Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒(PV)感染动物和人的上皮细胞，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类型的HPV有很强的相关性，最常见的是HPV16型。我们已经为HPV16型和其他由L1主要衣壳蛋白或L1+L2(次要衣壳蛋白)组成的PV产生了病毒样颗粒(VLP)。在动物模型中，静脉注射纯化的VLP可诱导高滴度的中和抗体并保护其免受实验攻击。基于这些结果，我们已经验证了GMP级VLP，最近完成了HPV16VLP疫苗的第一阶段和第二阶段临床试验。接种疫苗的人，即使是那些在没有佐剂的情况下接种的人，也会持续产生高滴度的HPV16假病毒中和抗体，并且只报告了很小的副作用。在动物免疫原性研究中，我们已经确定了一种HPV16变种的L1 VLP所激发的抗体有效地中和了其他HPV16变种的感染。因此，在设计预防性疫苗时，似乎只需要考虑一种HPV16血清型。 我们还在开发替代候选疫苗。为了提高VLP疫苗的治疗潜力，我们将非结构HPV蛋白作为L2融合蛋白引入VLP中。用HPV16E7或E7-E2嵌合VLP免疫小鼠，产生CD8限制性T细胞反应，保护小鼠免受肿瘤攻击，并诱导已建立的肿瘤的消退。一种为I期试验产生临床级嵌合VLP的方法已经开发出来，疫苗批次正在进行临床前评估。 为了打破B细胞的耐受性并诱导对自身蛋白的抗体反应，我们在VLP表面以有序的阵列展示了目标自体多肽。在小鼠中，在VLP上展示小鼠肿瘤坏死因子多肽可使肿瘤坏死因子抗体滴度增加1000倍。用结合的VLP接种小鼠，要么保护它们免受实验诱导的类风湿性关节炎，要么降低疾病的严重性。小鼠和猕猴接种显示猕猴CCR5 N末端的VLP后，产生高滴度的抗体，结合细胞表面CCR5并阻止培养细胞的HIV感染。这些发现提出了一种诱导自身抗体的一般方法，具有许多基础和应用研究应用。 为了开始确定VLP疫苗如何能够在没有佐剂和局部炎症的情况下诱导有效的B和T细胞反应，我们研究了VLP与未成熟的小鼠骨髓来源的树突状细胞(BMDCs)的相互作用。BMDCs快速结合和内化VLP。与VLPs的相互作用，但不是无序的衣壳亚单位，导致BMDCs的表型迅速成熟，但相对于一种具有良好特性的诱导物--细菌脂多糖，促炎症细胞因子的释放延迟。VLP激活的BMDCs在体外诱导以Th1为主的初级T细胞应答。这些结果表明，树突状细胞对病毒粒子表面的模式识别在抗病毒粒子免疫中起核心作用。 我们还研究了小衣壳蛋白L2在病毒粒子组装、感染和诱导中和抗体中的作用。我们发现L2与五聚体L1衣壳结合，但不与完整的L1 VLP结合，表明这两个衣壳蛋白在病毒粒子组装的中间阶段结合。L2被发现是基因组在体内封装所必需的。此外，突变分析表明，L2在感染过程中发挥作用。针对特定多肽HPV6、16或18L2的抗体能够交叉中和异型HPV。相反，L1/L2 VLP不能诱导交叉中和抗体。这些结果表明，HPV病毒粒子含有隐蔽的或亚显性的L2中和表位，L2多肽疫苗可能提供对生殖器HPV感染的广谱保护。

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 a phase 1 and phase 2 clinical trial of an HPV16 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. 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. 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 examine the interaction of VLPs with immature mouse bone marrow-derived dentritic 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 cell 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.