Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy-positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non-malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty-three individuals participated in the five cross-sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non-cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine-rich region II, DBP_II), we performed in vitro assays with mammalian cells expressing DBP_II sequences which were homologous or not to those from the outbreak isolate. In non-immune individuals, the results of a 12-month follow-up period provided evidence that naturally acquired inhibitory antibodies to DBP_II are short-lived and biased towards a specific allele.     

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein

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.     

Single-chain antibody fragment specific for Plasmodium vivax Duffy binding protein

Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected with Plasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of the P. vivax Duffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant = 3.62 x 10(-8) M) and the greatest inhibition activity (50% inhibitory concentration approximately 2.9 microg/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.     

Diversity and natural selection in Plasmodium vivax Duffy binding protein gene

The Plasmodium vivax Duffy binding protein (DBP) binds to the Duffy blood group antigen on the surface of erythrocytes and is essential for invasion. Natural immunity develops to this protein making it an important vaccine candidate. Genetic diversity within and between populations was compared in 100 dbp sequences from isolates obtained from Papua New Guinea, Colombia, and South Korea. The cysteine-rich region II, that contains the binding domain, has the highest diversity compared to the rest of the dbp gene and appears to be under strong selective pressure based on statistical tests comparing rates of non-synonymous (K(n)) to synonymous mutations (K(s)) among P. vivax isolates and to those of closely related species. By contrast, meiotic recombination was not found to be significant for maintaining genetic diversity. A comparison of the patterns of nucleotide diversity within dbpII to that of genes encoding homologous erythrocyte binding proteins of Plasmodium knowlesi predict critical binding residues juxtaposed to polymorphic B- and T-cell epitopes. Phylogenic analysis and measurement of nucleotide diversity between and within the different geographic populations support emergence of distinct allelic families suggestive of divergent selection of alleles between populations. Development of a P. vivax DBP-based vaccine must take into account regions of high diversity within the molecule and alleles that show distinct geographic differences.     

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.     

Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein.

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.     

Mapping Epitopes of the Plasmodium vivax Duffy Binding Protein with Naturally Acquired Inhibitory Antibodies

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.Plasmodium vivax is the major cause of malaria in most regions where this disease is endemic outside Africa, and it causes substantial morbidity worldwide (17). Plasmodium microneme proteins, such as Duffy binding protein (DBP), have important roles in the merozoite invasion of reticulocytes during asexual blood-stage infection (1, 5). DBP is a member of the Duffy binding-like erythrocyte binding protein (DBL-EBP) family expressed in the micronemes and on the surface of P. vivax merozoites and is associated with the decisive junction formation step during the invasion process (1). It is this critical interaction of DBP with its cognate receptor that makes DBP an important antimalaria vaccine candidate. The critical erythrocyte binding motif of DBP is in a 330-amino-acid cysteine-rich domain referred to as DBP region II (DBPII) or the DBL domain, which is the minimal domain responsible for binding to Duffy-positive human erythrocytes (2, 6). The central portion of the DBP domain is hypervariable compared to other DBP regions, and polymorphisms occur frequently at certain residues in a pattern consistent with selection pressure on DBP, suggesting that allelic variation functions as a mechanism for immune evasion (9, 15, 24).Naturally acquired antibodies to DBP are prevalent in residents of areas where malaria is highly endemic, but individuals show significant quantitative and qualitative differences in their anti-DBP serological responses (10, 12, 27, 28). Generally, serological responses to DBP and the inhibition of DBP-erythrocyte binding activity increase with a person''s age, suggesting that there is a boosting effect due to repeated exposure through recurrent infection (13, 16, 18). The initial antibody response to a single P. vivax infection is a response to conformational epitopes and is not broadly protective, while an immunity that transcends strain specificity develops only after repeated exposure (10, 28). Repeated exposure of residents of the areas of Papua New Guinea (PNG) where P. vivax is endemic was observed to correlate with development of antibodies that are reactive to linear epitopes in the critical binding region of DBP. In this study, we compared the reactivity of inhibitory human immune sera to the reactivity of noninhibitory immune sera to identify linear epitopes in DBPII that may serve as a target for vaccine-induced protective humoral immunity.     

Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi alpha protein, which binds human and rhesus Duffy antigens, and P. knowlesi beta and gamma proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi alpha protein (Pk(alpha)RII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant Pk(alpha)RII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against Pk(alpha)RII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175.     

Cloning of the Plasmodium vivax Duffy receptor.

Plasmodium vivax and Plasmodium knowlesi merozoites invade only Duffy blood group-positive human erythrocytes. Soluble P. vivax and P. knowlesi merozoite proteins of 135 kDa bind specifically to Duffy blood group determinants. The gene encoding a member of the Duffy receptor gene family of P. knowlesi has been cloned. We report here the molecular cloning of the presumptive Duffy receptor gene of P. vivax, using the P. knowlesi gene as a probe. There is a single gene in P. vivax which codes for a protein of 1115 amino acids. The deduced amino acid sequence predicts a putative signal sequence at the amino-terminus and a transmembrane region followed by 45 amino acids at the carboxy-terminus. The three introns found at the 3' end of the P. knowlesi gene were conserved in P. vivax, including high homology for the sequences of the introns. Comparison of the portion of the proteins amino to the transmembrane region between P. vivax and the partial sequence of P. knowlesi indicated at least three domains. Two homologous regions were separated by a non-homologous region. The cysteines in the homologous regions were conserved in number and position, indicating that the folding is similar and suggesting that these regions may be the Duffy blood group binding domains. In both P. vivax and P. knowlesi, the non-homologous region is hydrophilic and proline-rich, although the position of the prolines is not conserved. As prolines tend to stiffen a protein, this region may act as a 'hinge region' similar to those in the immunoglobulin gene family.     

