Protective Glycopeptide Immunodeterminants of Ehrlichia chaffeensis

描述（由申请人提供）：本研究项目的长期目标是了解埃立克体糖蛋白在沙菲埃立克体保护性免疫中的作用。人类嗜单核细胞埃里希体病（HME）是一种危及生命的新发蜱传人畜共患病，由专性细胞内细菌沙菲埃希体引起。抗体在对沙非叶虫的免疫中起着重要作用，但保护性抗体反应中涉及的保护性抗原和表位仍然相对未知。在沙非沙蚤中已经发现了几种引起强烈抗体反应的糖蛋白，包括gp120、gp200和gp47，我们已经确定了聚糖是重要的表位决定因子，对糖蛋白特异性抗体的产生有重要贡献。本研究的目的是对沙非沙螨gp47糖肽表位进行分子定位，并确定糖蛋白特异性抗体在免疫中的作用。我们假设，许多引发沙非叶虫保护性抗体的分子决定因素是由主要免疫反应性糖蛋白（包括gp47）中的o -链己糖肽定义的。为了验证这一中心假设，我们提出了以下具体目标：1)确定沙非叶蝉gp47的糖链组成、连锁和附着位点；2)确定gp47糖肽抗体的体内保护作用；3)确定糖蛋白组在抗体识别和保护沙非叶蝉感染中的作用。gp47聚糖的组成、连接和附着位点将通过气相色谱和质谱测定。gp47中存在的一个主要糖肽抗体表位在抗体介导的沙非叶蝉免疫中的作用将通过体内药效实验来确定，实验采用沙非叶蝉感染的SCID小鼠模型，以确定抗糖肽和抗肽抗体提供的免疫。在SCID小鼠模型中，我们将确定沙非沙蚤糖蛋白组在保护性抗体形成中的作用，以评估针对天然沙非沙蚤和糖修饰沙非沙蚤产生的多克隆抗体的保护作用。该提案旨在进一步了解埃氏体糖蛋白及其在沙非叶蝉免疫中的作用。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蜱传疾病。

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.