Search

Objectives Eravacycline is a novel synthetic halogenated tetracycline derivative with a broad antibacterial spectrum. Antibiotics, including tetracyclines, have been used for prophylaxis and, more rarely, for the treatment of malaria for several decades. The rise in drug-resistant malaria parasites renders the search for new treatment candidates urgent. We determined the in vitro potency of eravacycline against Plasmodium falciparum and investigated the apicoplast as a potential drug target. Methods Four tetracyclines, including eravacycline, tetracycline, tigecycline, and doxycycline, and the lincosamide clindamycin, were tested in 3-day and 6-day in vitro susceptibility assays of P. falciparum laboratory strain 3D7 and/or of clinical isolates obtained from 33 P. falciparum infected individuals from Gabon in 2018. Assays with isopentenyl pyrophosphate substitution were performed to investigate whether apicoplast-encoded isoprenoid biosynthesis is inhibited by these antibiotics. Results Eravacycline showed the highest activity of all tetracyclines tested in clinical isolates in the 3-day and 6-day assays. Substitution of isopentenyl pyrophosphate in vitro using the laboratory strain 3D7 reversed the activity of eravacycline and comparator antibiotics, indicating the apicoplast to be the main target organelle. Conclusions These results demonstrate the potential of novel antibiotics, and eravacycline, as candidate antimalarial therapies.

SummaryMedical research in sub-Saharan Africa is of high priority for societies to respond adequately to local health needs. Often enough it remains a challenge to build up capacity in infrastructure and human resources to highest international standards and to sustain this over mid-term to long-term periods due to difficulties in obtaining long-term institutional core funding, attracting highly qualified scientists for medical research and coping with ever changing structural and political environments. The Centre de Recherches Médicales de Lambaréné (CERMEL) serves as model for how to overcome such challenges and to continuously increase its impact on medical care in Central Africa and beyond. Starting off as a research annex to the Albert Schweitzer Hospital in Lambaréné, Gabon, it has since then expanded its activities to academic and regulatory clinical trials for drugs, vaccines and diagnostics in the field of malaria, tuberculosis, and a wide range of poverty related and neglected tropical infectious diseases. Advancing bioethics in medical research in Africa and steadily improving its global networks and infrastructures, CERMEL serves as a reference centre for several international consortia. In close collaboration with national authorities, CERMEL has become one of the main training hubs for medical research in Central Africa. It is hoped that CERMEL and its leitmotiv “to improve medical care for local populations” will serve as an inspiration to other institutions in sub-Saharan Africa to further increase African capacity to advance medicine.

Ivermectin is safe and widely used for treating helminth infections. It also kills arthropods feeding on treated subjects, including malaria vectors. Thus, ivermectin mass drug administration as an additional tool for malaria control is being evaluated by WHO. As in vitro data, animal experiments and epidemiological observations suggest that ivermectin has a direct effect on the liver stages of the malaria parasite, this study was designed to assess the prophylactic effect of ivermectin on Plasmodium falciparum controlled human malaria infection.A total of 4 volunteers were randomised to placebo, and 8 volunteers were randomised to receive ivermectin 0.4 mg/kg, orally, once 2 h before being experimentally infected intravenously with 3200 P. falciparum sporozoites. The primary endpoint was time to parasitaemia detected by positive thick blood smear; RT-qPCR was performed in parallel.All but one volunteer became thick blood smear positive between day 11 and day 12 after infection, and there was no significant effect of ivermectin on parasitaemia.Ivermectin - at the dose used - has no clinically relevant activity against the pre-erythrocytic stages of P. falciparum.L'ivermectine est sûr et largement utilisé pour traiter les helminthiases. Il tue également les arthropodes se nourrissant sur les sujets traités, y compris les vecteurs du paludisme. Ainsi, l'administration en masse d'ivermectine en tant qu'outil supplémentaire de lutte contre le paludisme est actuellement évaluée par l'OMS. Comme les données in vitro, les expériences sur animaux et les observations épidémiologiques suggèrent que l'ivermectine a un effet direct sur les stades hépatiques du parasite du paludisme, cette étude a été conçue pour évaluer l'effet prophylactique de l'ivermectine sur l'infection paludéenne humaine par Plasmodium falciparum contrôlée. MÉTHODES: Quatre volontaires ont été randomisés pour un placebo et 8 volontaires ont été randomisés pour recevoir de l'ivermectine à 0,4 mg/kg en une fois par voie orale, 2 heures avant d'être expérimentalement infectés par voie intraveineuse avec 3.200 sporozoïtes de P. falciparum. Le critère d'évaluation principal était le temps à la parasitémie détectée par un frottis sanguin épais positif. Une RT-qPCR a été réalisée en parallèle. RÉSULTATS: Tous les volontaires sauf un sont devenus positifs pour les frottis sanguins épais entre le jour 11 et le jour 12 après l'infection et il n'y avait aucun effet significatif de l'ivermectine sur la parasitémie.L'ivermectine - à la dose utilisée - n'a aucune activité cliniquement pertinente contre les stades pré-érythrocytaires de P. falciparum.

