Continuous Supply of Plasmodium vivax Sporozoites from Colonized Anopheles darlingi in the Peruvian Amazon.

In vitro culture of Plasmodium vivax liver stages underlies key understandings of the fundamental biology of this parasite, particularly the latent, hyponozoite stage, toward drug and vaccine development. Here, we report systematic production of Plasmodium vivax sporozoites in colonized Anopheles darlingi mosquitoes in the Peruvian Amazon. Human subject-derived P. vivax-infected blood was fed to Anopheles darlingi females using standard membrane feedings assays. Optimizing A. darlingi infection and sporozoite production included replacement of infected patient donor serum with naïve donor serum, comparing anticoagulants in processing blood samples, and addition of penicillin-streptomycin and ATP to infectious blood meals. Replacement of donor serum by naïve serum in the P. vivax donor blood increased oocysts in the mosquito midgut, and heparin, as anticoagulant, was associated with the highest sporozoite yields. Maintaining blood-fed mosquitoes on penicillin-streptomycin in sugar significantly extended mosquito survival which enabled greater sporozoite yield. In this study, we have shown that a robust P. vivax sporozoite production is feasible in a malaria-endemic setting where infected subjects and a stable A. darlingi colony are brought together, with optimized laboratory conditions

Exploring a Tetrahydroquinoline Antimalarial Hit from the Medicines for Malaria Pathogen Box and Identification of its Mode of Resistance as PfeEF2

New antimalarial treatments with novel mechanism of action are needed to tackle Plasmodium falciparum infections that are resistant to first-line therapeutics. Here we report the exploration of MMV692140 ( 2 ) from the Pathogen Box, a collection of 400 compounds that was made available by Medicines for Malaria Venture (MMV) in 2015. Compound 2 was profiled in in vitro models of malaria and was found to be active against multiple life-cycle stages of Plasmodium parasites. The mode of resistance, and putatively its mode of action, was identified as Plasmodium falciparum translation elongation factor 2 ( Pf eEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA. The compound maintains activity against a series of drug-resistant parasite strains. The structural motif of the tetrahydroquinoline ( 2 ) was explored in a chemistry program with its structure-activity relationships examined, resulting in the identification of an analog with 30-fold improvement of antimalarial asexual blood stage potency

Genetic diversity of Plasmodium vivax populations: analysis of neutral genetic markers and genes potentially associated with drug resistance.

Através da análise de microssatélites e SNPs, incluindo tanto marcadores neutros como sujeitos a seleção, neste trabalho se demonstrou que: 1) As populações brasileiras de P. vivax (Pv) são mais diversas que as populações simpátricas de P. falciparum (Pf), porém ambas apresentam desequilíbrio de ligação. 2) Os polimorfismos neutros apresentam alta variabilidade ao longo do tempo em ambas as espécies, em contraste com a estabilidade observada nos marcadores sob seleção. 3) As altas taxas de recorrências por Pv na Amazônia brasileira são causadas, em sua maioria, por parasitas geneticamente não relacionados. 4) A recombinação não meiótica contribui substancialmente à diversidade dos domínios repetitivos de PvCSP. 5) Os SNPs nos genes pvmdr1 e pvcrt-o de isolados de Pv resistentes à cloroquina não se associam ao seu fenótipo in vivo. Finalmente, através da genotipagem de 57 SNPs do cromossomo 8 de Pv em 234 isolados do Brasil e da Ásia observou-se que, em Pv, a diversidade e a recombinação mitótica não se associam aos níveis de endemicidade como acontece em Pf e que Pv apresenta estrutura populacional continental e subcontinental no Brasil.Through the analysis of microsatellites and SNPs, including both neutral and under selection markers, this work showed that: 1) The Brazilian populations of P. vivax (Pv) are more diverse than sympatric P. falciparum (Pf) populations, but both exhibit linkage disequilibrium. 2) For both species, neutral polymorphisms showed high variability over time, contrasting with the stability of under markers under selection. 3) The high rate of Pv recurrences in the Brazilian Amazon region is mostly due to genetically unrelated parasites. 4) Non-meiotic recombination substantially contributes to PvCSP repetitive domain diversity. 5) SNPs at pvmdr1 and pvcrt-o genes of chloroquine resistant isolates of Pv are not associated with the in vivo phenotype. Finally, 57 SNP genotyping of chromosome 8 of Pv among 234 isolates from Brazil and Asia showed that, in Pv, diversity and mitotic recombination are not associated with levels of endemicity as in Pf and that Pv presents continental population structure and subcontinental structure in Brazil

