CANINE ORAL PAPILLOMAVIRUS--MODEL FOR A VACCINE

这项提议的最终目标是生产一种疫苗，用于预防 嗜粘性病毒，犬口腔乳头瘤病毒(COPV)，将服务于 作为人乳头瘤病毒(HPV)疫苗研制的典范。 感染生殖道粘膜的人乳头瘤病毒起着重要作用 在宫颈癌的发生发展中起着关键作用。例如， 大多数人宫颈癌(95%)含有和表达HPV DNA 它是两种病毒癌蛋白E6和E7的表达， 似乎是细胞转化的关键。尽管我们 关于这些物质的分子作用机制的详细知识 癌蛋白，目前关于肿瘤生物学方面的信息很少。 乳头瘤病毒感染包括识别病毒受体， 控制病毒复制和组装，以及宿主免疫反应 病毒和病毒转化的细胞。这一缺陷源于 缺乏体外繁殖乳头状瘤病毒的系统(这 需要高度分化的角质形成细胞)以及缺乏 可用于分析宿主因素参与的动物模型 对乳头瘤病毒黏膜感染的反应。 我们建议在比格犬群体中研究冠状病毒感染的生物学。 其结果是口腔尖锐湿疣的发生率很高 病毒感染的可能性。我们的目的是描述抗体的作用 动物对这种感染的抵抗力，这种感染可以由 注射疣提取物(已知含有病毒颗粒)。我们 将对COPV的L1和L2基因进行测序，并将这些基因克隆到 已知(根据初步结果)可产生病毒的表达载体 保持构象的蛋白质存在于完整的病毒颗粒中。 这些病毒衣壳蛋白将用于筛选免疫动物血清 对于L1和L2特异性抗体的存在以及诱导 对易感动物的免疫力。将开发出最佳条件 以诱导免疫力。L1和L2抗体抑制的能力 将使用纯化的病毒粒子对COPV诱导的肿瘤进行评估 来自疣组织，或者，可能来自生长在 裸鼠。最后，将产生针对该病毒的单抗 完整的病毒粒子，以确定分子位置(L1或L2，或 两者)COPV上的构象中和表位。由于 COPV和HPV在病毒遗传组织方面的相似之处， 病毒结构、衣壳蛋白序列和粘膜部位 感染，很可能是小猎犬身上产生的结果 系统将直接适用于人类的发展 乳头瘤病毒疫苗。

The ultimate goal of this proposal is to produce a vaccine for the mucosotropic virus, canine oral papillomavirus (COPV), which will serve as a model for the development of human papillomavirus (HPV) vaccine. Human papillomaviruses which infect the genital tract mucosa play a critical role in the development of cervical cancer. For example, the majority of human cervical carcinomas (95%) contain and express HPV DNA and it is the expression of two viral oncoproteins, E6 and E7, which appears to be critical for cellular transformation. Despite our detailed knowledge concerning the molecular mechanism of action of these oncoproteins, there is little information available on the biology of papillomavirus infection including the identity viral receptors, the control of viral replication and assembly, and the host immune response to virus and virally-transformed cells. This deficiency derives from a lack of an in-vitro system for propagating the papillomaviruses (which require highly differentiated keratinocytes) as well as the lack of an available animal model to analyze the host factors participating in the response to mucosal infection by papillomavirus. We propose to study the biology of COPV infection in a beagle colony which exhibits a high incidence of oral wart formation as a consequence of viral infection. Our intent is to delineate the role of antibodies in the resistance of animals to this infection which can be produced by the injection of wart extracts (known to contain viral particles). We will sequence the L1 and L2 genes of COPV and clone these genes into expression vectors known (from preliminary results) to produce viral proteins which retain a conformation present in intact viral particles. These viral capsid proteins will be used to screen immune animal sera for the presence of L1 and L2-specific antibodies as well as to induce immunity in susceptible animals. Optimal conditions will be developed for inducing immunity. The ability of L1 and L2 antibodies to inhibit COPV-induced tumors will be evaluated using purified virions derived from wart tissue or, potentially, from viral-producing tumors grown in nude mice. Finally, monoclonal antibodies will be generated against intact virions in order to define the molecular location (L1 or L2, or both) of conformational, neutralizing epitopes on COPV. Due to similarities of COPV and HPV with respect to viral genetic organization, viral structure, capsid protein sequences, and mucosal site of infection, it is very likely that the findings generated with the beagle system will have direct applicability to the development of a human papillomavirus vaccine.