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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of Fy antigen (DARC) on red blood cells determines the susceptibility of individuals to infection by P. vivax asexual blood forms. This blood group was first detected in a hemophilic patient in 1950 by Cutbush and Mollison,7 and three different phenotypes have been defined in Caucasians using antibodies to Fy antigen.8 Miller and others9 and Gelpi and King10 later showed that the Fy antigen was necessary for invasion of red blood cells by P. vivax and P. knowlesi and that most west Africans do not express DARC on their erythrocytes. More recently, the lack of this receptor in African populations is believed to be due to selective pressure induced by P. vivax in those populations in earlier times.11 Because erythrocytes from Fy-negative individuals cannot be invaded by merozoites, such individuals do not have the clinical manifestations of malaria associated with development of the Plasmodium asexual erythrocytic cycle. They also do not contribute to the transmission of the parasite by mosquitoes because they do not develop gametocytes.
Until now, there has been little interest in assessing the immune response induced in Fy-negative individuals who have been frequently inoculated with P. vivax sporozoites and little is known about the characteristics of their immune responses. To understand the extent of the anti-P. vivax humoral immune responses in Fy-negative individuals, we studied individuals in several villages in the Colombian Pacific coastal region. We hypothesized that because the population is mainly composed of Afro-Colombian individuals, prevalence of individuals expressing the Fy-negative phenotype might vary. We typed the study populations for Fy and assessed the presence of antibodies to defined P. vivax antigens from the pre-erythrocytic and asexual blood forms. Understanding the immune response of Fy-negative individuals is an important window into the mechanisms of immune responses induced naturally to different phases of the P. vivax life cycle.
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MATERIALS AND METHODS |
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). We studied communities located in Quibdó (Chocó State) and La Delfina, Punta Soldado, and Zacarías (Valle State). These four rural communities focus on fishing, agriculture, logging, and mining activities.
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Antigens. Three P. vivax antigens were available to assess the antibody response: circumsporozoite protein (CS), merozoite surface protein 1 (MSP-1), and Duffy binding protein (DBP). These are among the most widely studied P. vivax antigens from the pre-erythrocytic and erythrocytic stages. Two long synthetic peptides corresponding to different regions of the PvCS were used to assess the response to the pre-erythrocytic phase. Peptide N, which encompasses the non-repeat amino flank of PvCS (amino acids 22–125), and a 48-mer peptide composed of tandem repeats of the p11 peptide (amino acids 96–104) were chemically synthesized as described and used as antigens for the ELISA.13 Recombinant proteins containing region II of the PvDBP, (rPvRII) and a fragment of the amino region of the PvMSP-1 (200L fragment), both expressed on the P. vivax asexual blood forms, were used to assess the response to the erythrocytic phase.
The rPvRII fragment was produced at the International Center for Genetic Engineering and Biotechnology (New Delhi, India) as previously described.14 The 200L fragment corresponding to the N-terminal fragment of PvMSP-1 was produced at the Malaria Vaccine and Drug Development Center (Cali, Colombia) as a chimeric recombinant protein fused with a six-histidine tag (6-his) in Escherichia coli and was purified to < 1,000 endotoxin units (EU)/50 µg of protein.
Duffy antigen typing. Duffy antigen expression was typed by a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique using genomic DNA obtained from the blood cell fraction of the patients. The DNA was extracted by a salting-out technique, and 1 µL was used for a nested PCR as previously described.15
Antibody response to P. vivax antigens. To determine whether Fy-negative individuals responded to specific blood antigens and whether immune antibody response to pre-erythrocytic forms of the parasite differed between Fy-positive and Fy-negative individuals from P. vivax–endemic areas, we conducted an IFAT using P. vivax sporozoites and blood forms (schizonts), fixed-parasite preparations, and an ELISA for specific antigens.
Immunofluorescent antibody test. Sporozoites and parasite blood forms were collected and used as antigen sources. Glass slides were previously coated with P. vivax sporozoites or parasite blood forms. Sporozoites were produced by infection of laboratory-reared Anopheles albimanus mosquitoes that fed on P. vivax–infected blood. Two weeks after feeding, sporozoites were collected by dissection of the mosquito salivary gland and deposited in a multiwell IFAT slide at a concentration of 2,000 sporozoites/well. Slides were kept frozen at –70°C until use as described elsewhere.16
Plasmodium vivax blood forms were obtained from patients with parasitemia levels > 0.5%. These were cultured for eight hours to improve their maturation and to allow schizonts to develop. A pool of sera from healthy volunteers without a history of malaria exposure was used as negative controls. Sera from semi-immune volunteers from malaria-endemic areas were used as positive controls. Fluorescence was read using an epifluorescence microscope (Laborlux 2; Leitz GmbH, Wetzlar, Germany). Antibody titers < 1:80 were considered negative.
