Tamirat Gebru

Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ (‘PfSPZ Vaccine’); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ (‘PfSPZ Challenge’) to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 104 PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, and prevented infection in 9 out of 9 (100%) volunteers who underwent controlled human malaria infection ten weeks after the last dose (group III). Protective efficacy was dependent on dose and regimen. Immunization with 3.2 × 103 (group I) or 1.28 × 104 (group II) PfSPZ protected 3 out of 9 (33%) or 6 out of 9 (67%) volunteers, respectively. Three doses of 5.12 × 104 PfSPZ at five-day intervals protected 5 out of 8 (63%) volunteers. The frequency of Pf-specific polyfunctional CD4 memory T cells was associated with protection. On a 7,455 peptide Pf proteome array, immune sera from at least 5 out of 9 group III vaccinees recognized each of 22 proteins. PfSPZ-CVac is a highly efficacious vaccine candidate; when we are able to optimize the immunization regimen (dose, interval between doses, and drug partner), this vaccine could be used for combination mass drug administration and a mass vaccination program approach to eliminate malaria from geographically defined areas.

Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia

BackgroundIncreased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia.MethodsOf the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates.ResultsThe pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y pfmdr1 polymorphisms. All investigated P. falciparum clinical isolates from Southern and Eastern Ethiopia carried only a single copy of the mutant pfmdr1 gene.ConclusionThe study reports for the first time the return of chloroquine sensitive P. falciparum in Ethiopia. These findings support the rationale for the use of CQ-based combination drugs as a possible future alternative.

DSM265 for Plasmodium falciparum chemoprophylaxis: a randomised, double blinded, phase 1 trial with controlled human malaria infection

Summary Background A drug for causal (ie, pre-erythrocytic) prophylaxis of Plasmodium falciparum malaria with prolonged activity would substantially advance malaria control. DSM265 is an experimental antimalarial that selectively inhibits the parasite dihydroorotate dehydrogenase. DSM265 shows in vitro activity against liver and blood stages of P falciparum. We assessed the prophylactic activity of DSM265 against controlled human malaria infection (CHMI). Methods At the Institute of Tropical Medicine, Eberhard Karls University (Tübingen, Germany), healthy, malaria-naive adults were allocated to receive 400 mg DSM265 or placebo either 1 day (cohort 1A) or 7 days (cohort 2) before CHMI by direct venous inoculation (DVI) of 3200 aseptic, purified, cryopreserved P falciparum sporozoites (PfSPZ Challenge; Sanaria Inc, Rockville, MD, USA). An additional group received daily atovaquone-proguanil (250-100 mg) for 9 days, starting 1 day before CHMI (cohort 1B). Allocation to DSM265, atovaquone-proguanil, or placebo was randomised by an interactive web response system. Allocation to cohort 1A and 1B was open-label, within cohorts 1A and 2, allocation to DSM265 and placebo was double-blinded. All treatments were given orally. Volunteers were treated with an antimalarial on day 28, or when parasitaemic, as detected by thick blood smear (TBS) microscopy. The primary efficacy endpoint was time-to-parasitaemia, assessed by TBS. All participants receiving at least one dose of chemoprophylaxis or placebo were considered for safety, those receiving PfSPZ Challenge for efficacy analyses. Log-rank test was used to compare time-to-parasitemia between interventions. The trial was registered with ClinicalTrials.gov, number NCT02450578. Findings 22 participants were enrolled between Oct 23, 2015, and Jan 18, 2016. Five participants received 400 mg DSM265 and two participants received placebo 1 day before CHMI (cohort 1A), six participants received daily atovaquone-proguanil 1 day before CHMI (cohort 1B), and six participants received 400 mg DSM265 and two participants received placebo 7 days before CHMI (cohort 2). Five of five participants receiving DSM265 1 day before CHMI and six of six in the atovaquone-proguanil cohort were protected, whereas placebo recipients (two of two) developed malaria on days 11 and 14. When given 7 days before CHMI, three of six volunteers receiving DSM265 became TBS positive on days 11, 13, and 24. The remaining three DSM265-treated, TBS-negative participants of cohort 2 developed transient submicroscopic parasitaemia. Both participants receiving placebo 7 days before CHMI became TBS positive on day 11. The only possible DSM265-related adverse event was a moderate transient elevation in serum bilirubin in one participant. Interpretation A single dose of 400 mg DSM265 was well tolerated and had causal prophylactic activity when given 1 day before CHMI. Future trials are needed to investigate further the use of DSM265 for the prophylaxis of malaria. Funding Global Health Innovative Technology Fund, Wellcome Trust, Bill & Melinda Gates Foundation through Medicines for Malaria Venture, and the German Center for Infection Research.

