Genetic diversity of Plasmodium falciparum AMA-1 antigen from the Northeast Indian state of Tripura and comparison with global sequences: implications for vaccine development.

BACKGROUND: Malaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen. METHODS: Patients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique. RESULTS: Among 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation. CONCLUSIONS: Besides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite.

Genetic polymorphism of Plasmodium falciparum msp-1, msp-2 and glurp vaccine candidate genes in pre-artemisinin era clinical isolates from Lakhimpur district in Assam, Northeast India.

Background: Northeast India shares its international border with Southeast Asia and has a number of malaria endemic zones. Monitoring genetic diversity of malaria parasites is important in this area as drug resistance and increasing genetic diversity form a vicious cycle in which one favours the development of the other. This retrospective study was done to evaluate the genetic diversity patterns in Plasmodium falciparum strains circulating in North Lakhimpur area of Assam in the pre-artemisinin era and compare the findings with current diversity patterns. Methods: Genomic DNA extraction was done from archived blood spot samples collected in 2006 from malaria-positive cases in Lakhimpur district of Assam, Northeast India. Three antigenic markers of genetic diversity were studied - msp-1 (block-2), msp-2 (block-3) and the glurp RII region of P. falciparum using nested PCR. Results: Allelic diversity was examined in 71 isolates and high polymorphism was observed. In msp-1, eight genotypes were detected; K1 (single allele), MAD20 (six different alleles) and RO33 (single allele) allelic families were noted. Among msp-2 genotypes, 22 distinct alleles were observed out of which FC27 had six alleles and IC/3D7 had 16 alleles. In RII region of glurp, nine genotypes were obtained. Expected heterozygosity (H E) values of the three antigenic markers were 0.72, 0.81 and 0.88, respectively. Multiplicity of infection (MOI) values noted were 1.28, 1.84 and 1.04 for msp-1, msp-2 and glurp, respectively. Conclusion: Results suggest a high level of genetic diversity in P. falciparum msp (block-2 of msp-1 and block-3 of msp-2) and the glurp RII region in Northeast India in the pre-artemisinin era when chloroqunine was the primary drug used for uncomplicated falciparum malaria. Comparison with current studies have revealed that the genetic diversity in these genes is still high in this region, complicating malaria vaccine research.

Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections.

The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual- and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections.