P. vivax depends on the interaction of the DBP with its cognate receptor for efficient invasion of human reticulocytes. The vital nature of this interaction makes DBP an ideal target for vaccine development. Potential impediments to the use of DBP as an effective vaccine have been attributed to the low frequency of development of naturally acquired DBP binding inhibitory antibodies, the lack of knowledge of DBP contact residues necessary for receptor recognition, and the polymorphic nature of the DBP ligand. Understanding the specificity of the protective anti-DBP responses is critical for effective vaccine development. Therefore, two purposes of this study were to obtain a clearer understanding of anti-DBP serological responses from natural exposure and to initiate identification of epitopes targeted by functionally inhibitory anti-DBP antibodies. Here we present evidence identifying potential inhibitory B-cell linear epitopes in the critical receptor-binding region of the DBP ligand.
When we screened residents of an area of PNG where malaria is highly endemic, we discovered that naturally acquired antibodies that effectively inhibit DBPII-erythrocyte binding are infrequent. There was great variability among these residents, and for a substantial proportion (~50%) there was a significant change in the titer from one transmission season to the next (Table ). The observation that there are relatively rapid changes in serological responses is similar to the previous observation that in PNG temporal variation was the primary source of variation in the IgG response to Plasmodium
). The small number of high-titer, highly inhibitory sera remained relatively stable, which is consistent with the results of another study performed in this area where malaria is endemic (16
), but low-responder individuals whose responses were mostly ineffective for functional inhibition of DBP binding to red blood cells also exhibited a poor-quality antibody response in all sampling periods.
In the high-titer group only 3 of 10 individuals exhibited effective inhibition of DBP function, while only 1 of the 23 low responders exhibited a high level of inhibition. Such marked individual differences in the naturally acquired anti-DBP responses and the functional inhibition of DBP-erythrocyte binding indicated that epitope specificity and antibody avidity are both critically important for effective inhibition. In addition, our results indicate that DBP inhibitory antibodies are relatively infrequent and most antibody responses are relatively unstable. These discoveries for anti-DBP serological responses contrast with acquisition of immunity to the homologous VAR2CSA DBL domain, where there is a better correlation between development of protective immunity and repeated exposure (21
). However, a longitudinal study to closely monitor the development of antibodies and B-cell memory for DBP is necessary to adequately determine the stability of naturally acquired anti-DBP antibodies.
A comparison of the epitope specificity of the highly inhibitory anti-DBP sera with that of the noninhibitory immune sera identified potential targets associated with protective anti-DBP immunity. Nearly all of the putative inhibitory B-cell linear peptides identified in this study are localized in the central region of the DBP ligand domain. This finding is similar to the finding for naturally acquired antibodies to VAR2CSA-DBL that are directed toward surface-exposed epitopes in the S1/S2 domains, a region homologous to the central region of DBPII, and the F1/F2 domains of EBA-175 (3
). This central region is known to be important for receptor recognition, but precisely how the Duffy receptor (DARC), interacts with this region is not yet understood. Mutagenesis studies have identified numerous residues in this region as residues that are important or essential for receptor recognition, including many residues absent from the 3D model based on the crystal structure of Plasmodium knowlesi
). Much attention has been focused on a receptor-binding pocket that is proposed to recognize a sulfated tyrosine thought to be part of the N-terminal domain of the DARC receptor similar to CXC-chemokine receptors (8
). This DARC binding site is on the surface opposite the residues of the homologous DBL domains of Plasmodium falciparum
EBA-175 identified as important for interactions with its receptor, glycophorin A. Even though these homologous domains are structurally conserved, this finding highlights a significant difference in how these ligands bind their cognate receptors; for EBA-175 the mechanism is “handshake” dimerization of the tandem F1/F2 DBL to form central channels that bind glycophorin A (23
), compared to a proposed monomeric interaction for DBP (20
The most significant neutralizing epitopes identified in our study occur in two areas of the DBL domain that are potentially important for receptor recognition (H1 and H3) and binding the DARC sulfated tyrosine (H2). Epitopes H1 and H3 are in regions containing variant residues with radical substitutions, while the position of H2 varies minimally. Epitopes that are considered moderately inhibitory (M1, M2, and M3) occupy a region parallel to the H2 epitope flanking the other side of the putative tyrosine recognition motif. The location of multiple putative epitopes (H2 and M1 to M3) that are targets of protective immunity does not support the “just-in-time” release model of immune evasion (7
), since identification of multiple targets for neutralizing antibodies in the area flanking the DARC sulfated tyrosine-binding site shows that this site is accessible to inhibitory antibodies. The relative lack of variability in this area may be a consequence of other functional requirements necessary for receptor recognition and a somewhat weaker effect of immune selection on these epitopes.
Antibody reactivity to the H1, H2, and H3 epitopes, which contain clusters of polymorphic residues, was significantly correlated with inhibition of DBPII-erythrocyte binding. For this reason, these epitopes do not appear to be simply associated with a nonprotective immune evasion mechanism misdirecting antibodies away from crucial functional sites elsewhere in the ligand domain. Instead, the pattern of polymorphisms of DBPII is similar to the pattern of variability observed for the P. falciparum
vaccine candidate AMA1, where polymorphisms are concentrated adjacent to the putative receptor-binding site (4
). It appears likely that the receptor-binding site of DBPII is larger than that identified so far for the DARC sulfated tyrosine.
Our results identified critical DBP epitopes that are the targets of inhibitory antibodies naturally acquired in P. vivax infection and represent an important advance in understanding part of blood-stage immunity to P. vivax. These linear epitopes, which are recognized as targets of a functionally inhibitory antibody, may represent potential correlates of immunity for a DBP vaccine, although further characterization is necessary to confirm their relative importance compared to the importance of other epitopes (e.g., conformational epitopes). The identification of specific epitope targets of inhibitory immunity against DBP opens the way for optimization of DBP immunogenicity for protection against diverse P. vivax strains.