Role of DM in Generation of Immunodominant Epitope(s)

DM 在免疫显性表位生成中的作用

基本信息

批准号：
8089851
负责人：
Scheherazade Sadegh-Nasseri
金额：
$ 20.82万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8089851
关键词：
Acids Amino Acid Sequence Aminopeptidase Antigen Presentation Pathway Antigens Binding Biological Assay Biological Preservation Biological Testing Carboxypeptidase Cathepsins Collagen Complex DR1 gene Data Digestion Dissociation Enzymes Epitopes Generations Goals HLA-DR Antigens HLA-DR1 Antigen Hemagglutinin High Pressure Liquid Chromatography Histocompatibility Antigens Class II Immunity Immunization Immunodominant Epitopes In Vitro Incubated Individual Infectious Agent Influenza Hemagglutinin Left MHC Class II Genes Methods Modeling Molecular Conformation Molecular Weight Muscle Rigidity National Institute of Allergy and Infectious Disease Organism Peptide/MHC Complex Peptides Proteins Regulation Research Role Scanning Source Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Staining method Stains T-Lymphocyte T-Lymphocyte Epitopes Testing Transgenic Mice Vaccine Design antigen processing biodefense design flexibility hemagglutinin (306-318)in vitro Assay in vivo protein complex

项目摘要

DESCRIPTION (provided by applicant): The goals of this proposal are to evaluate the role of DM, a non-classical MHC class II heterodimer, in the selection of immunodominant epitopes from a given protein. Our recent data strongly suggested that DM selectively dissociates complexes of peptide-MHC that have floppy conformation and leaves compact peptide/MHC complexes unaffected. We propose that this preferred interaction with a specific conformation of MHC class II, might influence epitope selection from the pool of all other epitopes that can bind to MHC class II. A new assay is designed here to investigate the role of DM in determinant selection by a given MHC class II. Using purified empty HLA-DR1, soluble HLA-DM, and purified Influenza Hemagglutinin (HA) as a model antigen, we will generate antigenic epitopes that remain bound to DR1 in the presence of DM. We would begin with purified influenza hemagglutinin that has a known immunodominant epitope of HA306- 318 in DR1 expressing individuals. Using enzymes with known activity in antigen processing, such as cathepsins, HA protein will be subjected to enzymatic digestion and DR1 binding in the presence of DR1 and DM in Aim I. Mass spectrometric analyzes of the HPLC fractionated peptides eluted from DR1 under the above condition will reveal whether HA306-318 is selected as a predominant peptide. Aim 2 will investigate effects of HLA-DO on regulation of DM and selection of immunodominant epitopes using similar approach to Aim I. Aim 3 will examine interactions of purified DO with DM in vitro and Aim IV will test biological activity of identified epitopes in vivo. Once the feasibility of the method is established, it should be applicable for identification of antigenic epitopes of any complex proteins from pathogenic organisms. The antigenic epitopes might be used in class II tetramer assays for staining of specific activated T cells in vivo and in design of vaccines and as correlates of immunity for immunization efficacy. This research has great impacts on discovering immunodominant epitopes of infectious agents relevant to biodefense.

描述（由申请人提供）：该提案的目标是评估非经典MHC MHC II类异二聚体DM的作用，在从给定蛋白质中选择免疫主流表位中。我们最近的数据强烈表明，DM有选择地解离具有软构的肽-MHC的复合物，并留下了不受影响的紧凑肽/MHC复合物。我们建议，这种与MHC II类的特定构象的这种优选相互作用可能会影响所有其他可以与MHC II类结合的表位的表位选择。这里设计了一种新测定，以调查DM通过给定MHC II类选择的决定性选择。使用纯化的空HLA-DR1，可溶性HLA-DM和纯化的流感血凝素（HA）作为模型抗原，我们将生成在DM存在下保持与DR1结合的抗原表位。我们将从纯化的流感血凝素蛋白开始，该血凝集素在DR1表达个体中具有HA306-318的已知免疫主流表位。在抗原加工中使用具有已知活性的酶，例如组织蛋白蛋白，HA蛋白将在AIM I中进行酶促消化和DR1结合。将揭示HA306-318是否被选为主要肽。 AIM 2将研究HLA-DO对使用类似的AIM I的调节的影响和免疫主导表位的选择。AIM3将检查纯化的纯化与DM体外的相互作用，AIM IV将测试体内鉴定表位的生物学活性。一旦建立了该方法的可行性，应适用于鉴定病原生物的任何复杂蛋白质的抗原表位。抗原表位可以用于II类四聚体测定中，用于在体内和疫苗设计中染色特定活化的T细胞，并作为免疫功能的免疫相关性。这项研究对发现与Biodefense相关的传染性药物的免疫主导表现有很大的影响。