Sanaria PfSPZ Vaccine Functional T Cell Assay-Hepatocyte Cytotoxicity Assay

描述(申请人提供)：Sanaria正在开发的PfSPZ减毒疫苗旨在激发强大的适应性免疫反应，防止蚊子传播子孢子在接种疫苗的个人中建立临床疟疾。基于几十年来对活的和减毒的子孢子的研究，预计这种成功的免疫反应将包括体液和细胞介导的机制，其中后者严重依赖于被疫苗启动的激活的CD8+T细胞，随后甚至能够摧毁罕见的感染的肝细胞。我们建议开发强有力地检测抗P-P存在的检测方法。恶性疟原虫子孢子和肝期CTL具有高度敏感性。针对亚单位疫苗的抗原特异性T细胞通常是通过间接测量细胞因子分泌来检测的，使用已建立的分析方法来评估针对这些抗原的特定抗原或表位的免疫反应。但Sanaria的减毒全机体PfSPZ疫苗可能会引发T细胞对仅受寄生虫基因组大小限制的多种抗原的反应。此外，基因组谱系中相关的免疫原性和保护性表位的一致性目前尚不清楚。因此，一种对特定多肽没有偏见并直接测试T细胞对受感染靶细胞的功能活性的检测方法，在这种情况下，从长远来看，肝细胞更有意义，最终可以更有力地预测疫苗的效果。为此，我们将开发一种具有四个特定目标的改进的肝期发育抑制试验(ILSDA)：1)增加ILSDA的稳健性；2)建立使用特定CTL克隆显示肝细胞杀伤的能力；3)使改进的ILSDA适应于合适的高通量平台；4)使用免疫个体的PBMC验证改进的高通量ILSDA。在它的最终版本中，我们希望通过耗尽特定的T细胞亚群来证明T细胞亚群对活性的依赖，通过与特定的人类白细胞抗原分子的抗体孵育来证明MHC的限制，以及通过感染间日疟原虫的子孢子来证明抗原特异性。这种测量T细胞对整个寄生虫的活性的功能性测试对Sanaria的PfSPZ疫苗的开发至关重要，并可能对其他旨在诱导保护性T细胞反应的红细胞前期疟疾疫苗起到同样重要的作用。 公共卫生相关性：这项建议旨在开发一种强大的高通量检测方法，可以检测由Sanaria的PfSPZ疫苗或任何其他前红细胞疟疾疫苗启动的高度特异的细胞毒T细胞。在Sanaria首席科学官兼这笔赠款的合作研究员Stephen Hoffman博士最初描述的基本抑制肝期发育试验(ILSDA)的基础上，肝细胞细胞毒性试验将使用对受感染肝细胞内寄生虫生长的生物抑制作为对免疫个体中预置淋巴细胞功能活性的直接且可靠的衡量标准。

DESCRIPTION (provided by applicant): The attenuated PfSPZ vaccine being developed by Sanaria is designed to evoke a robust adaptive immune response that will prevent mosquito transmitted sporozoites from establishing clinical malaria in vaccinated individuals. Based on decades of studies with live and attenuated sporozoites, it is expected that such a successful immune response will involve both humoral and cell mediated mechanisms where the latter component is critically dependent on activated CD8+ T cells that are primed by the vaccine and are subsequently able to destroy even the rare infected liver cell. We propose to develop assays that will robustly detect the presence of anti-P. falciparum sporozoite and liver stage CTLs with a high degree of sensitivity. Antigen-specific T cells against subunit vaccines are routinely detected by indirect measures of cytokine secretion using established assays that assess immune responses against defined antigens or epitopes from these antigens. But Sanaria's attenuated whole-organism PfSPZ vaccine is likely to elicit T cell responses against a multitude of antigens limited only by the size of the parasite genome. Moreover, the identity of the relevant immunogenic and protective epitopes within the genomic repertoire is currently unknown. Therefore, an assay that is unbiased to specific peptides and that directly tests the functional activity of T cells against infected target cells, in this case, a hepatocyte is more meaningful in the long run and can eventually be more robustly predictive of vaccine efficacy. Towards this goal we will develop a modified Inhibition of Liver-Stage Development Assay (ILSDA) with four specific aims 1) Increase robustness of ILSDA 2) Establish the capacity to demonstrate hepatocyte killing using specific CTL clones 3) Adapt the improved ILSDA to a suitable high-throughput platform and 4) Validate the improved high throughput ILSDA using PBMCs from immunized individuals. In its final version we expect to demonstrate T cell subset dependence of the activity by depleting specific T cell subsets, the MHC restriction by incubating with antibodies to specific HLA molecules, and the antigen specificity by infecting with P. vivax sporozoites. This functional assay which measures T cell activity against whole parasites is critical for the development of Sanaria's PfSPZ Vaccine, and could play an equally important role for other pre-erythrocytic stage malaria vaccines designed to induce protective T cell responses. PUBLIC HEALTH RELEVANCE: This proposal aims to develop a robust high throughput assay that can detect highly specific cytotoxic T cells primed by Sanaria's PfSPZ vaccine or any other pre-erythrocytic malaria vaccine. Building on the essential Inhibition of Liver stage Development Assay (ILSDA) that was originally described by Sanaria's Chief Scientific Officer and co-investigator on this grant, Dr. Stephen Hoffman, the hepatocyte cytotoxicity assay will use the biological inhibition of parasite growth within infected liver cells as a direct and reliable measure of the functional activity of primed lymphocytes in immunized individuals.