Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro

AbstractImproved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

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Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro

10.21203/rs.3.rs-185180/v1 ◽

2021 ◽

Author(s):

Steven Maher ◽

Amélie Vantaux ◽

Victor Chaumeau ◽

Adeline C. Y. Chua ◽

Caitlin A. Cooper ◽

...

Keyword(s):

Plasmodium Vivax ◽

Single Point ◽

Radical Cure ◽

Activity Data ◽

Liver Stage ◽

Compound Libraries ◽

Post Hoc ◽

Prevention Of Relapse

Abstract Improved control of malaria caused by Plasmodium vivax can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of vivax patients. We screened several compound libraries against P. vivax liver stages, including 1566 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically-relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support the screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals little activity overlap, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent, focused drug development test cascade.

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KAI407, a Potent Non-8-Aminoquinoline Compound That Kills Plasmodium cynomolgi Early Dormant Liver Stage ParasitesIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01927-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1586-1595 ◽

Cited By ~ 45

Author(s):

Anne-Marie Zeeman ◽

Sandra M. van Amsterdam ◽

Case W. McNamara ◽

Annemarie Voorberg-van der Wel ◽

Els J. Klooster ◽

...

Keyword(s):

Plasmodium Vivax ◽

Inhibitory Concentration ◽

Radical Cure ◽

Malaria Prophylaxis ◽

Activity Profile ◽

Liver Stage ◽

Content Type ◽

Plasmodium Cynomolgi ◽

Malaria Eradication

ABSTRACTPreventing relapses ofPlasmodium vivaxmalaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay ofin vitro-cultured hypnozoite forms ofPlasmodium cynomolgi(an excellent and accessible model forPlasmodium vivax). In this assay, primary rhesus hepatocytes are infected withP. cynomolgisporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class forP. vivaxmalaria prophylaxis and potentially a radical cure.

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A robust in vitro liver cell model for long-term study of Plasmodium vivax hypnozoites

10.21203/rs.2.17895/v1 ◽

2019 ◽

Author(s):

Araya Jivapetthai ◽

Yongyut Pewkliang ◽

Sreekanth Kokkonda ◽

Paviga Limudompon ◽

Phongthon Kanjanasirirat ◽

...

Keyword(s):

Plasmodium Vivax ◽

Cell Model ◽

Screening Methods ◽

Radical Cure ◽

Liver Stage ◽

Long Term Study ◽

Biological Studies ◽

Single Nucleus

Abstract Background Plasmodium vivax is the most prevalent species of human malaria parasites, affecting nearly half of the world’s population. Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax to tolerate most antimalarials, and later to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites, including hypnozoites. Experimentally, this form has previously been studied in the primary hepatocyte cell model, but this has some limitations due to its high cost and failures when used for long-term biological studies and high throughput drug screening. Methods Here, an immortalized human hepatocyte-like cell line (imHC) was infected with P. vivax sporozoites. The infected cultures were maintained for up to 28 days to obtain small liver-stage forms. A novel system to quickly enrich pure small liver-stage forms in culture was also developed. The susceptibility of the enriched small liver-stage forms, presumably hypnozoites, to known antimalarial drugs, atovaquone, primaquine, and tafenoquine, was examined. Results Small liver-stage forms of P. vivax could persist in long-term imHCs culture. These small forms had a single nucleus and could be enriched by treatment with DSM265, a compound active against growing parasites but not hypnozoites. Resistance to inhibition by atovaquone was consistent with the interpretation that the enriched small parasites represent hypnozoites. Primaquine and tafenoquine displayed poor activity at clearing these putative hypnozoites in vitro. Conclusions A robust cell-based model, with well-defined dormant liver-stage parasites in long-term stable human liver cells, allows us to follow hypnozoite formation and eventual reactivation to dividing parasites. This model is also well-suited to test radical cure efficacy of compounds against P. vivax hypnozoites. Thus, it will be of value for understanding hypnozoite biology and for drug discovery to eliminate dormant malaria. Keywords Malaria, Plasmodium vivax, Sporozoite, Liver stage, Hypnozoite, imHCs

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Influence of calcium hydroxide points on the quality of intracanal dressing filling

Journal of Applied Oral Science ◽

10.1590/s1678-77572006000300014 ◽

2006 ◽

Vol 14 (3) ◽

pp. 219-223 ◽

Cited By ~ 2

Author(s):

Nicole de Mello Rahde ◽

José Antonio Poli Figueiredo ◽

Elias Pandonor Motcy Oliveira

Keyword(s):

Calcium Hydroxide ◽

Root Canal Filling ◽

Human Teeth ◽

Significance Level ◽

Root Canals ◽

Gutta Percha ◽

Group 2 ◽

Post Hoc

PURPOSE: The aim of this study was to evaluate, in vitro, the quality of calcium hydroxide [Ca(OH)2] paste filling (Ultracal, Ultradent) associated or not with Ca(OH)2-containing gutta-percha points (Calcium Hydroxide Plus PointsTM, Roeko) in curved root canals. MATERIAL AND METHODS: One hundred and twenty roots of extracted human teeth, randomly divided into three curvature ranges (mild - 0 to 14º; moderate - 15º to 29º; severe - >30º) were used. After chemomechanical preparation, the roots were assigned to 4 groups (n=30), according to the technique of intracanal dressing placement: group 1 - Ca(OH)2 paste was applied with a lentulo spiral; group 2 - Ca(OH)2 paste was applied with a lentulo spiral and a Ca(OH)2 point was inserted into the canal; group 3 - Ca(OH)2 paste was applied with a NavitipTM tip (supplied with Ultracal system); group 4 - Ca(OH)2 paste was applied with a NavitipTM tip and a Ca(OH)2 point was inserted into the canal. The roots were cleared and the quality of apical third filling was assessed by a calibrated experienced examiner. The specimens were examined under stereomicroscopy and scored 1 to 4 (i.e., from inadequate to complete root canal filling). The results were analyzed statistically by ANOVA and Duncan's post hoc test at 5% significance level. RESULTS: There were no statistically significant differences (p>0.05) among the curvature degrees in groups 1, 3 and 4. Severely curved roots in group 2 presented bordering significance (p=0.05). The groups that associated the use of Ca(OH)2 paste and points (2 and 4) showed better apical filling than the other groups, but this difference was statistically significant (p<0.001) only for roots with severe curvature. CONCLUSION: According to the results of this study, the curvature degree did not influence the quality of filling. The techniques that used Ca(OH)2-containing gutta-percha points yielded better filling of the apical third in roots with severe curvature.

