 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 CRADA with Aura Biosciences was initiated to facilitate further development and clinical testing of this approach to tumor therapy. Based on earlier antibody-based studies by CCR's Peter Choyke, we are investigating tumor imaging and killing by capsids coupled to an infrared dye, IR700. Tumor therapy studies of the dye-coupled VLPs in several mouse models and particularly in a rabbit zenograft model of human uveal melanoma have produced exceptionally encouraging results. These studies have led to an FDA approved phase 1 and 2 trials for treatment of ocular melanoma. The treatment phase of the dose and frequency escalation trial has been completed, and we are now collecting follow-up safety and efficacy data. In other preclinical studies, for which we a obtained an NCI Director's Innovation Award, we are testing the hypothesis that preexisting immune responses to CMV can be effectively direct to tumors by expressing viral antigens specifically in the tumors via pseudovirus transduction. In order to further investigate the molecular mechanisms of HPV virion assembly and develop a psuedovirus production system that is amenable to industrial scale GMP production, we devised and refined methods for generating DNA plasmid transducing pseudovirions in a cell-free system. The initial basic mechanistic findings were first published. More recently, we have published methods to generate titers of pseudovirions in chemically defined cell-free reactions that rival the titers generated in our optimized intracellular assembly reactions. In unpublished studies, we have discovered methods to efficiently encapsiding RNA molecules. These advances should facilitate the translation of our preclinical vaccine and tumor treatment studies into additional human trials. A long-standing collaboration with DCEG colleagues has resulted in many publications related to the NCI-sponsored prophylactic HPV vaccine clinical trial in Costa Rica (CVT), in the past year focusing on further refinements of the efficacy and safety analyses. Based on recently published post hoc analyses from CVT indicating that even a single dose of Cervarix induces long term protection (now 11 years), we have worked over the last year with our DCEG colleagues to design, secure NCI and Gates funding, and initiate an efficacy trial of one versus two doses of Cervarix and Gardasil-9 in Costa Rican girls. In addition an immunogenicity non-inferiority trial comparing one dose of Cervarix to three doses of Gardasil is underway and has been fully enrolled. In collaboration with colleagues at McGill University, we have published the results of the first randomized control clinical study demonstrating that topical application of a carrageenan gel protects young women from genital HPV infection.