Enzyme-linked immunosorbent assay. The PvCSP, PvMSP-1 (200L fragment), and PvDBP (rPvRII) antigens were evaluated by an ELISA as previously described17 with modifications: blocking and diluting buffers contained skim milk (Becton-Dickinson) and bovine serum albumin (BSA) for PvCSP and PvMSP-1, respectively. Absorbance was read at 405 nm in a multichannel spectrophotometer (MRX; Dynex Technologies, Inc., Chantilly, VA). The rPvRII was used at a concentration of 2 µg/mL in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Plates were blocked and incubated with phosphate-buffered saline (PBS), 2.5% BSA for two hours. Serial dilutions were prepared in diluting buffer PBS, 0.1% BSA. Horseradish peroxidase–conjugated antihuman polyvalent immunoglobulin was used at a dilution of 1:1 x 106 in diluting buffer. The plates were incubated for 60 minutes with tetramethylbenzidine/hydrogen peroxide and read at 630 nm. Fifty microliters of stopping solution (2 N H2SO4) were added to each well and after 60 minutes they were read again at 450–550 nm.
To define the cut-off value, the antigen was tested using sera from 10 human volunteers with no history of malaria infection. Antibody titers were considered positive when absorbance of the test sera was greater than or equal to the mean absorbance of human control plasma plus 3 standard deviations. ELISA titers < 1:100 were considered negative.
Statistical analysis. To evaluate the association between presence of the Fy (DARC) antigen and elicitation of antibodies against parasite forms or antigens, we calculated prevalence ratios of antibody response for Fy-negative versus Fy-positive individuals using Stata version 8.0 software (Stat Corporation, College Station, TX). Prevalence ratios were adjusted by age as a marker of immune status and village, a proxy for intensity of transmission to control for potential confounders. Sample size was calculated estimating a 50% prevalence of DARC with a variation of 10% and an alpha error = 0.05.
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RESULTS |
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). Individuals from the different villages were tested by IFAT to determine their previous exposure to malaria. A total of 39% of the study volunteers showed responses to P. falciparum and 60% to P. vivax as determined using as antigen asexual blood parasite preparations from both parasite species. At the clinical interview, 182 (88%) reported previously having malaria. The proportions of individuals with antibodies to blood stages by IFAT were similar (43% and 36%) for P. falciparum and (74% and 52%) and P. vivax in Fy-positive and Fy-negative individuals, respectively.
Prevalence of Fy-negative phenotype.
Overall, 74.3% of the 214 individuals we studied were Afro-Colombian, ranging from 64% to 100% of the volunteers in each community. Ninety-six percent of Fy-negative individuals were Afro-Colombian. Among all the individuals studied, only three Fy-negative individuals were from the Mestizo ethnic group. The community with the highest percentage of Afro-Colombians was Quibdó; the majority of Mestizo and indigenous individuals were found in La Delfina. In both groups there were more women than men, and most participants were 20–39 years of age (Table 2).
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). Responses to the N-terminal peptide in Fy-negative and Fy-positive individuals were equal in frequency (adjusted odds ratio = 1.01, 95% CI = 0.75–1.36) and level of antibody titer reached. Also, responses to P11 were not significantly different between the two groups (adjusted odds ratio = 1.18, 95% CI = 0.91–1.55). However, in this group a larger percentage of Fy-negative individuals had each positive antibody titer compared with Fy-positive individuals. Additionally, only one individual in the Fy-negative group reached the highest antibody titer (1:1,600); most of the responders in this group reached titers of 1:200 and 1:400.
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). However, as expected, responses were less frequent in this group than in Fy-positive individuals (adjusted odds ratio = 0.71, 95% CI = 0.57–0.86). Similarly, Fy-negative individuals had lower titers than Fy-positive individuals.
The Fy-negative individuals also showed antibody responses to blood-stage antigens in concordance with their positive responses to blood parasite forms. For PvMSP-1 (200L fragment), the number of responders (adjusted odds ratio = 0.94, 95% CI = 0.78–1.10) and their maximum antibody titers (1:1,600 for both groups) were not different from those of Fy-positive individuals. However, for PvDBP (rPvRII), Fy-negative individuals responded less frequently than Fy-positive individuals (adjusted odds ratio = 0.61, 95% CI = 0.42–0.91). A lower percentage of responders in the Fy-negative group and one Fy-positive individual had the highest titer (1:6,400) observed in this study (Figure 2).