Effect of Fluorescent Dyes on In Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

ABSTRACT Plasmodium falciparum gametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates of P. falciparum with a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly high in vitro activity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds.

Antimalarial Activity of the Myxobacterial Macrolide Chlorotonil A

ABSTRACT Myxobacteria are Gram-negative soil-dwelling bacteria belonging to the phylum Proteobacteria. They are a rich source of promising compounds for clinical application, such as epothilones for cancer therapy and several new antibiotics. In the course of a bioactivity screening program of secondary metabolites produced by Sorangium cellulosum strains, the macrolide chlorotonil A was found to exhibit promising antimalarial activity. Subsequently, we evaluated chlorotonil A against Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Chlorotonil A was highly active, with a 50% inhibitory concentration between 4 and 32 nM; additionally, no correlations between the activities of chlorotonil A and artesunate (rho, 0.208) or chloroquine (rho, −0.046) were observed. Per os treatment of Plasmodium berghei-infected mice with four doses of as little as 36 mg of chlorotonil A per kg of body weight led to the suppression of parasitemia with no obvious signs of toxicity. Chlorotonil A acts against all stages of intraerythrocytic parasite development, including ring-stage parasites and stage IV to V gametocytes, and it requires only a very short exposure to the parasite to exert its antimalarial action. Conclusively, chlorotonil A has an exceptional and unprecedented profile of action and represents an urgently required novel antimalarial chemical scaffold. Therefore, we propose it as a lead structure for further development as an antimalarial chemotherapeutic.

Molecular surveillance of mutations in the cytochrome b gene of Plasmodium falciparum in Gabon and Ethiopia

BackgroundAtovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone®) and inhibits the cytochrome bc1 complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance.MethodsThe prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed.ResultsFour/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found.ConclusionIn the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare.

Demography, maternal health and the epidemiology of malaria and other major infectious diseases in the rural department Tsamba-Magotsi, Ngounie Province, in central African Gabon

BackgroundSub-Saharan Africa is undergoing an epidemiological transition from a predominance of infectious diseases to non-communicable and lifestyle related conditions. However, the pace of this transition and the pattern of disease epidemiology are uneven between affluent urban and rural poor populations. To address this question for a remote rural region located in the central African rainforest region of Gabon, this study was conducted to assess reasons for health care attendance and to characterize the epidemiology of malaria and other major infectious diseases for the department of Tsamba Magotsi.MethodsMajor causes for health care attendance were collected from local hospital records. Cross sectional population based surveys were performed for the assessment of local malaria epidemiology. Pregnant women attending antenatal care services were surveyed as a sentinel population for the characterization of chronic viral and parasitic infections in the community.ResultsInfectious diseases were responsible for 71% (7469) of a total of 10,580 consultations at the formal health care sector in 2010. Overall, malaria – defined by clinical syndrome – remained the most frequent cause for health care attendance. A cross sectional malaria survey in 840 asymptomatic individuals residing in Tsamba Magotsi resulted in a Plasmodium spp. infection prevalence of 37%. The infection rate in 2–10 year old asymptomatic children – a standard measure for malaria endemicity – was 46% (100 of 217) with P. falciparum as predominant species (79%). Infection with other plasmodial species (P. ovale and P. malariae) presented most commonly as coinfections (23.2%). Prevalence of HIV, HBV, and syphilis were 6.2, 7.3, and 2.5%, respectively, in cross-sectional assessments of antenatal care visits of pregnant women. Urogenital schistosomiasis and the filarial pathogens Loa loa and Mansonella perstans are highly prevalent chronic parasitic infections affecting the local population.ConclusionsDespite major improvements in the accessibility of Tsamba Magotsi over the past decade the epidemiological transition does not appear to have majorly changed on the spectrum of diseases in this rural Gabonese population. The high prevalence of Plasmodium infection indicates a high burden of malaria related morbidity. Infectious diseases remain one of the most important health issues and further research activities in the field of tropical medicine and infectious diseases could help improve health care for the local population.