Background:In a phase 3 trial in African infants and children, the RTS,S/AS01 vaccine (GSK) showed moderate efficacy against clinical malaria. We sought to further understand RTS,S/AS01-induced immune responses associated with vaccine protection.Methods:Applying the blood transcriptional module (BTM) framework, we characterized the transcriptomic response to RTS,S/AS01 vaccination in antigen-stimulated (and vehicle control) peripheral blood mononuclear cells sampled from a subset of trial participants at baseline and month 3 (1-month post-third dose). Using a matched case–control study design, we evaluated which of these ‘RTS,S/AS01 signature BTMs’ associated with malaria case status in RTS,S/AS01 vaccinees. Antigen-specific T-cell responses were analyzed by flow cytometry. We also performed a cross-study correlates analysis where we assessed the generalizability of our findings across three controlled human malaria infection studies of healthy, malaria-naive adult RTS,S/AS01 recipients.Results:RTS,S/AS01 vaccination was associated with downregulation of B-cell and monocyte-related BTMs and upregulation of T-cell-related BTMs, as well as higher month 3 (vs. baseline) circumsporozoite protein-specific CD4+ T-cell responses. There were few RTS,S/AS01-associated BTMs whose month 3 levels correlated with malaria risk. In contrast, baseline levels of BTMs associated with dendritic cells and with monocytes (among others) correlated with malaria risk. The baseline dendritic cell- and monocyte-related BTM correlations with malaria risk appeared to generalize to healthy, malaria-naive adults.Conclusions:A prevaccination transcriptomic signature associates with malaria in RTS,S/AS01-vaccinated African children, and elements of this signature may be broadly generalizable. The consistent presence of monocyte-related modules suggests that certain monocyte subsets may inhibit protective RTS,S/AS01-induced responses.Funding:Funding was obtained from the NIH-NIAID (R01AI095789), NIH-NIAID (U19AI128914), PATH Malaria Vaccine Initiative (MVI), and Ministerio de Economía y Competitividad (Instituto de Salud Carlos III, PI11/00423 and PI14/01422). The RNA-seq project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute. This study was also supported by the Vaccine Statistical Support (Bill and Melinda Gates Foundation award INV-008576/OPP1154739 to R.G.). C.D. was the recipient of a Ramon y Cajal Contract from the Ministerio de Economía y Competitividad (RYC-2008-02631). G.M. was the recipient of a Sara Borrell–ISCIII fellowship (CD010/00156) and work was performed with the support of Department of Health, Catalan Government grant (SLT006/17/00109). This research is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces and we acknowledge support from the Spanish Ministry of Science and Innovation through the ‘Centro de Excelencia Severo Ochoa 2019–2023’ Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

