Development of a recombinant protein vaccine against Plasmodium falciparum malaria

Sammanfattning: Malaria, caused by Plasmodium falciparum is one of the world´s deadliest diseases, killing one to two million children, while another 500 million suffer from clinical attacks every year. Infections caused by P. falciparum and culminating in death or in asymptomatic parasitaemia along with the intervening spectrum of severe clinical disease result from the sequestration of infected erythrocytes (iRBCs) in the microvasculature of vital organs. Adhesion to erythrocytes and endothelial receptors is mediated predominantly by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), while the variation exhibited by this molecule is also responsible for immune evasion. The iRBC s spectrum of interactions with endothelial receptors and erythrocytes is determined by Duffy-binding like (DBL1alpha ) and cysteine-rich interdomain region (CIDR1alpha ) domains, among others, harboured on the PfEMP1 molecule. DBL1 a mediated binding to endothelial receptors and erythrocytes, the latter enabling rosetting, is a major virulence factor. Adult residents of endemic regions are semi-immune, wherein much of this protection is afforded by variant and cross-reactive immune responses to PfEMP1. Immunization with recombinant PfEMP1 domains is thus an attractive option for attenuating sequestration and thereby severity of disease. PfEMP1 domains are notoriously difficult to express as recombinant proteins and thereby constitute a bottleneck for functional and vaccine studies. The parameters that have an impact on the quality and yield of DBL1 a domains on recombinant expression in E. coli were studied. Induction of recombinant expression at late log phase of E. coli growth substantially increases the yield of soluble protein. Additionally, the correlation of sequence specific features to their likelihood for expression as soluble proteins on recombinant expression in E. coli was studied. It was experimentally shown that the CIDR and acidic terminal sequence domains are appropriate candidates for recombinant expression in E. coli, while the remaining domain types including the DBL domains, constitute a poor choice for obtaining soluble protein on recombinant expression in E. coli. These studies provide guidelines for assigning candidates for structural and functional studies to appropriate expression systems and thereby potentially expedite the development of a vaccine against P. falciparum malaria. A prototype recombinant DBL1 a vaccine against P. falciparum malaria was developed and characterized. It was shown that cross-reactive antibody responses, believed to be an effective counter to antigenic variation, are elicited to heterologous DBL1 a variants on immunization with a single variant. Additionally, immunization with phylogenetically distant DBL1 a variants, can elicit partial cross-protection in in vivo to challenge with parasite strains harbouring a distant variant. It was demonstrated that immunization with DBL1 a in the context of its native domain structure, as provided by Semliki forest virus particles elicit antibody responses that are surface reactive, disrupt rosettes and attenuate sequestration in in vivo. The stage is thus set for DBL1 a to enter clinical trials.