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。在刚刚发表的一项研究中，我们进一步表明，在模拟伤口愈合环境的特定条件下，衣壳也可以利用特定修饰形式的硫酸软骨素蛋白聚糖作为初始附着因子。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，这已被证明是我们开发有效的，我们相信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。该公司正将我们的研究结果推向临床试验，但这些计划被COVID-19疫情所阻碍，部分原因是他们的疫苗基于相同的腺病毒载体。为了更广泛地评估HPV假病毒作为基因转移载体的潜力，我们进行了广泛的体内结合感染倾向调查。我们证明，完整的小鼠上皮在所有部位，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，NCI-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。我们现在已经发表了120多种癌症系的结合研究。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。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，我们正在研究基于HPV衣壳与红外染料IR700偶联的肿瘤治疗方法。在几种小鼠模型中，特别是在人类葡萄膜黑色素瘤的兔移植物模型中，静脉注射染料偶联VLPs并进行局部肿瘤照明的肿瘤治疗研究产生了非常令人鼓舞的结果。我们发表了这种治疗引起的肿瘤微环境的深刻变化。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。基于获得的积极安全性和有效性结果，公司赞助的3期研究将于2023年第四季度开始。在临床前小鼠研究中，我们获得了NCI主任创新奖，我们已经测试了假设，即先前存在的对巨细胞病毒的免疫反应可以有效地直接作用于肿瘤，杀死肿瘤细胞并启动/放大对肿瘤新抗原的保护反应，最初是通过假病毒转导在肿瘤中特异性表达病毒抗原。虽然观察到显著的肿瘤消退，但我们发现肿瘤内注射免疫优势的最小CD8和/或CD4限制性CMV肽可诱导更有效的肿瘤杀伤和抗原扩散，因此，在优化的方案下，它可诱导几乎所有治疗小鼠的完全长期消退和肿瘤再攻击保护。在这些研究的延伸中，我们已经确定疫苗特异性T细胞也可以用于治疗癌症。与CCR的比较肿瘤学部门一起，我们获得了CCR的资金，将于2023年第四季度开始在伴侣犬中进行概念验证癌症治疗试验。与DCEG同事的长期合作导致了许多与哥斯达黎加nci赞助的预防性HPV疫苗临床试验相关的出版物。去年，我们发表了关于对非靶向高风险类型的长期保护，1次和3次剂量后长期抗体亲和成熟的论文，并量化了非靶向疫苗类型引起的CIN3“揭膜”的程度。根据我们发表的来自CVT的临时分析，即使是单剂量的Cervarix也能产生长期保护（现在是11年），我们与DCEG的同事一起设计，获得NCI和盖茨基金会的资助，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已经基本完成，我们现在正在进行ELISA和体外中和试验来评估抗体反应。我们还刚刚启动了一项试验，以评估“老年”妇女（即26岁以上）对单剂量抗体的反应。

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. In a just published study we further shown that under certain conditions mimicking a wound healing environment, the capsid can also utilize specifically modified forms of chondroitan sulfate proteoglycans as initial attachment factors. 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 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. The company was moving our findings toward a clinical trial but these plans were waylaid by the COVID-19 epidemic, in part because their vaccine is based on the same adenovirus vectors. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we conducted a broad in vivio binding infection tropism survey. 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 NCI-60 panel were highly susceptible to VLP/psuedovirus binding/infection in vitro. We have now published binding studies on over 120 cancer lines. 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. 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 therapies based on HPV capsids coupled to an infrared dye, IR700. Tumor treatment studies of the intravenous delivery of the dye-coupled VLPs followed by local tumor illumination in several mouse models and particularly in a rabbit zenograft model of human uveal melanoma have produced exceptionally encouraging results. We published on the profound change in the tumor microenvironment induced by the this treatment. These studies have led to an FDA approved phase 1 and 2 trials for treatment of ocular melanoma. Based up the positive safety and efficacy results obtained, a company-sponsored phase 3 study should commence in Q4 2023. In preclinical murine studies, for which we a obtained an NCI Director's Innovation Award, we have testing the hypothesis that preexisting immune responses to CMV can be effectively direct to tumors to both kill tumor cells and initiate/amplify protect responses to tumor neoantigens, initially by expressing viral antigens specifically in the tumors via pseudovirus transduction. While significant tumor regression was observed, we found that intratumoral injection of immunodominant minimal CD8 and/or CD4 restricted CMV peptides induced more potent tumor killing and antigen spreading, such that, under an optimized protocol, it induced complete long term regression and protection from tumor rechallenge in almost all treated mice. In an extension of these studies we have determined that vaccine-specific T cells can also be recruited to treat cancer. Together with CCR's Comparative oncology unit, we secured CCR funding to conduct a proof-of concept cancer therapy trial in companion dogs which will commence in Q4 2023. 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 last year, we published papers on long term protection against non-targeted high risk types, on long term antibody avidity maturation after one vs 3 doses, and quantified the extent of "unmasking" of CIN3 arising from non-targeted vaccine types. Based on our published post hoc analyses from CVT indicating that even a single dose of Cervarix induces long term protection (now 11 years), we have worked with our DCEG colleagues to design, secure NCI and Gates Foundation 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 has largely been completed and we now conducting the ELISA and in vitro neutralization assays to assess the antibody responses. We have also just initiated a trial to evaluate the the antibody response to a single dose in "older" women, i.e. over 26 years of age.