Scientific argument base on article

Fya/Fyb antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria Christopher L. Kinga,b,1, John H. Adamsc, Jia Xianlia, Brian T. Grimberga, Amy M. McHenryc, Lior J. Greenberga, Asim Siddiquia, Rosalind E. Howesd, Monica da Silva-Nunese, Marcelo U. Ferreirae, and Peter A. Zimmermana

aCenter for Global Health and Diseases, Case Western Reserve University, Cleveland, OH 44106; bVeterans Affairs Medical Center, Cleveland, OH 44106; cCollege of Public Health, University of South Florida, Tampa, FL 33612; dDepartment of Zoology, University of Oxford, Oxford OX1 2JD, United Kingdom; and eDepartamento de Parasitologia, Instituto de Ciencias Biomédicas, Universidade de São Paulo, 05508-900, São Paulo, Brazil

Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved November 4, 2011 (received for review June 18, 2011)

Plasmodium vivax (Pv) is a major cause of human malaria and is in- creasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell’s surface re- ceptor, known as the Duffy blood-group antigen (Fy). Fy is an impor- tant minor blood-group antigen that has two immunologically distinct alleles, referred to as Fya or Fyb, resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite’s red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fya, compared with Fyb, significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fya had 41–50% lower binding compared with Fyb cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Indi- viduals with the Fya+b− phenotype demonstrated a 30–80% reduced risk of clinical vivax, but not falciparum malaria in a prospective co- hort study in the Brazilian Amazon. The Fya+b− phenotype, predom- inant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes.

Duffy binding protein | resistance

The parasite Plasmodium vivax plays a major role in the overallburden of malaria, causing severe morbidity and death (1). At least 80 million individuals worldwide suffer from vivax malaria; indeed, it is the most widely distributed malarial species outside of sub-Saharan Africa (2). Global efforts to eliminate malaria, largely based on reducing transmission, have been considerably less effective with P. vivax than with Plasmodium falciparum (3, 4), in part because of the former’s efficient transmission in diverse ecological settings and its ability to reinitiate blood-stage infection from a dormant liver hypnozoite phase (5). Thus, success at P. vivax elimination may depend more on developing vaccines to prevent infection and suppress re- emergent blood-stage parasites. P. falciparum demonstrates capacity to invade erythrocytes

through multiple receptor pathways (6). In contrast, P. vivax red cell invasion appears to be primarily dependent on the Duffy antigen (Fy) (7). Although Duffy-independent P. vivax infection and disease can occur (8), alternative invasion pathways are not understood. As detailed understanding of host and parasite ge- netic polymorphisms and immune response inhibition of re- ceptor-ligand interaction is of critical importance for vaccine development, here we have investigated the relevance of the Fya→Fyb antigen polymorphism on susceptibility to clinical P. vivax malaria. The gene that encodes the Duffy antigen has two major

polymorphisms. A Asp→Gly amino acid substitution (codon 42)

in the N-terminal region is associated with the Fyb and Fya

blood-group antigens, respectively (Fig. 1A). The second poly- morphism T→C transition at nucleotide −33 in the Duffy gene promoter ablates Duffy expression on erythrocytes (ES; eryth- rocyte silent). The blood group and expression phenotypes as- sociated with these polymorphisms have been well characterized; nomenclature and biological properties of Duffy have been summarized previously (8, 9) (Table S1). Because of the critical role played by the Duffy antigen in

P. vivax erythrocyte invasion, the corresponding parasite ligand, the Duffy binding protein (PvDBP), which is expressed at the parasite’s cellular surface upon invasion, is a major vaccine can- didate (10). The binding domain of PvDBP to Fy has been indentified in a 330-aa cysteine-rich region referred to as region II, designated PvDBPII (11, 12). Naturally acquired and artifi- cially induced antibodies to PvDBPII inhibit parasite invasion in vitro (13) and protect against clinical malaria in children (14), supporting PvDBPII as a leading vaccine candidate. The critical residues of Fy, to which PvDBPII binds, map to N-terminal re- gion amino acids 8–42 (Fig. 1A) (15, 16). Given studies on non- human primates indicating that Fyb is the ancestral allele (17, 18), we hypothesized that Fya decreased the efficiency of PvDBPII binding, thereby reducing susceptibility to P. vivax malaria. Indeed, cross sectional association studies performed in the Brazilian Amazon region suggested that individuals expressing the Fyb

compared with Fya antigen may be more susceptible to P. vivax in- fection (19). Additionally, prior studies showed that an ortholo- gous protein expressed by the simian malaria parasite, Plasmodium knowlesi, which infects human erythrocytes in a Duffy-dependent manner, preferentially bound Fyb- compared with Fya-expressing erythrocytes, both in vivo and in vitro (20).

