Plasmodium vivax invasion into human reticulocytes is a complex process. The Duffy binding protein (DBP) dimerization with its cognate receptor is vital for junction formation in the invasion process. Due to its functional importance, DBP is considered a prime vaccine candidate, but variation in B-cell epitopes at the dimer interface of DBP leads to induction of strain-limited immunity. We believe that the polymorphic residues tend to divert immune responses away from functionally conserved epitopes important for receptor binding or DBP dimerization. As a proof of concept, we engineered the vaccine DEKnull to ablate the dominant Bc epitope to partially overcome strain-specific immune antibody responses. Additional surface engineering on the next generation immunogen, DEKnull-2, provides an immunogenicity breakthrough to conserved protective epitopes. DEKnull-2 elicits a stronger broadly neutralizing response and reactivity with long-term persistent antibody responses of acquired natural immunity. By using novel engineered DBP immunogens, we validate that the prime targets of protective immunity are conformational epitopes at the dimer interface. These successful results indicate a potential approach that can be used generally to improve efficacy of other malaria vaccine candidates.

The Duffy Binding protein (DBP) of Plasmodium vivax is vital for host erythrocyte invasion. DBP region II (DBPII) contains critical residues for receptor recognition and anti-DBPII antibodies have been shown to inhibit erythrocyte binding and invasion, thereby making the molecule an attractive vaccine candidate against P. vivax blood stages. Similar to other blood-stage antigens, allelic variation within the DBPII and associated strain-specific immunity is a major challenge for development of a broadly effective vaccine against P. vivax malaria. We hypothesized that immunization with a vaccine composed of multiple DBP alleles or a modified epitope DBP (DEKnull) will be more effective in producing a broadly reactive and inhibitory antibody response to diverse DBPII alleles than a single allele vaccine. In this study, we compared single, naturally occurring DBPII allele immunizations (Sal1, 7.18, P) and DEKnull with a combination of (Sal1, 7.18, P) alleles. Quantitative analysis by ELISA demonstrated that the multiple allele vaccine tend to be more immunogenic than any of the single allele vaccines when tested for reactivity against a panel of DBPII allelic variants whereas DEKnull was less immunogenic than the mixed-allele vaccine but similar in reactivity to the single allele vaccines. Further analysis for functional efficacy by in vitro erythrocyte-binding inhibition assays demonstrated that the multiple allele immunization produced a stronger strain-neutralizing response than the other vaccination strategies even though inhibition remained biased toward some alleles. Overall, there was no correlation between antibody titer and functional inhibition. These data suggest that a multiple allele vaccine may enhance immunogenicity of a DBPII vaccine but further investigation is required to optimize this vaccine strategy to achieve broader coverage against global P. vivax strains.

The Plasmodium vivax Duffy binding protein region II (DBPII) is a vital ligand for the parasite’s invasion of reticulocytes, thereby making this molecule an attractive vaccine candidate against vivax malaria. However, strain-specific immunity due to DBPII allelic variation in Bc epitopes may complicate vaccine efficacy, suggesting that an effective DBPII vaccine needs to target conserved epitopes that are potential targets of strain-transcending neutralizing immunity. The minimal epitopes reactive with functionally inhibitory anti-DBPII monoclonal antibody (MAb) 3C9 and noninhibitory anti-DBPII MAb 3D10 were mapped using phage display expression libraries, since previous attempts to deduce the 3C9 epitope by cocrystallographic methods failed. Inhibitory MAb 3C9 binds to a conserved conformation-dependent epitope in subdomain 3, while noninhibitory MAb 3D10 binds to a linear epitope in subdomain 1 of DBPII, consistent with previous studies. Immunogenicity studies using synthetic linear peptides of the minimal epitopes determined that the 3C9 epitope, but not the 3D10 epitope, could induce functionally inhibitory anti-DBPII antibodies. Therefore, the highly conserved binding-inhibitory 3C9 epitope offers the potential as a component in a broadly inhibitory, strain-transcending DBP subunit vaccine. IMPORTANCE Vivax malaria is the second leading cause of malaria worldwide and the major cause of non-African malaria. Unfortunately, efforts to develop antimalarial vaccines specifically targeting Plasmodium vivax have been largely neglected, and few candidates have progressed into clinical trials. The Duffy binding protein is considered a leading blood-stage vaccine candidate because this ligand’s recognition of the Duffy blood group reticulocyte surface receptor is considered essential for infection. This study identifies a new target epitope on the ligand’s surface that may serve as the target of vaccine-induced binding-inhibitory antibody (BIAb). Understanding the potential targets of vaccine protection will be important for development of an effective vaccine.