ANALYSIS OF PAPILLOMAVIRUSES

乳头瘤病毒（PV）感染动物和人的上皮细胞， 它们通常在感染部位诱导良性增殖。 然而，恶性进展与肿瘤的发生有很强的相关性。 人类生殖器病变和某些HPV类型，最常见的是HPV 16。我们 已经产生了针对HPV 16和其他PV的病毒样颗粒（VLP）， 由L1主要衣壳蛋白或L1加L2，次要衣壳蛋白组成 蛋白纯化的VLP的胃肠外注射诱导高滴度的 中和抗体和保护免受实验挑战， 动物模型根据这些结果，我们目前正在组织一个 HPV 16 VLP疫苗的临床试验。此外，我们正在开发 替代疫苗候选人。为了增加治疗潜力 在基于VLP的疫苗中，我们已经将非结构性HPV蛋白 作为L2融合蛋白。接种HPV 16 E7 嵌合VLP产生了CD 8限制性T细胞应答， 使用表达E7的肿瘤系从肿瘤攻击中获得小鼠。增加 粘膜抗体应答并降低疫苗生产的费用， 我们已经产生了L1重组沙门氏菌。与预期相反， VLP在细菌中的组装和活病毒的鼻内滴注 小鼠中的重组体产生高滴度的中和抗体。 与VLP的肠胃外接种相反，该方案还 在女性生殖道中引起分泌型伊加。根据我们 最近开发了一种在体外产生感染性PV的方法， 我们已经开始分析以确定病毒粒子组装的机制。 尽管L1单独自组装成VLP，但L2次要衣壳蛋白 和E2转录/复制因子也需要 使基因组易位并产生感染性病毒体。一系列 检测病毒粒子的亚细胞定位的实验 组件和E2支持一种基于 对L2募集其他重要病毒成分的能力， 不同的核结构，以前称为POD。特异性 病毒基因组的重叠很可能是由于 E2与特定病毒基因组序列的结合， E2和L2/POD复合物之间的蛋白质/蛋白质相互作用。使用我们 以前验证的HPV 16 VLP为基础的ELISA，我们比较了病毒粒子 抗体反应的男性和女性，研究了这种关系， 对宫颈疾病进展和消退的反应，并进行量化 生殖器局部粘膜抗体对HPV感染的反应。在 此外，我们还使用该测定来证实和扩展我们先前的 观察到对HPV 16的血清学应答与 增加患宫颈癌、外阴癌和食道癌的风险。

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 are currently organizing a clinical trial of an HPV16 VLP vaccine. In addition, we are 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 chimeric VLP generated a CD8 restricted T cell response that protected mice from tumor challenge using an E7 expressing tumor line. To increase mucosal antibody responses and reduce the expense of vaccine production, we have generated L1 recombinant Salmonella. Contrary to expectations, VLPs assembled in the bacteria and intranasal instillation of the live recombinants in mice produced high titers of neutralizing antibodies. In contrast to parenteral inoculation of VLPs, this protocol also elicited secretory IgA in the female genital tract. Based upon our recent development of a procedure to generate infectious PVs in vitro, we have begun an analysis to determine the mechanism of virion assembly. Although L1 alone self-assembles into VLPs, the L2 minor capsid proteins and the E2 transcription/replication factor were also required to encapsidate the genome and generate infectious virions. A series of experiments examining the subcellular localization of the virion components and E2 supported a model for virion assembly that is based on the ability of L2 to recruit the other essential viral components to distinct nuclear structures previously designated PODs. The specificity of viral genome encapsidation is likely to result from the high affinity binding of E2 to specific viral genome sequences coupled with protein/protein interactions between E2 and an L2/POD complex. Using our previously validated HPV16 VLP-based ELISA, we have compared the virion antibody respose in men and women, examined the relationship of this response to cervical disease progression and regression, and quantitated the local genital mucosal antibody response to HPV infection. In addition, we have use the assay to confirm and extend our previous observations that a serological response to HPV16 is associated with an increased risk of cervical, vulvar and esophageal cancer.