Results Fya/Fyb Polymorphism Affects Binding of PvDBPII to Erythrocytes. To examine whether human erythrocytes expressing Fya showed differential binding of recombinant PvDBPII compared with those expressing Fyb, we screened blood samples from a group of healthy North American volunteers. Using a PCR assay, in- dividual Fy genotypes were established (Table S1). Samples were assayed for degree of recombinant PvDBPII binding to red cells using flow cytometry. We found 40–50% lower binding of

Author contributions: C.L.K., J.H.A., B.T.G., M.U.F., and P.A.Z. designed research; C.L.K., J.X., B.T.G., A.M.M., L.J.G., A.S., R.E.H., M.d.S.-N., and M.U.F. performed research; C.L.K. contrib- uted new reagents/analytic tools; C.L.K., M.U.F., and P.A.Z. analyzed data; and C.L.K., J.H.A., M.U.F., and P.A.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. 1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1109621108/-/DCSupplemental.

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PvDBPII (0.2 μg per 106 red cells) (Fig. S2) to erythrocytes from FY*A/FY*A (i.e., phenotypically Fya+b−) compared with FY*B/ FY*B (i.e., phenotypically Fya−b+) blood donors (Fig. 1B) (P < 0.0001). Erythrocytes from FY*A/FY*B donors displayed in- termediate binding (Fya+b+). Our observed differences in PvDBPII binding could not be attributed to levels of Fy ex- pression, which were similar for FY*B/FY*B, FY*A/FY*A, and FY*A/FY*B genotypes (Fig. 1C). FY*A/FY*BES cells expressed approximately half the levels of Fy compared with FY*A/FY*A cells; as expected, their binding was significantly reduced com- pared with cells from corresponding FY*A homozygotes. Duffy- negative erythrocytes (FY*BES/FY*BES) failed to express Fy and did not bind PvDBPII. We also performed erythrocyte rosetting assays, where PvDBPII was surface-expressed on COS cells. In these studies, cells from FY*A/FY*A donors bound COS cells at a 50% lower level compared with erythrocytes from FY*B/FY*B donors (Fig. 2D and Fig. S2). To investigate mechanisms responsible for the differential bind-

ing, we looked at differences in electrostatic charge, as well as ty- rosine (Tyr) sulfation between Fya and Fyb (Fig. 1A). The less- efficient parasite binding of Fya may be a result of the charge neutrality of Gly42 (replacing Asp42), because the N-terminal re- gion of Fy is negatively charged but PvDBPII is positively charged. Prior studies have shown that the degree of sulfation of Tyr41

markedly affected binding of PvDBPII to Fy (21). Although Choe et al. observed no substantial difference in sulfation of their Fya vs. Fyb constructs [60 codons Duffy amino terminus joined to human IgG1 Fc domain (20)], no data were provided to compare PvDBPII interaction with their constructs correspond- ing to Fya vs. Fyb or native Fya vs. Fyb antigens on the red cell surface. To make these comparisons, we treated erythrocytes with the enzyme arylsulfatase, which selectively and partially

removes sulfate groups from Tyr (Fig. S2) (22). Interestingly, en- zymatic treatment of FY*A/FY*A erythrocytes reduced PvDBPII binding by 42% (Fig. 2A) (P = 0.0004), but had no effect on PvDBPII binding to FY*B/FY*B erythrocytes (Fig. 2C). Enzy- matic treatment did not affect quantitative expression of Fy on erythrocytes (Fig. 2 C and D, and Fig. S2B). Arylsulfatase con- centrations that would result in complete removal of sulfate groups could not be used because it caused erythrocyte lysis. These results suggest that Fya may be more susceptible to loss of sulfate groups from tyrosines compared with Fyb.

