SEROLOGICAL EVALUATION OF CHLAMYDIAL EYE INFECTIONS

衣原体眼部感染的血清学评估

• 批准号: 3264813
• 负责人: Richard S Stephens
• 金额: $ 19.68万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3264813
• 关键词: Africa; Chlamydia trachomatis; Egypt; antibacterial antibody; antigens; bacterial antigens; bacterial genetics; bacterial proteins; bactericidal immunity; enzyme linked immunosorbent assay; genetic manipulation; genetic strain; host organism interaction; human tissue; interleukin 1; laboratory mouse; laboratory rabbit; membrane proteins; microorganism immunology; molecular cloning; nucleic acid sequence; protein engineering; protein sequence; relapse /recurrence; serology /serodiagnosis; synthetic peptide; tear; trachomas

Chlamydia trachomatis is the cause of trachoma, and following repeated infections the most damaging sequela is blindness. Over 15 serovariants of C. trachomatis have been described, and serovar-specific antigens are associated with protective immunity while broadly reacting antigens are associated with immunopathology. The major outer membrane protein (MOMP) of chlamydiae contains the serovar-specific antigen, however, this is an antigenically complex molecule that also contains subspecies- and species-specific antigens. Immunity to C. trachomatis eye infections is poorly understood, although animal models and human vaccine trials have demonstrated antibody mediated immune protection. The broad, long-term objectives of this proposal are 1) the evaluation of the human humoral response of trachoma tear samples to sequence-specific chlamydial epitopes and correlation of this data to clinical status, and 2) the development of a new amino acid sequence-defined immunoassay suitable for rapid seroepidemiological evaluations and assessments of immune status. These objectives will be of value for developing a basic understanding of immune mechanisms elicited during natural infection, and for developing and evaluating strategies for immune prophylactic intervention and for monitoring epidemiological intervention approaches. The specific aims are: 1) Map the antigenic determinants for the 60K outer membrane protein using recombinant DNA expression in E. coli and peptide synthesis. Expression proteins and synthetic peptides will be identified and characterized using immune human and rabbit sera. 2) Evaluation of strain diversity for the MOMP gene of serovar-specific isolates obtained in trachoma endemic areas. This is accomplished using primer extension sequencing of mRNA isolated from infected tissue cultures. 3) Definition of the molecular basis of antigenic relatedness among serovariants. The DNA sequences of each of the 15 prototype serovars for the portion of the gene which encodes the serovar-specific epitope will determined and compared. Antisera will be obtained in rabbits immunized with synthetic peptides representing these epitopes, and the specificities of the antibody reactivities will define the molecular complexities of these relationships. 4) Develop an epidemiological immunoassay using serovar-specific DNA and amino acid sequence information to clone and express sequence-defined reagents for each of the 15 serovariants. These reagents will be constructed from synthetic oligonucleotides and expressed as fusion products of glutathione transferase. One-step affinity purification of these soluble fusion proteins will provide unlimited quantities of standardized material. 5) Determination of the human humoral response to antigenic domains and sequence-specific epitopes for the MOMP and 60K proteins. Tears obtained from trachoma studies will be evaluated for immunoglobulin classes and isotypes, and their quantity, avidity, and epitopic specificities will be evaluated.

沙眼衣原体是沙眼的病因， 反复感染最严重的后遗症是失明。 超过 15株C.已经描述了沙眼， 血清型特异性抗原与保护性免疫有关 而广泛反应的抗原与 免疫病理学 主要外膜蛋白（MOMP） 衣原体含有血清型特异性抗原，然而， 一种抗原复合物分子，它也包含亚种， 和物种特异性抗原。 免疫C。沙眼眼 感染知之甚少，尽管动物模型和 人类疫苗试验已经证明抗体介导的 免疫保护。 这一广泛的长期目标 建议是：1）评价人的体液反应， 沙眼泪液样品与序列特异性衣原体表位 以及该数据与临床状态的相关性，以及2） 一种新的氨基酸序列确定免疫分析方法的建立 适用于快速血清流行病学评价和评估 免疫状态。 这些目标将具有价值， 对引发的免疫机制有基本了解 在自然感染过程中， 免疫预防性干预和 监测流行病干预办法。 具体目标是：1）定位用于免疫缺陷病毒的抗原决定簇。 利用重组DNA表达60K外膜蛋白 在大肠大肠杆菌和肽合成。 表达蛋白和合成 肽将使用免疫人 和兔血清。 2)MOMP菌株多样性评价 沙眼流行病中血清型特异性分离株的基因 地区 这是使用引物延伸测序完成的。 从感染的组织培养物中分离的mRNA。 3)定义 血清变体之间抗原相关性的分子基础。 的 15个原型血清型中每一个的DNA序列 编码血清型特异性表位的基因部分 将被确定和比较。 抗血清将在 用代表这些的合成肽免疫的兔 表位，抗体反应性的特异性将 定义了这些关系的分子复杂性。 四、 开发一种流行病学免疫测定方法， 用于克隆和表达的DNA和氨基酸序列信息 用于15种血清变异体中每一种的序列确定的试剂。 这些 试剂将由合成的寡核苷酸构建， 表达为谷胱甘肽转移酶的融合产物。 一步 这些可溶性融合蛋白的亲和纯化将提供 无限量的标准化材料。 5)测定 人类体液对抗原域的反应， MOMP和60K蛋白的序列特异性表位。 将对从沙眼研究中获得的泪液进行评价， 免疫球蛋白种类和同种型，以及它们的数量，亲合力， 并评价表位特异性。