Molecular Analysis of Antigenic Variation in Malaria

描述（由申请人提供）：该项目的长期目标是揭示控制疟原虫抗原变异的分子和免疫生物学机制。疟原虫的抗原变异是逃避宿主保护性免疫反应的一种基本适应，也是导致慢性疟疾血液感染的主要因素之一。当恶性疟原虫在被感染的红细胞中发展时，它会产生暴露在红细胞表面的高分子量抗原。这些抗原在免疫反应过程中通过改变一个叫做var基因家族的大基因家族中大约50个成员的表达而发生变化。调控这一过程的确切机制在很大程度上仍然未知。该项目利用诺氏疟原虫类人猿疟疾模型和恒河猴感染来研究当表型表达发生变化时，体内DNA、RNA和蛋白质水平上发生的事件。诺氏疟原虫表面暴露的变异抗原最初被发现，并被称为分裂体感染细胞凝集抗原（siica）。它们由相关的SICAvar多基因家族编码。该模型系统已被很好地表征，并能够开发和研究体内衍生的等基因相关克隆，显示出明显稳定的SICA[+]表型；以及SICA[-]在去脾动物传代后的表型。这些克隆的诺氏疟原虫种群允许对变异抗原表达、转换以及与宿主免疫反应的体内相互作用进行仔细控制的研究。使用该模型系统，我们确定了Pk1(B+)1+寄生虫中205 kea SICA抗原的表达是涉及3'编码序列和非编码序列重排的结果。进一步的研究将在几个等基因克隆系中进行表达基因及其转录本的研究。拟制研究的具体目的是：1)确定在诺氏疟原虫体内转换过程中，在Pk1(B+)1+ 205 SICAvar表达等位基因中观察到的3'基因组DNA改变是否与SICAvar基因激活相关，并进一步表征SICAvar多基因家族的基因组组织；2)在连续开关/激活事件衍生的稳定等基因无性系中，研究表达和非表达SICAvar基因的转录模式，表征转录rna，并评估5‘和3’调控区在转录激活和转录后基因沉默（PTGS）中的贡献。

DESCRIPTION (provided by the applicant): The broad, long-term objective of this project is to unravel the molecular and immunobiological mechanisms that govern antigenic variation in Plasmodium. Antigenic variation in Plasmodium is a fundamental adaptation to evade a host protective immune response and one of the major factors contributing to the establishment of chronic malaria blood infections. As Plasmodium falciparum develops in an infected red blood cell it produces high molecular weight antigens that become exposed at the erythrocyte surface. These antigens vary in the course of an immune response by switching the expression of about 50 members of a large gene family, called the var gene family. The precise mechanisms regulating this process remain largely unknown. This project utilizes the P. knowlesi simian malaria model and rhesus monkey infections to investigate what events occur in vivo at the DNA, RNA and protein levels as changes in phenotypic expression occurs. The surface exposed variant antigens of P. knowlesi were originally discovered and called the SICA (Schizont Infected Cell Agglutination) antigens. They are encoded by the related SICAvar multigene family. This model system has been well characterized and enables the development and investigation of in-vivo derived, isogenic related clones exhibiting distinct stable SICA[+] phenotypes; as well as SICA[-] phenotypes after passaged in splenectomized animals. These cloned P. knowlesi parasite populations allow for carefully controlled studies related to variant antigen expression, switching, and the in vivo interplay with the host immune response. Using this model system we determined that the expression of the 205 kea SICA antigen in Pk1(B+)1+ parasites is the result of a rearrangement involving 3' coding and non-coding sequences. Further studies will be pursued to study the expressed genes and their transcripts in several isogenic cloned lines. The specific aims of the proposed studies are to: 1) Determine if a 3' genomic DNA alteration as observed in the Pk1(B+)1+ 205 SICAvar expressed allele is a typical event associated with SICAvar gene activation during in vivo switching in P. knowlesi, as well as further characterize the genomic organization of the SICAvar multigene family; and 2) Investigate the pattern of transcription of expressed and non-expressed SICAvar genes in stable isogenic clonal parasite lines derived from successive switching/activation events, characterize the transcribed RNAs, and evaluate the contribution of 5' and 3' regulatory regions in transcriptional activation and post-transcriptional gene silencing (PTGS).