MAPPING OF PSPA PROTECTION ELICITING EPITOPES

PSPA 保护诱导表位的作图

基本信息

批准号：
2068201
负责人：
LARRY S MCDANIEL
金额：
$ 12.07万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-04-01 至 1998-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2068201
关键词：
Streptococcus pneumoniae Streptococcus pneumoniae vaccine antibacterial antibody bacterial antigens bacterial genetics bactericidal immunity drug design /synthesis /production epitope mapping laboratory mouse monoclonal antibody southern blotting virulence

项目摘要

DESCRIPTION (adapted from investigator's abstract): Pneumococcal surface protein A(PspA) is a virulence factor that slows the clearance of Streptococcus pneumoniae from the blood. PspA is, also, highly immunogenic and elicits antibodies that can protect against fatal infections with S. pneumoniae in mice. Although PspAs are serologically variable, antisera raised against individual PspAs are sufficiently cross-reactive, that they can frequently protect against infections with pneumococci possessing serologically distinguished PspAs. The complete sequence has been determine for pspA of pneumococcal strain Rx1. The N-terminal 49 percent of the inferred protein sequence is highly charged and consistent with an alpha- helix. This region of the molecule is serologically variable and contains the epitopes recognized by all known protective monoclonal antibodies (MAbs) to PspA. The alpha-helical domain of PspA is made up of three coiled-coil motifs of about 90 amino acids each separated by two short sequences expected to interrupt the coiled-coil conformation. Just C-terminal to the alpha-helical domain is a proline-rich domain followed by a large repeat domain. PCR analysis and Southern blots have shown more variability in the alpha-helical domain that in the C-terminal half of PspA. MAbs reactive with surface exposed Rx1 PspA recognize epitopes in the most N-terminal and most C-terminal of the three coiled-coil regions. The middle coiled-coil region appears, so far, to be immunologically silent. The epitopes of the C-terminal coiled-coil region are able to elicit protective antibodies and are much more cross- reactive with other PspAs than epitopes in the N-terminal coiled-coil region. The proposed studies will attempt to confirm and extend this finding by mapping additional protection-eliciting epitopes in Rx1 PspA and in other serologically different PspAs. The investigator plans to determine the structural basis for the variability of PspAs by sequencing serologically different PspAs (another complete pspA and 3-5 additional alpha-helical coding regions). For each of the 4-6 new alpha-helical sequences, he will also attempt to determine whether, like Rx1 PspA, the C-terminal third of each alpha-helical region contains the most highly cross-reactive protection- eliciting epitopes. In addition, the investigator plans to make a more complete map of protection-eliciting epitopes of Rx1 and possibly one of the new alpha-helical sequences. Together, these experiments will allow the investigator to identify the relationship between 1) conserved and variable regions of PspA; 2) protection-eliciting area of PspA; and 3) the structural domains revealed by the Rx1 pspA sequence. Finally, these data will allow Dr. McDaniel to identify those regions of PspA most appropriate for inclusion in a protein based vaccine to protect against infection with S. pneumoniae.

描述（改编自研究者的摘要）：肺炎球菌表面蛋白 A (PspA) 是一种毒力因子，可减缓从血液中清除肺炎链球菌。 PspA 也是，具有高度免疫原性，并引发抗体，可以预防小鼠肺炎链球菌的致命感染。尽管 PspAs 是血清学上存在差异，针对个体 PspAs 的抗血清是具有足够的交叉反应性，因此它们可以经常防止具有血清学特征的肺炎球菌感染 PspAs。 pspA 的完整序列已确定肺炎球菌菌株 Rx1。 N 端 49% 的推断蛋白质序列是高度带电的并且与α-一致螺旋。该分子区域在血清学上是可变的并且包含所有已知的保护性单克隆抗体识别的表位 PspA 抗体 (MAb)。 PspA 的 α 螺旋结构域被制成由三个卷曲螺旋基序组成，每个基序包含约 90 个氨基酸通过预计会中断线圈的两个短序列构象。 α 螺旋结构域的 C 末端是富含脯氨酸的结构域，后面跟着一个大的重复结构域。 PCR分析和 Southern 印迹显示 α 螺旋有更大的变异性域位于 PspA 的 C 端一半。单克隆抗体与表面暴露的 Rx1 PspA 识别最 N 端和三个卷曲线圈区域的大部分 C 末端。中间到目前为止，卷曲螺旋区域似乎处于免疫沉默状态。这 C 末端卷曲螺旋区域的表位能够引发保护性抗体，与其他抗体的交叉反应性更强 PspAs 比 N 末端卷曲螺旋区域中的表位更重要。拟议的研究将尝试通过绘制地图来证实和扩展这一发现 Rx1 PspA 和其他中的额外保护诱导表位血清学上不同的 PspAs。调查人员计划确定通过测序分析 PspAs 变异的结构基础血清学上不同的 PspA（另一个完整的 pspA 和 3-5 额外的α螺旋编码区）。对于每 4-6 个新 α-螺旋序列，他还将尝试确定是否，与 Rx1 PspA 一样，每个 α 螺旋区域的 C 末端三分之一包含最高程度的交叉反应保护 - 引发表位。此外，研究人员计划制定更完整的 Rx1 的保护诱导表位图谱，可能是其中之一新的α螺旋序列。总之，这些实验将允许研究者确定 1) 保守和 PspA的可变区； 2) PspA的保护诱导区；和 3) Rx1 pspA 序列揭示的结构域。最后，这些数据将使 McDaniel 博士能够识别 PspA 的那些区域最适合包含在基于蛋白质的疫苗中以保护预防肺炎链球菌感染。