Boromycin is a boron-containing macrolide antibiotic produced by Streptomyces antibioticus with potent activity against certain viruses, Gram-positive bacteria and protozoan parasites. Most antimalarial antibiotics affect plasmodial organelles of prokaryotic origin and have a relatively slow onset of action. They are used for malaria prophylaxis and for the treatment of malaria when combined to a fast-acting drug. Despite the success of artemisinin combination therapies, the current gold standard treatment, new alternatives are constantly needed due to the ability of malaria parasites to become resistant to almost all drugs that are in heavy clinical use. In vitro antiplasmodial activity screens of tetracyclines (omadacycline, sarecycline, methacycline, demeclocycline, lymecycline, meclocycline), macrolides (oleandomycin, boromycin, josamycin, troleandomycin), and control drugs (chloroquine, clindamycin, doxycycline, minocycline, eravacycline) revealed boromycin as highly potent against Plasmodium falciparum and the zoonotic Plasmodium knowlesi. In contrast to tetracyclines, boromycin rapidly killed asexual stages of both Plasmodium species already at low concentrations (~ 1 nM) including multidrug resistant P. falciparum strains (Dd2, K1, 7G8). In addition, boromycin was active against P. falciparum stage V gametocytes at a low nanomolar range (IC50: 8.5 ± 3.6 nM). Assessment of the mode of action excluded the apicoplast as the main target. Although there was an ionophoric activity on potassium channels, the effect was too low to explain the drug´s antiplasmodial activity. Boromycin is a promising antimalarial candidate with activity against multiple life cycle stages of the parasite.

Contains fulltext : 252097.pdf (Publisher’s version ) (Open Access) Peptidylarginine deiminase 4 (PAD4) is a key regulator of inflammation but its function in infections remains incompletely understood. We investigate PAD4 in the context of malaria and demonstrate a role in regulation of immune cell trafficking and chemokine production. PAD4 regulates liver immunopathology by promoting neutrophil trafficking in a Plasmodium chabaudi mouse malaria model. In human macrophages, PAD4 regulates expression of CXCL chemokines in response to stimulation with TLR ligands and P. falciparum. Using patient samples, we show that CXCL1 may be a biomarker for severe malaria. PAD4 inhibition promotes disease tolerance and may represent a therapeutic avenue in malaria.

The RTS,S/AS01E vaccine targets the circumsporozoite protein (CSP) of the Plasmodium falciparum parasite. Using protein microarrays, levels of IgG to 1,000 P. falciparum antigens were measured in 2,138 infants (age 6-12 weeks) and children (age 5-17 months) from 6 African sites of the phase 3 trial, sampled before and at four longitudinal visits after vaccination. One month post-vaccination, IgG responses to 17% of all probed antigens showed differences between RTS,S/AS01E and comparator vaccination groups, whereas no pre-vaccination differences were found. A small subset of antigens presented IgG levels reaching 4- to 8-fold increases in the RTS,S/AS01E group, comparable in magnitude to anti-CSP IgG levels (∼11-fold increase). They were strongly cross-correlated and correlated with anti-CSP levels, waning similarly over time and re-increasing with the booster dose. Such an intriguing phenomenon may be due to cross-reactivity of anti-CSP antibodies with these antigens. RTS,S/AS01E vaccinees with strong off-target IgG responses had an estimated lower clinical malaria incidence after adjusting for age group, site and post-vaccination anti-CSP levels. RTS,S/AS01E-induced IgG may bind strongly not only to CSP, but to unrelated malaria antigens, and this seems to either confer, or at least be a marker of, increased protection from clinical malaria.

