Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（PV）感染动物和人类的上皮，其中它们通常在感染部位诱导良性增殖。此外，一些人乳头瘤病毒（HPV），特别是HPV 16的持续感染可诱导人类生殖器和口咽部病变的恶性进展。我们的研究主要涉及乳头瘤病毒感染的生物学，PV生命周期的阐明，以及疫苗和其他抑制HPV感染的策略的开发。我们以前开发了一种简单而有效的策略，用于产生高滴度的感染性乳头瘤病毒颗粒，该颗粒包裹标记质粒，即假病毒体。我们已经在我们的基础病毒学和转化研究工作中利用了这项技术，从而开发了HPV的小鼠宫颈阴道攻击模型。使用该模型，我们确定了HPV感染所需的上皮完整性的瞬时破坏，病毒生命周期的第一步涉及病毒与基底膜的结合，并且只有在病毒位于基底膜上时衣壳中的L2蛋白经历酶促裂解后，病毒才转移到靶上皮细胞。L2的切割与衣壳中的构象变化相关，导致位于切割位点C-末端的高度保守的L2交叉中和表位暴露。在需要几个小时的过程中，病毒体从基底膜转移到角质形成细胞的表面，角质形成细胞使暴露的基底膜再上皮化，然后病毒体被内化。小鼠模型还使我们能够确定由L1 VLP（病毒样颗粒）疫苗和L2疫苗诱导的抗体通过不同的机制阻断体内感染。在高浓度下，L1 VLP诱导的抗体阻断病毒体与基底膜的结合，而在低浓度下，它们允许基底膜结合，但阻止与靶角质形成细胞的结合。由于L2交叉中和表位是隐蔽的，并且直到病毒体已经结合到基底膜之后才暴露，因此广泛交叉中和HPV类型的L2抗体不干扰初始基底膜结合。然而，一旦L2表位暴露，而病毒体保留在基底膜上，L2抗体可以结合病毒体并通过阻止病毒体转移到靶角质形成细胞来消除感染过程。在小鼠模型和为恒河猴开发的类似模型中，通过局部应用角叉菜胶来预防感染，角叉菜胶是一种广泛用于加工食品和化妆品的藻类多糖，并且是一些非处方润滑剂中的主要胶凝剂。根据我们的数据，卡拉胶作为杀微生物剂预防年轻女性生殖器HPV感染的临床试验正在进行中。使用HPV假病毒，我们以前开发了一种高通量体外HPV中和试验，该试验已成为该领域的金标准。基于对体内感染过程和L2疫苗提供保护的机制的更好理解，我们开发了一种替代的体外中和试验，其在检测L2中和抗体方面比标准试验灵敏约三个数量级。该替代检测方法在确定候选L2疫苗的免疫原性和L2疫苗的临床试验方面具有相当大的潜力。我们开发了一种方法，在用壬苯醇醚-9（N-9）短暂破坏上皮后诱导有效的HPV假病毒感染女性生殖道，这被证明是我们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N-9处理的小鼠的阴道内假病毒疫苗接种诱导对由假病毒体转导的靶抗原的强烈的全身和粘膜T和B细胞应答。系统性应答与先前优化的Ad 5载体诱导的应答相媲美。阴道内反应非常强烈，高达80%的阴道内CD 8 T细胞对靶抗原呈四聚体染色阳性。大多数诱导的T细胞似乎是上皮内的，并在接种后至少100天维持在阴道中。阴道内假病毒疫苗接种是一种很有前途的方法，用于将免疫应答集中于女性生殖道，这可能会增加针对单纯疱疹病毒感染、HIV感染和HPV感染的治疗性疫苗的有效性。我们还组织并参与了NCI病毒样颗粒疫苗的人体临床试验，此前我们开发了商业HPV疫苗的基础技术。我们最初在哥斯达黎加测试了二价HPV疫苗（由葛兰素史克公司生产），对接受少于标准3剂方案的妇女进行的事后分析表明，在7年的研究期间，她们与接受所有3剂疫苗的妇女一样，可以预防疫苗靶向HPV类型（HPV 16/18）的感染。此外，仅接受一次剂量的妇女在第1年至第7年期间血清HPV 16/18抗体水平稳定，这表明强有力的保护可能是持久的。这一令人惊讶的结果提出了单剂量疫苗可能提供长期保护的可能性。哥斯达黎加现已开始进行一项严格控制的临床试验，测试一剂疫苗与两剂FDA批准的疫苗的效力。

Papillomaviruses (PVs) infect the epithelia of animals and humans, where they generally induce benign proliferations at sites of infection. In addition, persistent infection by some human papillomaviruses (HPV), especially HPV16, can induce malignant progression of human genital and oropharynx lesions. Our research is mainly concerned with the biology of papillomavirus infection, elucidation of the PV life cycle, and development of vaccines and other strategies to inhibit HPV infection. We previously developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited this technology in our basic virologic and translational research efforts, leading to developing a mouse cervicovaginal challenge model for HPVs. Using this model, we determined that transient disruption of epithelial integrity is required for HPV infection, that the first step in the virus life cycle involves binding of the virus to the basement membrane, and that the virus transfers to target epithelial cells only after the L2 protein in the capsid has undergone an enzymatic cleavage while the virus is on the basement membrane. The cleavage of L2 is associated with a conformational change in the capsid, leading to exposure of highly conserved L2 cross-neutralization epitopes that lie C-terminal to the cleavage site. In a process that takes several hours, the virions transfer from the basement membrane to the surface of keratinocytes that are re-epithelializing the exposed basement membrane, and the virions are then internalized. The mouse model also enabled us to determine that antibodies induced by L1 VLP (virus-like particle) vaccines and L2 vaccines block in vivo infection by distinct mechanisms. At high concentrations, L1 