Plasmodium vivax: Microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections. (C) 2008 Elsevier Inc. All rights reserved.Fundacao de Apoio a Pesquisa do Estado de Sao Paulo (FAPESP)[07/51199-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)[470570/2006-7

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.Fundacao de Apoio a Pesquisa do Estado de Sao Paulo, Brazil (FAPESP)[07/51199-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil (CNPq)[470570/2006-7]U.S. National Institutes of Health (NIH)National Institutes of Health of USA (NIH)[AI 075416-01

Recurrent Parasitemias and Population Dynamics of Plasmodium vivax Polymorphisms in Rural Amazonia

Clinical trials documented alarming post-treatment Plasmodium vivax recurrence rates caused by recrudescence of surviving asexual blood stages, relapse from hypnozoites, or new infections. Here we describe high rates of P vivax recurrence (26-40% 180 days after treatment) in two cohorts of rural Amazonians exposed to low levels of malaria transmission after a vivax malaria episode treated with chloroquine-primaquine. Microsatellite analysis of 28 paired acute infection and recurrence parasites showed only two pairs of identical haplotypes (consistent with recrudescences or reactivation of homologous hypnozoites) and four pairs of related haplotypes (sharing alleles at 11-13 of 14 microsatellites analyzed). Local isolates of P vivax were extraordinarily diverse and rarely shared the same haplotype, indicating that frequent recurrences did not favor the persistence or reappearance of clonal lineages of parasites in the Population. This fast haplotype replacement rate may represent the typical population dynamics Of neutral polymorphisms in parasites from low-endemicity areas.National Institutes of Health (NIH)[RO1 AI 075416-01]U.S. National Institutes of Health (NIH)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico c Tecnologico (CNPq)[470570/2006-7]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[05//51988-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[07/51199-0

Slow and continuous delivery of a low dose of nimodipine improves survival and electrocardiogram parameters in rescue therapy of mice with experimental cerebral malaria

Submitted by sandra infurna ([email protected]) on 2016-02-02T15:24:00Z No. of bitstreams: 1 leonardo2_carvalho_etal_IOC_2013.pdf: 1312016 bytes, checksum: 1d0000f743d99eba7d206bada2bc59e5 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-02-02T15:46:53Z (GMT) No. of bitstreams: 1 leonardo2_carvalho_etal_IOC_2013.pdf: 1312016 bytes, checksum: 1d0000f743d99eba7d206bada2bc59e5 (MD5)Made available in DSpace on 2016-02-02T15:46:53Z (GMT). No. of bitstreams: 1 leonardo2_carvalho_etal_IOC_2013.pdf: 1312016 bytes, checksum: 1d0000f743d99eba7d206bada2bc59e5 (MD5) Previous issue date: 2013La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA / Universidade Federal do Rio de Janeiro (UFRJ). Instituto de Microbiologia Professor Paulo de Góes. Laboratório de Inflamação e Imunologia. Rio de Janeiro, RJ, Brasil.La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA.La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA.La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA /Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Serviço de Parasitologia. Rio de Janeiro, RJ, Brasil.La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA.La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA.La Jolla Bioengineering Institute. Center for Malaria Research. San Diego, CA, USA / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Background: Human cerebral malaria (HCM) is a life-threatening complication caused by Plasmodium falciparum infection that continues to be a major global health problem despite optimal anti-malarial treatment. In the experimental model of cerebral malaria (ECM) by Plasmodium berghei ANKA, bolus administration of nimodipine at high doses together with artemether, increases survival of mice with ECM. However, the dose and administration route used is associated with cardiovascular side effects such as hypotension and bradycardia in humans and mice, which could preclude its potential use as adjunctive treatment in HCM. Methods: In the present study, alternative delivery systems for nimodipine during late-stage ECM in association with artesunate were searched to define optimal protocols to achieve maximum efficacy in increasing survival in rescue therapy while causing the least cardiac side effects. The baseline electrocardiogram (ECG) and arterial pressure characteristics of uninfected control animals and of mice with ECM and its response upon rescue treatment with artesunate associated or not with nimodipine is also analysed. Results: Nimodipine, given at 0.5 mg/kg/day via a slow and continuous delivery system by osmotic pumps, increases survival of mice with ECM when used as adjunctive treatment to artesunate. Mice with ECM showed hypotension and ECG changes, including bradycardia and increases in PR, QRS, QTc and ST interval duration. ECM mice also show increased QTc dispersion, heart rate variability (HRV), RMSSD, low frequency (LF) and high frequency (HF) bands of the power spectrum. Both sympathetic and parasympathetic inputs to the heart were increased, but there was a predominance of sympathetic tone as demonstrated by an increased LF/HF ratio. Nimodipine potentiated bradycardia when given by bolus injection, but not when via osmotic pumps. In addition, nimodipine shortened PR duration and improved HRV, RMSSD, LF and HF powers in mice with ECM. In addition, nimodipine did not increased hypotension or decreased the speed of arterial pressure recovery when used in rescue therapy with artesunate. Conclusions: These data show that slow and continuous delivery of lower doses of nimodipine improves survival of mice with ECM in rescue therapy with artesunate while showing a safer profile in terms of cardiovascular effects