Controlled human malaria infection (CHMI) has supported Plasmodium falciparum (Pf) malaria vaccine development by providing preliminary estimates of vaccine efficacy (VE). Because CHMIs generally use Pf strains similar to vaccine strains, VE against antigenically heterogeneous Pf in the field has been required to establish VE. We increased the stringency of CHMI by selecting a Brazilian isolate, Pf7G8, which is genetically distant from the West African parasite (PfNF54) in our PfSPZ vaccines. Using two regimens to identically immunize US and Malian adults, VE over 24 weeks in the field was as good as or better than against CHMI at 24 weeks in the US. To explain this finding, we quantified differences in the genome, proteome and predicted CD8 T cell epitopes of PfNF54 relative to 709 Pf isolates from Africa and Pf7G8. Pf7G8 is more distant from PfNF54 than any African isolates tested. We propose VE against Pf7G8 CHMI for providing pivotal data for malaria vaccine licensure for travelers to Africa, and potentially for endemic populations, because the genetic distance of Pf7G8 from the Pf vaccine strain makes it a stringent surrogate for Pf parasites in Africa.

Boromycin is a boron-containing macrolide antibiotic produced by Streptomyces antibioticus with potent activity against certain viruses, Gram-positive bacteria and protozoan parasites. Most antimalarial antibiotics affect plasmodial organelles of prokaryotic origin and have a relatively slow onset of action. They are used for malaria prophylaxis and for the treatment of malaria when combined to a fast-acting drug. Despite the success of artemisinin combination therapies, the current gold standard treatment, new alternatives are constantly needed due to the ability of malaria parasites to become resistant to almost all drugs that are in heavy clinical use. In vitro antiplasmodial activity screens of tetracyclines (omadacycline, sarecycline, methacycline, demeclocycline, lymecycline, meclocycline), macrolides (oleandomycin, boromycin, josamycin, troleandomycin), and control drugs (chloroquine, clindamycin, doxycycline, minocycline, eravacycline) revealed boromycin as highly potent against Plasmodium falciparum and the zoonotic Plasmodium knowlesi. In contrast to tetracyclines, boromycin rapidly killed asexual stages of both Plasmodium species already at low concentrations (~ 1 nM) including multidrug resistant P. falciparum strains (Dd2, K1, 7G8). In addition, boromycin was active against P. falciparum stage V gametocytes at a low nanomolar range (IC50: 8.5 ± 3.6 nM). Assessment of the mode of action excluded the apicoplast as the main target. Although there was an ionophoric activity on potassium channels, the effect was too low to explain the drug´s antiplasmodial activity. Boromycin is a promising antimalarial candidate with activity against multiple life cycle stages of the parasite.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria., Here, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to differences in the ability to induce FcγRIIIa-dependent natural killer cell degranulation.

Malaria reflects not only a state of immune activation, but also a state of general immune defect or immunosuppression, of complex etiology that can last longer than the actual episode. Inhabitants of malaria-endemic regions with lifelong exposure to the parasite show an exhausted or immune regulatory profile compared to non- or minimally exposed subjects. Several studies and experiments to identify and characterize the cause of this malaria-related immunosuppression have shown that malaria suppresses humoral and cellular responses to both homologous (Plasmodium) and heterologous antigens (e.g., vaccines). However, neither the underlying mechanisms nor the relative involvement of different types of immune cells in immunosuppression during malaria is well understood. Moreover, the implication of the parasite during the different stages of the modulation of immunity has not been addressed in detail. There is growing evidence of a role of immune regulators and cellular components in malaria that may lead to immunosuppression that needs further research. In this review, we summarize the current evidence on how malaria parasites may directly and indirectly induce immunosuppression and investigate the potential role of specific cell types, effector molecules and other immunoregulatory factors.

Malaria reflects not only a state of immune activation, but also a state of general immune defect or immunosuppression, of complex etiology that can last longer than the actual episode. Inhabitants of malaria-endemic regions with lifelong exposure to the parasite show an exhausted or immune regulatory profile compared to non- or minimally exposed subjects. Several studies and experiments to identify and characterize the cause of this malaria-related immunosuppression have shown that malaria suppresses humoral and cellular responses to both homologous (Plasmodium) and heterologous antigens (e.g., vaccines). However, neither the underlying mechanisms nor the relative involvement of different types of immune cells in immunosuppression during malaria is well understood. Moreover, the implication of the parasite during the different stages of the modulation of immunity has not been addressed in detail. There is growing evidence of a role of immune regulators and cellular components in malaria that may lead to immunosuppression that needs further research. In this review, we summarize the current evidence on how malaria parasites may directly and indirectly induce immunosuppression and investigate the potential role of specific cell types, effector molecules and other immunoregulatory factors.

