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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.

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 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.

MAEBL is a type 1 membrane protein that is implicated in the merozoite invasion of erythrocytes and sporozoite invasion of mosquito salivary glands. This apical organelle protein is structurally similar to the ebl erythrocyte binding proteins, such as EBA-175, except that the tandem ligand domains of MAEBL are similar to part of the extracellular domain of apical membrane antigen 1 and not the Duffy binding-like domain. Although midgut and salivary gland sporozoites are morphologically similar, salivary gland sporozoites undergo a period of new gene expression after infecting the salivary glands, display distinct phenotypic differences, and are more infectious for the mammalian host. The objectives of this project were to determine the molecular form of MAEBL in the infectious salivary gland sporozoites and whether the ligand has a role in the sporozoite development to exoerythrocytic stages in hepatocytes. We determined that MAEBL is newly expressed in salivary gland sporozoites and in a form distinct from what is present in the midgut sporozoites or present in erythrocytic stages. Both ligand domains (M1 and M2) were expressed as part of a full-length membrane form of MAEBL in the salivary gland sporozoites in contrast to the other stages that retain only the M2 ligand domain as part of the membrane form of the protein. Antisera developed against the cysteine-rich regions of the extracellular portion of MAEBL inhibited sporozoite development to exoerythrocytic forms in vitro. Together these data indicate that MAEBL has a role in this third developmental stage in the life cycle of the malaria parasite. Thus, MAEBL is another target for pre-erythrocytic-stage vaccine development against malaria parasites.

Region II of the 175-kDa erythrocyte-binding antigen (EBA-175RII) of Plasmodium falciparum is functionally important in sialic acid-dependent erythrocyte invasion and is considered a prime target for an invasion-blocking vaccine. The objectives of this study were to (i) determine the prevalence of anti-EBA-175RII antibodies in a naturally exposed population, (ii) determine whether naturally acquired antibodies have a functional role by inhibiting binding of EBA-175RII to erythrocytes, and (iii) determine whether antibodies against EBA-175RII correlate with immunity to clinical malaria. We treated 301 lifelong residents of an area of malaria holoendemicity in western Kenya for malaria, monitored them during a high-transmission season, and identified 33 individuals who were asymptomatic despite parasitemia (clinically immune). We also identified 50 clinically susceptible individuals to serve as controls. These 83 individuals were treated and monitored again during the subsequent low-transmission season. Anti-EBA-175RII antibodies were present in 98.7% of the individuals studied. The antibody levels were relatively stable between the beginning and end of the high-transmission season and correlated with the plasma EBA-175RII erythrocyte-binding-inhibitory activity. There was no difference in anti-EBA-175RII levels or plasma EBA-175RII erythrocyte-binding-inhibitory activity between clinically immune and clinically susceptible groups. However, these parameters were higher in nonparasitemic than in parasitemic individuals at enrollment. These results suggest that although antibodies against EBA-175RII may be effective in suppressing some of the wild parasite strains, EBA-175RII is unlikely to be effective as a monovalent vaccine against malaria, perhaps due to allelic heterogeneity and/or presence of sialic acid-independent strains.

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.

Malaria merozoites require the presence of specific surface receptors on the red blood cell for invasion. Plasmodium vivax, requires the Duffy blood group antigen as an obligate receptor for invasion. The parasite Duffy binding protein (DBP) is the ligand involved in this process, making the DBP a potential vaccine candidate. A preliminary objective was to study whether people exposed to vivax malaria acquire antibodies that have the ability to block erythrocyte cytoadherence to the PvDBP. In comparison, we studied the immunogenicity of various recombinant DBP vaccines and investigated their potential to induct antifunctional antibodies. In order to do so, recombinant proteins to different regions of the putative ectodomain of the DBP and a DNA vaccine were used to immunize laboratory animals. An in vitro cytoadherence assay was used to investigate the presence of antifunctional antibodies in plasmas from people naturally exposed to vivax malaria, as well as in antisera obtained by animal vaccination. Our results showed that human plasma from populations naturally exposed to vivax malaria, as well as antisera obtained by vaccination using recombinant proteins, a DNA vaccine, and a synthetic peptide to DBP, inhibited in vitro binding of human erythrocytes to the DBP ligand domain (DBP(II)) in correlation to their previously measured antibody titer. Our results provide further evidence for the vaccine potential of this essential parasite adhesion molecule.

