Improved Diagnosis of Kaposi's Sarcoma-Associated Herpesvirus Infection

项目总结/摘要 卡波西肉瘤疱疹病毒（KSHV）是一种致癌病毒，可导致包括卡波西肉瘤（KS）在内的多种癌症。是 据估计，3%至5%的美国献血者感染了KSHV，但在一些高危人群中， 男性同性恋者，患病率高达65%。尽管有HAART，KS仍然是第二大最常见的艾滋病- 相关恶性肿瘤。三分之一的AIDS-KS病例现在出现在具有相对高的CD 4+细胞计数和低的CD 4+细胞计数的个体中。 HIV-1病毒载量。最近的一项研究观察到，在艾滋病毒感染者中，KSHV的流行率持续高企， 1+接受HAART治疗的感染者。这些数据清楚地表明，KS仍然发生在艾滋病毒感染者中，即使在 成功的HAART的背景。在免疫抑制人群中，大约每200名移植患者中就有1名 发展KS。KSHV的流行是导致KS持续发生及其相关的 艾滋病毒阳性者的预期寿命。尽管它与疾病有很强的联系，特别是在艾滋病毒+和 免疫抑制人群，没有FDA批准的KSHV临床诊断测试，因此， 对于一种简单的、经济的商业上可行的KSHV诊断试剂盒， 控制病毒传播和监测已感染者血清状况的任何努力的重要组成部分。的 评估HIV+个体的KSHV血清状态的能力将有助于临床医生进行适当的干预， 预防KS发作的选择。本项目的总体目标是开发一种血清学多抗原检测方法， 检测KSHV抗体，可用于商业诊断高危患者和识别血液/组织供体 KSHV感染在之前的SBIR I期和II期工作中，Epiphany开发了一种单孔、多抗原的KSHV， 酶联免疫吸附试验（ELISA）称为“4-Combo”。在筛选含有KSHV真- 阳性和正常，低风险的献血者，4-Combo是唯一的分析了7个其他独立的ELISA检测所有 一致的KSHV样品具有优异的特异性和灵敏度（分别估计> 99%，98%）。为下一 在试剂盒开发的阶段，关键是检查4-Combo对大型血清库的性能， 流行病学验证的KSHV阳性和阴性对照品，以及相关的确证性试验，以确定 分析的有效性。一个将主显节与生物医学和临床研究相结合的合作联盟 迈阿密大学米勒医学院、迈阿密艾滋病研究中心（CFAR）的能力 和恩里克·A·卡耐基博士的实验室Mesri（CFAR）已创建。主显节和迈阿密CFAR是独一无二的 装备，以实现以下目标。在目标1中，合成、生产和分析工艺开发将 实施，随后进行一套4-Combo诊断ELISA试剂盒的中试生产。在目标2中，这些套件将 通过筛选回顾性临床血液样本和血清学表征的血浆/PMBC进行评估和优化 并通过western blot验证。在目标3中，将开始大规模的成套工具生产。测定稳定性和验证研究 将被执行。UofM CFAR、其核心和移植中心可获得更多HIV+血清和组织，以及 将对国家储存库进行大规模筛选，以确定敏感性和特异性。实现本项目的目标 将建立4-Combo的临床和商业价值，最终导致FDA批准的第一个临床试验。 KSHV的诊断试验。

Project Summary/Abstract Kaposi's sarcoma herpes virus (KSHV) is an oncovirus that causes several cancers including Kaposi's sarcoma (KS). It is estimated that 3 to 5% of U.S. blood donors have been infected with KSHV, yet in some high-risk populations such as homosexual men, prevalence is as high as 65%. In spite of HAART, KS remains the second most common AIDS- associated malignancy. A third of AIDS-KS cases now arise in individuals with relatively high CD4+ cell counts and low HIV-1 viral load. A recent study has observed a continuing high prevalence and increased acquisition of KSHV in HIV- 1+ infected persons on HAART. The data clearly indicate that KS is still occurring in HIV-infected individuals, even in the context of successful HAART. In immunosuppressed populations, approximately 1 in 200 transplant patients will develop KS. The prevalence of KSHV is responsible for the continuing incidence of KS and its associated reduction in life expectancy of HIV+ individuals. Despite its strong disease association, particularly in the HIV+ and immunosuppressed populations, there is no FDA approved clinical diagnostic test for KSHV and consequently a significant unmet need remains for a simple, cost-effective commercially viable KSHV diagnostic kit that would be an essential component of any effort to control virus transmission and monitor serostatus of those already infected. The ability to gauge the KSHV serostatus of an HIV+ individual would help clinicians make appropriate interventional choices to prevent KS onset. The overall goal of this project is to develop a serological multi-antigen assay for the detection of KSHV antibodies that can be used commercially to diagnose at-risk patients and identify blood/tissue donors with KSHV infection. In prior SBIR Phase I and II work, Epiphany developed a single-well, multi-antigenic KSHV enzyme linked immunosorbent assay (ELISA) called “4-Combo.” In screening a sera panel containing both KSHV true- positives and normal, low-risk blood donors, 4-Combo was the only assay out of 7 other independent ELISAs to detect all consensus KSHV samples with excellent specificity and sensitivity (estimated >99%, 98%, respectively). For the next stage of the kit development, it is critical to examine 4-Combo's performance against large sera banks of epidemiologically validated KSHV positive and negative controls with relevant confirmatory assays to establish the true effectiveness of the assay. A collaborative consortium that merges Epiphany with the biomedical and clinical research capabilities of the University of Miami (UofM) Miller School of Medicine, the Miami Center for AIDS Research (CFAR) and the labs of Dr. Enrique A. Mesri (CFAR) has been created. Together, Epiphany and Miami CFAR are uniquely equipped to achieve the following Aims. In Aim 1, synthetic, manufacturing, and analytical process development will be implemented followed by pilot production of a set of 4-Combo diagnostic ELISA kits. In Aim 2, these kits will be assessed and optimized by screening retrospective clinical blood samples and serologically characterized plasma/ PMBCs and validated by western blot. In Aim 3, large-scale kit production will commence. Assay stability and validation studies will be performed. A larger panel of HIV+ sera and tissues available to UofM CFAR, its cores and transplant centers, and national repositories will be massively screened to establish sensitivity and specificity. Achieving the goals of this project will establish the clinical and commercial value of 4-Combo ultimately resulting in the first FDA-approved clinical diagnostic assay for KSHV.