Dear Editor,Viruses represent the major cause of febrile consultations in children across sub-Saharan Africa [1,2], but considering their high number and the need of specific molecular tools target...

Background: The transcriptome of Plasmodium falciparum clinical isolates varies according to strain, mosquito bites, disease severity and clinical history. Therefore, it remains a challenge to directly interpret the parasite's transcriptomic information into a more general biological signature in a natural human malaria infection. These confounding variations can be potentially overcome with parasites derived from controlled-human malaria infection (CHMI) studies. Methods: We performed CHMI studies in healthy and immunologically naïve volunteers receiving the same P. falciparum strain ((Sanaria® PfSPZ Challenge (NF54)), but with different sporozoite dosage and route of infection. Parasites isolated from these volunteers at the day of patency were subjected to in vitro culture for several generations and synchronized ring-stage parasites were subjected to transcriptome profiling. Findings: We observed clear deviations between CHMI-derived parasites from volunteer groups receiving different PfSPZ dose and route. CHMI-derived parasites and the pre-mosquito strain used for PfSPZ generation showed significant transcriptional variability for gene clusters associated with malaria pathogenesis, immune evasion and transmission. These transcriptional variation signature clusters were also observed in the transcriptome of P. falciparum isolates from acute clinical infections. Interpretation: Our work identifies a previously unrecognized transcriptional pattern in malaria infections in a non-immune background. Significant transcriptome heterogeneity exits between parasites derived from human infections and the pre-mosquito strain, implying that the malaria parasites undergo a change in functional state to adapt to its host environment. Our work also highlights the potential use of transcriptomics data from CHMI study advance our understanding of malaria parasite adaptation and transmission in humans. Fund: This work is supported by German Israeli Foundation, German ministry for education and research, MOE Tier 1 from the Singapore Ministry of Education Academic Research Fund, Singapore Ministry of Health's National Medical Research Council, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA and the German Centre for Infection Research (Deutsches Zentrum für Infektionsforschung-DZIF). Keywords: Malaria, CHMI, Sporozoite, Transcriptome

RTS,S/AS01E has been tested in a phase 3 malaria vaccine study with partial efficacy in African children and infants. In a cohort of 1028 subjects from one low (Bagomoyo) and two high (Nanoro, Kintampo) malaria transmission sites, we analysed IgG plasma/serum concentration and avidity to CSP (NANP-repeat and C-terminal domains) after a 3-dose vaccination against time to clinical malaria events during 12-months. Here we report that RTS,S/AS01E induces substantial increases in IgG levels from pre- to post-vaccination (p, RTS,S/AS01E has been tested in a phase 3 malaria vaccine trial and has shown partial efficacy in children and infants. Here, the authors analyze IgG concentration and avidity to CSP in ~1000 participants and show that IgG avidity to the C-terminus of CSP is significantly associated with vaccine-mediated protection.

Background Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual “blood-stage” parasites in the placenta, the major virulence mechanism. Methods The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 μg (n = 9) or 50 μg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. Results All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. Conclusions PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. Clinical Trials Registration EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489., The pregnancy-associated malaria-vaccine candidate PAMVAC is safe and well tolerated with all three tested formulations and induces functional binding-inhibitory antibodies. The vaccine with glucopyranosyl lipid adjuvant (GLA) in stable emulsion is superior to Alhydrogel and a GLA/QS21 liposomal formulation.