Recognition of Plasmodium falciparum mature gametocyte-infected erythrocytes by antibodies of semi-immune adults and malaria-exposed children from Gabon

BackgroundTransmission of malaria from man to mosquito depends on the presence of gametocytes, the sexual stage of Plasmodium parasites in the infected host. Naturally acquired antibodies against gametocytes exist and may play a role in controlling transmission by limiting the gametocyte development in the circulation or by interrupting gamete development and fertilization in the mosquito following ingestion. So far, most studies on antibody responses to sexual stage antigens have focused on a subset of gametocyte-surface antigens, even though inhibitory Ab responses to other gametocyte antigens might also play a role in controlling gametocyte density and fertility. Limited information is available on natural antibody response to the surfaces of gametocyte-infected erythrocytes.MethodsAb responses to surface antigens of erythrocytes infected by in vitro differentiated Plasmodium falciparum mature gametocytes were investigated in sera of semi-immune adults and malaria-exposed children. In addition, the effect of immunization with GMZ2, a blood stage malaria vaccine candidate, and the effect of intestinal helminth infection on the development of immunity to gametocytes of P. falciparum was evaluated in malaria-exposed children and adults from Gabon. Serum samples from two Phase I clinical trials conducted in Gabon were analysed by microscopic and flow-cytometric immunofluorescence assay.ResultsAdults had a higher Ab response compared to children. Ab reactivity was significantly higher after fixation and permeabilization of parasitized erythrocytes. Following vaccination with the malaria vaccine candidate GMZ2, anti-gametocyte Ab concentration decreased in adults compared to baseline. Ab response to whole asexual stage antigens had a significant but weak positive correlation to anti-gametocyte Ab responses in adults, but not in children. Children infected with Ascaris lumbricoides had a significantly higher anti-gametocyte Ab response compared to non-infected children.ConclusionThe current data suggest that antigens exposed on the gametocyte-infected red blood cells are recognized by serum antibodies from malaria-exposed children and semi-immune adults. This anti-gametocyte immune response may be influenced by natural exposure and vaccination. Modulation of the natural immune response to gametocytes by co-infecting parasites should be investigated further and may have an important impact on malaria control strategies.

Behavioural and clinical predictors for Loiasis

Background Loiasis is a vector-borne disease in Central and West Africa. While there is still uncertainty to what extent loiasis is responsible for population morbidity, individuals having both loiasis and onchocerciasis have a high risk of fatal encephalopathy when treatment (ie, ivermectin) for onchocerciasis is given. Therefore it is current policy that communities of high loiasis-burden are excluded from mass drug administration programmes of ivermectin. To address this treatment gap we present diagnostic scores, based on clinical and behavioural predictors that may help to rapidly identify sub-groups with loiasis within high-burden communities. Methods A cross-sectional survey was performed in the province of la Ngounie, Gabon between December 2015 and Februrary 2016 and 947 participants of all ages were recruited. Clinical parameters and behavioural exposure factors were ascertained by questionnaire-based interviews. Parasitological analysis of blood samples was performed for L. loa detection. Diagnostic scores consisting of clinical and behavioural factors were modelled to predict loiasis in sub-groups residing in endemic regions. Results Increasing sylvan exposure was identified as important risk factor for loiasis with adjusted odds ratios of 5.1 (95% confidence interval CI 2.6-9.9) for occasional forest exposure, 11.1 (95% CI 5.4-22.6) for frequent forest exposure and 25.7 (95% CI 12.5-52.9) for intensive forest exposure. Individuals with loiasis were 7.7 (95% CI 5.4-11.0) times more likely to report recurrent pruritus than those without loiasis. Reporting of regular daily exposure to the deep rain forest and recurrent pruritus was 9-fold (positive likelihood ratio 9.18; 95% CI: 6.39-13.18) more prevalent in individuals with loiasis than in controls. Concordantly, the absence of regular weekly forest exposure was associated with extremely low disease-likelihood (negative likelihood ratio 0.09; 95% CI 0.05-0.16). Conclusions These composite scores may serve as a simple tool to rapidly identify both those most and those least at risk of disease and may simplify loiasis control activities as well as screening procedures for studies on loiasis. Further, they may aid policy-makers to tailor the delivery of ivermectin mass drug administration for onchocerciasis control programmes more effectively and safely in regions of high loiasis-burden.