ANTIBODIES TO PLASMODIUM VIVAX APICAL MEMBRANE ANTIGEN 1: PERSISTENCE AND CORRELATION WITH MALARIA TRANSMISSION INTENSITY CRISTIANE G. MORAIS, IRENE S. SOARES, LUZIA H. CARVALHO, COR J. F. FONTES, ANTONIANA U. KRETTLI, AND ÉRIKA M. BRAGA* Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Departamento de Análises Clínicas e Toxicológicas, Universidade de Sao Paulo, Sao Paulo, Brazil; Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil; Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil Abstract. The antibody responses to the apical membrane antigen 1 of the Plasmodium vivax (PvAMA-1) were investigated in subjects living in areas of Brazil with different levels of malaria transmission. The prevalence and the levels of IgG to PvAMA-1 increased with the time of exposure. The frequency of a positive response and the mean IgG level were higher in areas where malaria prevalence was more intense, especially among non-infected subjects exposed to moderate transmission over a period of 20 years. The proportions and levels of IgG1and IgG3 isotypes were significantly higher among those subjects with long-term exposure. Antibodies, mainly IgG1, to PvAMA-1 persisted for seven years among subjects briefly exposed to malaria in an outbreak outside the Brazilian malaria-endemic area. These data show the highly immunogenic properties of PvAMA-1 and emphasize its possible use as a malaria vaccine candi- date. INTRODUCTION Antigens of Plasmodium located on the surface or in the apical organelles of merozoites have been characterized as targets for protection or as possible vaccines against malaria. Among several vaccine candidates, apical membrane antigen 1 (AMA-1), an asexual blood stage antigen, is considered to be an important candidate malaria vaccine antigen. 1 This pro- tein is present in all Plasmodium species examined, as well as in other Apicomplexa. 2–4 This antigen is a type I integral membrane protein and in all characterized Plasmodium vivax AMA-1 (PvAMA-1) genes 16 invariant Cys residues are en- coded in the ectoplasmic region; analysis of the disulfide bond pattern suggests a division into three distinct domains. 5 Analysis of the crystal structure of the ectoplasmic region of the PvAMA-1 confirmed the division of this region into three structural domains. 6 Comparison of the PvAMA-1 structure with other known three-dimensional structures showed that domains I and II are structurally similar to each other and belong to the PAN module superfamily. 6 this superfamily is related to proteins with diverse adhesion functions binding to protein or carbohydrates receptors. 7 AMA-1 is synthesized late during the development of para- site schizonts as a precursor of an 83-kD protein that is ini- tially located in the micronemes of the apical complex of merozoites and sporozoites. 7 It is processed to a 66-kD form before its relocation into the surface of mature merozoites. 7 Once on the surface, this form is redistributed and undergoes two C-terminal cleavages, giving rise to 48-kD and 44-kD soluble forms. 8–11 The function of AMA-1 is not well under- stood, but its stage specificity and location suggest that this protein is involved in the process of invasion of host erythro- cytes. 12,13 The inclusion of AMA-1 as malaria vaccine candidate is supported by evidence from studies of malaria in animal mod- els. 14,15 Furthermore, in malaria-endemic African popula- tions, a considerable proportion of individuals has naturally acquired antibodies and T cell reactivities to AMA-1. 16–18 In spite of the critical importance of the AMA-1, few se- roepidemiologic studies have been conducted to understand the dynamics of naturally acquired humoral immune re- sponses to this molecule in populations exposed to different patterns of malaria transmission. Most of those studies have been conducted in holo-hyperendemic areas and P. falci- parum AMA-1 has been used as a target for antibody detec- tion. 16–18 Although P. vivax causes less mortality than P. falciparum, it has an enormous socioeconomic impact, particularly in South America and Asia. In Brazil, P. vivax was responsible for approximately 80% of the 459,013 cases of malaria re- ported in 2004 (Brazilian Ministry of Health). Given the criti- cal importance of understanding naturally acquired immunity to P. vivax antigens, we conducted an immunoepidemiologic study focusing on the antibody response to PvAMA-1 in dis- tinct areas within the Brazilian Amazon malaria-endemic re- gion. We also evaluated the persistence of specific antibodies to PvAMA-1 among subjects briefly exposed to P. vivax transmission outside the malaria-endemic area. MATERIALS AND METHODS Study areas, subjects, and blood sample collection. We ana- lyzed four different groups of non-infected adults who had been exposed to malaria transmission in the Brazilian Ama- zon area (Table 1). A complete health questionnaire was ap- plied to all study participants. Ethical clearance for this study was obtained from the institutional review board of the Fed- eral University of Minas Gerais, Brazil (April 15, 1998; #007/ 98 and May 16, 2001; #096/01). All participants reported at least one malaria infection diagnosed by microscopy; the date and species involved in this most recent episode were re- corded. Since most subjects were migrants from areas in Bra- zil not endemic for malaria, their ages do not correlate with cumulative exposure to malaria. Past malaria exposure was therefore estimated as the length of residence or exposure to malaria in malaria-endemic areas in Brazil. These parameters were used to classify the four groups. The first group lived in Belém, the capital of Pará State. It was composed of 59 persons (median age  29 years) who had acquired P. vivax malaria after a brief exposure (a few * Address correspondence to Érika M. Braga, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 31279-901 Belo Hori- zonte, Minais Gerais, Brazil. E-mail: embraga@icb.ufmg.br Am. J. Trop. Med. Hyg., 75(4), 2006, pp. 582–587 Copyright © 2006 by The American Society of Tropical Medicine and Hygiene 582