This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objectives of this research are focused on the molecular mechanisms that govern variant antigen gene expression in Plasmodium. Antigenic variation is a fundamental adaptation to evade a host protective immune response and is one of the major factors contributing to the establishment of chronic blood infections. The classic P. knowlesi-rhesus monkey model is amenable to both in vitro and in vivo studies and unique stable clones of the P. knowlesi H strain expressing distinct SICA (Schizont Infected Cell Agglutination) variant antigen phenotypes after induced sequential switchings can be maintained after numerous in vivo passages (60 generations) in naive rhesus monkeys. These isogenic clonal lines provide a special tool for studies of the cellular and genetic mechanisms underlying clonal antigenic variation. We hypothesized that studies of the clonal phenotypes provide insights towards understanding the regulation of antigenic variation in vivo. This year, we published an important report on the redefined SICAvar prototype gene and protein, which emphasizes the need for further attention to the closure of the Pk genome data. We have also produced definitive data using proteomics (LC-MS/MS), real time RT-PCR and northern blot experiments defining the predominant transcripts and proteins produced by the classical P. knowlesi Pk1(A+)1+, Pk1(B+)1+ and Pk1(C+)1+ (i.e. SICA+) clones and show strong downregulation in the SICA- clones. Analyses are also underway with collaborators and a manuscript in preparation on a comprehensive P. knowlesi blood-stage transcriptome from RNA samples collected from an in vivo rhesus infection and in vitro adapted parasites. Proteomic data is also being generated, and compared with the transcriptome, with SICA expression and other hypotheses being tested. We also published a review emphasizing the importance of P. knowlesi not only as a model system, but also as a parasite shown recently to cause hundreds of cases of malaria in humans.