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1.
Malar J ; 21(1): 393, 2022 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-36564750

RESUMO

BACKGROUND: The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. The transcriptional profiling of P. cynomolgi liver stages was previously reported and revealed many important biological features of the parasite but left out the host response to malaria infection. METHODS: Previously published RNA sequencing data were used to quantify the expression of host genes in rhesus macaque hepatocytes infected with P. cynomolgi in comparison to either cells from uninfected samples or uninfected bystander cells. RESULTS: Although the dataset could not be used to resolve the transcriptional profile of hypnozoite-infected hepatocytes, it provided a snapshot of the host response to liver stage schizonts at 9-10 day post-infection and identified specific host pathways that are modulated during the exo-erythrocytic stage of P. cynomolgi. CONCLUSIONS: This study constitutes a valuable resource characterizing the hepatocyte response to P. cynomolgi infection and provides a framework to build on future research that aims at understanding hepatocyte-parasite interactions during relapsing malaria infection.


Assuntos
Malária , Parasitos , Plasmodium cynomolgi , Animais , Plasmodium cynomolgi/genética , Macaca mulatta/parasitologia , Hepatócitos/parasitologia , Malária/parasitologia , Fígado/parasitologia
2.
Anal Chem ; 92(9): 6667-6675, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32267675

RESUMO

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.


Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Luciferases/metabolismo , Malária/tratamento farmacológico , Plasmodium/efeitos dos fármacos , Animais , Células Cultivadas , Hepatócitos/efeitos dos fármacos , Hepatócitos/parasitologia , Medições Luminescentes , Macaca mulatta , Malária/diagnóstico por imagem , Imagem Óptica , Testes de Sensibilidade Parasitária
3.
Parasitology ; 145(1): 56-70, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27938428

RESUMO

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence that P. knowlesi is an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.


Assuntos
Modelos Animais de Doenças , Haplorrinos , Interações Hospedeiro-Parasita , Malária/parasitologia , Plasmodium knowlesi/fisiologia , Adaptação Biológica , Animais , Eritrócitos/parasitologia , Humanos , Macaca fascicularis , Macaca mulatta , Malária/imunologia , Malária/prevenção & controle , Malária/veterinária , Vacinas Antimaláricas/análise , Vacinas Antimaláricas/farmacologia , Doenças dos Macacos/imunologia , Doenças dos Macacos/parasitologia , Doenças dos Macacos/prevenção & controle , Plasmodium knowlesi/imunologia , Zoonoses/imunologia , Zoonoses/parasitologia , Zoonoses/prevenção & controle
4.
Antimicrob Agents Chemother ; 60(5): 2858-63, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26926645

RESUMO

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo.


Assuntos
Antimaláricos/uso terapêutico , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Plasmodium vivax/patogenicidade , Animais , Macaca mulatta , Camundongos , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Plasmodium vivax/efeitos dos fármacos , Esporozoítos/efeitos dos fármacos
5.
Antimicrob Agents Chemother ; 58(3): 1586-95, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24366744

RESUMO

Preventing relapses of Plasmodium vivax malaria 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 of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, 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 for P. vivax malaria prophylaxis and potentially a radical cure.


Assuntos
Antimaláricos/farmacologia , Imidazóis/farmacologia , Malária/tratamento farmacológico , Plasmodium cynomolgi/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Antimaláricos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Hepatócitos/parasitologia , Imidazóis/uso terapêutico , Técnicas In Vitro , Fígado/parasitologia , Macaca mulatta/parasitologia , Malária/parasitologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos ICR , Pirazinas/uso terapêutico , Esporozoítos/efeitos dos fármacos
6.
Vaccine ; 42(7): 1785-1792, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38365484

RESUMO

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax-induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10.


Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária Vivax , Parasitos , Animais , Coelhos , Proteínas de Protozoários/genética , Plasmodium vivax , Rafinose , Sulfatos , Proteínas de Membrana/genética , Antígenos de Protozoários/genética , Adjuvantes Imunológicos , Malária Falciparum/prevenção & controle , Plasmodium falciparum , Malária Vivax/prevenção & controle , Anticorpos Antiprotozoários
7.
Pathogens ; 12(9)2023 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-37764878

RESUMO

Plasmodium vivax causes the second highest number of malaria morbidity and mortality cases in humans. Several biological traits of this parasite species, including the formation of dormant stages (hypnozoites) that persist inside the liver for prolonged periods of time, present an obstacle for intervention measures and create a barrier for the elimination of malaria. Research into the biology of hypnozoites requires efficient systems for parasite transmission, liver stage cultivation and genetic modification. However, P. vivax research is hampered by the lack of an in vitro blood stage culture system, rendering it reliant on in vivo-derived, mainly patient, material for transmission and liver stage culture. This has also resulted in limited capability for genetic modification, creating a bottleneck in investigations into the mechanisms underlying the persistence of the parasite inside the liver. This bottleneck can be overcome through optimal use of the closely related and experimentally more amenable nonhuman primate (NHP) parasite, Plasmodium cynomolgi, as a model system. In this review, we discuss the genetic modification tools and liver stage cultivation platforms available for studying P. vivax persistent stages and highlight how their combined use may advance our understanding of hypnozoite biology.

8.
Cell Rep ; 42(7): 112727, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37392389

RESUMO

Dormancy enables relapsing malaria parasites, such as Plasmodium vivax and cynomolgi, to survive unfavorable conditions. It is enabled by hypnozoites, parasites remaining quiescent inside hepatocytes before reactivating and establishing blood-stage infection. We integrate omics approaches to explore gene-regulatory mechanisms underlying hypnozoite dormancy. Genome-wide profiling of activating and repressing histone marks identifies a few genes that get silenced by heterochromatin during hepatic infection of relapsing parasites. By combining single-cell transcriptomics, chromatin accessibility profiling, and fluorescent in situ RNA hybridization, we show that these genes are expressed in hypnozoites and that their silencing precedes parasite development. Intriguingly, these hypnozoite-specific genes mainly encode proteins with RNA-binding domains. We hence hypothesize that these likely repressive RNA-binding proteins keep hypnozoites in a developmentally competent but dormant state and that heterochromatin-mediated silencing of the corresponding genes aids reactivation. Exploring the regulation and exact function of these proteins hence could provide clues for targeted reactivation and killing of these latent pathogens.


Assuntos
Malária , Plasmodium cynomolgi , Humanos , Heterocromatina , Plasmodium cynomolgi/genética , Malária/parasitologia , Hepatócitos/parasitologia , Perfilação da Expressão Gênica
9.
Methods Mol Biol ; 2524: 397-408, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35821489

RESUMO

Malaria hypnozoites are dormant parasite stages that reside inside hepatocytes. Upon activation, these stages can resume growth, causing new episodes of blood stage malaria infection. This chapter describes a fast and sensitive protocol for the detection of bioluminescent (BL) hypnozoites in vitro. Using transgenic Plasmodium cynomolgi parasites that differentially express the BL reporter proteins firefly luciferase and the ultrabright NanoLuc, hypnozoites can be distinguished from liver stage schizonts. This robust method sets the stage for implementation in large-scale drug screening platforms with the aim to find new compounds that eliminate hypnozoites.


