MATERNAL MALARIA--PARASITE ADHESION AND ANTIBODIES

这个应用程序提出了描述变量和不变式 疟原虫表达的CSA结合蛋白的性质， 并了解女性产生体液免疫的机制 其防止寄生虫粘附到CSA。具体来说，我们将确定 var基因产物、独特转录物和CSA结合蛋白 疟疾寄生虫所表达的：这些方法中的每一种都提供了 有机会鉴定参与寄生虫-CSA相互作用的分子。 采用RT-PCR方法从胎盘组织中扩增var基因产物 定义为CSA结合的田间分离株;这些扩增的转录本将 与从野生型获得的田间分离物表达的var基因进行比较， 未怀孕的捐赠者 减法将采用减法PCR， 消减文库，以鉴定CSA结合特异性基因转录物 来自胎盘的寄生虫（因此不常见于CD 36结合 来自非妊娠供体的寄生虫）;这些和其他表达 文库可以用标记的CSA或用来自多胎孕妇的血清进行筛选， 妇女CSA结合蛋白将在CSA亲和柱上纯化， 然后用于测定以表征功能以及获得 肽序列;这些信息将用于设计 用寡核苷酸探针筛选文库的全长序列。 然后，我们将确定这些的保守和变异性质， 分子，探索它们对寄生虫粘附的贡献， 胎盘，并确定天然存在的 已知可抑制寄生虫粘附至CSA的抗体。 序列 变异和保守的基序将通过比对 通过上述方法鉴定的分子。 此信息将 在结合功能的背景下， 蛋白质碎片作为粘附的证据。 我们假设CSA- 由疟原虫表达的结合分子可以采取变体或 不同的形式，但参与结合的基序是保守的，或 抗粘附抗体靶向的表位是保守的，或 限制了它们的变化程度。 因此，与 这些分子的序列和功能特征，我们将 确定阻断寄生虫的抗体的表位特异性 对CSA有约束力。 这些信息将提供第一个完整的 表征可接近组织中的组织-寄生虫粘附， 并可能提供一个模型来检查其他疟疾综合征。 此外，本发明还 结合结构域和B细胞表位的描绘将提供 为设计抗粘连疫苗保护妊娠妇女提供了依据， 其他妇女易受胎盘感染与CSA结合 寄生虫

This application proposes to describe the variant and invariant properties of CSA-binding proteins expressed by the malaria parasite, and to understand the mechanisms by which women develop humoral immunity which prevents parasite adhesion to CSA. Specifically, we will identify var gene products, unique transcripts, and CSA-binding proteins expressed by the malaria parasite: each of these approaches provides the opportunity to identify molecules involved in parasite-CSA interactions. RT-PCR will be employed to amplify var gene products from placental field isolates defined as CSA-binding; these amplified transcripts will be compared with var genes expressed by field isolated obtained from non-pregnant donors. Subtraction will employ subtractive PCR and subtraction libraries to identify gene transcripts unique to CSA-binding parasites from the placenta (and therefore not common with CD36-binding parasites from non-pregnant donors); these and other expression libraries can be screened with labeled CSA or with sera from multigravid women. CSA-binding proteins will be purified on CSA affinity columns, then employed in assays to characterize function as well as obtain peptide sequence; this information will be used to design oligonucleotide probes to screen libraries for full length sequence. We will then determine conserved and variant properties of these molecules, explore their contribution to parasite adhesion in the placenta, and identify epitopes targeted by naturally occurring antibodies known to inhibit parasite adhesion to CSA. Sequence variation and conserved motifs will be explored by aligning the molecules identified by the above methods. This information will be placed in the context of binding function by examining recombinant protein fragments for evidence of adhesion. We hypothesize that CSA- binding molecules expressed by the malaria parasite can take variant or distinct forms, but that motifs involved in binding are conserved, or that epitopes targeted by anti-adhesion antibodies are conserved or limited in their degree of variation. Therefore, in conjunction with the sequence and functional characterization of these molecules, we will determine the epitopic specificity of antibodies which block parasite binding to CSA. This information will provide the first full characterization of tissue-parasite adhesion in an accessible tissue, and may provide a model to examine other malaria syndromes. Further, the delineation of binding domains and B cell epitopes will provide the basis to design an anti-adhesion vaccine to protect primigravid and other women susceptible to placental infection with the CSA-binding parasites.