Sanaria PfSPZ Vaccine Functional T Cell Assay-Hepatocyte Cytotoxicity Assay

描述（由申请方提供）：Sanaria正在开发的减毒PfSPZ疫苗旨在引起强大的适应性免疫应答，防止蚊子传播的子孢子在接种疫苗的个体中建立临床疟疾。基于几十年来对活的和减毒的子孢子的研究，预期这种成功的免疫应答将涉及体液和细胞介导的机制，其中后一种组分严重依赖于活化的CD 8 + T细胞，所述活化的CD 8 + T细胞由疫苗引发并且随后能够破坏甚至罕见的感染的肝细胞。我们建议开发一种检测方法，以高度的灵敏度稳健地检测抗恶性疟原虫子孢子和肝期CTL的存在。针对亚单位疫苗的抗原特异性T细胞通常通过使用已建立的测定法间接测量细胞因子分泌来检测，所述测定法评估针对限定抗原或来自这些抗原的表位的免疫应答。但是Sanaria的减毒全生物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，基因组库内相关免疫原性和保护性表位的身份目前尚不清楚。因此，对特定肽无偏倚并且直接测试T细胞针对感染的靶细胞（在这种情况下，肝细胞）的功能活性的测定从长远来看更有意义，并且最终可以更稳健地预测疫苗功效。为了实现这一目标，我们将开发具有四个特定目标的改良的肝脏阶段发育抑制测定（ILSDA）：1）增加ILSDA的稳健性; 2）建立使用特异性CTL克隆证明肝细胞杀伤的能力; 3）使改良的ILSDA适应合适的高通量平台;以及4）使用来自免疫个体的PBMC验证改良的高通量ILSDA。在其最终版本中，我们期望通过耗尽特异性T细胞亚群来证明活性的T细胞亚群依赖性，通过与特异性HLA分子的抗体一起孵育来证明MHC限制性，以及通过感染间日疟原虫子孢子来证明抗原特异性。这种测量针对整个寄生虫的T细胞活性的功能测定对于Sanaria的PfSPZ疫苗的开发至关重要，并且可以对设计用于诱导保护性T细胞应答的其他红细胞前期疟疾疫苗发挥同样重要的作用。 公共卫生相关性：该提案旨在开发一种稳健的高通量测定，其可以检测由Sanaria的PfSPZ疫苗或任何其他红细胞前疟疾疫苗引发的高度特异性细胞毒性T细胞。基于Sanaria首席科学官和该资助的共同研究者Stephen霍夫曼博士最初描述的肝脏阶段发育基本抑制试验（ILSDA），肝细胞毒性试验将使用感染肝细胞内寄生虫生长的生物抑制作为免疫个体中引发淋巴细胞功能活性的直接和可靠测量。

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.