Recombinant expression of "Plasmodium falciparum" erythrocyte membrane protein 1 fragments and serological studies

Abstract

The intraerythrocytic stages of the Plasmodium falciparum life cycle are exclusively responsible for all clinical symptoms of malaria. Both children and adults that are infected with P. falciparum can either have symptoms of variable severity or be asymptomatic. However, it is mostly young children who suffer from severe symptoms ranging from severe anaemia to cerebral malaria, and it is mostly adults from endemic areas who experience comparatively mild episodes with headache and sometimes fever. The observed morbidity is largely associated with sequestration of parasitized erythrocytes (iRBCs) on endothelial cells of host blood capillaries. This cytoadherence prevents late stage iRBCs from being cleared by the spleen. Instead, iRBCs bind to various host cell receptors such as CD36, ICAM, or CSA leading to obstruction of blood vessels, impaired oxygen delivery in affected host organs and immunological reactions of the affected tissues. The key mediator of sequestration found is the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1). This large parasite derived protein is exported from the parasite and trafficked through various membranes and through the host cell cytosol until becoming inserted into the erythrocyte cell membrane. It is located at the interface between parasite and host immune system, and undergoes antigenic variation. PfEMP1 is encoded by approximately 60 var genes per haploid genome, and is expressed in a mutually exclusive manner, i.e. only one gene is expressed at any one time. As one of its sophisticated immune evasion strategies, the parasite can switch to another PfEMP1 variant and thus becomes no more recognizable by the host immune system. It is believed that protection against severe malaria is the result of the development of immune responses against various variants of PfEMP1. However, immunity to malaria is never sterile but instead only reduces parasite density and morbidity. We have based our work on the hypothesis that not all variants of PfEMP1 are equally pathogenic i.e. have the same affinity to host cell receptors. We believe that only a certain subset of PfEMP1 variants is able to confer solid cytoadherence, and consequently is responsible for severe malaria. Possessing an antibody repertoire against these specific variants therefore will protect from severe episodes. In this work we have chosen a multiple approach to generate molecular tools and to test this hypothesis. Firstly, we elaborated on the generation of pan-specific noncross reactive PfEMP1 antibodies using both recombinantly expressed domains both from the molecule’s head structure (NTS domain) and synthetic peptides corresponding to the semi-conserved intracellular part of PfEMP1 (ATS peptides). By means of various molecular methods, however, we found that none of the generated sera recognized full length endogenous PfEMP1 exclusively. Secondly, we attempted expression of large fragments of PfEMP1 in E.coli to test the recognition of sera from different malaria cases. At the same time we wanted to exploit the possibility to express random fragments of PfEMP1 in a bacterial library to similarly test these sera on. Insuperable obstacles with large recombinant protein expression forced us to divert our approach towards smaller domains. For this we isolated var mRNA from samples from several individuals presenting either with asymptomatic infections or experiencing severe malaria episodes. 14 var DBL domains were recombinantly expressed in E. coli and used to measure antibody titers in sera from 100 semi-immune Papua New Guinean adults. The frequency of recognition (FoR) for these antigens of was assessed and compared between FoR of DBL domains deriving from severe cases and from asymptomatic samples. We found that DBL domains deriving from severe cases were significantly more often recognized by sera from semi immune Papua New Guinean adults than DBL domains derived from asymptomatic samples. This is indicative for semiimmune adults not suffering from clinical malaria because being better protected against parasites expressing “severe” DBL domains of PfEMP1. We also tested 34 sera from children with asymptomatic infections collected during a longitudinal study in Tanzania. We selected sera that were collected at two time points 6 months apart to assess the development and dynamics of antibodies against those DBL domains. FoR increased significantly over time in these children but only for DBL domains deriving from severe cases. As these children did not suffer from clinical episodes between the two sampling dates, these results also indicate that acquisition of antibodies against “severe” DBL domains is faster and can confer protection. In summary, our findings support the notion that development of antibodies against PfEMP1 variants (in this case against DBL domains) is associated with protection against severe disease and thus contributes as an important factor to the acquired clinical immunity to severe malaria. These findings raise hope in the feasibility of a putative protective vaccine against the major virulence factor PfEMP1.