Molecular Analysis of Antigenic Variation in Malaria

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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).