Binding Inhibitory Antibodies Show Greater Blocking of PvDBPII to Fya- Compared with Fyb-Expressing Erythrocytes. Previous studies have shown that PvDBPII-specific antibodies inhibit P. vivax erythrocyte invasion in vitro (13) and correlate with protection against blood-stage infection in vivo (14). To determine whether antibodies that inhibit P. vivax invasion would bind differentially to Fya- vs. Fyb-expressing cells, a binding-competition assay was performed using antibodies directed against PvDBPII. We found that similar concentrations of either naturally acquired (Fig. 3 A and B) or artificially induced inhibitory antibodies (Fig. 3 C and D) effected 200–300% greater inhibition of PvDBPII binding to erythrocytes from FY*A/FY*A compared with FY*B/FY*B do- nors. For these experiments, PvDBPII-specific antibodies were affinity-purified from human and rabbit sera. Therefore, the concentration of these PvDBPII antibodies would be higher than in circulating blood. Of note, antibody preparations were affinity- purified to enrich for antibodies directed to PvDBPII, and thus antibody concentrations used are unlikely to represent circulating antibody levels in individuals. Overall, these results suggest that binding inhibitory antibodies directed against PvDBPII may

A B

DC

Fig. 1. Relationship of FY genotype on binding to PvDBP. (A) N-terminal binding domain (black residues) of PvDBP to Fy. Fy6 is a mAb and the corresponding epitope. Fya→Fyb is the only polymorphism in the N-terminal region. Red highlights indicate negatively charged amino acids; the overall pI is 3.4 for the N- terminal region. In contrast, the binding domain of PvDBPII [consisting of 330 aa (32)] has a pI of 9.6. (B) Flow cytometry assessment of recombinant PvDBPII binding levels to erythrocytes from people differing by Duffy genotype. (C) Level of Duffy expression using mAb Fy6. (D) Relative binding of PvDBPII expressing COS-cells to erythrocytes of FY*A/FY*A (n = 12) vs. FY*B/FY*B (n = 12, P < 0.0001) blood donors; combines results of three separate experiments. For example, the mean number of rosettes per 30 high-powered field was 83 ± 11 for FY*B/FY*B erythrocytes compared with 46 ± 5 for FY*A/FY*A (P = 0.007) for one experiment (Fig. S1).

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inhibit more efficiently when P. vivax is accessing the red blood cell through contact with Fya compared with Fyb.

Fya/Fyb Polymorphism Is Associated with Reduced Risk to Clinical P. vivax. We then sought to determine if our in vitro findings associated with FY genotype correlated with in vivo susceptibility to uncomplicated clinical P. vivax malaria. For this study, we analyzed data from 400 individuals (5–74 y of age) living in a P. vivax-endemic region of the Brazilian Amazon studied pre- viously (Table S2). All individuals were actively followed for clinical malaria over 14 mo, as determined by blood-smear mi- croscopy and PCR-confirmation during a time when there was a surge in malaria infection (23). Overall, 124 cases of P. vivax and 66 cases of P. falciparum malaria were diagnosed, with annual incidence rates of 0.31 and 0.17, respectively. Mixed infection was found in 31 cases. Prior analysis of the complete dataset suggested that duration

of residence in the community and distance from the Iquiri River influenced risk for malaria (23). Here we observed a slight re- duction in risk for P. vivax malaria, with longer duration of res- idence in the transmission area [risk ratio 0.94, 95% confidence interval (CI) 0.91–0.98, P = 0.01, negative binomial analysis], consistent with development of acquired immunity. Individuals who lived in the high-transmission area close to the river were at increased risk of P. vivax malaria (risk ratio 3.24, 95% CI 1.60– 6.05, P = 0.001). Similar risk ratios for development of P. fal- ciparum malaria were associated with time and location (risk ratio 2.89, 95% CI 1.72–5.67, P < 0.001). There was no signifi- cant association for FY genotype and prevalence of individuals living in high vs. low transmission areas, nor for FY genotype and duration of residence (Table S2) (Pearson’s χ2 test, P = 0.12). Individuals with FY*A/FY*BES and FY*A/FY*A genotypes

showed the lowest incidence of clinical P. vivax (Fig. 4). Negative binominal analysis adjusting for duration of local residence and transmission areas showed respectively, 80% and 29% reduced risk of clinical vivax malaria for individuals with FY*A/FY*BES

and FY*A/FY*A compared with the FY*A/FY*B genotype (Table 1). In contrast, individuals with FY*B/FY*BES and FY*B/FY*B genotypes had 220–270% greater risk of vivax malaria compared with the FY*A/FY*B genotype (Fig. 3B). Interestingly, we ob- served no significant difference in the percent of subjects with antibody responses against PvDBPII in association with FY ge- notype (see Table 1 for sample sizes, FY*A/FY*BES 16.7%, FY*A/ FY*A 11.9%, FY*A/FY*B 24.4%, FY*B/FY*BES 21.4%, FY*B/ FY*B 11.3%). Results show a consistent decreased susceptibility

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Fig. 2. Effect of arysulfatase treatment on binding of PvDBPII to eryth- rocytes from blood donors with different FY genotypes. (A) Treatment of erythrocytes from FY*A/FY*A donors selectively and partially removes sul- fonate groups from tyrosine residues (Fig. S3) and reduced binding by PvDBPII. (C) Identical treatment of erythrocytes from FY*B/FY*B donors did not reduce PvDBPII binding. (B and D) Enzyme treatment of erythrocytes does not affect Fy expression on erythrocytes. Symbols (circle to triangle) paired by lines compare red blood cells from each individual before and after treatment with 500 million units of arylsulfatase.

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Fig. 3. Fya/Fyb polymorphism affects binding inhibitory antibody blocking activity of recombinant PvDBPII to erythrocytes. (A) Inhibition of PvDBPII by different serum dilutions of pooled affinity-purified human binding in- hibitory Abs of PvDBPII binding to erythrocytes from an FY*A/FY*A com- pared with an FY*B/FY*B blood donor. (B) Binding inhibitory Abs from human serum (1:50 dilution) show consistently greater blocking of PvDBPII binding to erythrocytes from FY*A/FY*A (n = 7) compared with FY*B/FY*B (n = 8) blood donors. (C and D) Affinity-purified rabbit serum generated against PvDBPII also shows a greater capacity to inhibit binding of PvDBPII to erythrocytes from FY*A/FY*A (n = 5) compared with the FY*B/FY*B (n = 5) individuals at a serum dilution of 1:200. Each datapoint (A and C) represents mean (± SD) percent inhibition for individuals with Fya vs. Fyb. Each dot (B and D) represents means of duplicate or triplicate binding assays for one donor. Statistical comparison uses a Student t test.

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Fig. 4. Effect of FY genotype on the unadjusted annual incidence of clinical P. vivax (black bars) and P. falciparum malaria (striped bars). Incidence is expressed as mean number of clinical episodes per person-years of follow- up. Using a negative binomial analysis, the overall effect was highly signif- icant, P < 0.001. The adjusted effect of specific genotype on P. vivax risk is shown in Table 1.

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to vivax malaria associated with Fya and increased susceptibility to vivax malaria with increased expression of Fyb. There was no association between FY genotype and risk for P. falciparum in the multivariate analysis (overall risk ratio 1.08, 95% CI 0.87–2.38, P = 0.42). The analysis was repeated excluding individuals with mixed P. vivax and P. falciparum infections; FY*A/FY*BES and FY*A/FY*A had risk ratios of 0.197 (CI 0.07–0.98), P = 0.03 and 0.684 (CI 0.28–1.45), P = 0.09 compared with the FY*A/FY*B genotype. These results imply that differences in binding of PvDBPII to Fya compared with Fyb translate to decreased P. vivax erythrocyte invasion efficiency, and lower parasitemias in Fya+b− compared with Fya−b+ individuals. It should be noted that parasitemia levels were not specifically determined in this study.

Geographical Distribution of the Duffy Allelle Frequencies. The global distribution of the three major FY alleles, FY*A, FY*B, and FY*BES, are shown in Fig. 5. This map was derived from a suite of allele-frequency maps assembled from a database of Duffy blood-typing surveys from 1950 to 2010 (24) (Table S3). The FY*A allele appears to be advancing to fixation in many Asian populations. In contrast, FY*BES has followed a different pattern and has achieved fixation in Africa. The FY*B allele predominates in European populations. Admixture of FY*A and FY*B usually occurs in populations from relatively recent European

migrations into North Africa and the Americas following the much early migration of populations from Asia.

