Novel KSHV-specific CTL epitopes for development

描述（由申请人提供）：卡波西肉瘤相关疱疹病毒-8 （KSHV）在卡波西肉瘤（KS）的发病机制中起明确的致病作用。KS最常见于免疫抑制患者，如获得性免疫缺陷综合征患者。KSHV在健康成人中引起比hiv感染者更强的HLA CD8+细胞毒性t淋巴细胞（CTL）反应，支持疫苗作为控制KSHV相关疾病策略的潜力。我们提出了一种基于比自然感染更有效的CD8+ CTL刺激的KSHV疫苗的开发方法。这将通过使用合成组合文库（SCLs）来鉴定肽，这些肽将有效地作为免疫原刺激t细胞介导的免疫反应来对抗KSHV感染。SCL方法，特别是以位置扫描（PS）格式生成时，允许从文库筛选中直接识别活性肽序列的关键残基。由非肽和十肽PS-SCLs组成的每种混合物将通过hla - a2阳性患者衍生的CTL克隆筛选其刺激细胞因子产生和/或细胞溶解活性的能力，这些克隆识别kaposin （ORF K12）、LANA （ORF 73）和SCIP （ORF 65）蛋白中的靶标，并对kshv感染的细胞具有裂解特异性。筛选结果还将与数据库中病毒或人类蛋白质中的非肽或十肽进行系统比较，以使用生物计量学分析预测和识别刺激性天然肽。基于筛选结果和生物计量学分析，将合成一系列单独的肽，并确定其刺激t细胞介导的免疫反应的能力。利用HLA-A2.1 Kb小鼠模型进行体内免疫，并通过体外刺激实验进一步评价肽的免疫原性。最有效的多肽的交叉识别将在一组hla - a2阳性患者中进行研究，它们的交叉反应性将通过免疫或刺激研究产生的t细胞来确定。PS-SCL方法结合生物统计学分析将允许1)生成最佳表位模拟物，2)鉴定最佳表位未知的蛋白质中的天然配体，以及3)深入了解不同病毒或人类蛋白质序列与KSHV表位之间潜在的交叉反应性。

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus-8 (KSHV) plays a clear causative role in the pathogenesis of Kaposi's sarcoma (KS). KS is most frequently found in immunosuppressed patients such as patients with acquired immunodeficiency syndrome. KSHV elicits stronger HLA CD8+ cytotoxic T-lymphocyte (CTL) responses in healthy adults than in HIV-infected individuals, supporting the potential of vaccines as a strategy for controlling KSHV-related diseases. We propose an approach toward the development of a KSHV vaccine based on the stimulation of CD8+ CTL responses more efficiently than the natural infection. This will be accomplished by using synthetic combinatorial libraries (SCLs) to identify peptides that would be effective as immunogens in stimulating T-cell mediated immune responses against KSHV infection. The SCL approach, particularly when generated in a positional scanning (PS) format, allows the direct identification of the key residue(s) of active peptide sequence(s) from the library screening. Each mixture making up nonapeptide and decapeptide PS-SCLs will be screened for their ability to stimulate cytokine production and/or cytolytic activity by CTL clones derived from HLA-A2-positive patients that recognize targets in kaposin (ORF K12), LANA (ORF 73), and SCIP (ORF 65) proteins and have lytic specificity for KSHV-infected cells. The screening results will also be systematically compared to nonapeptides or decapeptides in viral or human proteins in databases to predict and identify stimulatory natural peptides using a biometrical analysis. Based on the screening results and biometrical analysis, series of individual peptides will be synthesized and their ability to stimulate T-cell mediated immune response determined. The peptides immunogenicity will be assessed by performing in vivo immunization using the HLA-A2.1 Kb mouse model, and further evaluated through in vitro stimulation experiments. The cross-recognition of the most potent peptides will then be investigated using a cohort of HLA-A2-positive patients and their cross-reactivity determined by challenging T-cells generated through the immunization or stimulation studies to the native ligand. The PS-SCL approach combined with the biometrical analysis will allow 1) the generation of optimal epitope mimics, 2) the identification of natural ligands in those proteins for which the optimal epitope is unknown, and 3) provide insight into potential cross-reactivity between different viral or human protein sequences and KSHV epitopes.