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New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.

Plasmodium cynomolgi, a malaria parasite of Asian Old World monkeys, is the sister taxon of Plasmodium vivax, the most prevalent human malaria species outside Africa. Since P. cynomolgi shares many phenotypic, biologic and genetic characteristics of P. vivax, we generated draft genome sequences of three P. cynomolgi strains and performed comparative genomic analysis between them and P. vivax, as well as a third previously sequenced simian parasite, Plasmodium knowlesi. Here we show that genomes of the monkey malaria clade can be characterized by CNVs in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites, and CNVs in the P. cynomolgi genome, providing a map of genetic variation for mapping parasite traits and studying parasite populations. The P. cynomolgi genome is a critical step in developing a model system for P. vivax research, and to counteract the neglect of P. vivax.

The organization and allelic recombination of the merozoite surface protein-1 gene of Plasmodium vivax (PvMsp-1), the most widely prevalent human malaria parasite, were evaluated in complete nucleotide sequences of 40 isolates from various geographic areas. Alignment of 31 distinct alleles revealed the mosaic organization of PvMsp-1, consisting of seven interallele conserved blocks flanked by six variable blocks. The variable blocks showed extensive variation in repeats and nonrepeat unique sequences. Numerous recombination sites were distributed throughout PvMsp-1, in both conserved blocks and variable block unique sequences, and the distribution was not uniform. Heterozygosity of PvMsp-1 alleles was higher in Asia (0.953 ؎ 0.009) than in Brazil (0.813 ؎ 0.047). No identical alleles were shared between Asia and Brazil, whereas all but one variable block nonrepeat sequence found in Brazil occurred in Asia. These observations suggest that P. vivax populations in Asia are ancestral to Brazilian populations, and that PvMsp-1 has heterogeneity in frequency of allelic recombination events. Recurrent origins of new PvMsp-1 alleles by repeated recombination events were supported by a rapid decline in linkage disequilibrium between pairs of synonymous sites with increasing nucleotide distance, with little linkage disequilibrium at a distance of over 3 kb in a P. vivax population from Thailand, evidence for an effectively high recombination rate of the parasite. Meanwhile, highly reduced nucleotide diversity was noted in a region encoding the 19-kDa C-terminal epidermal growth factorlike domain of merozoite surface protein-1, a vaccine candidate. T he human malaria parasite Plasmodium vivax is prevalent worldwide, and accounts for 70-80 million cases annually, mostly in Asia and Latin America (1). Growing resistance of P. vivax strains to chloroquine is spurring the development of a vaccine against P. vivax malaria. One current vaccine candidate is merozoite surface protein-1 (MSP-1), a 200-kDa protein expressed on the surface of the P. vivax merozoite (2). MSP-1 of Plasmodium species is synthesized as a high-molecular-weight precursor and then processed into several fragments (3). At the time of red cell invasion by the merozoite, only the 19-kDa C-terminal fragment (MSP-1 19 ), which contains two epidermal growth factor-like domains, remains on the surface. Antibodies against MSP-1 19 inhibit merozoite entry into red cells (4), and immunization with MSP-1 19 protects monkeys from challenging infections (5, 6). Hence, MSP-1 19 is considered a promising vaccine candidate.Importantly, there is extensive allelic diversity of MSP-1 among isolates (7), and this polymorphism may hamper development of effective vaccines. In Plasmodium falciparum, the most virulent malaria parasite, polymorphism in PfMsp-1 is well characterized. PfMsp-1 consists of several interallele variable blocks flanked by conserved or semiconserved blocks. Variation in this gene is basically dimorphic; i.e., one or the other of two different residues (8, ...

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