Discussion Our study demonstrates that PvDBPII binding is significantly lower to Fya than the ancestral Fyb antigen (Fig. 1B). Concor- dantly, we found that Fya was associated with protection, but Fyb

was associated with increased infection and disease (Table 1). Although P. vivax parasitemia has been shown to correlate with risk of clinical vivax malaria (25), we did not evaluate the re- lationship of the FY genotype with parasitemia, as this feature of infection was not recorded in the Brazilian longitudinal study. Despite our recent findings that P. vivax is able to infect human red cells through a Duffy-independent mechanism (8), it is well known that Duffy-negativity (FY*BES/FY*BES) is responsible for high-level resistance to P. vivax blood-stage infection (7), sug- gesting that this parasite’s invasion mechanism is heavily reliant on access to Fy. Given the burden of illness and death associated with vivax malaria (1), and proposals that P. vivax originated in Asia following lateral transfers of simian parasites from Old World monkeys (26), the emergence of a major vivax-resistance allele in African populations alone (FY*BES) is puzzling. There- fore, we hypothesize that FY*A has also been positively selected to reduce efficiency of P. vivax red cell invasion to improve hu- man fitness to P. vivax malaria. The observation that FY*A has advanced to fixation in many Asian and American pop- ulations where vivax malaria is most highly endemic (Fig. 5 and SI Methods) supports this conclusion. Although historical re- cord of vivax malaria in Africa is scanty, FY*BES predominates in most African ethnicities and holds vivax malaria at very low prevalence. Prior studies investigating Fy and other red cell surface pro-

teins as receptors for P. knowlesi/P. vivax red cell invasion have reported results that both support and vary with our present findings. Although evidence is limited, in vitro infection studies by Miller et al. suggested that P. knowlesi displayed lower effi- ciency in infecting human Fya+b− compared with Fya−b+ red cells (27). Additional studies did not compare parasite invasion be- tween Fya+b− and Fya−b+ red cells directly (20, 28, 29) but did

Table 1. Effect of Different FY genotype on risk of clinical vivax malaria

Genotype n Risk ratios (95% CI) P

FY*A/FY*BES 35 0.204 (0.09–0.87) 0.005 FY*A/FY*A 52 0.715 (0.31–1.21) 0.06 FY*A/FY*B 140 Comparator FY*B/FY*BES 76 2.17 (0.91–4.77) 0.09 FY*B/FY*B 87 2.70 (1.36–5.49) 0.002

Rate ratios are adjusted for location and duration of residence in endemic area using FY*A/FY*B as the comparator groups based on a negative bi- nomial analysis. Boldface indicates statistical significance.

Fig. 5. Global frequencies of the FY alleles. Areas predominated by a single allele (frequency ≥ 50%) are represented by a color gradient (blue, FY*A; green, FY*B; red/yellow, FY*BES). Areas of allelic heterogeneity where no single allele predominates, but two or more alleles each have frequencies ≥ 20%, are shown in gray-scale: palest for heterogeneity between the silent FY*BES allele and either FY*A or FY*B (when coinherited, these do not generate new phenotypes), and darkest being co-occurrence of all three alleles (and correspondingly the greatest genotypic and phenotypic diversity). Overall percentage surface area of each class is listed in the legend. Refer to SI Methods for a methodological summary and further detail about the map surface.

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compare binding of parasite proteins to human Fya+b− and Fya−b+

red cells. Haynes et al. showed that a 135-kDa protein from P. knowlesi culture supernatants bound to Fya−b+ erythrocytes much better than to Fya+b− cells (20). Similar studies found that a 135– 140 “native” protein corresponding to the full-length Duffy binding protein from P. vivax culture supernatants showed similar binding to Fya+b− vs. Fya−b+ red cells (30). A subsequent study using the same recombinant PvDBPII construct as the current study and a similar flow cytometry erythrocyte-binding assay showed no significant difference in PvDBPII binding to Fya+b− vs. Fya−b+ erythrocytes (31). Experiments in this latter study used PvDBPII at concen- trations 50-fold higher that those shown here that reveal preferen- tial binding to Fya−b+ vs. Fya+b− erythrocytes. High-concentration PvDBPII obscure differential binding to Fya−b+ vs. Fya+b−

erythrocytes (Fig. S1). Overall differences and similarities suggest that outcomes of in vitro studies may be sensitive to variation in parasite species/strains and their parasitemias, as well as differ- ences in antigen polymorphism and concentration. Results from our in vitro studies were consistent with our

in vivo observations, suggesting a relationship between Fya com- pared with Fyb expressed on erythrocytes with the amounts of PvDBPII erythrocyte binding in vitro and further susceptibility to clinical vivax malaria in vivo. Individuals who were Fya+b−

