Sanaria PfSPZ Vaccine Functional T Cell Assay-Hepatocyte Cytotoxicity Assay

Sanaria PfSPZ疫苗功能性T细胞检测-肝细胞细胞毒性检测

基本信息

批准号：
7908110
负责人：
Sumana Chakravarty
金额：
$ 30万
依托单位：
SANARIA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-12 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7908110
关键词：
Alleles Antibodies Antigens Attenuated Biological Biological Assay CD8B1 gene Cells Clinical Culicidae Custom Cytotoxic T-Lymphocytes Dependence Detection Development Epitopes Erythrocytes Genomics Goals Grant Growth Hepatocyte Human Human Resources Image Image Analysis Immune response Incubated Individual Intervention Life Liver Lymphocyte MHC Class I Genes Malaria Malaria Vaccines Measures Mediating Parasites Peptides Phase Plasmodium Plasmodium falciparum Play Reproducibility Research Personnel Rodent Role Running Sampling Small Business Innovation Research Grant Software Design Specificity Sporozoites Staging Subunit Vaccines System T cell response T-Lymphocyte T-Lymphocyte Subsets Testing Training Vaccinated Vaccine Design Vaccines Viral Vector Whole Organism assay development base circumsporozoite protein cytokine cytotoxic cytotoxicity design high throughput analysis high throughput screening immunogenic improved indexing killings parasite genome prevent public health relevance vaccine efficacy

Alleles Antibodies Antigens Attenuated Biological Biological Assay CD8B1 gene Cells Clinical Culicidae Custom Cytotoxic T-Lymphocytes Dependence Detection Development Epitopes Erythrocytes Genomics Goals Grant Growth Hepatocyte Human Human Resources Image Image Analysis Immune response Incubated Individual Intervention Life Liver Lymphocyte MHC Class I Genes Malaria Malaria Vaccines Measures Mediating Parasites Peptides Phase Plasmodium Plasmodium falciparum Play Reproducibility Research Personnel Rodent Role Running Sampling Small Business Innovation Research Grant Software Design Specificity Sporozoites Staging Subunit Vaccines System T cell response T-Lymphocyte T-Lymphocyte Subsets Testing Training Vaccinated Vaccine Design Vaccines Viral Vector Whole Organism assay development base circumsporozoite protein cytokine cytotoxic cytotoxicity design high throughput analysis high throughput screening immunogenic improved indexing killings parasite genome prevent public health relevance vaccine efficacy

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项目摘要

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.

描述（由申请人提供）：由Sanaria开发的减毒PFSPZ疫苗旨在引起强大的适应性免疫反应，可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。基于数十年来对活和减弱的孢子菌的研究，预计如此成功的免疫反应将涉及体液和细胞介导的机制，在这些机制中，后者的成分严格依赖于疫苗引发的活化的CD8+ T细胞，并随后能够破坏稀有感染的liver liver细胞。我们建议开发将鲁棒检测抗P的存在的测定。恶性孢子虫和肝脏阶段CTL具有高度灵敏度。抗原特异性T细胞针对亚基疫苗通常通过固定的测定法常规检测到细胞因子分泌的测量，这些测定法评估了针对这些抗原的定义抗原或表位的免疫反应。但是，萨纳里亚（Sanaria）减毒的全生物PFSPZ疫苗可能会引起T细胞反应，仅受寄生虫基因组大小限制的多种抗原。此外，目前尚不清楚基因组库中相关免疫原性和保护性表位的身份。因此，一种对特定肽无偏的测定，直接测试T细胞对感染靶细胞的功能活性，在这种情况下，肝细胞在长期中更有意义，最终可以更强大地预测疫苗功效。 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.在其最终版本中，我们希望通过耗尽特定T细胞亚群来证明活性的T细胞子集依赖性，通过与特定HLA分子的抗体孵育，通过与Vivax Sporozoite感染抗原特异性来证明MHC限制。该功能性测定测量对整个寄生虫的T细胞活性对于开发Sanaria PFSPZ疫苗至关重要，并且对于其他旨在诱导保护性T细胞反应的其他前炎性阶段疟疾疫苗也可能发挥同样重要的作用。公共卫生相关性：该提案旨在开发出强大的高通量测定法，该测定法可以检测出由Sanaria的PFSPZ疫苗或任何其他近肉毒杆菌前疟疾疫苗启动的高度特异性细胞毒性T细胞。基于Sanaria首席科学官和该赠款中最初描述的肝脏阶段开发测定法（ILSDA）的基础，该赠款中的肝细胞细胞毒性测定法将使用肝细胞毒性测定法将使用直接措施和可靠性的措施，将在感染肝细胞中的生态抑制作用，将其用于PRIPISER的措施，并将其置于PRIPISED的措施中。