Mapping Epitopes of the Plasmodium vivax Duffy Binding Protein with Naturally Acquired Inhibitory Antibodies

Plasmodium vivax Duffy binding protein (DBP) is a merozoite microneme ligand vital for blood-stage infection, which makes it an important candidate vaccine for antibody-mediated immunity against vivax malaria. A differential screen with a linear peptide array compared the reactivities of noninhibitory and inhibitory high-titer human immune sera to identify target epitopes associated with protective immunity. Naturally acquired anti-DBP-specific serologic responses observed in the residents of a region of Papua New Guinea where P. vivax is highly endemic exhibited significant changes in DBP-specific titers over time. The anti-DBP functional inhibition for each serum ranged from complete inhibition to no inhibition even for high-titer responders to the DBP, indicating that epitope specificity is important. Inhibitory immune human antibodies identified specific B-cell linear epitopes on the DBP (SalI) ligand domain that showed significant correlations with inhibitory responses. Affinity-purified naturally acquired antibodies on these epitopes inhibited the DBP erythrocyte binding function greatly, confirming the protective value of specific epitopes. These results represent an important advance in our understanding of part of blood-stage immunity to P. vivax and some of the specific targets for vaccine-elicited antibody protection.Plasmodium vivax is the major cause of malaria in most regions where this disease is endemic outside Africa, and it causes substantial morbidity worldwide (17). Plasmodium microneme proteins, such as Duffy binding protein (DBP), have important roles in the merozoite invasion of reticulocytes during asexual blood-stage infection (1, 5). DBP is a member of the Duffy binding-like erythrocyte binding protein (DBL-EBP) family expressed in the micronemes and on the surface of P. vivax merozoites and is associated with the decisive junction formation step during the invasion process (1). It is this critical interaction of DBP with its cognate receptor that makes DBP an important antimalaria vaccine candidate. The critical erythrocyte binding motif of DBP is in a 330-amino-acid cysteine-rich domain referred to as DBP region II (DBPII) or the DBL domain, which is the minimal domain responsible for binding to Duffy-positive human erythrocytes (2, 6). The central portion of the DBP domain is hypervariable compared to other DBP regions, and polymorphisms occur frequently at certain residues in a pattern consistent with selection pressure on DBP, suggesting that allelic variation functions as a mechanism for immune evasion (9, 15, 24).Naturally acquired antibodies to DBP are prevalent in residents of areas where malaria is highly endemic, but individuals show significant quantitative and qualitative differences in their anti-DBP serological responses (10, 12, 27, 28). Generally, serological responses to DBP and the inhibition of DBP-erythrocyte binding activity increase with a person''s age, suggesting that there is a boosting effect due to repeated exposure through recurrent infection (13, 16, 18). The initial antibody response to a single P. vivax infection is a response to conformational epitopes and is not broadly protective, while an immunity that transcends strain specificity develops only after repeated exposure (10, 28). Repeated exposure of residents of the areas of Papua New Guinea (PNG) where P. vivax is endemic was observed to correlate with development of antibodies that are reactive to linear epitopes in the critical binding region of DBP. In this study, we compared the reactivity of inhibitory human immune sera to the reactivity of noninhibitory immune sera to identify linear epitopes in DBPII that may serve as a target for vaccine-induced protective humoral immunity.