Plasmodium vivax Duffy binding protein (DBP) is a conserved functionally important protein. P. vivax DBP is an asexual blood-stage malaria vaccine candidate because adhesion of P. vivax DBP to its erythrocyte receptor is essential for the parasite to continue development in human blood. We developed a soluble recombinant protein of P. vivax DBP (rDBP) and examined serologic activity to it in residents of a region of high endemicity. This soluble rDBP product contained the cysteine-rich ligand domain and most of the contiguous proline-rich hydrophilic region. rDBP was expressed as a glutathione S-transferase (GST) fusion protein and was isolated from GST by thrombin treatment of the purified fusion protein bound on glutathione agarose beads. P. vivax rDBP was immunogenic in rabbits and induced antibodies that reacted with P. vivax and Plasmodium knowlesi merozoites. Human sera from adult residents of a region of Papua New Guinea where malaria is highly endemic or P. vivax-infected North American residents reacted with rDBP in an immunoblot and an enzyme-linked immunosorbent assay. The reactivity to reduced, denatured P. vivax rDBP and the cross-reactivity with P. knowlesi indicated the presence of immunogenic conserved linear B-cell epitopes. A more extensive serologic survey of Papua New Guinea residents showed that antibody response to P. vivax DBP is common and increases with age, suggesting a possible boosting of the antibody response in some by repeated exposure to P. vivax. A positive humoral response to P. vivax DBP correlated with a significantly higher response to P. vivax MSP-1(19). The natural immunogenicity of this DBP should strengthen its usefulness as a vaccine.

The blood-stage development of malaria parasites is initiated by the invasion of merozoites into susceptible erythrocytes. Specific receptor-ligand interactions must occur for the merozoites to first attach to and then invade erythrocytes. Because the invasion process is essential for the parasite's survival and the merozoite adhesion molecules are exposed on the merozoite surface during invasion, these adhesion molecules are candidates for antibody-dependent malaria vaccines. The Duffy binding protein of Plasmodium vivax belongs to a family of erythrocyte-binding proteins that contain functionally conserved cysteine-rich regions. The amino cysteine-rich regions of these homologous erythrocyte-binding proteins were recently identified for P. vivax, Plasmodium knowlesi, and Plasmodium falciparum as the principal erythrocyte-binding domains (C. Chitnis and L. H. Miller, J. Exp. Med. 180:497-506, 1994, and B. K. L. Sim, C. E. Chitnis, K. Wasniowska, T. J. Hadley, and L. H. Miller, Science 264:1941-1944, 1994). We report that amino acids in this critical ligand domain of the P. vivax Duffy binding protein are hypervariable, but this variability is limited. Hypervariability of the erythrocyte-binding domain suggests that this domain is the target of an effective immune response, but conservation of amino acid substitutions indicates that functional constraints limit this variation. In addition, the amino cysteine-rich region and part of the hydrophilic region immediately following it were the site of repeated homologous recombinations as represented by tandem repeat sequence polymorphisms. Similar polymorphisms have been identified in the same region of the homologous genes of P. falciparum and P. knowlesi, suggesting that there is a common mechanism of recombination or gene conversion that occurs in these Plasmodium genes.

We used immunoelectron microscopy to study the fate of dense granules during the invasion of erythrocytes by Plasmodium knowlesi merozoites. When merozoites entered host cells, dense granules moved to the pellicle, released their contents into the parasitophorous vacuole space, and then moved into fingerlike channels of the vacuole membrane. This is the first report showing that the content of dense granules of P. knowlesi is different from the contents of rhoptries and micronemes and is associated with the formation of channels from the parasitophorous vacuole.