VLP-induced antibodies block virion binding to the basement membrane, while at low concentrations, they permit basement membrane binding but prevent binding to target keratinocytes. Since the L2 cross-neutralization epitopes are cryptic, and are not exposed until after the virions have been bound to the basement membrane, L2 antibodies that broadly cross-neutralize HPV types do not interfere with initial basement membrane binding. Once the L2 epitopes are exposed, however, while the virions remain on the basement membrane, the L2 antibodies can bind to the virions and abrogate the infectious process by preventing transfer of the virions to target keratinocytes. Both in the mouse model and in an analogous model developed for the rhesus macaque, infection is prevented by local application of carrageenan, which is an algal polysaccharide widely used in processed food and cosmetics and is the main gelling agent in some over-the-counter lubricants. Based on our data, clinical trials of carrageenan as a microbicide to prevent genital HPV infection in young women are underway. Using HPV pseudoviruses, we previously developed a high throughput in vitro HPV neutralization assay that has become the gold standard for the field. Based on the improved understanding of the in vivo infectious process and the mechanism by which L2 vaccines confer protection, we developed an alternative in vitro neutralization assay that is approximately three orders of magnitude more sensitive than the standard assay in detecting L2 neutralizing antibodies. The alternative assay has considerable potential for determining the immunogenicity of candidate L2 vaccines and for clinical trials of L2 vaccines. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient epithelial disruption with nonoxynol-9 (N-9) proved to be the key to our development of an effective intravaginal vaccination strategy. In patent pending studies, we 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 intravaginal CD8 T cells staining tetramer positive for the targeted antigen. Most of the induced T cells appear to be intraepithelial and are maintained in the vaginal tract at least 100 days after vaccination. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract that may increase the effectiveness of therapeutic vaccines directed at herpes simplex virus infection, HIV infection, and HPV infection. We have also organized and participated in NCI human clinical trials of the virus-like particle vaccines, following our earlier development of the technology that underlies the commercial HPV vaccines. We initially tested the bivalent HPV vaccine (manufactured by GlaxoSmithKline) in Costa Rica, where post-hoc analyses of women who received fewer than the standard 3 dose regimen indicated they were as protected, during 7 years of study, against infection by the HPV types targeted by the vaccine (HPV16/18) as the women who received all 3 doses. In addition, the women who received only one dose had stable serum HPV16/18 antibody levels between years 1 and 7, suggesting the strong protection may be durable. This surprising result raised the possibility that a single vaccine dose might confer long-term protection. A rigorously controlled clinical trial that tests the efficacy of one vaccine dose vs. two doses with two FDA-approved vaccines has now started in Costa Rica.