Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（pv）感染动物和人的上皮，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之间有很强的联系。我们已经为HPV 16和其他由L1主衣壳蛋白或L1加L2（次要衣壳蛋白）组成的pv生成了病毒样颗粒（VLPs）。在动物模型中，经肠外注射纯化的VLPs可诱导高滴度的中和抗体和对实验性攻击的保护。基于这些结果，我们已经验证了GMP级VLP，并且最近完成了HPV16 VLP疫苗的1期和2期临床试验。疫苗接种者，即使是那些在没有佐剂的情况下接种疫苗的人，也会持续产生高滴度的HPV16伪病毒粒子中和抗体，并且只有轻微的副作用。我们也在开发备选疫苗。为了增加基于vlp的疫苗的治疗潜力，我们将非结构性HPV蛋白作为L2融合蛋白加入到vlp中。接种HPV16 E7或E7- e2嵌合VLP产生CD8限制性T细胞反应，保护小鼠免受表达E7的肿瘤细胞系的肿瘤攻击，并诱导已建立的肿瘤消退。用于I期临床试验的临床级嵌合VLPs目前正在生产中。为了增加粘膜免疫反应，我们在小鼠中进行了鼻内滴注纯化的VLPs的试验。与肠外接种VLPs相比，该方案在女性生殖道中诱导分泌IgA和转体IgG。中和抗体在鼻内接种的小鼠整个发情周期中检测到，但在肠外接种的小鼠中没有检测到。经鼻给药后也检测到对VLPs有效的抗肿瘤细胞介导反应。为了打破b细胞的耐受性并诱导抗体对自蛋白的反应，我们在VLP表面上以有序阵列显示了目标自多肽。显示TNF肽诱导小鼠高滴度TNF IgG，部分保护小鼠免受实验诱导的类风湿关节炎。小鼠和猕猴接种了显示猕猴CCR5 n端的VLPs，产生高滴度的抗体，结合细胞表面CCR5并阻断培养细胞的HIV感染。这些发现提示了一种诱导自身抗体的通用方法，具有许多基础和应用研究的应用价值。为了开始确定VLP疫苗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细胞和T细胞反应，我们研究了VLP与未成熟小鼠骨髓源树突状细胞（bmdc）的相互作用。BMDCs快速结合和内化VLPs。与VLPs的相互作用，而不是与无组织衣壳亚基的相互作用，导致BMDCs的表型快速成熟，但与一种特性良好的诱导剂细菌脂多糖相比，促炎细胞因子的释放延迟。VLP激活的BMDCs诱导Th1主导的原代T细胞反应。这些结果证明树突状细胞对病毒粒子表面的模式识别在抗病毒免疫中起核心作用。

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. 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. Clinical grade chimeric VLPs for a phase I clinical trial are now being produced. To increase mucosal immune responses, we have tested intranasal instillation of purified VLPs in mice. In contrast to parenteral inoculation of VLPs, this protocol elicited both secretory IgA and transudated IgG in the female genital tract. Neutralizing antibodies were detected throughout the estus cycle in intranasally vaccinated mice, but not in those vaccinated parenterally. Potent anti-tumor cell mediated responses to VLPs were also detected after intranasal administration.To break B-cell tolerance and induce antibody responses to a self-protein, we have displayed the target self-polypeptide in an ordered array on a VLP surface. Display of a TNF peptide induced high titers of TNF IgG in mice that partially protected the mice from experimentally induced rheumatoid arthritis. 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 research 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.