In a phase 3 trial in African infants/children, the RTS,S/AS01 (GSK) vaccine showed moderate efficacy against clinical malaria. We aimed to identify RTS,S/AS01-induced signatures associated with clinical malaria by analyzing antigen-stimulated peripheral blood mononuclear cells sampled from a subset of trial participants at baseline and month 3 (one month post-third dose). RTS,S/AS01 vaccination was associated with downregulation of B-cell and monocyte-related blood transcriptional modules (BTMs) and upregulation of T-cell related BTMs, as well as higher month 3 (vs baseline) circumsporozoite protein (CSP)-specific CD4+T-cell responses. There were few RTS,S/AS01-associated BTMs whose month 3 levels correlated with malaria risk. In contrast, baseline levels of BTMs associated with dendritic cells and with monocytes (among others) correlated with malaria risk. A cross-study analysis supported generalizability of the baseline dendritic cell- and monocyte-related BTM correlations with malaria risk to healthy, malaria-naïve adults, suggesting inflammatory monocytes may inhibit protective RTS,S/AS01-induced responses.

3-Hydroxypropanamidines are a new promising class of highly active antiplasmodial agents. The most active compound 22 exhibited excellent antiplasmodial in vitro activity with nanomolar inhibition of chloroquine-sensitive and multidrug-resistant parasite strains of Plasmodium falciparum (with IC(50) values of 5 and 12 nM against 3D7 and Dd2 strains, respectively) as well as low cytotoxicity in human cells. In addition, 22 showed strong in vivo activity in the Plasmodium berghei mouse model with a cure rate of 66% at 50 mg/kg and a cure rate of 33% at 30 mg/kg in the Peters test after once daily oral administration for 4 consecutive days. A quick onset of action was indicated by the fast drug absorption shown in mice. The new lead compound was also characterized by a high barrier to resistance and inhibited the heme detoxification machinery in P. falciparum.

Malaria can cause life-threatening complications which are often associated with inflammatory reactions. More subtle, but also contributing to the burden of disease are chronic, often subclinical infections, which result in conditions like anemia and immunologic hyporesponsiveness. Although very frequent, such infections are difficult to study in endemic regions because of interaction with concurrent infections and immune responses. In particular, knowledge about mechanisms of malaria-induced immunosuppression is scarce. We measured circulating immune cells by cytometry in healthy, malaria-naïve, adult volunteers undergoing controlled human malaria infection (CHMI) with a focus on potentially immunosuppressive cells. Infectious Plasmodium falciparum (Pf) sporozoites (SPZ) (PfSPZ Challenge) were inoculated during two independent studies to assess malaria vaccine efficacy. Volunteers were followed daily until parasites were detected in the circulation by RT-qPCR. This allowed us to analyze immune responses during pre-patency and at very low parasite densities in malaria-naïve healthy adults. We observed a consistent increase in circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in volunteers who developed P. falciparum blood stage parasitemia. The increase was independent of preceding vaccination with a pre-erythrocytic malaria vaccine. PMN-MDSC were functional, they suppressed CD4+ and CD8+ T cell proliferation as shown by ex-vivo co-cultivation with stimulated T cells. PMN-MDSC reduced T cell proliferation upon stimulation by about 50%. Interestingly, high circulating PMN-MDSC numbers were associated with lymphocytopenia. The number of circulating regulatory T cells (Treg) and monocytic MDSC (M-MDSC) showed no significant parasitemia-dependent variation. These results highlight PMN-MDSC in the peripheral circulation as an early indicator of infection during malaria. They suppress CD4+ and CD8+ T cell proliferation in vitro. Their contribution to immunosuppression in vivo in subclinical and uncomplicated malaria will be the subject of further research. Pre-emptive antimalarial pre-treatment of vaccinees to reverse malaria-associated PMN-MDSC immunosuppression could improve vaccine response in exposed individuals.

