In P. vivax
, DBP is associated with the decisive and irreversible step of junction formation during merozoite invasion and, unlike P. falciparum
, there are not any obvious alternate ligand pathways (1
). The virtual absence of P. vivax
in populations lacking the Duffy blood group presents overwhelming evidence for the vital nature of this interaction and marks DBP as an ideal target for vaccine development. Even though DBP's biological importance justifies its strong consideration as a vaccine candidate, the polymorphic nature of its ligand domain may pose a challenge of effectively developing DBP as a vaccine target against vivax malaria. Previous studies have shown that individuals can acquire robust naturally acquired immunity to P. vivax
infections. However, relatively few individuals are capable of developing broadly inhibitory anti-DBP immune antibody responses. Therefore, similar to blood-stage vaccine candidates of P. falciparum
, some concern is justified about whether vaccine-induced immunity will be strain specific and limit effectiveness against different alleles of the DBP. Based on this concern, one strategy is to identify conserved neutralizing epitopes that are suitable targets and optimize vaccine development toward those conserved epitopes while avoiding presenting immunogenic variant epitopes in a vaccine. The main objective of this study was to evaluate feasibility for vaccine-elicited immunization to potentially target conserved epitopes of DBP with highly inhibitory antibody desirable for protection against diverse P. vivax
strains. Toward that goal, we successfully developed a set of monoclonal antibodies raised against the DBPII that are high titer and broadly inhibitory.
Expressed as a type I membrane protein, DBP is sequestered in the micronemes along with other ligands until required by merozoites for host cell invasion. This “just-in-time” release on the merozoite surface presumably limits exposure to inhibitory antibody targeting the N-terminal 330 amino acid cysteine-rich ligand domain (1
). Structural analysis identified six disulfide bonds, which define three subdomains of region II, as critical for maintenance of the native conformation needed for erythrocyte receptor recognition in DBL domains (28
). Comparative analysis of the deduced three-dimensional structure with site-directed mutation analysis reveals that many residues important for receptor recognition are surface exposed and lie adjacent to polymorphic residues responsible for DBP allelic variation (4
). Together, these data are suggestive of a selection mechanism driven by antibody responses to nonessential variant epitopes adjacent to surface-exposed functionally sensitive areas of the ligand domain required for receptor recognition. Fortunately, this immune escape process does appear to have limits since some individuals exposed to P. vivax
in countries where malaria is endemic are capable of producing broadly reactive invasion inhibitory antibodies. Focusing antibody responses on the epitopes recognized by these elite responders requires developing the reagents to validate and characterize the conserved neutralizing epitopes.
Our study developed a panel of murine monoclonal antibodies that reacted to conserved and variant epitopes. To determine the binding specificity of the various monoclonal antibodies to different DBPII alleles, we performed ELISA studies with five refolded recombinant variant DBPII alleles previously identified as genetically or antigenically distinct. The results demonstrated differential reactivity profiles among the antibodies for different DBP variants, confirming the existence of conserved epitopes as well as antigenic variability in the different DBP alleles, which has previously been suggested with naturally acquired human anti-DBP antibodies (10
). Reactivity of all monoclonal antibodies was reduction sensitive, indicating conformation of both types of epitopes were disulfide bond dependent. Only the weakly inhibitory monoclonal antibody 3D10 was able to recognize both denatured and refolded antigens, although there was >5-fold reduction in reactivity of the reduced antigens compared to the refolded antigens.
The COS7 in vitro
binding assay was used to determine the efficacy of the anti-DBPII monoclonal antibodies to inhibit DBPII-erythrocyte interaction. As observed in the ELISA, some of the monoclonal antibodies (3C9, 2D10, 2C6, 2A6) showed significant differential inhibitory responses to the different alleles of the COS7-expressed DBPII, indicating that these antibodies might be binding to different epitopes within the different alleles. Monoclonal 3C9 showed the highest level of inhibition (IC50
= 0.19 and 0.16 μg/ml), followed by 2D10 (0.35 and 0.18 μg/ml), 2H2 (0.53 and 1.5 μg/ml), and 2C6 (2.25 and 0.75 μg/ml) for 7.18 and SalI, respectively. Interestingly, 2C6 and 2D10, which were produced by immunizing with 7.18, showed a higher anti-SalI inhibitory response compared to that of the homologous 7.18 allele (3-fold for 2C6 and ~2-fold for 2D10). This is in line with previous studies, which observed that some changes in variant residues on DBPII enhanced sensitivity to heterologous anti-SalI antibodies and other amino acid changes in the same residues increased refractoriness to antibody inhibition (31
). Similarly, some human immune antisera have been found to have an enhancing effect on DBP-erythrocyte binding (10
). Whether it is by binding to the receptor site or by steric hindrance, the mechanism by which these monoclonal antibodies inhibit binding is yet to be determined. However, it is clear from our results that optimizing antibody specificity is critical for effective inhibition of DBP-erythrocyte binding and a high-titer antibody alone is not sufficient for protection.
The data presented here represent the first study to use monoclonal antibodies to evaluate binding specificity to different DBPII alleles and inhibition of erythrocyte binding to DBP. The development and characterization of antibody reagents, especially those capable of binding and inhibiting parasite-erythrocyte interaction and subsequent invasion, are critical and are powerful tools for identifying specific targets on the DBP ligand to inhibit invasion. Optimizing the design of DBP immunogenicity to target such conserved epitopes will be important for development of a broadly effective vaccine against P. vivax.