Biomarkers of immunity to Orthopox Viruses

正痘病毒免疫的生物标志物

• 批准号: 7698906
• 负责人: ELLIS L REINHERZ
• 金额: $ 43.41万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-20 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7698906
• 关键词: Address; Affinity; Alleles; Antibodies; Antibody Formation; Antigens; Autologous; Binding; Bioinformatics; Biological Assay; Biological Markers; Blood; CD4 Positive T Lymphocytes; CD8B1 gene; CTL assay; Cell Death; Cell Line; Cell Maturation; Cells; Characteristics; Chromates; Class; DNA Sequence Analysis; Dendritic Cells; Development; Epitopes; Genome; HLA-A2 Antigen; Human; Human Characteristics; Human Genome; Human Identifications; Immune; Immune Targeting; Immune response; Immunity; Immunization; In Vitro; Individual; Infection; Informatics; Kinetics; Ligands; Link; MHC Class I Genes; MHC Class II Genes; Mass Spectrum Analysis; Memory; Memory B-Lymphocyte; Methods; Monitor; Monoclonal Antibodies; Mononuclear; Mus; Nature; Normal tissue morphology; Patients; Peptides; Phenotype; Polymerase Chain Reaction; Polysaccharides; Positioning Attribute; Recombinants; Relative (related person); Score; Serum; Site; Smallpox; Specificity; Stimulus; T memory cell; T-Lymphocyte; T-Lymphocyte Epitopes; Technology; Testing; Time; Vaccinated; Vaccinia; Viral; Viral Proteins; Virus; adduct; base; design; dimer; doxorubicin/mitomycin/vinblastine protocol; enzyme linked immunospot assay; multicatalytic endopeptidase complex; neutralizing antibody; neutralizing monoclonal antibodies; receptor; response

This project will address elicitation of long-term human immune T and B cell memory to orthopox viruses following immunization of normal individuals and immunodeficient patients. Recent advances in understanding of mechanisms shunting TCR-based triggering away from activation-induced cell death have identified memory phenotype biomarkers. The murine TL MHC class Ib molecule, expressed during dendritic cell maturation, and its inducible CDSc_ homodimeric receptor on T cells serve as a "memory switch". The present proposal has three aims. First, as bioinformatic analysis of the human genome strongly suggests that HLA-F is the human orthologue of murine TL, we will express, refold and purify recombinant HLA-F, demonstrate its capacity to serve as a CDSaa ligand and investigate receptor-ligand inducibility, using both monoclonal antibodies and HLA-F tetramers. The CD4 and CD8 subset distribution and stimuli for expression of CDSc_ and its ligand will be examined and linkage to memory phenotype established. Second, possible HLA-A2-restricted (and other) T cell epitopes shared by vaccinia, MVA and smallpox have been identified through genome-wide comparison in conjunction with position-specific scoring matrices, further parsed by proteosome cleavage site predictors. Those 1008 potential antigens will be synthesized, tested functionally using blood from vaccinated individuals by CFC assays, ELISPOT, proliferation and CTL assays as well as mass spectrometry. HLA-A2 tetramers containing the identified functional peptide epitopes will monitor elicitation of antigen-specific T cells, including induction, expansion and memory/effector phenotype development. Single cellbased PCR sequencing technology will detail the course of TCR usage during the immune response, ascertaining whether the antigen-specific repertoire becomes more focused or diverse and identify emergence of any HLA-A2- restricted public clonotypes. Once completed, other class I and class I! MHC-restricted epitopes will be identified. Third, the nature of human neutralizing antibody responses elicited to orthopox immunization will be defined. Neutralization targets will be determined and the relative contribution of antibodies directed at individual viral proteins ascertained. Biophysical characterization of elicited human neutralizing monoclonal antibodies will be conducted. In this way, the rules regarding both the kinetics of B and T cell responses to their respective epitopes and concordance or discordance in immune targets will be defined.

本研究将探讨人类免疫T细胞和B细胞对正痘病毒的长期记忆 在正常个体和免疫缺陷患者的免疫之后。认识的最新进展 将基于TCR的触发从激活诱导的细胞死亡分流的机制已经确定了记忆 表型生物标志物。在树突状细胞成熟过程中表达的鼠TL MHC Ib类分子及其表达 T细胞上可诱导CDSc_2同二聚体受体充当“记忆开关”。本建议有三个目标。 首先，对人类基因组的生物信息学分析强烈表明HLA-F是小鼠的人类直系同源物 TL，我们将表达，重折叠和纯化重组HLA-F，证明其作为CDSaa配体的能力， 研究受体-配体诱导，使用单克隆抗体和HLA-F四聚体。cd 4和cd 8 将检查CDSc_1及其配体表达的亚群分布和刺激，并将其与记忆联系起来 表型建立。第二，可能的HLA-A2限制性（和其他）T细胞表位由牛痘，MVA和 通过全基因组比较结合位置特异性评分矩阵， 通过蛋白体切割位点预测物进一步解析。这1008种潜在的抗原将被合成，测试 通过CFC测定、ELISPOT、增殖和CTL测定， as mass质量spectrometry光谱.含有鉴定的功能性肽表位的HLA-A2四聚体将监测激发 抗原特异性T细胞，包括诱导，扩增和记忆/效应表型的发展。单细胞 PCR测序技术将详细描述免疫应答过程中TCR的使用过程， 抗原特异性库是否变得更加集中或多样化，并确定任何HLA-A2- 受限制的公共克隆型。一旦完成，其他I类和I类！将鉴定MHC限制性表位。 第三，将定义正痘免疫引起的人中和抗体应答的性质。 将确定中和靶点，并确定针对单个病毒蛋白的抗体的相对贡献 确定。将对引发的人中和单克隆抗体进行生物物理表征。在 这样，关于B和T细胞对其各自表位的反应动力学和一致性或 将定义免疫靶标的不一致性。