Objectives This study investigated the molecular epidemiology of respiratory syncytial virus (RSV) among febrile children with acute respiratory tract infection in Ghana, Gabon, Tanzania and Burkina Faso between 2014 and 2017 as well as the evolution and diversification of RSV strains from other sub-Saharan countries. Methods Pharyngeal swabs were collected at four study sites (Agogo, Ghana: n = 490; Lambarene, Gabon: n = 182; Mbeya, Tanzania: n = 293; Nouna, Burkina Faso: n = 115) and analysed for RSV and other respiratory viruses using rtPCR. For RSV-positive samples, sequence analysis of the second hypervariable region of the G gene was performed. A dataset of RSV strains from sub-Saharan Africa (2011-2017) currently available in GenBank was compiled. Phylogenetic analysis was conducted to identify the diversity of circulating RSV genotypes. Results In total, 46 samples were tested RSV positive (Ghana n = 31 (6.3%), Gabon n = 4 (2.2%), Tanzania n = 9 (3.1%) and Burkina Faso n = 2 (1.7%)). The most common RSV co-infection was with rhinovirus. All RSV A strains clustered with genotype ON1 strains with a 72-nucleotide duplication and all RSV B strains belonged to genotype BAIX. Phylogenetic analysis of amino acid sequences from sub-Saharan Africa revealed the diversification into 11 different ON1 and 22 different BAIX lineages and differentiation of ON1 and BAIX strains into potential new sub-genotypes, provisionally named ON1-NGR, BAIX-KEN1, BAIX-KEN2 and BAIX-KEN3. Conclusion The study contributes to an improved understanding of the molecular epidemiology of RSV infection in sub-Saharan Africa. It provides the first phylogenetic data for RSV from Tanzania, Gabon and Burkina Faso and combines it with RSV strains from all other sub-Saharan countries currently available in GenBank.

Diarrheal disease is the second most frequent cause of mortality in children younger than 5 years worldwide, causing more than half a million deaths each year. Our knowledge of the epidemiology of potentially pathogenic agents found in children suffering from diarrhea in sub-Saharan African countries is still patchy, and thereby hinders implementation of effective preventative interventions. The lack of cheap, easy-to-use diagnostic tools leads to mostly symptomatic and empirical case management. An observational study with a total of 241 participants was conducted from February 2017 to August 2018 among children younger than 5 years with diarrhea in Lambaréné, Gabon. Clinical and demographic data were recorded, and a stool sample was collected. The samples were examined using a commercial rapid immunoassay to detect Rotavirus/adenovirus, conventional bacterial culture for Salmonella spp., and multiplex real-time PCR for Cryptosporidium spp., Giardia lamblia, Cyclospora cayetanensis, enterotoxigenic Escherichia coli (ETEC), and enteroinvasive Escherichia coli (EIEC)/Shigella. At least one infectious agent was present in 121 of 241 (50%) samples. The most frequently isolated pathogens were EIEC/Shigella and ETEC (54/179; 30.2% and 44/179; 24.6%, respectively), followed by G. lamblia (33/241; 13.7%), Cryptosporidium spp. (31/241; 12.9%), and Rotavirus (23/241; 9.5%). Coinfection with multiple pathogens was observed in 33% (40/121) of the positive cases with EIEC/Shigella, ETEC, and Cryptosporidium spp. most frequently identified. Our results provide new insight into the possible causes of diarrheal disease in the Moyen-Ogooué region of Gabon and motivate further research on possible modes of infection and targeted preventive measures.

Controlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune signatures of malaria-naive Europeans and of Africans with lifelong malaria exposure using mass cytometry, RNA sequencing and data integration, before and 5 and 11 days after venous inoculation with Plasmodium falciparum sporozoites. We observed differences in immune cell populations, antigen-specific responses and gene expression profiles between Europeans and Africans and among Africans with differing degrees of immunity. Before inoculation, an activated/differentiated state of both innate and adaptive cells, including elevated CD161(+)CD4(+) T cells and interferon-gamma production, predicted Africans capable of controlling parasitemia. After inoculation, the rapidity of the transcriptional response and clusters of CD4(+) T cells, plasmacytoid dendritic cells and innate T cells were among the features distinguishing Africans capable of controlling parasitemia from susceptible individuals. These findings can guide the development of a vaccine effective in malaria-endemic regions.Malaria immunity can be acquired through natural infection, but the correlates of protection are still being determined. Yazdanbakhsh and colleagues combine experimental infection of volunteers with Plasmodium falciparum with systems analysis to throw light on the nature of protective immune responses.

