Role of variant antigens in the immunity against murine Plasmodium: Estimating the influence of variant proteins BIR in cytoadherence and their potential as vaccine using novel means of administration.

Abstract

All species of the malaria causing protozoan Plasmodium possess gene families which encode antigens that are translocated to the hosts infected red blood cell. In Plasmodium falciparum, the var gene family encodes the PfEMP1 proteins which function as ligands that lead to the sequestration (cytoadherence) of the infected red blood cell in deep venules. Adhesive properties were also recently reported for P. vivax and members of the pir (Plasmodium interspersed repeats) superfamily were suspected as ligands. The rodent species P. berghei possesses the bir gene family which encode the BIR antigens and this species also shows cytoadherence and development of a cerebral malaria-like phenotype in susceptible mice. However, the ligand for this interaction is unknown. Also, the control of expression of the bir gene family is unknown. In this project, we hypothesize that I) there is a participation of BIR in the observed virulence/pathology in mice and ii) that bir genes are expressed in a way that there is a crosstalk between active and silent bir loci. In order to address these issues, we propose to transfect P. berghei parasites with an artificial chromosome containing a bir promoter which controls a reporter gene (GFP) or a fusion of reporter and antibiotic resistance gene (GFP-TgDHFR) which confers resistance against Pyrimethamin. Transfectants of these parasites will then be used to infect C57Bl/6 mice and transgenic parasites will be selected by pyrimethamin treatment. Bir and gfp transcription will be monitored by RNA seq or massive sequencing of RT-PCR amplified/cloned bir transcripts. In parallel, we will try to monitor if GFP expressing parasites lead to the development of cerebral malaria in mice and if green fluorescent parasites accumulate or evade specifically the brain vessels. By the use of GFP-TgDHFR expressing parasites under constant drug pressure which may be unable to cause cerebral malaria we would then show indirectly i) a participation of BIR in the pathogenic process and ii) that forced bir transcription from an artificial bir locus interferes with the transcription of other bir loci. In parallel, GFP expressing parasites will also be used for the infection of transgenic GFP-expressing mice. Following our hypothesis, the transcription of gfp should be modulated differently in animals which are tolerant to GFP as compared to wildtype mice. All the constructs can be also used to explore the placental malaria model which was developed by a group from the department. Additionally we propose to test if directable nanoparticles are superior as vaccine formulations when compared to conventional delivery systems. For this, vaccine antigens are expressed as GPI anchored versions in an eucaryotic system and the GPI anchored proteins are then inserted in lipossomes. These can be turned into directable liposomes by the insertion of ligands for specific receptors. Multilamellar liposomes may also be loaded with effector reagents. Initially, we will use the PfRh5 protein which is a strong malaria vaccine candidate. As a loading control GPI anchored GFP will be used. In case we identify a specific BIR protein associated to cerebral or placental malaria, we will express the corresponding BIR ectodomain and insert it as GPI anchored version in liposomes. The success of liposome based vaccination will be compared to conventional delivery systems such as AlOH3, Freund's adjuvant or monophosphoryl lipid A formulated vaccines. The type of response will be monitored by cytometry, western blots and ELISA, employing also subtype specific secondary antibodies.