A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole.

Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late pre-divisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. An hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. An hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize that HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.

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.

Strain-specific duffy binding protein antibodies correlate with protection against infection with homologous compared to heterologous plasmodium vivax strains in Papua New Guinean children.

Individuals repeatedly infected with malaria acquire protection from infection and disease; immunity is thought to be primarily antibody-mediated and directed to blood-stage infection. Merozoite surface proteins involved in the invasion of host erythrocytes are likely targets of protective antibodies. We hypothesized that Papua New Guinean children (n = 206) who acquire high antibody levels to two Plasmodium vivax merozoite proteins, Duffy binding protein region II (PvDBPII) and the 19-kDa C-terminal region of P. vivax merozoite surface protein 1 (PvMSP1(19)), would have a delay in the time to reinfection following treatment to clear all blood-stage malaria infections. Ninety-four percent of the children were reinfected with P. vivax during biweekly follow-ups for 6 months. Since PvDBPII is polymorphic, we examined whether individuals acquired strain-specific immunity to PvDBPII. Children with high antibody levels to a prevalent PvDBPII allele (O) were associated with a delay in the time to reinfection with the same variant of P. vivax by 25% compared to parasites expressing other PvDBPII alleles (age-adjusted hazard ratio, 0.75 [95% confidence interval, 0.56 to 1.00 by Cox regression]) and 39% lower incidence density parasitemia (P = 0.01). Two other prevalent alleles (AH and P) showed a similar trend of 16% and 18% protection, respectively, against parasites with the same PvDBPII allele and reduced incidence density parasitemia. Antibodies directed to PvDBPII PNG-P and -O were both associated with a 21 to 26% reduction in the risk of P. vivax infections with higher levels of parasitemia (>150 parasites/mul), respectively. There was no association with high antibody levels to PvMSP1(19) and a delay in the time to P. vivax reinfection. Thus, anti-PvDBPII antibodies are associated with strain-specific immunity to P. vivax and support the use of PvDBPII for a vaccine against P. vivax.

Naturally acquired Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage Plasmodium vivax infection.

Individuals residing in malaria-endemic regions acquire protective immunity after repeated infection with malaria parasites; however, mechanisms of protective immunity and their immune correlates are poorly understood. Blood-stage infection with Plasmodium vivax depends completely on interaction of P. vivax Duffy-binding protein (PvDBP) with the Duffy antigen on host erythrocytes. Here, we performed a prospective cohort treatment/reinfection study of children (5-14 years) residing in a P. vivax-endemic region of Papua New Guinea (PNG) in which children were cleared of blood-stage infection and then examined biweekly for reinfection for 25 weeks. To test the hypothesis that naturally acquired binding inhibitory antibodies (BIAbs) targeting PvDBP region II (PvDBPII) provide protection against P. vivax infection, we used a quantitative receptor-binding assay to distinguish between antibodies that merely recognize PvDBP and those that inhibit binding to Duffy. The presence of high-level BIAbs (>90% inhibition of PvDBPII-Duffy binding, n = 18) before treatment was associated with delayed time to P. vivax reinfection diagnosed by light microscopy (P = 0.02), 55% reduced risk of P. vivax reinfection (Hazard's ratio = 0.45, P = 0.04), and 48% reduction in geometric mean P. vivax parasitemia (P < 0.001) when compared with children with low-level BIAbs (n = 148). Further, we found that stable, high-level BIAbs displayed strain-transcending inhibition by reducing reinfection with similar efficiency of PNG P. vivax strains characterized by six diverse PvDBPII haplotypes. These observations demonstrate a functional correlate of protective immunity in vivo and provide support for developing a vaccine against P. vivax malaria based on PvDBPII.

Dynamics of asymptomatic Plasmodium vivax infections and Duffy binding protein polymorphisms in relation to parasitemia levels in Papua New Guinean children.

The interaction between Plasmodium vivax Duffy binding protein II (PvDBPII) and human erythrocyte Duffy antigen is necessary for blood stage infections. However, PvDBPII is highly polymorphic. We recently observed that certain recombinant DBPII variants bind better to erythrocytes in vitro. To examine the hypothesis that haplotypes with enhanced binding have increased parasitemia levels, we followed 206 Papua New Guinean children biweekly for six months with a total of 713 P. vivax samples genotyped. Twenty-seven PvDBPII haplotypes were identified, and 3 haplotypes accounted for 57% of the infections. The relative frequencies of dominant haplotypes remained stable throughout the study. There was no significant association with PvDBPII alleles or haplotypes with P. vivax parasitemia. The dominant haplotype (26% of samples), however, corresponded to a high-binding haplotype. Thus, common haplotypes are not likely to have arisen from increased fitness as measured by greater parasitemia levels. The restricted number of common haplotypes increases the feasibility of a PvDBPII-based vaccine.

The risk of malarial infections and disease in Papua New Guinean children.