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DISCUSSION |
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Theoretically, Fy-negative individuals are equally exposed to the P. vivax pre-erythrocytic cycle as Fy-positive persons in malaria-endemic areas. However, since they do not proceed to the blood phase, FY-negative individuals do not develop malaria symptoms characteristic of this phase of the parasite cycle. They are never exposed to P. vivax gametocytes and likely do not have most of the antigens expressed in the asexual blood forms.
The Fy-negative individuals exposed to sporozoite inoculation would proceed to the liver cycle and release merozoites that would be exposed to the immune system. Although the antigens already expressed on the merozoite surface would induce immune responses, the immune system of those individuals will be selectively stimulated by a limited number of P. vivax antigens. A model similar to this one has been used by Guerin-Marchand and others to identify P. falciparum liver-stage antigens (LSAs).18 They used sera from individuals exposed for long periods to P. falciparum, but who had not entered the blood phase of the cycle because of chemoprophylaxis. Sera form these individuals allowed the selection of multiple LSAs, which are currently being tested as vaccine candidates in clinical trials.
A finding that deserves further analysis is that PvMSP-1 is similarly recognized by Fy-positive and Fy-negative individuals, whereas the PvDBP is mainly recognized by Fy-positive individuals. This may indirectly indicate that MSP-1 is expressed during liver schizogony, as has been shown for P. falciparum, and that it might be expressed abundantly enough to stimulate the immune system as much as the repeated merozoite release from the blood cycle does in Fy-positive individuals. Conversely, infection with Plasmodium blood parasite forms has been shown to modulate the liver stage immunity to P. yoelii and to specific antigens including a fragment of the MSP-1 protein by inducing apoptosis of specific CD4+ T helper cells.19,20 This modulation might result in lower specific T helper activity to B lymphocytes and consequently lower antibody production in Fy-positive individuals to blood-stage antigens. Additionally, the poor recognition of PvDBP by Fy-negative individuals may be explained by the late expression of this ligand, whose expression appears to be triggered by merozoite binding to the merozoite Fy receptor.
A relationship between expression of DARC and the age of red blood cells has been previously reported21; however, previous data about their expression and immune response are not available. In this study, we report a relationship between the expression of DARC and the humoral immune response to different parasite forms of P. vivax, as well as to specific antigens in individuals chronically exposed to malaria parasites.
In conclusion, Fy-negative individuals exhibit antibody responses to pre-erythrocytic parasite forms and to a limited number of blood-stage antigens. Selected anti-P. vivax responses could be used for continued study of the mechanisms of immunity and for selection of vaccine candidates.
Received April 12, 2005. Accepted for publication June 17, 2005.
Acknowledgments: We thank Anilza Bonelo and Ana Milena Lenis for scientific and technical support in the assessment of the humoral response, Rosalie Dominik and Hugo Hurtado for statistical advice, and Lina González for collection of field samples.
Financial support: This work was supported by the National Institute of Allergy and Infectious Diseases (Bethesda, MD) through a Tropical Medicine Research Center grant (no. 5 P50 AI049486-01). Chetan E. Chitnis is supported by an International Senior Research Fellowship from The Wellcome Trust, United Kingdom and an International Research Scholarship from the Howard Hughes Medical Institute.
* Address correspondence to Sócrates Herrera, Malaria Vaccine and Drug Development Center, Carrera 35 # 4A-53, AA 26020, Cali, Colombia. E-mail: sherrera{at}inmuno.org 
Authors’ addresses: Sócrates Herrera, Andrés Gómez, Omaira Vera, Juana Vergara, Augusto Valderrama-Aguirre, Fabián Méndez, and Myriam Arévalo-Herrera, Instituto de Inmunología, Edificio de Microbiología, Tercer Piso, Facultad de Salud, Universidad del Valle, Sede San Fernando, AA 25574, Cali, Colombia, Telephone: 57-2-558-1931, Fax: 57-2-557-0449 and Malaria Vaccine and Drug Development Center, Carrera 35 # 4A-53, AA 26020, Cali, Colombia, Telephone: 57-2-558-3937, Fax: 57-2-556-0141, E-mail: sherrera{at}inmuno.org. Amanda Maestre, Grupo de Malaria, Universidad de Antioquia, Calle 62 # 52-59, Medellín, Colombia. Ruobing Wang, The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, Telephone: 301-315-2524. Chetan E. Chitnis and Syed S. Yazdani, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg 110 067, New Delhi, India, Telephone: 91-11-2619-5007.
Reprint requests: Sócrates Herrera, Malaria Vaccine and Drug Development Center, Carrera 35 No 4A-53, Cali, Colombia.
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