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Background Crucial gaps in our understanding of Plasmodium vivax reticulocyte invasion and protective immunity have hampered development of vivax vaccines. P. vivax exclusively invades reticulocytes that is mediated by the P. vivax reticulocyte-binding proteins (PvRBPs) specifically PvRBP2c and PvRBP1a. Vivax infections in Duffy-null individuals have suggested the evolution of alternate invasion pathways that may be mediated by the PvRBPs. Thus, PvRBPs appear as potential targets for efficacious P. vivax neutralization. However, there are limited data validating their vaccine efficacy. In the absence of vivax invasion assays, binding-inhibitory activity of antibodies has been reported to be associated with protection and a measure of vaccine potential. Methods -based analysis was performed of the PvRBP reticulocyte-binding properties and binding-inhibitory activity of specific anti-PvRBP2c/PvRBP1a human antibodies. Results PvRBP2c and PvRBP1a displayed a distinct reticulocyte-binding specificity, and their specific reticulocyte-binding domains were mapped within their N-terminal regions. Importantly, naturally acquired antibodies against the reticulocyte-binding domains efficaciously blocked reticulocyte binding of native PvRBPs, suggesting that the human immune system produced functional binding-inhibitory antibodies through exposure to vivax malaria. Conclusions Reticulocyte-binding domains of PvRBP2c/PvRBP1a are targets of naturally acquired binding-inhibitory antibodies, substantiating their promise as candidate antigens against which vaccine-inducible immunity could potentially be boosted through natural infections.

Background Tryptophan-rich antigens of malarial parasites have been proposed to be the potential vaccine candidate antigens. Plasmodium vivax contains the largest number of such antigens, which need to be evaluated for their immune responses. Methods Recombinant proteins of 15 P. vivax tryptophan-rich antigens (PvTRAgs) were expressed, purified, and used for the human humoral and cellular immune responses. Genetic polymorphism of these 15 genes was also determined among clinical P. vivax isolates. Results The T lymphocytes of P. vivax exposed individuals expressed higher level of CD69 against all 15 PvTRAgs. These antigens also activated the large population of CD4(+) T cells and produced higher level of intracellular IL-2, INF-γ and IL-4. Although there was a mixed Th1 and Th2 response against these antigens, this response was biased toward Th2. The majority of P. vivax patients (75.7%-100%, n = 33) produced IgG antibodies against these antigens. Most of these antigens showed conserved T- and B-cell epitopes in the parasite population. Conclusions These results suggest the presence of memory T cells in humans against these antigens to generate faster and more specific immune responses to minimize the P. vivax infection. Further characterization of these PvTRAgs may lead to the identification of a potential therapeutic target.

Effective malaria control programs require continuous monitoring of drug pressure in the field, using molecular markers.We used sequence analysis to investigate the pfcrt and pfmdr1 mutations in Indian Plasmodium falciparum isolates. To evaluate the chloroquine drug pressure in the field, isolates were collected from 5 different areas at 2 time points, with an interval of 2 years.In 265 P. falciparum isolates, pfcrt mutations were observed at codons 72, 74, 75, 76, and 220, resulting in 8 different genotypes: SMNTS (61.89%), CIETS (12.08%), CMNKS (0.38%), CMNTA (2.64%), CMNTS (4.91%), SMNTA (0.38%), CIDTS (2.26%), and wild-type CMNKA (15.47%). During the 2-year period, there was a significant decrease in the number of isolates with the SMNTS genotype and an increase in the number of isolates with the highly chloroquine-resistant pfcrt genotype CIETS (P.05). The N86Y mutation was less prevalent (30.13%) than the Y184F mutation (99.16%) in the pfmdr1 gene in 239 isolates, but the number of isolates with the N86Y mutation increased significantly during the 2-year period (P.05). The number of isolates with higher total numbers of pfcrt and pfmdr1 2-loci mutations, therefore, increased significantly during this period. There was a regional bias in the mutation rate of these genes, because isolates from areas where chloroquine resistance was high had higher numbers of 2-loci mutations, and areas where chloroquine resistance was low had isolates with lower numbers of 2-loci mutations.There was a temporal increase in the number of pfcrt and pfmdr1 2-loci mutations, and this led to the higher level of chloroquine resistance. This is a cause for concern for the antimalarial drug policy in India.

Background: Enormous amounts of drugs were used to contain the outbreak of infectious diseases in areas of India affected by the tsunami in December 2004. The impact of this drug use on the Plasmodium falciparum population needs to be investigated. Methods: The nucleotide sequence of the pfcrt, pfdhps and pfdhfr genes was determined for 229 clinical P. falciparum isolates collected from patients on Car Nicobar Island at 6 different time points between May 2004 and May 2008. Results: Over time, there was an increase in the proportion of the P. falciparum population that had mutations in the pfcrt, pfdhps and pfdhfr genes associated with higher levels of chloroquine, sulfadoxine, and pyrimethamine resistance, respectively. However, the parasites collected during October 2005 had mutations associated with a lower level of pyrimethamine resistance and a higher level of sulfadoxine resistance (a rare combination), as well as a novel K540N mutation in P. falciparum dihydropteroate synthetase (PfDHPS). The emergence of this parasite population coincided with the widespread use of an additional antifolate drug, trimethoprim-sulfamethoxazole, to treat other infections during January-March 2005. Molecular modeling revealed that the sulfadoxine binding affinity of the new PfDHPS triple mutant A 436 G 437 N 540 A 581 A 613 was similar to that of A 436 G 437 E 540 A 581 A 613 (bold type indicates mutated amino acids). Conclusions: The use of 2 antifolate drugs in combination should be avoided to prevent the selection of parasites with newer mutations and altered drug susceptibilities.