Human nuclear hormone receptor activity contributes to malaria parasite liver stage development.

Chemical genetic approaches have had a transformative impact on discovery of drug targets for malaria but have primarily been used for parasite targets. To identify human pathways required for intrahepatic development of parasite, we implemented multiplex cytological profiling of malaria infected hepatocytes treated with liver stage active compounds. Some compounds, including MMV1088447 and MMV1346624, exhibited profiles similar to cells treated with nuclear hormone receptor (NHR) agonist/antagonists. siRNAs targeting human NHRs, or their signaling partners identified eight genes that were critical for Plasmodium berghei infection. Knockdown of NR1D2, a host NHR, significantly impaired parasite growth by downregulation of host lipid metabolism. Importantly, treatment with MMV1088447 and MMV1346624 but not other antimalarials, phenocopied the lipid metabolism defect of NR1D2 knockdown. Our data underlines the use of high-content imaging for host-cellular pathway deconvolution, highlights host lipid metabolism as a drug-able human pathway and provides new chemical biology tools for studying host-parasite interactions

Continuous Supply of <i>Plasmodium vivax</i> Sporozoites from Colonized <i>Anopheles darlingi</i> in the Peruvian Amazon

<i>In vitro</i> culture of <i>Plasmodium vivax</i> liver stages underlies key understandings of the fundamental biology of this parasite, particularly the latent, hyponozoite stage, toward drug and vaccine development. Here, we report systematic production of <i>Plasmodium vivax</i> sporozoites in colonized <i>Anopheles darlingi</i> mosquitoes in the Peruvian Amazon. Human subject-derived <i>P. vivax</i>-infected blood was fed to <i>Anopheles darlingi</i> females using standard membrane feedings assays. Optimizing <i>A. darlingi</i> infection and sporozoite production included replacement of infected patient donor serum with naïve donor serum, comparing anticoagulants in processing blood samples, and addition of penicillin–streptomycin and ATP to infectious blood meals. Replacement of donor serum by naïve serum in the <i>P. vivax</i> donor blood increased oocysts in the mosquito midgut, and heparin, as anticoagulant, was associated with the highest sporozoite yields. Maintaining blood-fed mosquitoes on penicillin–streptomycin in sugar significantly extended mosquito survival which enabled greater sporozoite yield. In this study, we have shown that a robust <i>P. vivax</i> sporozoite production is feasible in a malaria-endemic setting where infected subjects and a stable <i>A. darlingi</i> colony are brought together, with optimized laboratory conditions