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Am. J. Trop. Med. Hyg., 59(4), 1998, pp. 597–599 Copyright q 1998 by The American Society of Tropical Medicine and Hygiene

SEROLOGIC RESPONSES TO RECOMBINANT PLASMODIUM VIVAX DUFFY BINDING PROTEIN IN A COLOMBIAN VILLAGE PASCAL A. MICHON, MYRIAM AREVALO-HERRERA, TRESA FRASER, SOCRATES HERRERA, AND JOHN H. ADAMS Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana; Instituto de Inmunologia, Universidad del Valle Sede San Fernando, Cali, Colombia

Abstract. The Plasmodium vivax Duffy binding protein (DBP) is essential during merozoite invasion into human erythrocytes. Because of its biological importance, the DBP is also seen to have potential use as a malaria bloodstage vaccine. We have used a soluble recombinant DBP (rDBP) containing the functional ligand domain to assess the natural immunogenicity of DBP in a low-endemic vivax malaria region. Human sera from adult residents from a Colombian village with unstable vivax malaria transmission reacted specifically with the rDBP as determined by ELISA. There was a significant positive correlation between increased antibody response (average, median, and percent positives) and age of patients, although the level of responses did vary considerably in their reactivity to the rDBP from negative to very high level within each age group. These data confirm previous findings on the serologic reactivity of the DBP in exposed populations and that immunologic boosting to the DBP occurs in malaria-endemic regions even with low-level transmission. erage rainfall of 6,895 mm with 87% relative humidity and a temperature of 268C. The village was composed of 577 inhabitants at the time of the study (from three months to 96 years old), in which 93% of the residents are blacks of African origin, 5.6% are Spanish-Amerindians and 0.7% are Indians. Malaria transmission is present throughout the year as recorded by Malaria Eradication Service data. Epidemiologic studies carried out in the village reported a malaria prevalence of 48% for P. vivax, 41% for P. falciparum, and 3.7% for P. malariae, with 7.4% mixed infections.6 No individual had malaria symptoms when examined, but 4% were positive for P. vivax (range 5 70–2,275 parasites/ ml by thick blood film) and 3% for P. falciparum (range 5 70–12,600 parasites/ml). Blood samples were collected in tubes containing heparin as an anticoagulant. Plasma were separated by centrifugation and kept at 2708C until used. Fifteen sera were collected from North American residents never exposed to malaria, pooled, and used as negative controls for an ELISA. Enzyme-linked immunosorbent assay. Analysis by ELISA was performed as described earlier.3 Negative sera from 15 North American residents were used to calculate the cutoff value. Statistical analysis. The antibody unit (AU) averages were transformed into log values for statistical comparisons of means, standard deviations (SDs), and medians. Statistical analysis was performed using Systat 5.2 for MacIntosh software package (Systat, Inc., Evanston, IL). Multivariate analysis of variance and Tukey’s high significance degree post hoc test were used for analysis of the relatedness of antibody response with age groups. Two different age groupings were used to assess age-related immunoreactivity. Positive sera were determined after a cut-off value (3.12) that corresponds to mean 1 2 SD of the AU from the 15 nonexposed North American residents used as negative controls.

All malaria parasites undergo repeated cycles of asexual development to ensure their maintenance in the blood. During the obligatory process of invasion into a new erythrocyte, the merozoite of Plasmodium requires interaction with a specific set of erythrocyte surface receptors.1 These receptor-ligand interactions constitute a target of choice for chemotherapy or vaccination-based approaches to reduce or eliminate blood-stage malaria infections.2 The P. vivax Duffy binding protein (DBP) is essential in invasion. Its localization in the merozoite and gene structure have already been reviewed.1,3 Evidence that the DBP is the target of an effective immune response was suggested by hypervariability of amino acids in the critical ligand domain of the cysteinerich DBL erythrocyte-binding domain.4,5 However, direct evidence for naturally occurring antibodies to the DBP from malaria-exposed people was only recently demonstrated.3 A recombinant protein corresponding to regions II-IV of the DBP was designed and was immunogenic in laboratory animals, generating antisera that reacted to P. knolewsi and P. vivax parasites. Additionally, human sera from a highly endemic region of Papua New Guinea reacted specifically to DBPII–IV, demonstrating an age-related increase in responsiveness. Here we present data showing that human sera from 92 people indigenous to regions endemic for low-level vivax malaria in Colombia reacted to this recombinant protein, and that a positive correlation was observed between antibody response and age. MATERIALS AND METHODS

Recombinant protein expression and purification. The recombinant protein rDBP was expressed and purified as described earlier.3 Plasma samples. Protocols for this research were cleared by the University of Notre Dame Human Subjects Institutional Review Board and by the Ethical Committee of the School of Health at the Universidad del Valle (Cali, Colombia). After informed consent was obtained, a medical examination was performed on 92 outpatients from Zacarı´as, Colombia. Zacarı´as is a village situated in a malaria-endemic area located 8 km from Buenaventura, the main seaport on the Colombian Pacific coast. This region has an annual av-


