Mapping epitopes of the Plasmodium vivax Duffy binding protein with naturally acquired inhibitory antibodies

Abstract

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.

FIG. 1.

Inhibition of DBPII binding to Duffy-positive erythrocytes by human sera from Papua New Guinea. Individual sera were tested at a dilution of 1:10 for inhibition of binding in an in vitro assay (COS7 cell assay). Sera were classified based on their responses to recombinant DBP as determined by ELISA, as follows: high responders (black bars), low responders (dark gray bars), and nonresponders (light gray bars). Sera showed marked differences in inhibitory efficacy, and the differences in inhibition did not correlate with antibody titers. Binding inhibitory effects were classified as follows: major effect, ≥80% inhibition; moderate effect, 40 to 80% inhibition; and minor or no effect, 0 to 40% inhibition. The sample numbers used are not the same as those in Table 1.

FIG. 2.

Reactivity of human sera to overlapping DBPII peptide array. The average OD values for the peptides with highly inhibitory sera (n = 4) and noninhibitory sera (n = 22) are indicated by red and green lines, respectively, while the gray line indicates the average OD values for the nonexposed North American sera (n = 6) that served as a background control. An epitope is considered a B-cell epitope when the difference in OD value between the highly inhibitory sera (HI) and the noninhibitory sera (NI) is greater than the mean plus 2 standard deviations of the values for the North American control sera.

FIG. 3.

3D model of DBPII. The molecule is depicted as a space-filling model showing localization of B-cell epitopes. The structure is rotated in the panels by 90° around a horizontal axis. (A) Front (0°). (B) Top (90°). (C) Back (180°). (D) 270°. High-inhibition (H1 to H3), medium-inhibition (M1 to M3), and low-inhibition (L1 to L4) B-cell epitopes are indicated by red, yellow, and green, respectively, against a gray background, while residues of the predicted DARC binding site are indicated by dark blue.

FIG. 4.

Inhibition of DBPII binding to human erythrocytes. Human antibodies specific to the H1, H2, H3, L3, L4, and NI peptides were tested to determine their abilities to inhibit DBP binding to Duffy-positive erythrocytes. Different concentrations of the antibodies were preincubated with transfected COS7 cells prior to addition of erythrocytes, and the numbers of rosettes in 30 microscopic fields at a magnification of ×200 were determined. The symbols indicate the mean percentages of binding for three independent experiments compared to the results of a control experiment with no antibody, and the error bars indicate the standard deviations. For an antibody concentration of 10 μg/ml the P value was <0.0001 for comparisons of H and L peptides, H and NI peptides, and L and NI peptides. P values were adjusted for multiple comparisons.