Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（PV）感染动物和人的上皮，其中它们通常在感染部位诱导良性增殖。然而，人类生殖器病变的恶性进展与某些HPV类型（最常见的是HPV 16）之间存在很强的相关性。我们已经产生了HPV 16和其他PV的病毒样颗粒（VLP），这些颗粒由L1主要衣壳蛋白或L1加L2次要衣壳蛋白组成。在动物模型中，经纯化的VLP的胃肠外注射诱导高滴度的中和抗体和对实验攻击的保护。基于这些结果，我们已经验证了GMP级VLP，最近完成了HPV 16 L1 VLP疫苗的1期和2期临床试验。接种者，即使是在没有佐剂的情况下接种的那些，也持续产生高滴度的HPV 16假病毒中和抗体，并且仅报告了轻微的副作用。特异性IgG抗体在子宫颈处发现。虽然口服避孕药的妇女宫颈滴度保持不变，但宫颈滴度在月经周期中变化很大，宫颈总IgG也是如此。这种变化的临床意义仍有待确定。在动物免疫原性研究中，我们已经确定由一种HPV 16变体的L1 VLP引发的抗体有效地中和了其他HPV 16变体的感染。因此，似乎在设计预防性疫苗时仅需要考虑单一HPV 16血清型。 我们还在开发备选疫苗。为了增加基于VLP的疫苗的治疗潜力，我们将非结构HPV蛋白作为L2融合蛋白掺入VLP中。用HPV 16 E7或E7-E2嵌合VLP接种产生CD 8限制性T细胞应答，其保护小鼠免受使用表达E7的肿瘤系的肿瘤攻击，并且还诱导已建立的肿瘤消退。已经开发了用于产生用于I期试验的临床级嵌合VLP的方法，并且疫苗批次正在进行临床前评价。 为了打破B细胞耐受性并诱导对自身蛋白的抗体应答，我们在VLP表面上以有序阵列展示了靶自身多肽。在小鼠中，在VLP上展示小鼠TNF肽使TNF IgG滴度增加1000倍。用缀合的VLP接种小鼠，保护它们免受实验诱导的类风湿性关节炎或降低疾病的严重程度。高密度的自身抗原似乎是消除B细胞耐受性的关键决定因素。用展示猕猴CCR 5的N末端的VLP接种的小鼠和猕猴产生高滴度的抗体，所述抗体结合细胞表面CCR 5并阻断培养细胞的HIV感染。这些发现表明了诱导自身抗体的一般方法，具有许多基本和应用的再研究应用。 为了开始确定VLP疫苗接种如何能够在没有佐剂和没有局部炎症的情况下诱导有效的B和T细胞应答，我们检查了VLP与未成熟小鼠骨髓来源的树突状细胞（BMDC）的相互作用。BMDC迅速结合和内化VLP。与VLP的相互作用，但不是无序的衣壳亚基，导致BMDC的快速表型成熟，但延迟释放的促炎细胞因子，相对于一个良好的特征性诱导剂，细菌脂多糖。VLP激活的BMDC在体外诱导Th 1主导的初级T细胞反应。结果提供证据表明，树突状细胞对病毒体表面的模式识别在抗病毒体免疫中起着重要作用。 我们还研究了次要衣壳蛋白L2在病毒体组装、感染和诱导中和抗体中的作用。我们发现，L2与五聚体L1衣壳，但不是完整的L1 VLP，表明这两个衣壳蛋白关联在病毒体组装的中间阶段。发现L2是体内基因组扩增所必需的。此外，突变分析表明L2在感染过程中发挥作用。特异性肽HPV 6、16或18 L2的抗体能够交叉中和异质性HPV类型。相反，L1/L2 VLP不诱导交叉中和抗体。这些结果表明，HPV病毒体含有隐蔽的或亚显性的L2中和表位，L2多肽疫苗可提供广谱保护生殖器HPV感染。此外，我们已经确定乳头瘤病毒通过网格蛋白依赖性途径感染细胞，VLP遵循与感染性病毒粒子相同的途径。

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 HPV types, most frequently HPV 16. We have generated virus-like particles (VLPs) for HPV 16 and other PVs that consist of the L1 major capsid protein or L1 plus L2, the minor capsid protein. Parenteral injection of purifed VLPs induced high titers of neutralizing antibodies and protection from experimental challenge in animal models. Based upon these results, we have validated GMP grade VLPs and have recently completed phase 1 and phase 2 clinical trials of an HPV16 L1 VLP vaccine. Vaccinees, even those vaccinated in the absence of adjuvant, consistantly produced high titers of HPV16 psuedovirion neutralizing antibodies and reported only minor side effects. Specific IgG antibodies are found at the cervix. Although the titers at the cervix remain constant in women taking oral contraceptives, cervical titers vary considerably during the menstrual cycle, as does total cervical IgG. The clinical implications of this variation remain to be determined. In animal immunogenicity studies we have determined that antibodies elicited by L1 VLPs of one HPV16 variant effectively neutralized infection by other HPV16 variants. Therefore, it appears that that only a single HPV16 serotype needs to be considered in designing a prophylactic vaccine. We are also developing alternative vaccine candidates. To increase the therapeutic potential of a VLP-based vaccine, we have incorporated non-structural HPV proteins into the VLPs