Assuntos
Malária , Plasmodium cynomolgi , Hepatócitos , Humanos , Luciferases/genética , Malária/diagnóstico , Malária/parasitologia , Plasmodium cynomolgi/fisiologia , Recidiva
10.
Infect Immun ; 78(3): 1032-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20048045

RESUMO

Both Plasmodium and Babesia species are intraerythrocytic protozoans that infect a wide range of hosts, including humans, and they elicit similar inflammatory responses and clinical manifestations that differ markedly in severity. We recently reported that a rhesus macaque that was chronically infected with Babesia microti was able to control infection with Plasmodium cynomolgi (a parasite of macaques with characteristics very similar to those of Plasmodium vivax) better than naïve monkeys. To confirm this and to investigate the underlying immunopathology, six naïve rhesus monkeys were infected with B. microti. After 24 days, four of these monkeys and four naïve rhesus monkeys were challenged with P. cynomolgi blood-stage parasites. B. microti persisted at low levels in all monkeys, and the clinical parameters were comparable to those of noninfected controls. There was a significant decrease in P. cynomolgi parasitemia in animals coinfected with B. microti compared to the parasitemia in animals infected with P. cynomolgi alone. This decrease in P. cynomolgi parasitemia correlated with increases in the levels of proinflammatory monocytes at the time of P. cynomolgi infection and with higher C-reactive protein (CRP) serum levels 1 week after malaria infection. Therefore, we conclude that ongoing infection with B. microti parasites leads to suppression of malaria infection.


Assuntos
Babesia microti/patogenicidade , Babesiose/complicações , Babesiose/patologia , Malária/complicações , Malária/patologia , Doenças dos Macacos/parasitologia , Plasmodium cynomolgi/patogenicidade , Animais , Babesiose/parasitologia , Sangue/imunologia , Sangue/parasitologia , Análise Química do Sangue , Proteína C-Reativa/análise , Modelos Animais de Doenças , Feminino , Humanos , Macaca mulatta , Malária/parasitologia , Monócitos/imunologia , Parasitemia
11.
Front Cell Infect Microbiol ; 10: 606033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33585277

RESUMO

Recent studies of liver stage malaria parasite-host interactions have provided exciting new insights on the cross-talk between parasite and its mammalian (predominantly rodent) host. We review the latest state of the art and and zoom in on new technologies that will provide the tools necessary to investigate host-parasite interactions of relapsing parasites. Interactions between hypnozoites and hepatocytes are particularly interesting because the parasite can remain in a quiescent state for prolonged periods of time and triggers for reactivation have not been irrefutably identified. If we learn more about the cross-talk between hypnozoite and host we may be able to identify factors that encourage waking up these dormant parasite reservoirs and help to achieve the total eradication of malaria.


Assuntos
Malária , Plasmodium cynomolgi , Animais , Hepatócitos , Interações Hospedeiro-Parasita , Fígado
12.
Commun Biol ; 3: 7, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31909199

RESUMO

Plasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches.


Assuntos
Genes Reporter , Malária/parasitologia , Plasmodium cynomolgi/fisiologia , Reinfecção/parasitologia , Proteínas de Fluorescência Verde/genética , Fígado/parasitologia , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/fisiologia , Plasmodium cynomolgi/genética
13.
Elife ; 82019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31094679

RESUMO

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.


Assuntos
Malária Vivax/genética , Plasmodium cynomolgi/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Aminoquinolinas/farmacologia , Animais , Antimaláricos/farmacologia , Biomarcadores/metabolismo , Biomarcadores Farmacológicos , Hepatócitos/metabolismo , Hepatócitos/parasitologia , Interações Hospedeiro-Parasita/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/parasitologia , Macaca mulatta/genética , Macaca mulatta/parasitologia , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Plasmodium cynomolgi/parasitologia , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/patogenicidade , Proteínas de Protozoários/metabolismo , Esporozoítos/genética , Transcriptoma/efeitos dos fármacos
14.
Elife ; 72018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30589413

RESUMO

Relapses of Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.


