Protein Biomarkers for a New Human Polyomavirus in AIDS-related Malignancies

描述(申请人提供)：默克尔细胞癌(MCC)是一种与艾滋病相关的神经外胚层癌症，怀疑有病毒病因。MCC在艾滋病患者中的发病率是预期的13倍，发生在移植后和其他免疫抑制患者群体中。虽然罕见，但它是最具侵袭性的皮肤癌，晚期疾病患者中只有50%能存活9个月或更长时间。目前尚无早期诊断标记物，常被误诊为其他小圆细胞肿瘤。我们利用数字转录组消减技术(DTS)在MCC中发现了一种新的多瘤病毒。默克尔癌多瘤病毒(MCPyV)与非洲绿猴嗜淋巴多瘤病毒(LPyV)关系最密切。15%-30%的人有LPyV抗体，这导致调查人员推测存在一种密切相关但未被发现的人类病毒。MCPyV基因组全长5387个核苷酸，在MCC肿瘤中表达T抗原蛋白。大多数MCC对整合的MCPyV呈阳性，这种病毒很容易通过PCR和Southern blotting检测到。相比之下，只有8-16%的对照组织含有可检测到的病毒基因组。在Southern blotting上，大多数MCPyV阳性肿瘤显示出单克隆体插入模式，表明该病毒有助于克隆性细胞扩张。我们已经开发了研究这种新病毒所需的试剂，包括受感染的细胞系、特异性抗体和VP1蛋白的重叠多肽库。初步研究表明，MCPyV是一种常见但并不普遍的感染，当病毒突变并整合到宿主基因组中时，它会导致MCC。识别MCPyV感染的特定生物标志物对于确定MCPyV在人类癌症中的作用至关重要。我们建议使用已建立的多瘤病毒免疫诊断技术以及新的免疫诊断方法来开发MCPyV感染的筛查血液检测方法。我们将使用基于细胞的IFA、MCPyV多肽图谱和病毒样颗粒ELISA法，以明确定义的人群的血清来鉴定和表征人类对MCPyV感染的抗体反应。将审查这些检测方法与已知人类多瘤病毒的交叉反应，以确保检测的特异性。我们还建议产生特定的单抗，用于检测组织微阵列中的病毒抗原，以便对常见肿瘤进行感染筛查。病毒蛋白的表达也可以与细胞蛋白生物标记物的表达相关联，以确定临床病程和对治疗的反应。这是我们实验室发现的一种可能导致默克尔细胞癌的新型人类肿瘤病毒MCPyV的研究资金的第一份提案。默克尔细胞癌是一种老年癌症、艾滋病、移植瘤等免疫抑制人群。它是皮肤癌中总死亡率最高的，目前还没有针对MCPyV感染的具体预防措施或诊断测试。如果获得资金，这项提案将开发准确的血液检测方法，以测量标记MCPyV感染的抗体蛋白质，并测量组织中的病毒蛋白质。这些是了解人类感染这种病毒以及开发有效的MCPyV疫苗和药物所必需的。

DESCRIPTION (provided by applicant): Merkel cell carcinoma (MCC) is a neuroectodermal, AIDS-related cancer suspected to have a viral etiology. MCC is 13 times more common than expected among AIDS patients and occurs in post-transplant and other immunosuppressed patient populations. While rare, it is the most aggressive skin cancer and only 50% of patients with advanced disease survive 9 months or longer. There are no early detection markers and it is frequently misdiagnosed with other small round cell tumors. We identified a novel polyomavirus in MCC using digital transcriptome subtraction (DTS). This agent, Merkel carcinoma polyomavirus (MCPyV), is most closely related to African green monkey lymphotropic polyomavirus (LPyV). Between 15-30% of persons have antibodies to LPyV, which has led investigators to speculate on the existence of a closely related but undiscovered human virus. MCPyV has a 5387 bp genome and expresses T antigen proteins in MCC tumors. Most MCC are positive for an integrated MCPyV that is readily detected by PCR and by Southern blotting. In contrast only 8-16% of control tissues harbor detectable viral genome. On Southern blotting, the majority of MCPyV positive tumors show a monoclonal somatic insertion pattern suggesting that the virus contributes to clonal cell expansion. We have developed reagents required to study this new virus including infected cell lines, specific antibodies and an overlapping peptide library for the VP1 protein. Preliminary studies suggest that MCPyV is a common but not ubiquitous infection that contributes to MCC when the virus mutates and integrates into the host genome. Identification of specific biomarkers for MCPyV infection is essential to determine MCPyV's role in human cancers. We propose to develop screening blood tests for MCPyV infection using established polyomavirus immunodiagnostic techniques as well as new immunodiagnostic methods. We will identify and characterize human antibody responses to MCPyV infection using cell-based IFA, MCPyV peptide mapping and virus-like particle ELISA with sera from well-defined populations. These assays will be vetted for cross-reactivity to known human polyomaviruses to ensure assay specificity. We also propose generating specific monoclonal antibodies to be used to detect viral antigens in tissue microarrays so that common tumors can be screened for infection. Expression of viral proteins can also be correlated with cellular protein biomarker expression to determine clinical course and responsiveness to treatment. This is the first proposal for funding to study a new human tumor virus found in our laboratory, MCPyV, likely to cause Merkel cell carcinoma. Merkel cell carcinoma is a cancer of the elderly, AIDS, transplant and other immunosuppressed persons. It has the highest overall mortality rate among skin cancers and there are no specific preventive measures or diagnostic tests for MCPyV infection. If funded, this proposal will develop accurate blood assays to measure antibody proteins marking MCPyV infection and to measure viral proteins in tissues. These are required to understand human infection with this virus and to develop effective vaccines and drugs against MCPyV.