The Blood Stage Antigen RBP2-P1 of Plasmodium vivax Binds Reticulocytes and Is a Target of Naturally Acquired Immunity.

The interactions between Plasmodium parasites and human erythrocytes are prime targets of blood stage malaria vaccine development. The reticulocyte binding protein 2-P1 (RBP2-P1) of Plasmodium vivax, a member of the reticulocyte binding protein family, has recently been shown to be highly antigenic in several settings endemic for malaria. Yet, its functional characteristics and the relevance of its antibody response in human malaria have not been examined. In this study, the potential function of RBP2-P1 as an invasion ligand of P. vivax was evaluated. The protein was found to be expressed in schizonts, be localized at the apical end of the merozoite, and preferentially bind reticulocytes over normocytes. Human antibodies to this protein also exhibit erythrocyte binding inhibition at physiologically relevant concentrations. Furthermore, RBP2-P1 antibodies are associated with lower parasitemia and tend to be higher in asymptomatic carriers than in patients. This study provides evidence supporting a role of RBP2-P1 as an invasion ligand and its consideration as a vaccine target.

Declining Genetic Polymorphisms of the C-terminus Merozoite Surface Protein-1 Amidst Increased Plasmodium knowlesi Transmission in Thailand.

Recent reports from Thailand reveal a substantial surge in Plasmodium knowlesi cases over the past decade, with a more than eightfold increase in incidence by 2023 compared to 2018. This study investigates temporal changes in genetic polymorphism associated with the escalating transmission of P. knowlesi malaria in Thailand over the past two decades. Twenty-five P. knowlesi samples collected in 2018-2023 were sequenced for the 42-kDa region of pkmsp1 and compared with 24 samples collected in 2000-2009, focusing on nucleotide diversity, natural selection, recombination rate, and population differentiation. Seven unique haplotypes were identified in recent samples, compared to 15 in earlier samples. Nucleotide and haplotype diversities were lower in recent samples (π = 0.016, Hd = 0.817) than in earlier samples (π = 0.018, Hd = 0.942). Significantly higher synonymous substitution rates were observed in both sample sets (dS - dN = 2.77 and 2.43, p < 0.05), indicating purifying selection and reduced genetic diversity over time. Additionally, 8 out of 17 mutation points were located on B-cell epitopes, suggesting an adaptive response by the parasites to evade immune recognition. Population differentiation analysis using the fixation index (Fst) revealed high genetic differentiation between parasite populations in central and southern Thailand or Malaysia. Conversely, the relatively lower Fst value between southern Thailand and Malaysia suggests a closer genetic relationship, possibly reflecting historical gene flow. In conclusion, our findings highlight a decline in genetic diversity and evidence of purifying selection associated with the recently increased incidence of P. knowlesi malaria in Thailand. The minor genetic differentiation between P. knowlesi populations from southern Thailand and Malaysia suggests a shared recent ancestry of these parasites and underscores the need for coordinated efforts between the two countries for the elimination of P. knowlesi.

Genetic diversity and molecular evolution of Plasmodium vivax Duffy Binding Protein and Merozoite Surface Protein-1 in northwestern Thailand.

The local diversity and population structure of malaria parasites vary across different regions of the world, reflecting variations in transmission intensity, host immunity, and vector species. This study aimed to use amplicon sequencing to investigate the genotypic patterns and population structure of P. vivax isolates from a highly endemic province of Thailand in recent years. Amplicon deep sequencing was performed on 70 samples for the 42-kDa region of pvmsp1 and domain II of pvdbp. Unique haplotypes were identified and a network constructed to illustrate genetic relatedness in northwestern Thailand. Based on this dataset of 70 samples collected between 2015 and 2021, 16 and 40 unique haplotypes were identified in pvdbpII and pvmsp142kDa, respectively. Nucleotide diversity was higher in pvmsp142kDa than in pvdbpII (π = 0.027 and 0.012), as was haplotype diversity (Hd = 0.962 and 0.849). pvmsp142kDa also showed a higher recombination rate and higher levels of genetic differentiation (Fst) in northwestern Thailand versus other regions (0.2761-0.4881). These data together suggested that the genetic diversity of P. vivax in northwestern Thailand at these two studied loci evolved under a balancing selection, most likely host immunity. The lower genetic diversity of pvdbpII may reflect its stronger functional constrain. In addition, despite the balancing selection, a decrease in genetic diversity was observed. Hd of pvdbpII decreased from 0.874 in 2015-2016 to 0.778 in 2018-2021; π of pvmsp142kDa decreased from 0.030 to 0.022 over the same period. Thus, the control activities must have had a strong impact on the parasite population size. The findings from this study provide an understanding of P. vivax population structure and the evolutionary force on vaccine candidates. They also established a new baseline for tracking future changes in P. vivax diversity in the most malarious area of Thailand.

