Identification of an Immunogenic Broadly Inhibitory Surface Epitope of the Plasmodium vivax Duffy Binding Protein Ligand Domain.

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.

Immunogenicity of a synthetic vaccine based on Plasmodium vivax Duffy binding protein region II.

Molecules that play a role in Plasmodium merozoite invasion of host red blood cells represent attractive targets for blood-stage vaccine development against malaria. In Plasmodium vivax, merozoite invasion of reticulocytes is mediated by the Duffy binding protein (DBP), which interacts with its cognate receptor, the Duffy antigen receptor for chemokines, on the surface of reticulocytes. The DBP ligand domain, known as region II (DBPII), contains the critical residues for receptor recognition, making it a prime target for vaccine development against blood-stage vivax malaria. In natural infections, DBP is weakly immunogenic and DBPII allelic variation is associated with strain-specific immunity, which may compromise vaccine efficacy. In a previous study, a synthetic vaccine termed DEKnull that lacked an immunodominant variant epitope in DBPII induced functional antibodies to shared neutralizing epitopes on the native Sal1 allele. Anti-DEKnull antibody titers were lower than anti-Sal1 titers but produced more consistent, strain-transcending anti-DBPII inhibitory responses. In this study, we further characterized the immunogenicity of DEKnull, finding that immunization with recombinant DEKnull produced an immune response comparable to that obtained with native recombinant DBP alleles. Further investigation of DEKnull is necessary to enhance its immunogenicity and broaden its specificity.

Immunogenicity of single versus mixed allele vaccines of Plasmodium vivax Duffy binding protein region II.

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.

Fine specificity of Plasmodium vivax Duffy binding protein binding engagement of the Duffy antigen on human erythrocytes.

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax.

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies.

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils.

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying beta-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.