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型(KSHV)在卡波西肉瘤(KS)的发病机制中起着明显的致病作用。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在健康成人中比HIV感染者诱导更强的人类白细胞抗原CD8+细胞毒性T淋巴细胞(CTL)反应，支持疫苗作为控制KSHV相关疾病的策略的潜力。我们提出了一种基于刺激CD8+CTL反应的KSHV疫苗的开发方法，该方法比自然感染更有效。这将通过使用合成组合库(SCL)来识别在刺激T细胞介导的针对KSHV感染的免疫反应中有效的免疫原肽来完成。特别是当以位置扫描(PS)格式生成时，SCL方法允许从文库筛选中直接鉴定活性多肽序列(S)的关键残基(S)。每一种由非肽和十肽PS-SCLS组成的混合物都将通过来自HLA-A2阳性患者的CTL克隆进行筛选，以确定它们是否能够刺激细胞因子的产生和/或细胞溶解活性，这些克隆识别kaposin(ORF K12)、LANA(ORF 73)和SCIP(ORF 65)蛋白的靶标，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多肽或十肽进行系统比较，以使用生物测定分析来预测和识别刺激性天然多肽。根据筛选结果和生物计量学分析，将合成一系列单肽，并测定其刺激T细胞介导的免疫反应的能力。多肽的免疫原性将通过使用HLA-A2.1KB小鼠模型进行体内免疫来评估，并通过体外刺激实验进一步评估。然后，将使用一组HLA-A2阳性患者来研究最有效的多肽的交叉识别，并通过将免疫或刺激研究中产生的T细胞与天然配体相结合来确定它们的交叉反应。PS-SCL方法与生物测定分析相结合，将允许1)产生最佳表位模拟，2)识别那些最佳表位未知的蛋白质中的天然配体，以及3)洞察不同病毒或人类蛋白序列与KSHV表位之间潜在的交叉反应。

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.