Sanaria PfSPZ Vaccine Functional T Cell Assay-Hepatocyte Cytotoxicity Assay

描述（由申请人提供）：Sanaria正在开发的PfSPZ减毒疫苗旨在引起强大的适应性免疫反应，以防止蚊子传播的孢子虫在接种疫苗的个体中建立临床疟疾。根据数十年来对活孢子和减毒孢子的研究，预计这种成功的免疫反应将涉及体液和细胞介导的机制，其中后一种成分严重依赖于活化的CD8+ T细胞，这些细胞由疫苗启动，随后能够破坏甚至罕见的感染肝细胞。我们建议开发检测抗p存在的检测方法。恶性疟原虫孢子子和肝期ctl具有高度敏感性。针对亚单位疫苗的抗原特异性T细胞通常通过间接测量细胞因子分泌来检测，使用已建立的检测方法来评估针对特定抗原或来自这些抗原的表位的免疫反应。但是，Sanaria的减毒全生物体PfSPZ疫苗可能会引发T细胞对多种抗原的反应，这些抗原仅受寄生虫基因组大小的限制。此外，基因组库中相关免疫原性和保护性表位的身份目前尚不清楚。因此，一种不偏向于特定肽并直接测试T细胞对感染靶细胞（在这种情况下，肝细胞）的功能活性的检测从长远来看更有意义，最终可以更有力地预测疫苗的疗效。为了实现这一目标，我们将开发一种改良的肝期发育抑制试验（ILSDA），有四个具体目标：1)增加ILSDA的鲁棒性；2)建立使用特异性CTL克隆证明肝细胞杀伤的能力；3)将改进的ILSDA适应合适的高通量平台；4)使用免疫个体的PBMCs验证改进的高通量ILSDA。在其最终版本中，我们希望通过消耗特定T细胞亚群来证明T细胞亚群的活性依赖性，通过与特定HLA分子的抗体孵育来限制MHC，以及通过感染间日疟原虫孢子体来证明抗原特异性。这种测量T细胞对整个寄生虫活性的功能试验对于Sanaria的PfSPZ疫苗的开发至关重要，并且可能对设计用于诱导保护性T细胞反应的其他红细胞前阶段疟疾疫苗发挥同样重要的作用。

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.