Ivermectin metabolites reduce Anopheles survival.

Ivermectin mass drug administration to humans or livestock is a potential vector control tool for malaria elimination. The mosquito-lethal effect of ivermectin in clinical trials exceeds that predicted from in vitro laboratory experiments, suggesting that ivermectin metabolites have mosquito-lethal effect. The three primary ivermectin metabolites in humans (i.e., M1 (3″-O-demethyl ivermectin), M3 (4-hydroxymethyl ivermectin), and M6 (3″-O-demethyl, 4-hydroxymethyl ivermectin) were obtained by chemical synthesis or bacterial modification/metabolism. Ivermectin and its metabolites were mixed in human blood at various concentrations, blood-fed to Anopheles dirus and Anopheles minimus mosquitoes, and mortality was observed daily for fourteen days. Ivermectin and metabolite concentrations were quantified by liquid chromatography linked with tandem mass spectrometry to confirm the concentrations in the blood matrix. Results revealed that neither the LC50 nor LC90 values differed between ivermectin and its major metabolites for An. dirus or An. minimus., Additionally, there was no substantial differences in the time to median mosquito mortality when comparing ivermectin and its metabolites, demonstrating an equal rate of mosquito killing between the compounds evaluated. These results demonstrate that ivermectin metabolites have a mosquito-lethal effect equal to the parent compound, contributing to Anopheles mortality after treatment of humans.

Overexpression of hepatocyte EphA2 enhances liver-stage infection by Plasmodium vivax.

The liver is the first destination of malaria parasites in humans. After reaching the liver by the blood stream, Plasmodium sporozoites cross the liver sinusoid epithelium, enter and exit several hepatocytes, and eventually invade a final hepatocyte host cell. At present, the mechanism of hepatocyte invasion is only partially understood, presenting a key research gap with opportunities for the development of new therapeutics. Recently, human EphA2, a membrane-bound receptor tyrosine kinase, was implicated in hepatocyte infection by the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium yoelii, but its role is not known for Plasmodium vivax, a major human parasite whose liver infection poses a specific challenge for malaria treatment and elimination. In this study, the role of EphA2 in P. vivax infection was investigated. It was found that surface expression of several recombinant fragments of EphA2 enhanced the parasite infection rate, thus establishing its role in P. vivax infection. Furthermore, a new permanent cell line (EphA2Extra-HC04) expressing the whole extracellular domain of EphA2 was generated. This cell line supports a higher rate of P. vivax infection and is a valuable tool for P. vivax liver-stage research.

Anopheles bionomics in a malaria endemic area of southern Thailand.

BACKGROUND: Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles in Surat Thani province, Thailand. METHODS: Mosquitoes were collected via human landing collections between February and October 2019. Anopheles mosquitoes were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status, and a subset were evaluated for molecular identification and Plasmodium detection. RESULTS: A total of 17,348 mosquitoes were collected during the study period; of these, 5777 were Anopheles mosquitoes. Morphological studies identified 15 Anopheles species, of which the most abundant were Anopheles minimus (s.l.) (87.16%, n = 5035), An. dirus s.l. (7.05%, n = 407) and An. barbirostris s.l. (2.86%, n = 165). Molecular identification confirmed that of the An. minimus s.l. mosquitoes collected, 99.80% were An. minimus (s.s.) (n = 484) and 0.2% were An. aconitus (n = 1), of the An. dirus (s.l.) collected, 100% were An. baimaii (n = 348), and of the An. maculatus (s.l.) collected, 93.62% were An. maculatus (s.s.) (n = 44) and 6.38% were An. sawadwongporni (n = 3). No Anopheles mosquito tested was Plasmodium positive (0/879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection (Kruskal-Wallis H-test: χ2 = 80.89, P < 0.0001, n = 5666), with more An. barbirostris (s.l.) and An. maculatus (s.l.) caught earlier compared to An. minimus (s.l.) (P = 0.0001 and P < 0.0001, respectively) and An. dirus (s.l.) (P = 0.0082 and P < 0.001, respectively). The proportion of parous An. minimus (s.l.) captured by hour increased throughout the night (Wald Chi-square: χ2 = 17.31, P = 0.000, odds ratio = 1.0535, 95% confidence interval 1.0279-1.0796, n = 3400). Overall, An. minimus (s.l.) parity was 67.68% (2375/3509) with an intra-cluster correlation of 0.0378. A power calculation determined that an An. minimus (s.l.) parity reduction treatment effect size = 34%, with four clusters per treatment arm and a minimum of 300 mosquitoes dissected per cluster, at an α = 0.05, will provide 82% power to detect a significant difference following ivermectin MDA. CONCLUSIONS: The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on An. minimus parity.