as L2 fusion proteins. Vaccination with an HPV16 E7 or E7-E2 chimeric VLP generated a CD8 restricted T cell response that protected mice from tumor challenge using an E7 expressing tumor line and also induced regression of established tumors. A method for generating clinical grade chimeric VLPs for a phase I trial has been developed, and the vaccine lots are undergoing preclinical evaluation. To break B-cell tolerance and induce antibody responses to a self-protein, we have displayed target self-polypeptides in an ordered array on a VLP surface. In mice, display of a mouse TNF peptide on the VLPs increased TNF IgG titers 1000-fold. Vaccination of mice with the conjugated VLPs, either protected them from experimentally induced rheumatoid arthritis or reduced the severity of disease. High density of self-antigen appears to be a critical determinant for abrogating B cell tolerance. Mice and macaques vaccinated with VLPs displaying the N-terminus of macaque CCR5 produced high titers of antibodies that bound cell surface CCR5 and blocked HIV infection of cultured cells. These findings suggest a general method for inducing auto-antibodies, with many basic and applied reasearch applications. To begin to determine how VLP vaccination is able to induce potent B and T cell responses in the absence of adjuvant and without local inflammation, we have examined the interaction of VLPs with immature mouse bone marrow-derived dendritic cells (BMDCs). BMDCs rapidly bound and internalized VLPs. Interaction with VLPs, but not disorganized capsid subunits, resulted in rapid phenotypic maturation of BMDCs, but delayed release of pro-inflammatory cytokines, relative to a well characterized inducer, bacterial lipopolysacharide. VLP activated BMDCs induced a Th1 dominated primary T cell response in vitro. The results provide evidence that pattern recognition of virion surfaces by dendritic cells can play a central role in anti-virion immunity. We have also examined the role of the minor capsid protein L2 in virion assembly, infection, and induction of neutralizing antibodies. We found that L2 associates with pentameric L1 capsomers but not intact L1 VLPs, indicating that the two capsid proteins associate at an intermediate stage of virion assembly. L2 was found to be required for genome encapsidation in vivo. In addition, mutation analysis indicated that L2 functions during the infectious process. Antibodies to specific peptides HPV6, 16, or 18 L2 were able to cross-neutralize heterlogous HPV types. In contrast L1/L2 VLPs did not induce cross-neutralizing antibodies. These results indicate that HPV virions contain cryptic or subdominant L2 neutralizing epitopes and that an L2 polypepdide vaccine may provide broad-spectrum protection from genital HPV infection. In addition, we have determined that papillomaviruses infect cells via a clathrin-dependent pathway, and that VLPs follow the same route as infectious virions.