(particularly the FY*A/FY*BES genotype) had the lowest binding of PvDBPII to their erythrocytes in vitro and the greatest re- sistance to vivax malaria. Those who were Fya−b+ displayed the highest binding to PvDBPII and the greatest sensitivity to clin- ical vivax malaria. Interestingly, even though erythrocytes from FY*B/FY*BES donors express approximately half the amount of Duffy antigen (Table S1) compared with FY*A/FY*A and FY*A/ FY*B, they were more susceptible to P. vivax malaria than the FY*A/FY*A and FY*A/FY*B genotypes (Table 1). The reasons for the relationships between these in vitro and in vivo results are unclear, and may be related to cohort sample size. However, our in vivo findings are consistent with a significant protective effect of the FY*A allele. The mechanism by which Fya-expressing red cells show re-

duced binding to PvDBPII and reduced susceptibility to vivax malaria needs to be fully elucidated. As the N-terminal region of Fy is negatively charged and PvDBPII is positive, less efficient parasite binding to Fya may be because of the electrostatic neutrality of Gly42 (pI = 6) vs. negatively charged Asp42 (pI = 3.1). Additionally, because sulfation of Fy appears to influence PvDBPII binding, observed increased lability of PvDBPII binding to Fya+b− vs. Fya−b+ following arylsulfatase treatment of donor cells suggests that P. vivax red cell invasion efficiency may be susceptible to differences in Fy sulfation. In conclusion, our observations related to P. vivax interaction

with Fya vs. Fyb and subsequent development of naturally ac- quired immunity has important implications for vaccine trials using PvDBPII. In vitro studies demonstrate that both naturally

acquired and artificially induced antibodies block erythrocyte binding of recombinant PvDBPII to Fya- better than Fyb-expressing erythrocytes. Although we observed that the FY genotype is not associated with any significant differences in PvDBPII-specific an- tibody responses, our results suggest that naturally acquired im- munity to P. vivax infection and disease may be more effective in populations where the FY*A allele predominates. Additionally, our findings indicate that it will be important to test PvDBPII-based vaccine in populations that carry combinations of both FY*A and FY*B alleles (Fig. 5).

Methods Detailed information is provided in SI Methods.

Participants. Erythrocytes for the binding experiments were obtained from malaria uninfected volunteers at Case Western Reserve University. Malaria- exposed subjects were recruited as part of longitudinal cohort study per- formed in the Brazilian Amazon in 2004–2005, as previously described (23). All work with human samples was performed in accordance with approved Institutional Review Board protocols of the Veterans Affairs Medical Center, Cleveland, OH, University Hospitals, Cleveland, OH, and Ethical Review Board of the Institute of Biomedical Sciences of the University of São Paulo, Brazil.

Binding Experiments. Binding experiments with recombinant Pv Duffy Binding protein and Fy6 mAb that recognizes N-terminal region of Duffy antigen used fresh human erythrocytes from subjects previously genotyped for Duffy and were performed as previously described (13). Binding inhibition levels by anti-PvDBPII were assessed as previously described (13).

Statistical Analysis. A negative binomial regression analysis (SAS version 9.2; SAS Institute) was used because of the overdispersion of the data. Sample size was 400, input risk variables were Duffy genotype, location, and duration of residence in the study area with the primary output being risk of clinical P. vivax. There was little interaction between Duffy genotype and location and duration of residence (Table S2).

Mapping Duffy Genotypes. The probability distribution based on a Bayesian model is summarized as a single statistic: in this case, the median value, as this corresponds best to the input dataset, as previously described (24). Median values of the predictions were generated for each allele frequency at a 10 × 10-km resolution on a global grid with GIS software (ArcMap 9.3; ESRI).

ACKNOWLEDGMENTS. We thank Chetan Chitnis for providing the nDARCIg; Kevin Moore for providing the monoclonal antibody that recognizes sulfonated tyrosine; Jennifer Cole-Tobian for help with statistical analysis; Christine J. Julian for help in preparing the manuscript; and Menachem Shoham and Martin Stone for advice in experimental design and insights into the interaction between Duffy antigen and Plasmodium vivax Duffy binding protein. This research was supported in part by Veterans Affairs Research Service, and US Public Health Service Grant R01 AI064478; field work was supported by the Brazilian agencies Fundação de Amparo à Pes- quisa do Estado de São Paulo (FAPESP, Grants 2003/09719-6 and 2005/51988- 0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant 470067/2004-7); M.d.S.-N. received a PhD scholarship from FAPESP and M.U.F. is supported by a research scholarship from CNPq.

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