Abstract
Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.
Highlights
A hallmark of Plasmodium falciparum is the ability of its mature intracellular blood stages to cytoadhere to microvascular endothelial cells or circulating blood cells, causing vascular obstruction and local inflammation [1, 2]
To maximize the chance of producing these cysteine-rich domains as correctly folded recombinant proteins, three expression systems were tested for some domains: i) baculovirus/insect cells (Sf9 or HiFive cells) [39], ii) Pichia pastoris [42] and iii) Escherichia coli [16, 42]
All expression products were obtained as soluble proteins except for eDBL0, which was produced mostly as an insoluble protein in E. coli that was solubilised in urea
Summary
A hallmark of Plasmodium falciparum is the ability of its mature intracellular blood stages to cytoadhere to microvascular endothelial cells or circulating blood cells, causing vascular obstruction and local inflammation [1, 2]. The major parasite adhesin implicated in cytoadherence is PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), a variant surface protein encoded by the approximately 60-member var gene family [3]. Rosetting (the capacity of iRBC to cytoadhere to uninfected RBC) is consistently associated with severe malaria in African children [5,6,7,8] and a high parasite burden in a non-human primate experimental model [9]. Rosetting is due to expression of a subset of var genes from the so called UpsA group [10,11,12] that code for adhesins capable of binding to a variety of receptors on the RBC surface [10, 13,14,15,16]. A reasonable strategy to prevent malaria pathology is to target rosette-forming PfEMP1 adhesins by either vaccination or soluble inhibitors
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