In vitro activity of rhinacanthin analogues against drug resistant Plasmodium falciparum isolates from Northeast Thailand.

BACKGROUND: New anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand. METHODS: Using a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates. RESULTS: The 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9-791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6-63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83-0.97, P < 0.001). There were no P. falciparum cytochrome b mutations observed in the field isolates, indicating that P. falciparum isolates from this area remained ATQ-sensitive. Pfkelch13 mutations and the ring-stage survival assay confirmed that most isolates were resistant to ART. CONCLUSIONS: Two rhinacanthin analogues showed parasiticidal activity against multi-drug resistant P. falciparum isolates, although less potent than ATQ. Rhinacanthin analogue 2 was more potent than analogue 1, and can be a lead compound for further optimization as an anti-malarial in areas with multidrug resistance.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry.

The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria.

The geographical distribution of allele polymorphisms of Plasmodium vivax in different regions of Thailand.

OBJECTIVE: To investigate the genetic structure of Plasmodium vivax circumsporozoite surface protein (PvCSP) and P. vivax merozoite surface protein 1 (PvMSP1) genes from field isolates of malaria parasites from four different regions of Thailand. MATERIAL AND METHOD: The data was collected by cross-sectional survey, consisting of 273 P. vivax infected blood samples from malaria clinics in 4 different border regions of Thailand from February 2008 to February 2009. The dried blood spots were extracted for DNA and Plasmodium species confirmed by species-specific primer sets. PvCSP and PvMSP1 genes were amplified and their population genetics were analyzed by using the Heterozygosity (H) formula, F-STAT and LIAN programs. RESULT: There was considerable variation in the PvMSP1 gene within 2 fragments for which HE was 0.8303, whereas PvCSP showed low HE at 0.1418. Significant differences in allele frequencies between sites were quantified by Fst, Linkage disequilibrium (LD). The results showed PvMSP1 F2; Fst = 0.063, p = 0.07; PvMSP1 F2 RFLP pattern; Fst = 0.154, p = 0.005; PvMSP1 F3; Fst = 0.23, p = 0.005 and the overall loci showed Fst = 0.151, p = 0.005 (Fisher's exact test). All values of Index association (I(S)A) were non-significant. There was no evidence of LD within the P. vivax populations. CONCLUSION: H(E) at each locus of the PvMSP1 gene showed significant differences in allele frequencies between sites.

Genetic diversity in new members of the reticulocyte binding protein family in Thai Plasmodium vivax isolates.

BACKGROUND: Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes. METHODOLOGY/PRINCIPAL FINDINGS: The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3∶1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates. CONCLUSIONS/SIGNIFICANCE: The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax.

The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand.

OBJECTIVE: To investigate the genetic polymorphism of Plasmodium vivax (P. vivax) PvCSP and PvMSP1 genes from field isolates at four endemic regions (North, East, West and South) of Thailand. METHODS: The 152 P. vivax infected cases from dried blood spots were DNA extracted and confirmed by species-specific primer sets using multiplex PCR method. PvMSP1 fragments F2 and F3; PvCSP were genotyped using RFLP-PCR method. RESULTS: Totally amplified DNA which was multiple genotypes for PvMSP1 F2 and PvMSP1 F3 were 12.50% and 8.55%, respectively while PvCSP was 3.95%. The overall frequency of multiple genotypes was 25%. There were 12 allele types of PvMSP1 F2 using AluI enzyme digestion and 8 size variations were found in PvMSP1 F3. The isolates from western region was highly genetic diverse when compare among all isolates. The predominant variant type of PvCSP gene was VK210 type. CONCLUSIONS: The multiple genotypes are common found in Thailand and it might hide the real genotype. PvCSP does not have extensive genetic diversity in this study. However, PvMSP1 marker due to multiple genotypes is difficult to be analyzed. The multiple genotypes findings might stem from population migration and vector species findings.

Plasmodium vivax trophozoites insensitive to chloroquine.

BACKGROUND: Plasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax. METHODS: The in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment. RESULTS: Plasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415 nM p < 0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100 nM. CONCLUSION: Data from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.

Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms.

BACKGROUND: Treatment failure of chloroquine for P. vivax infections has reached high levels in the eastern provinces of Indonesia, however, in vitro characterization of chloroquine resistance and its associated molecular profile have yet to be determined. METHODS: Using a modified schizont maturation assay we investigated the in vitro chloroquine susceptibility profile and molecular polymorphisms of P. vivax isolates collected from Papua, Indonesia, where high levels of clinical chloroquine treatment failure have been reported, and from Thailand, where chloroquine treatment is generally effective. RESULTS: The geometric mean chloroquine IC(50) for P. vivax isolates from Papua (n = 145) was 312 nM [95%CI: 237-411 nM] compared to 46.8 nM [95%CI: 34.7-63.1 nM] from Thailand (n = 81); p<0.001. Correlating with the known clinical efficacy of the area, a cut off for chloroquine resistance was defined as 220 nM, a level exceeded in 13.6% (11/81) of Thai isolates and 65% (94/145) of Papuan isolates; p<0.001. Several sequence polymorphisms in pvcrt-o and pvmdr1, and difference in pvmdr1 copy number were identified. A Y976F mutation in pvmdr1 was present in 96% (123/128) of Papuan isolates and 25% (17/69) of Thai isolates; p<0.001. Overall, the geometric mean chloroquine IC(50) in isolates with the Y976F mutation was 283 nM [95%CI: 211-379], compared to 44.5 nM [95%CI: 31.3-63.4] in isolates with the wild type; p< 0.001. Pvmdr1 amplification occurred in 23% (15/66) of Thai isolates compared to none (0/104) of Indonesian isolates (p<0.001), but was not associated with increased chloroquine resistance after controlling for geographical location. CONCLUSIONS: In vitro susceptibility testing of P. vivax discriminates between populations with differing levels of clinical efficacy of chloroquine. The pvmdr1 polymorphism at Y976F may provide a useful tool to highlight areas of emerging chloroquine resistance, although further studies defining its clinical correlates are needed.

Plasmodium vivax: isotopic, PicoGreen, and microscopic assays for measuring chloroquine sensitivity in fresh and cryopreserved isolates.

In vitro susceptibility tests provide information on the intrinsic response of Plasmodium vivax to antimalarials, free from confounding factors such as host immunity or relapse. This study examined the utility of radioisotope and PicoGreen assays as alternatives to the traditional microscopic examination for assessing response of P. vivax to antimalarial drugs. There was no significant difference in the mean chloroquine IC(50) of P. vivax (n=40) as determined by the microscopic (33.4 ng/ml), isotopic (33.6 ng/ml), and PicoGreen (39.1 ng/ml) assays, respectively (F=0.239, df=2, 51, and p=0.788). However measurement of IC(50)s by the microscopic method was slightly more successful in producing valid assays (57%), compared to the isotopic (32.5%) and PicoGreen (45.5%) methods. In a paired comparison of 20 fresh and cryopreserved isolates as examined by the microscopic method, there were no significant differences between the mean IC(50) responses (T=1.58, df=15, and p=0.34). Detailed methodologies for the short time culture of field and cryopreserved P. vivax are described. Although the microscopic in vitro assay provides a useful method for characterizing the drug susceptibility phenotype of P. vivax isolates, its utility is limited by a laborious methodology and need for highly skilled microscopists. Future efforts should focus on further development of high throughput assays such as the PicoGreen assay as described in this study.