In a treatment re-infection study of 206 Papua New Guinean school children, we examined risk of reinfection and symptomatic malaria caused by different Plasmodium species. Although children acquired a similar number of polymerase chain reaction-detectable Plasmodium falciparum and P. vivax infections in six months of active follow-up (P. falciparum = 5.00, P. vivax = 5.28), they were 21 times more likely to develop symptomatic P. falciparum malaria (1.17/year) than P. vivax malaria (0.06/year). Children greater than nine years of age had a reduced risk of acquiring P. vivax infections of low-to-moderate (>150/microL) density (adjusted hazard rate [AHR] = 0.65 and 0.42), whereas similar reductions in risk with age of P. falciparum infection was only seen for parasitemias > 5,000/microL (AHR = 0.49) and symptomatic episodes (AHR = 0.51). Infection and symptomatic episodes with P. malariae and P. ovale were rare. By nine years of age, children have thus acquired almost complete clinical immunity to P. vivax characterized by a very tight control of parasite density, whereas the acquisition of immunity to symptomatic P. falciparum malaria remained incomplete. These observations suggest that different mechanisms of immunity may be important for protection from these malaria species.

High-throughput identification of the predominant malaria parasite clone in complex blood stage infections using a multi-SNP molecular haplotyping assay.

Individuals living in malaria endemic areas are often infected with multiple parasite clones. Currently used single nucleotide polymorphism (SNP) genotyping methods for malaria parasites are cumbersome; furthermore, few methods currently exist that can rapidly determine the most abundant clone in these complex infections. Here we describe an oligonucleotide ligation assay (OLA) to distinguish SNPs in the Plasmodium vivax Duffy binding protein gene (Pvdbp) at 14 polymorphic residues simultaneously. Allele abundance is determined by the highest mean fluorescent intensity of each allele. Using mixtures of plasmids encoding known haplotypes of the Pvdbp, single clones of P. vivax parasites from infected Aotus monkeys, and well-defined mixed infections from field samples, we were able to identify the predominant Pvdbp genotype with > 93% accuracy when the dominant clone is twice as abundant as a lesser genotype and > 97% of the time if the ratio was 5:1 or greater. Thus, the OLA can accurately, reproducibly, and rapidly determine the predominant parasite haplotype in complex blood stage infections.

High complexity of Plasmodium vivax infections in Papua New Guinean children.

Although genetically distinct malaria parasites have been shown to simultaneously infect an individual, the total number of unique parasites has not been systematically studied. We examined multiple clones (8-38) from individual blood samples collected from Papua New Guinean children for polymorphisms in the Plasmodium vivax Duffy binding protein (dbpII) and the merozoite surface protein 3alpha (msp3alpha). We found a median of 4 (range = 2-6) and 12 (range = 2-23) unique genotypes based on dbpII and msp3alpha, respectively, per person at one time point and at least 12-33 unique genotypes per person over a four-month period. Control polymerase chain reactions (PCRs) detected 0-31% of clones with haplotypes that arose from PCR artifacts, indicating that caution must be taken when using PCR-based analysis to examine complex infections. To reduce artifacts from clones, analysis was based on haplotypes unlikely to have been generated by PCR artifacts or had been previously identified. Plasmodium vivax infections can be highly complex in disease-endemic areas, suggesting continual genetic mixing that could have significant implications for the use of antimalarial drugs and malaria vaccines.

Antigenic drift in the ligand domain of Plasmodium vivax duffy binding protein confers resistance to inhibitory antibodies.

Interaction of the Duffy binding protein (DBP) with its erythrocyte receptor is critical for maintaining Plasmodium vivax blood-stage infections, making DBP an appealing vaccine candidate. The cysteine-rich region II is the ligand domain of DBP and a target of vaccine development. Interestingly, most of the allelic diversity observed in DBP is due to the high rate of nonsynonymous polymorphisms in this critical domain for receptor recognition. Similar to the hypervariability in influenza hemagglutinin, this pattern of polymorphisms in the DBP ligand domain suggests that this variation is a mechanism to evade antibody neutralization. To evaluate the role that dbp allelic diversity plays in strain-specific immunity, we examined the ability of an anti-Sal1 DBP serum to inhibit the erythrocyte-binding function of variant dbp alleles expressed on COS cells. We observed that the PNG-7.18 allele was significantly less sensitive to immune inhibition of its erythrocyte-binding activity than were the Sal1 and PNG-27.16 alleles. This result suggested that the unique polymorphisms of resistant PNG-7.18 were part of a protective epitope on the DBP ligand. To confirm this, Sal1 was converted to the refractory phenotype by introduction of 3 polymorphisms unique to PNG-7.18, via site-directed mutagenesis. The results of the present study indicate that linked polymorphisms have an additive, synergistic effect on DBP antigenic character.

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.

Age-acquired immunity to a Plasmodium vivax invasion ligand, the duffy binding protein.

The interaction between the Plasmodium vivax merozoite Duffy binding protein region II (DBPII) and the human erythrocyte Duffy antigen leads to infection. Highly polymorphic regions of this protein may have arisen as a mechanism to avoid host immunity. To examine whether immunity to P. vivax is directed against these polymorphic regions of DBPII, age-associated changes in the frequency of specific DBPII alleles among 358 P. vivax-positive Papua New Guineans were examined. Although the overall number and diversity of DBPII haplotypes simultaneously infecting an individual decreased with increasing age, only certain alleles at particular loci declined in frequency, indicating preferential immune selection against these alleles. One such polymorphic locus formed part of a B cell epitope, and antibodies from exposed individuals differentially recognized alleles at this locus. Therefore, acquisition of strain-specific age-acquired immunity is partially directed against polymorphic motifs within P. vivax DBPII, suggesting that these polymorphisms are maintained and likely arose under immune pressure in the host.