The P. vivax DBP is a well-conserved molecule that plays an essential role during the blood-stage infection. The merozoite requires the presence of a Duffy blood group antigen as a receptor to invade human reticulocytes.7 Unlike P. fal-




TABLE 1 Seroprevalence to the Plasmodium vivax Duffy binding protein in residents of Zacarı´as, Colombia, segregated by two different age groupings* Age group (years)


% Positive

0–,20 20–,40 $40 ,25 25–,50 $50

28 37 27 39 32 21

14.3 40.5 66.7 18.0 56.3 57.1

2.83 3.22 3.55 2.88 3.43 3.45




3.20 6 0.59

Mean 6 SD†

6 6 6 6 6 6

0.42 (a) 0.57 (b) 0.56 (c) 0.42 (a) 0.64 (b) 0.54 (b)

expected because of people repeatedly exposed to the malaria parasite. Use of two different age groupings (Table 1) shows that there is a response transition in those in their late 40s, leading to a plateau in the antibody response to the DBP antigen.


2.68 3.00 3.60 2.81 3.23 3.34 2.99

* n 5 total number of patients analyzed; SD 5 standard deviation. † Group mean antibody responses are given as log transformed antibody units. Values $ 3.12 were considered positive. Different letters indicate significantly different means (P , 0.05) determined by multivariate analysis of variance with Tukey’s high significance degree post hoc test.

ciparum, no evidence has been shown in P. vivax for an alternate invasion pathway.8,9 Because of its absolute requirement, the DBP is a good potential candidate as an asexual-stage malaria vaccine.2 The hydrophobic, cysteine-rich ligand domain (region II) of the DBP was expressed as a recombinant protein (rDBP) as mentioned earlier.3 The rDBP fusion protein was immunogenic in laboratory animals and induced antibodies that reacted in an immunofluorescence assay with the native DBP in blood-stage schizonts of P. vivax and P. knowlesi. The presence of naturally occurring antibodies to the DBP in humans exposed to vivax malaria was demonstrated by ELISA and Western blotting in residents of a highly endemic region with year round transmission.3 These data indicated that repeated exposure of Papua New Guinean residents to P. vivax boosted antibody response to the DBP in some individuals. This is consistent with the conserved nature of the DBP, being the product of a single-copy gene with one major allele type and limited variation compared with other antigens. In the present study, 92 plasma samples from residents of a Colombian malaria-endemic area were screened by ELISA against the rDBP antigen. The village of Zacarias, where human blood samples for P. vivax-exposed patients were collected, is situated in an area of unstable malaria transmission in which P. vivax accounts for 50% of the malaria cases, with the rest being due to P. falciparum (P. malariae is present in a very low percentage: , 0.5%). Although vivax malaria is dominant in Colombia (65%), the lower prevalence on the Pacific coast is likely to be due to the probable high frequency of the Duffy negative phenotype, Fy (a-, b-), since about 80% of the population is of African origin. Although persons with the Duffy-negative phenotype are not expected to develop P. vivax blood-stage infections, they will be infected with the liver stage and thereby have exposure to merozoite antigens. There was a wide range in patients’ responsiveness to the recombinant DBP antigen that had a positive correlation with age. We found that 40% of the total patient population sampled reacted positively to the rDBP (Table 1), which is consistent with the unstable transmission in this area. The level of antibody response seen by the average and median responses increased significantly with age as


These results demonstrated that even low level and unstable transmission leads to a significant boosting of the antibody responses in residents of this region. Similar to the residents of a highly endemic region of Papua New Guinea, the residents of Zacarı´as demonstrated a transition to highlevel antibody response only at mature adulthood, suggesting that chronic subclinical infections are responsible for boosting the antibody response. Together, these studies confirm the consistency of the antibody responses to the DBP from natural exposure. This inherent ability to boost the antibody response should be beneficial in the development of the DBP as a blood-stage vaccine against vivax malaria. Acknowledgments: We thank all the study volunteers for participation, the people from the Immunology Institute in Cali for assistance, Dr. G. Lamberti for assistance in the interpretation of statistical test results, and Amy Noe and Stefan Kappe for advice. Financial support: This investigation received financial support from the UNDP/WORLD BANK/WHO Special Program for Research and Training in Tropical Diseases (TDR) and from NIH grant AI33656. Authors’ addresses: Pascal A. Michon, Tresa Fraser, and John H. Adams, Department of Biological Sciences, University of Notre Dame, PO Box 369, Notre Dame, IN 46556. Myriam Arevalo-Herrera and Socrates Herrera, Instituto de Inmunologı´a, Universidad del Valle Sede San Fernando, Calle 4B No. 36-00 Apartado Aereo 25360 Cali, Colombia. Reprint requests: John H. Adams, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556. REFERENCES

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