Assuntos
Perfilação da Expressão Gênica , Fígado/parasitologia , Plasmodium cynomolgi/crescimento & desenvolvimento , Plasmodium cynomolgi/genética , Animais , Primatas , Fatores de Tempo
15.
Wellcome Open Res ; 2: 42, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28748222

RESUMO

BACKGROUND: Plasmodium cynomolgi, a non-human primate malaria parasite species, has been an important model parasite since its discovery in 1907. Similarities in the biology of P. cynomolgi to the closely related, but less tractable, human malaria parasite P. vivax make it the model parasite of choice for liver biology and vaccine studies pertinent to P. vivax malaria. Molecular and genome-scale studies of P. cynomolgi have relied on the current reference genome sequence, which remains highly fragmented with 1,649 unassigned scaffolds and little representation of the subtelomeres.  Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated a new reference genome sequence, PcyM, sourced from an Indian rhesus monkey. We compare the newly assembled genome sequence with those of several other Plasmodium species, including a re-annotated P. coatneyi assembly. RESULTS: The new PcyM genome assembly is of significantly higher quality than the existing reference, comprising only 56 pieces, no gaps and an improved average gene length. Detailed manual curation has ensured a comprehensive annotation of the genome with 6,632 genes, nearly 1,000 more than previously attributed to P. cynomolgi. The new assembly also has an improved representation of the subtelomeric regions, which account for nearly 40% of the sequence. Within the subtelomeres, we identified more than 1300 Plasmodium interspersed repeat ( pir) genes, as well as a striking expansion of 36 methyltransferase pseudogenes that originated from a single copy on chromosome 9. CONCLUSIONS: The manually curated PcyM reference genome sequence is an important new resource for the malaria research community. The high quality and contiguity of the data have enabled the discovery of a novel expansion of methyltransferase in the subtelomeres, and illustrates the new comparative genomics capabilities that are being unlocked by complete reference genomes.

16.
Elife ; 62017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215331

RESUMO

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.


Assuntos
Perfilação da Expressão Gênica , Fígado/parasitologia , Macaca mulatta/parasitologia , Plasmodium cynomolgi/crescimento & desenvolvimento , Plasmodium cynomolgi/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/genética , Animais , Feminino , Masculino
17.
PLoS One ; 9(1): e83704, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24421900

RESUMO

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.


Assuntos
Formação de Anticorpos/imunologia , Haplorrinos/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Antígenos de Protozoários/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Haplorrinos/sangue , Haplorrinos/parasitologia , Humanos , Imunização , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Parasitemia/imunologia , Parasitemia/parasitologia , Proteínas Recombinantes/imunologia
18.
PLoS One ; 8(1): e54888, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23359816

RESUMO

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.


Assuntos
Antimaláricos/farmacologia , Fígado/parasitologia , Plasmodium cynomolgi/fisiologia , Animais , Animais Geneticamente Modificados , Atovaquona/farmacologia , Fluorescência , Humanos , Plasmodium cynomolgi/efeitos dos fármacos , Plasmodium cynomolgi/crescimento & desenvolvimento , Primaquina/farmacologia
19.
Trends Parasitol ; 25(8): 370-4, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19635679

RESUMO

Plasmodium knowlesi is a primate malaria parasite that is phylogenetically close to the major human parasite Plasmodium vivax. P. knowlesi causes life-threatening disease in humans, infects a wide range of non-human primates and is one of few malaria parasites amenable to cyclical in vitro propagation. A robust in vivo and in vitro genetic manipulation system has been developed for this parasite, enabling in vitro-in vivo shuttling of transgenes, which (together with recent characterization of its genome and that of its macaque experimental host) offers unique opportunities to gain insight in molecular function and parasite-host interactions.


Assuntos
Modelos Animais de Doenças , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Malária , Organismos Geneticamente Modificados , Plasmodium knowlesi , Projetos de Pesquisa , Animais , Genômica , Humanos , Macaca mulatta , Malária/parasitologia , Malária/fisiopatologia , Plasmodium knowlesi/genética , Plasmodium knowlesi/crescimento & desenvolvimento , Plasmodium knowlesi/patogenicidade , Plasmodium knowlesi/fisiologia , Proteômica
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