Naturally acquired antibody responses to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in Korea

We expressed a protein in Saccharomyces cerevisiae in order to evaluate the humoral immune responses to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax. This protein (Pv200(18)) had a molecular mass of 18 kDa and was reactive with the sera of individuals with patent vivax malaria on immunoblotting analysis. The levels of immunoglobulin M (IgM) and IgG antibodies against Pv200(18) were measured in 421 patients with vivax malaria (patient group), 528 healthy individuals from areas of nonendemicity (control group 1), and 470 healthy individuals from areas of endemicity (control group 2), using the indirect enzyme-linked immunosorbent assay (ELISA) method. To study the longevity of the antibodies, 20 subjects from the patient group were also tested for the antibody levels once a month for 1 year. When the cutoff values for seropositivity were determined as the mean + 3 x standard deviation of the antibody levels in control group 1, both IgG and IgM antibody levels were negative in 98.5% (465 of 472) of control group 2. The IgG and IgM antibodies were positive in 88.1% (371 of 421) and 94.5% (398 of 421) of the patient group, respectively. The IgM antibody became negative 2 to 4 months after the onset of symptoms, whereas the IgG antibody usually remained positive for more than 5 months. In conclusion, indirect ELISA using Pv200(18) expressed in S. cerevisiae may be a useful diagnostic method for vivax malaria.     

Inhibitory properties of the antibody response to Plasmodium vivax Duffy binding protein in an area with unstable malaria transmission

The function of the Plasmodium vivax Duffy binding protein (DBP) during the erythrocyte invasion process is critical for successful parasite growth and pathogenesis in human infections. Although DBP is the subject of intensive malaria vaccine research, investigations on the functional proprieties of anti-DBP antibodies in the human population have been limited [ Infect Immun 68 (2000) 3164]. In the present study, we examined the ability of sera from different populations of the Brazilian Amazon – an area of markedly unstable malaria transmission – to inhibit the erythrocyte-binding function of the DBP ligand domain (region II, DBP_II). We found that long-term exposure to malaria in the Amazon area elicits DBP-specific antibodies that inhibit the binding of different DBP_II variants to erythrocytes. Despite the great variability of inhibitory antibody responses observed among study participants, we observed a positive correlation between erythrocyte binding-inhibitory activity and enzyme-linked immunosorbent assay anti-DBP antibodies. Of importance, there was a non-significant tendency towards increased levels of anti-DBP antibodies among individuals with asymptomatic P. vivax infections.     

Plasmodium vivax and the Duffy antigen: A paradigm revisited

The Duffy blood group antigen is the portal of entry of the Plasmodium vivax malaria parasite into human red blood cells and the receptor for a number of CXC and CC chemokines. We review here epidemiological data and evidence derived from therapeutic or experimental human infections associating P. vivax and the Duffy glycoprotein and laboratory studies indicating that P. vivax uses the Duffy antigen as a receptor to invade the red cell. We then review recent field observations indicating that the conclusion of the absolute dependence on the presence of Duffy on the red cell for P. vivax infection and development into the red cell no longer holds true and that in some parts of the world, P. vivax infects and causes disease in Duffy-negative people.     

Antigenic polymorphism and naturally acquired antibodies to Plasmodium vivax merozoite surface protein 1 in rural Amazonians

Merozoite surface protein 1 of Plasmodium vivax (PvMSP-1), a major target for malaria vaccine development, contains six highly polymorphic domains interspersed with conserved sequences. Although there is evidence that the sequence divergence in PvMSP-1 has been maintained over 5 million years by balanced selection exerted by the host's acquired immunity, the variant specificity of naturally acquired antibodies to PvMSP-1 remains poorly investigated. Here, we show that 15 recombinant proteins corresponding to PvMSP-1 variants commonly found in local parasites were poorly recognized by 376 noninfected subjects aged 5 to 90 years exposed to malaria in rural Amazonia; less than one-third of them had detectable immunoglobulin G (IgG) antibodies to at least one variant of blocks 2, 6, and 10 that were expressed, although 54.3% recognized the invariant 19-kDa C-terminal domain PvMSP-1(19). Although the proportion of responders to PvMSP-1 variants increased substantially during subsequent acute P. vivax infections, the specificity of IgG antibodies did not necessarily match the PvMSP-1 variant(s) found in infecting parasites. We discuss the relative contribution of antigenic polymorphism, poor immunogenicity, and original antigenic sin (the skew in the specificity of antibodies elicited by exposure to new antigenic variants due to preexisting variant-specific responses) to the observed patterns of antibody recognition of PvMSP-1. We suggest that antibody responses to the repertoire of variable domains of PvMSP-1 to which subjects are continuously exposed are elicited only after several repeated infections and may require frequent boosting, with clear implications for the development of PvMSP-1-based subunit vaccines.     

The analysis of Plasmodium vivax Duffy receptor binding domain gene sequence from resurgent Korea isolates

The Duffy binding domain gene structures of Plasmodium vivax facilitate the invasion of erythrocytes. Human erythrocytes that lack Duffy blood group antigens are resistant to invasion by P. vivax. We have sequenced the Duffy binding domain gene from eight P. vivax isolates collected from malaria cases in South Korea. When compared to isolates from other regions in the world, the amino acid sequences of the Korean isolates showed unique variations in region II. From 606 sequenced amino acids, 32 variations were found. Of these, three variations were regularly found in positions 424, 437 and 503 of the Sal-1 amino acid sequence. In region III, six isolates had a loss of the 30 bp (FAESTKSAE) insert. However, six isolates had 6 bp (SD) inserts at the end of region III. Two cases had a reverse pattern. Our results suggest that the P. vivax currently found in South Korea are unique when compared to other isolates and can be divided, by the analysis of their molecular structure, into two strains.