Protective Glycopeptide Immunodeterminants of Ehrlichia chaffeensis

描述(申请人提供)：本研究项目的长期目标是了解埃立克体糖蛋白在对查菲埃立克体的保护性免疫中的作用。人单核细胞增多性埃立克体病(HME)是一种由胞内专性细菌查菲埃希氏菌引起的、威胁生命的新型人畜共患病。抗体在对查菲艾美耳球虫的免疫中起着重要的作用，但保护性抗体反应的保护性抗原和表位仍然相对未知。在查菲氏乳杆菌中已经鉴定出几种能引起强烈抗体反应的糖蛋白，包括gp120、gp200和gp47，我们已经确定多糖是重要的表位决定簇，对糖蛋白特异性抗体的产生有重要作用。这项建议的目的是从分子上确定查菲埃希氏菌gp47糖肽表位，并确定糖蛋白特异性抗体在免疫中的作用。我们假设，许多诱导对查菲埃希氏菌保护性抗体的分子决定因素是由主要免疫反应糖蛋白中的O-连接的己糖多肽定义的，包括gp47。1)确定查菲埃希菌gp47糖链的组成、连接和结合位点(S)；2)确定gp47糖肽抗体的体内保护作用；3)确定糖蛋白组体内对查菲埃希氏菌感染的抗体识别和保护作用。Gp47的糖链组成、连接和连接位点(S)将通过气相色谱和质谱仪进行测定。Gp47中的一个主要糖肽抗体表位在抗体介导的对查菲埃希氏菌免疫中的作用将通过体内效力实验来确定，该实验使用查菲埃希氏菌感染的SCID小鼠模型来确定由抗糖肽和抗肽抗体提供的免疫。将在SCID小鼠模型中确定查菲肠杆菌糖蛋白质组在保护性抗体发展中的作用，以评估针对天然和糖变性查菲肠杆菌产生的多克隆抗体所提供的保护。该提案旨在加深对埃立克糖蛋白及其在对查菲埃希氏杆菌免疫中的作用的有限了解。HME是一个新兴的公共卫生问题，这项研究将促进合理开发疫苗和治疗方法，以对抗北美最普遍的威胁生命的硬虱传播疾病。

DESCRIPTION (provided by applicant): The long term goal of this research project is to understand the role of ehrlichial glycoproteins in protective immunity to Ehrlichia chaffeensis. Human monocytotropic ehrlichiosis (HME) is a life-threatening emerging tick-borne zoonosis caused by the obligately intracellular bacterium, E. chaffeensis. Antibody plays a substantial role in immunity to E. chaffeensis, but the protective antigens and epitopes involved in the development of a protective antibody response remain relatively unknown. Several glycoproteins have been identified in E. chaffeensis that elicit strong antibody response including the gp120, gp200 and gp47, and we have determined that glycans are important epitope determinants that contribute substantially to the development of glycoprotein specific antibody. The objectives of this proposal are to molecularly define the E. chaffeensis gp47 glycopeptide epitopes, and to determine the role of glycoprotein-specific antibodies in immunity. We hypothesize that many of the molecular determinants that elicit protective antibodies to E. chaffeensis are defined by O-linked hexose glycopeptides within major immunoreactive glycoproteins, including gp47. We propose the following specific aims to test this central hypothesis: 1) define the E. chaffeensis gp47 glycan compostion, linkage and attachment site(s), 2) determine the in vivo protective efficacy of gp47 glycopeptide antibodies, and 3) determine the contribution of the glycoproteome in antibody recognition and protection against E. chaffeensis infection. The gp47 glycan composition, linkage, and attachment site(s) will be determined by gas chromatography and mass spectrometry. The role of a major glycopeptide antibody epitope present in the gp47 in antibody mediated immunity to E. chaffeensis will be determined by in vivo efficacy experiments using a SCID mouse model of E. chaffeensis infection to determine immunity provided by anti-glycopeptide and anti-peptide antibodies. The role of the E. chaffeensis glycoproteome in development of protective antibodies will be determined in a SCID mouse model to evaluate protection provided by polyclonal antibodies produced against native and glycan-altered E. chaffeensis. The proposal aims to further the limited understanding of ehrlichial glycoproteins and their role in immunity to E. chaffeensis. HME is an emerging public health concern, and this research will facilitate rational development of vaccines and therapeutics against the most prevalent life-threatening tick- borne disease in North America.