NEUTRALIZING EPITOPES OF CHLAMYDIA TRACHOMATIS

本提案的目标是识别和表征连续 主要外膜蛋白（MOMP）的中和表位 沙眼衣原体。 这种微生物是可预防的 失明和性传播疾病，因此通过 需要有效的疫苗。 过去的疫苗试验 使用整个生物体进行免疫的方法都失败了， 部分原因是过敏反应。 的替代方法 免疫是工程化亚单位疫苗， 引发保护性宿主反应。 识别这样的关键 表位MOMP被选为本研究的重点，因为它是 表面暴露，已被证明可以引发中和抗体和DNA 序列数据是可获得的，因此得到了氨基酸序列。 更 特别是可变结构域（VD）IV将是本研究的主要焦点。 由于中和B、B相关和C相关的抗体 综合体已被绘制到该地区。 然而，具有 映射到这个区域的蛋白质不能中和C复合体的成员 因此，除VD IV外，VD I和VD 11还将用 C复合体血清型，以便更成功地识别 具有广泛反应性和中和性的连续中和表位 这些物种的成员。 来自血清型的代表VD IV的肽 来自血清型C的L2和E以及VD I、II和IV将用于免疫 小鼠 多克隆血清以及由免疫球蛋白产生的单克隆抗体都可以被检测到。 将检查免疫接种的中和C的能力。 沙眼 这将通过在体外进行 中和测定。此外，Geysen技术将用于 使用重叠的六聚体肽通过ELISA定位识别位点， 疫苗接种中使用的VD 功能，即，可中和的， 网站将通过竞争实验确定， 中和抗体将被活的C竞争。沙眼衣原体和 将肽转移到感兴趣的区域。 中和血清型谱 将建立的抗体以及能力的 抗体以相加或协同方式起作用。 小鼠将被 用肽缀合物免疫或用MAb被动免疫， 在血清型方面被确定为广泛中和。 这些 然后用C.全身性沙眼， 以及粘膜]感染模型。 此外，研究旨在 确定广谱中和MAb的抑制机制 将被执行。 本次调查中提出的工作将提供 关于该蛋白的关键结构蛋白的结构-功能信息 生物体，并将在未来的疫苗设计，以控制 这种重要的人类病原体的传播和生长。

The goal of this proposal is to identify and characterize continuous neutralizing epitopes of the major outer membrane protein (MOMP) of Chlamydia trachomatis. This organism is the leading cause of preventable blindness and sexually transmitted diseases, therefore protection through an effective vaccine would be desirable. Vaccine trials in the past which have employed the whole organism for immunization have failed, partly due to hypersensitivity reactions. An alternate approach to immunization is to engineer a subunit vaccine where only epitopes that elicit a protective host response are included. To identify such key epitopes MOMP was chosen as the focus of this investigation since it is surface exposed, has been shown to elicit neutralizing antibodies and DNA sequence data is available and thus the derived amino add sequence. More specifically variable domain (VD) IV will be the main focus of this proposal since antibodies that neutralize the B-,B-related and C-related complexes have been mapped to this region. However, MAbs that have mapped to this region fail to neutralize members of the C-complex Therefore, in addition to VD IV, VD I and VD 11 will be examined with C-complex serovars in order to be more successful in identifying continuous neutralizing epitopes that are broadly reactive and neutralize these members of the species. Peptides representing VD IV from serovars L2 and E and VD I, II and IV from serovar C will be used to immunize mice. Both the polyclonal sera as well as MAbs resulting from the immunizations will be examined for their ability to neutralize C. trachomatis. This will be accomplished by performing in vitro neutralization assays. In addition, the Geysen technique will be used to map recognition sites by ELISA using overlapping hexameric peptides to the VD used in the immunizations. The functional, i.e., neutralizable, site will be determined by competition experiments in which the neutralizing antibodies will be competed for by viable C. trachomatis and peptides to the region of interest. The spectrum of serovars neutralized by the antibodies will be established as well as the ability of the antibodies to act in an additive or synergistic manner. Mice will be immunized with peptide conjugates or passively immunized with MAbs that are identified as broadly neutralizing in terms of serovars. These animals will then be challenged with C. trachomatis in both a systemic as well as a mucosa] model of infection. In addition, studies aimed at determining the mechanism(s) of inhibition of the broad neutralizing MAbs will be performed. The work proposed in this investigation will provide structure-function information about a key structural protein of this organism and will be useful in the future design of vaccines to control the spread and growth of this important human pathogen.