Malaria is one of the major global public health problems that affect most tropical regions of the world. Even though Plasmodium falciparum is the most virulent, it is estimated that Plasmodium vivax produces around 80 to 300 million clinical cases per year . Furthermore, there have been several reports of severe P. vivax malaria cases in the last few years [2–4]. In 2008, 560,221 malaria cases were reported in the Americas ; 74.2% of them caused by P. vivax and 25.7% by P. falciparum . About 90% of these malaria cases originated in the Amazon basin shared by Bolivia, Brazil, Colombia, Ecuador, French Guiana, Guyana, Venezuela, Suriname and Peru , whereas the other 10% was contributed by non-Amazon regions.
Developing a vaccine for P. vivax represents a major challenge especially considering the lack of in vitro cultures. Thus, current efforts focus on orthologs of P. falciparum. Over the past four decades, experiments performed in animals and human subjects have led to the development of several Plasmodium vaccine candidates. Antigenic surface proteins such as the Circumsporozoite protein (CSP), Thrombospondin related anonymous protein (TRAP), Duffy-binding protein (DBP), Merozoite surface protein-1 (MSP-1) and Apical membrane antigen-1 (AMA-1) are currently being evaluated as vaccine candidates in clinical trials [1, 6–8]. The CSP is one of the most extensively studied antigens and it is involved in the motility and invasion of the sporozoite during its entrance in the hepatocyte . PvCSP has an immunogenic central repeat domain flanked by amino and carboxyl sequences containing highly conserved protein stretches (Regions I and II-plus) . This protein displays two major types of nonapeptide repeats: type I (VK210), which contains repeats of GDRA(A/D)GQPA or related sequences and type II (VK247), which is composed of ANGAGNQPG repeats [11, 12]. A third type of variant, called P. vivax-like has repeat units of APGANQ(E/G)GAA, identical to that described for Plasmodium simiovale [13, 14]. Currently, the P. falciparum RTS, S vaccine, which is based on CSP have showed to protect semi-immune adults  and children from P. falciparum natural infection in endemic areas [16–18]; this vaccine is being tested in Phase III clinical trials [8, 19].
Another sporozoite surface protein that stands out as a good vaccine candidate is TRAP, which is a 90 kDa protein with six distinct regions. TRAP is essential for sporozoite gliding and hepatocyte invasion [20, 21]. Genetic analysis of PvTRAP reveals that most of the polymorphisms occur in Region II (von Willebrand factor A-domain) and III . The potential role of TRAP as a malaria vaccine candidate has been tested in mice and monkeys, inducing high levels of specific antibodies . Furthermore, a synthetic peptide-derived from a conserved region of the TRAP protein located in the N-terminal, proved to be immunogenic and provided partial protection in Aotus monkeys [1, 24].
On the other hand, the asexual P. vivax parasite antigens DBP, MSP-1 and AMA-1 have also received attention for their role in the invasion process and/or their generation of antibody response. DBP interacts with the Duffy blood group antigen of reticulocytes, MSP-1 is expressed abundantly on the merozoite surface and AMA-1 plays a role in apical reorientation of merozoites following initial attachment during invasion . Region II of DBP (DBPII) functions as a ligand domain and consists of 330 aa with 12 cysteines and numerous aromatic residues. Studies have identified the central region of the DBPII domain between cysteines 4 and 8, as the segment containing the conserved contact residues Tyr 94, Asn 95, Lys 96, Arg 103 and Ile 175 required for recognition of DARC (Duffy Antigen/Receptor for Chemokine) on human erythrocytes [25–28].
MSP-1 is part of a complex of proteins that is processed into smaller fragments by serine proteases during invasion of red blood cells . It has been suggested that fragments PvMSP-114 and PvMSP-120 could be included in a P. vivax multi-antigen vaccine since they contain high affinity reticulocyte binding (HARB) cluster regions and confer protection in monkeys [30–32]. AMA-1 has also been evaluated for inclusion in a multi- sub-unit vaccine for both P. falciparum and P. vivax [33, 34]. The complete AMA-1 protein contains three domains, from which a higher rate of mutations and level of diversifying selection has been shown in domain I [35, 36]. Indeed, AMA-1 is highly immunogenic, protecting against Plasmodium chabaudi infection in mice  and displaying significant antibody and T cell responses in endemic human populations [38–40].
Investigation of the sequence variation among malaria antigenic regions is necessary for the development of an effective malaria vaccine that includes haplotypes from different regions, considering that Plasmodium is highly diverse and these regions are under strong immunological pressure. In this study, important regions in PvCSP, PvTRAP, PvDBP, PvMSP-1 and PvAMA-1 were genotyped using P. vivax Peruvian isolates collected during years 2006-2007. In addition, since natural selection plays a role in structuring diversity of these highly polymorphic antigens, the worldwide distribution of the main variants was determined by comparing the Peruvian samples with sequences previously reported from South America, Asia and Oceania.