Evaluation of the genetic diversity of domain II of Plasmodium vivax Apical Membrane Antigen 1 (PvAMA-1) and the ensuing strain-specific immune responses in patients from Sri Lanka.

Antigenic polymorphism displayed by malaria parasites is a skewed schema to escape the host immune system. The prevailing genetic diversity at domain II of the Plasmodium vivax Apical Membrane Antigen-1 (Pvama-1DII) was characterized in 64 single clone P. vivax isolates from Sri Lanka, where unstable malaria prevails with low intensity. In Sri Lanka, the Pvama-1DII gene showed meager meiotic recombination with the enclosure of single nucleotide polymorphisms (SNPs). Eleven amino acid (a.a.) variant positions defined 21 a.a. haplotypes with 9 unique to the island, where the predominant haplotype, H1, was identical to the reference Salvador I strain. A further 376 globally dispersed isolates defined 38 a.a. haplotypes (H22-H59), with 4 and 26 haplotypes exclusive to India and Thailand, respectively. The phylogenetic tree revealed no clustering, where most isolates had a very recent common origin. The polymorphism detected in PvAMA-1DII B and T cell epitopes evidenced an immune evasion mechanism exploited by the parasite. Majority of Sri Lankan patients developed antibody responses to both conformational and linear B cell epitopes. The ensuing strain-specific immunity due to extensive antigenic polymorphism was evaluated by aligning a.a. sequences of PvAMA-1DII with the homologous total (IgM+IgG) antibody responses assayed by in-house established indirect ELISAs against 7 PvAMA-1DII overlapping synthetic peptides, P01-P07. While the antibody responses to P01-P03, P06, P07 harbouring P. vivax clinical isolates with polymorphic a.a. haplotype to Sal I was clearly strain-transcending (cross-reactive), individuals with isolates identical to the Sal I strain observed varying antibody prevalence against the seven PvAMA-1DII Sal-I synthetic peptides, with the highest prevalence detected against P04. Synthetic peptide P04, spanning a.a. positions 302-324 of the PvAMA-1DII of the Sal I strain that included the epitope recognized by the invasion inhibitory 4G2 monoclonal antibody of PfAMA-1, was highly conserved in all 440 local and global P. vivax isolates examined. A functional role for this region is reinforced by the highly immunogenic nature of P04, and could point towards a presumably "protective" anti-P04 antibody response that elicited an isotype switch from IgM to IgG, with increasing exposure to malaria exclusively in endemic residents. Thus the conserved and seemingly "protective" nature of the domain II loop of PvAMA-1 makes it a putative contender to be included in a cocktail vaccine against P. vivax asexual erythrocytic stages in Sri Lanka.

Plasmodium falciparum: genome wide perturbations in transcript profiles among mixed stage cultures after chloroquine treatment.

A genomic approach was taken to study the effect of chloroquine (CQ) on Plasmodium falciparum cultures in multiple cell states, following short and long exposures to drug at varying concentrations. Six hundred genes from numerous functional groups were responsive to CQ amongst all cell states assayed in a micro-array analysis; however, the amplitude of fold-change was low in the majority of cases. Moreover, alterations in specific, functionally related cascades could not be discerned, leading us to believe there is no single signature response to CQ at the transcript level in P. falciparum. Instead, cell cycle changes appear to have a more pronounced effect on gene expression; only a fraction of the drug responsive loci (approximately 5%) were shared between two separate starting cultures that varied in staging profile in the current study, as well as a previous published analysis using SAGE technology [Gunasekera, A.M., Patankar, S., Schug, J., Eisen, G.,Wirth, D.F., 2003. Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum. Molecular Microbiology 50, 1229-1239]. These findings are important to report, given the striking contrast to similar studies in other model eukaryotic organisms.

Regulatory motifs uncovered among gene expression clusters in Plasmodium falciparum.

Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum. More specifically, we applied bioinformatic tools to a previously characterized micro-array data set from drug-treated asexual stage cultures (Gunasekera et al., submitted). Cluster analysis of 600 drug responsive genes identified only a single 5' motif, GAGAGAA. Two additional 5' motifs, ACTATAAAGA and TGCAC, were also shared among loci displaying patterns of coordinate expression across varying asexual growth stages. Secondly and most importantly, the functional relevance of each motif was tested in two independent assays-transient transfection and gel-retardation experiments. The GAGAGAA and TGCAC motifs were both active in the former. The GAGAGAA and ACTATAAAGA elements formed specific RNA-protein, but not DNA-protein complexes in gel shift assays, suggesting a key level of control at the RNA level. This is the first report of functionally characterized motifs in P. falciparum that were uncovered following clustering analysis of its asexual stage transcriptome. Together, both the bioinformatic and functional data reported here imply that multiple forms of gene regulation, including post-transcriptional control, may be important in the malarial system.

Genetic diversity and selection at the Plasmodium vivax apical membrane antigen-1 (PvAMA-1) locus in a Sri Lankan population.

Plasmodium vivax apical membrane antigen 1 (PvAMA-1) is an important malaria vaccine candidate. We present the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Sri Lanka. In contrast to what has been observed at the AMA-1 locus of Plasmodium falciparum, the signature of diversifying selection is seen most strongly in Domain II of PvAMA-1, indicating that the different domains in each species may be subject to varying selective pressures and functional constraints. We also find that recombination plays an important role in generating haplotype diversity at this locus, even in a region of low endemicity such as Sri Lanka. Mapping of diversity and recombination hotspots onto a 3-dimensional structural model of the protein indicates that one surface of the molecule may be particularly likely to bear epitopes for antibody recognition. Regions of this surface that show constrained variability may prove to be promising vaccine targets.