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Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross

Nature Communications ◽

10.1038/s41467-019-12256-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Juliana M. Sá ◽

Sarah R. Kaslow ◽

Roberto R. Moraes Barros ◽

Nicholas F. Brazeau ◽

Christian M. Parobek ◽

...

Keyword(s):

Plasmodium Vivax ◽

Malaria Control ◽

Chloroquine Resistance ◽

Transcriptional Analysis ◽

Chromosome 1 ◽

In Vitro Cultivation ◽

Liver Stage ◽

Control Programs ◽

Parasite Populations

Abstract Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.

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A Novel Chimeric Plasmodium vivax Circumsporozoite Protein Induces Biologically Functional Antibodies That Recognize both VK210 and VK247 Sporozoites

Infection and Immunity ◽

10.1128/iai.01667-06 ◽

2006 ◽

Vol 75 (3) ◽

pp. 1177-1185 ◽

Cited By ~ 46

Author(s):

Anjali Yadava ◽

Jetsumon Sattabongkot ◽

Michael A. Washington ◽

Lisa A. Ware ◽

Victoria Majam ◽

...

Keyword(s):

Plasmodium Vivax ◽

Circumsporozoite Protein ◽

Biologically Active ◽

Histocompatibility Complex ◽

Successful Vaccine ◽

A Subunit ◽

Functional Antibodies

ABSTRACT A successful vaccine against Plasmodium vivax malaria would significantly improve the health and quality of the lives of more than 1 billion people around the world. A subunit vaccine is the only option in the absence of long-term culture of P. vivax parasites. The circumsporozoite protein that covers the surface of Plasmodium sporozoites is one of the best-studied malarial antigens and the most promising vaccine in clinical trials. We report here the development of a novel “immunologically optimal” recombinant vaccine expressed in Escherichia coli that encodes a chimeric CS protein encompassing repeats from the two major alleles, VK210 and VK247. This molecule is widely recognized by sera from patients naturally exposed to P. vivax infection and induces a highly potent immune response in genetically disparate strains of mice. Antibodies from immunized animals recognize both VK210 and VK247 sporozoites. Furthermore, these antibodies appear to be protective in nature since they cause the agglutination of live sporozoites, an in vitro surrogate of sporozoite infectivity. These results strongly suggest that recombinant CS is biologically active and highly immunogenic across major histocompatibility complex strains and raises the prospect that in humans this vaccine may induce protective immune responses.

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Safety, pharmacokinetics, and liver-stage Plasmodium cynomolgi effect of high-dose ivermectin and chloroquine in Rhesus Macaques

10.1101/2020.04.27.065409 ◽

2020 ◽

Author(s):

Pattaraporn Vanachayangkul ◽

Rawiwan Im-erbsin ◽

Anchalee Tungtaeng ◽

Chanikarn Kodchakorn ◽

Alison Roth ◽

...

Keyword(s):

Plasmodium Berghei ◽

Rhesus Macaques ◽

High Dose ◽

Radical Cure ◽

The Novel ◽

Liver Stage ◽

Plasmodium Cynomolgi ◽

Stage Development ◽

Pharmacokinetic Drug

AbstractPreviously, ivermectin (1–10 mg/kg) was shown to inhibit liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (IC50 = 10.42 μM) and hypnozoites (IC50 = 29.24 μM) in primary macaque hepatocytes when administered in high-dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for seven consecutive days was evaluated for prophylaxis or radical cure of Plasmodium cynomolgi liver-stages in Rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for seven days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

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Induction of specific immune responses against the Plasmodium vivax liver-stage via in vitro activation by dendritic cells

Parasitology International ◽

10.1016/j.parint.2006.04.001 ◽

2006 ◽

Vol 55 (3) ◽

pp. 187-193 ◽

Cited By ~ 10

Author(s):

Prachya Vichchathorn ◽

Rachaneeporn Jenwithisuk ◽

Surasak Leelaudomlipi ◽

Sumalee Tungpradabkul ◽

Suradej Hongeng ◽

...

Keyword(s):

Dendritic Cells ◽

Immune Responses ◽

Plasmodium Vivax ◽

Liver Stage ◽

In Vitro Activation

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Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00741-20 ◽

2020 ◽

Vol 64 (9) ◽

Author(s):

Pattaraporn Vanachayangkul ◽

Rawiwan Im-erbsin ◽

Anchalee Tungtaeng ◽

Chanikarn Kodchakorn ◽

Alison Roth ◽

...

Keyword(s):

Rhesus Macaques ◽

High Dose ◽

Radical Cure ◽

The Novel ◽

Liver Stage ◽

Content Type ◽

Plasmodium Cynomolgi ◽

Stage Development ◽

Pharmacokinetic Drug

ABSTRACT Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 μM) and hypnozoites (IC50, 29.24 μM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

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