Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein

Abstract

Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP(II) increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semi-immune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP(II) but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.

FIG. 1.

The molecular structure of the DBP shows that regions II and VI (dark shaded areas) are cysteine-rich regions of the molecule. There are 12 cysteines in region II. The region between cysteine 4 and cysteine 7 (aa 291 to 460) contains the critical binding motif to DARC (7, 8, 21).

FIG. 2.

(A) The relationship of age and P. vivax prevalence as determined by PCR for P. vivax dbp or by blood smear for the whole study population (n = 1,025). (B) Antibody responses to rDBP_II were measured in 551 of the subjects from the same samples used to measure parasite infection, and the levels are plotted with respect to age. Dark regions represent ODs of >0.51 (the highest tercile or third of antibody responses), shaded regions represent ODs of 0.178 to 0.5, and the open box shows the absence of a significant antibody responses (OD < 0.178, the mean + 3 SD of control plasma; n = 11). The number of individuals examined in each group is indicated in parentheses above each bar.

FIG. 3.

Plasma from individuals residing in areas endemic for P. vivax recognizes linear epitopes. Nineteen 15-mer peptides overlapping by 2 to 5 aa spanning the 170-aa critical binding motif of region II of DBP were used to examine antibody reactivity from individual plasma, as shown on the x axis. The upper left panel shows the mean values (dark bars) + 3 SD (hatched bars) for control plasma (n = 11) for each peptide. The remaining panels show representative antibody responses to all 19 peptides in three representative individuals. The OD of the same serum sample directed to rDBP_II-IV is shown for the three study subjects.

FIG. 4.

The cumulative number of individuals showing selective antibody reactivity to 15-mer peptides spanning the 170-aa binding motif of DBPII. Some serum samples recognized two or more epitopes. An individual was considered to preferentially recognize one peptide over others when the OD was greater than the mean + 3 SD of eight peptides with the lowest OD. This differed from criteria used to define a positive response to any peptide shown in Fig. 3.

FIG. 5.

Fine B-cell epitope mapping of the C-terminal region of the DBPII erythrocyte adhesion motif, using plasma from four subjects containing antibodies that recognized peptides 75 and/or 79. 15-mer peptides (offset by 1 or 2 aa) were bound to polyvinyl plates, which are described in detail in Materials and Methods.

FIG. 6.

Relationship between mean peptide-specific antibody levels with that to rDBP_II in residents of a village endemic for P. vivax in PNG. Peptide-specific antibody levels were estimated by taking the mean OD to all 19 peptides. Antibody levels to rDBP_II were determined as described in Materials and Methods at 1:400. Each point represents plasma from a single individual (n = 69; closed circles) and controls (n = 11; open circles). The dashed line indicates a positive peptide-specific antibody response (e.g., >0.242, which is the mean + 3 SD for all peptides in controls, as shown in Fig. 3). Plasma from some individuals had antibodies that recognized rDBP but little reactivity to peptides (spots at or below dashed line).

FIG. 7.

Affinity-purified antibody to peptide 5 binds correctly folded rDBP_II. Individual plasma samples with elevated levels of antibody directed to peptide 5 (S variant, n = 4) were affinity purified as described in Materials and Methods. Eluted antibody did not bind other peptides (55 and 78 are shown as examples) or P. vivax MSP1₁₉. Bars represent the mean ± SEM of triplicate wells.

FIG. 8.

Peptides containing the 308 and 333 residues form linear B-cell epitopes in which substitution by a serine (S) for an arginine (R; 308) or a leucine (L) for a phenylalanine (F; 333) is differentially recognized by antibodies in the same subject (samples connected by lines). Values represent mean ODs of duplicate measurements of serum samples at a 1:10 dilution unless otherwise noted. SD were all <8% of the mean. All OD differences were highly significant based on criterion described in the text. The dashed line represents cutoff values for a positive response, e.g., mean + 3 SD of the unexposed control (see Fig. 3). Only two individuals are the same between the two panels.