Immunisation with Recombinant PfEMP1 Domains Elicits Functional Rosette-Inhibiting and Phagocytosis-Inducing Antibodies to Plasmodium falciparum

Abstract

BackgroundRosetting is a Plasmodium falciparum virulence factor implicated in the pathogenesis of life-threatening malaria. Rosetting occurs when parasite–derived P. falciparum Erythrocyte Membrane Protein One (PfEMP1) on the surface of infected erythrocytes binds to human receptors on uninfected erythrocytes. PfEMP1 is a possible target for a vaccine to induce antibodies to inhibit rosetting and prevent severe malaria.Methodology/FindingsWe examined the vaccine potential of the six extracellular domains of a rosette-mediating PfEMP1 variant (ITvar9/R29var1 from the R29 parasite strain) by immunizing rabbits with recombinant proteins expressed in E. coli. Antibodies raised to each domain were tested for surface fluorescence with live infected erythrocytes, rosette inhibition and phagocytosis-induction. Antibodies to all PfEMP1 domains recognized the surface of live infected erythrocytes down to low concentrations (0.02–1.56 µg/ml of total IgG). Antibodies to all PfEMP1 domains except for the second Duffy-Binding-Like region inhibited rosetting (50% inhibitory concentration 0.04–4 µg/ml) and were able to opsonize and induce phagocytosis of infected erythrocytes at low concentrations (1.56–6.25 µg/ml). Antibodies to the N-terminal region (NTS-DBL1α) were the most effective in all assays. All antibodies were specific for the R29 parasite strain, and showed no functional activity against five other rosetting strains.Conclusions/SignificanceThese results are encouraging for vaccine development as they show that potent antibodies can be generated to recombinant PfEMP1 domains that will inhibit rosetting and induce phagocytosis of infected erythrocytes. However, further work is needed on rosetting mechanisms and cross-reactivity in field isolates to define a set of PfEMP1 variants that could induce functional antibodies against a broad range of P. falciparum rosetting parasites.

A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells

“…The majority of studies addressing the relationship between var genes and clinical disease have found an association between group A var gene transcription and severe malaria or CM (16)(17)(18)(19)51). Previously, the only known binding phenotype for group A PfEMP1 variants was rosetting (20)(21)(22)(23), with two group A domain cassettes (DC11 and DC16) being linked to rosette formation (24). DC16 was not associated with severe malaria in the study by Lavstsen et al (44) and DC11 was not tested.…”

“…IFA with rabbit preimmune and immune sera and live IEs were carried out as described (23). For each antigen, the rabbit serum giving the brightest IFA signal with the lowest background was chosen for purification of total IgG.…”

“…Antisera to HB3var3 NTS-DBLα1 and the di-domain showed punctate surface fluorescence of live HB3-HBEC IEs by immunofluorescence assay (IFA) (Fig. 5B) typical of PfEMP1 antibodies (23). Rabbit preimmune sera and nonimmunized rabbit control sera were negative by IFA.…”

“…We also tested antiserum raised against another recombinant domain of the HB3var3 variant (DBLδ5) produced in baculovirus (42). Previous work has shown that antibodies to multiple PfEMP1 domains are able to block adhesion in the case of rosetting parasites (23). The HB3var3 DBLδ5 antiserum showed punctate surface fluorescence with live IEs of HB3-HBEC parasites at 1/25 and 1/100 dilution, whereas the preimmune serum was negative at the same concentrations.…”

“…Group B and C var genes encode PfEMP1 variants that bind CD36 (14,15) and are linked to nonvirulent clinical disease, whereas group A var genes encode non-CD36-binding variants linked to severe clinical disease including CM (16)(17)(18)(19). Some group A var genes encode PfEMP1 variants that bind to uninfected erythrocytes to form rosettes (20)(21)(22)(23)(24)(25); however, the adhesion phenotype of the majority of group A variants is unknown currently.…”