Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒(PV)感染动物和人类的上皮细胞，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)的持续感染，特别是HPV16，可导致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，对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来产生高滴度的感染性乳头瘤病毒颗粒，该颗粒转导被包裹的标记质粒，即假病毒粒子。我们已经在我们的基础病毒学和翻译研究工作中利用了这项技术，导致开发了一种针对HPV的小鼠宫颈阴道挑战模型。利用这个模型，我们确定了HPV感染需要短暂的上皮完整性破坏，病毒生命周期的第一步涉及病毒与基底膜的结合，并且病毒只有在衣壳中的L2蛋白经历了酶裂解后才转移到靶上皮细胞，而病毒在基底膜上。L2的切割与衣壳的构象变化有关，导致位于C端的高度保守的L2交叉中和表位暴露在切割位置。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，角质形成细胞重新上皮化暴露的基底膜，然后病毒粒子被内化。小鼠模型也使我们能够确定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。在高浓度时，L1VLP诱导的抗体阻止病毒粒子与基底膜的结合，而在低浓度时，它们允许基底膜结合，但阻止与靶向角质形成细胞的结合。由于L2交叉中和表位是隐蔽的，直到病毒粒子与基底膜结合后才暴露，因此广泛交叉中和HPV类型的L2抗体不会干扰初始的基底膜结合。然而，一旦L2表位暴露，当病毒粒子留在基底膜上时，L2抗体可以与病毒粒子结合，通过阻止病毒粒子转移到靶向角质形成细胞来消除感染过程。无论是在小鼠模型中，还是在为猕猴开发的类似模型中，感染都是通过局部应用卡拉胶来预防的，卡拉胶是一种广泛用于加工食品和化妆品的藻类多糖，是一些非处方润滑剂的主要胶凝剂。根据我们的数据，卡拉胶作为一种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。利用HPV伪病毒，我们以前建立了高通量的体外HPV中和试验，该方法已成为该领域的金标准。基于对体内感染过程和L2疫苗提供保护机制的更好理解，我们开发了一种替代的体外中和试验，在检测L2中和抗体方面比标准试验灵敏约三个数量级。该替代试验在确定候选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，在用非氧合酶-9(N-9)短暂破坏上皮后，有效地诱导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策略的关键。在正在申请专利的研究中，我们发现，在N-9处理的小鼠阴道内接种伪病毒疫苗，可以诱导对伪病毒转导的靶抗原产生强烈的全身和粘膜T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CD8T细胞对靶抗原四聚体染色呈阳性。大多数诱导的T细胞似乎是上皮内的，并在接种疫苗后至少100天保持在阴道内。阴道内接种伪病毒疫苗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，从而提高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。我们还组织和参与了NCI病毒样颗粒疫苗的人体临床试验，此前我们开发了作为商业HPV疫苗基础的技术。我们最初在哥斯达黎加测试了双价HPV疫苗(由葛兰素史克生产)，在那里，对接种少于标准3剂方案的妇女进行的特别分析表明，在7年的研究中，她们与接种全部3剂的妇女一样，对疫苗(HPV16/18)所针对的HPV类型的感染具有保护作用。此外，只接受一剂疫苗的女性在1至7岁之间血清HPV16/18抗体水平稳定，这表明强大的保护可能是持久的。这一令人惊讶的结果增加了单剂疫苗可能提供长期保护的可能性。一项严格控制的临床试验现已在哥斯达黎加启动，该试验测试一剂疫苗与两剂FDA批准的疫苗的疗效。

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.