Background: Antimicrobial resistance (AMR) thwarts the curative power of drugs and is a present-time global problem. We present data on antimicrobial susceptibility and resistance determinants of bacteria the WHO has highlighted as being key antimicrobial resistance concerns in Africa, to strengthen knowledge of AMR patterns in the region. Methods: Blood, stool, and urine specimens of febrile patients, aged between ≥ 30 days and ≤ 15 years and hospitalized in Burkina Faso, Gabon, Ghana, and Tanzania were cultured from November 2013 to March 2017 (Patients > 15 years were included in Tanzania). Antimicrobial susceptibility testing was performed for all Enterobacterales and Staphylococcus aureus isolates using disk diffusion method. Extended-spectrum beta-lactamase (ESBL) production was confirmed by double-disk diffusion test and the detection of blaCTX–M, blaTEM and blaSHV. Multilocus sequence typing was conducted for ESBL-producing Escherichia coli and Klebsiella pneumoniae, ciprofloxacin-resistant Salmonella enterica and S. aureus. Ciprofloxacin-resistant Salmonella enterica were screened for plasmid-mediated resistance genes and mutations in gyrA, gyrB, parC, and parE. S. aureus isolates were tested for the presence of mecA and Panton-Valentine Leukocidin (PVL) and further genotyped by spa typing. Results: Among 4,052 specimens from 3,012 patients, 219 cultures were positive of which 88.1% (n = 193) were Enterobacterales and 7.3% (n = 16) S. aureus. The prevalence of ESBL-producing Enterobacterales (all CTX-M15 genotype) was 45.2% (14/31; 95% CI: 27.3, 64.0) in Burkina Faso, 25.8% (8/31; 95% CI: 11.9, 44.6) in Gabon, 15.1% (18/119; 95% CI: 9.2, 22.8) in Ghana and 0.0% (0/12; 95% CI: 0.0, 26.5) in Tanzania. ESBL positive non-typhoid Salmonella (n = 3) were detected in Burkina Faso only and methicillin-resistant S. aureus (n = 2) were detected in Ghana only. While sequence type (ST)131 predominated among ESBL E. coli (39.1%;9/23), STs among ESBL K. pneumoniae were highly heterogenous. Ciprofloxacin resistant nt Salmonella were commonest in Burkina Faso (50.0%; 6/12) and all harbored qnrB genes. PVL were found in 81.3% S. aureus. Conclusion: Our findings reveal a distinct susceptibility pattern across the various study regions in Africa, with notably high rates of ESBL-producing Enterobacterales and ciprofloxacin-resistant nt Salmonella in Burkina Faso. This highlights the need for local AMR surveillance and reporting of resistances to support appropriate action.

Background Helminths can modulate the host immune response to Plasmodium falciparum and can therefore affect the risk of clinical malaria. We assessed here the effect of helminth infections on both the immunogenicity and efficacy of the GMZ2 malaria vaccine candidate, a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of P. falciparum. Controlled human malaria infection (CHMI) was used to assess the efficacy of the vaccine. Methodology In a randomized, double-blind Phase I clinical trial, fifty, healthy, lifelong malaria-exposed adult volunteers received three doses of GMZ2 adjuvanted with either Cationic Adjuvant Formulation (CAF) 01 or Alhydrogel, or a control vaccine (Rabies) on days (D) 0, D28 and D56, followed by direct venous inoculation (DVI) of 3,200 P. falciparum sporozoites (PfSPZ Challenge) approximately 13 weeks after last vaccination to assess vaccine efficacy. Participants were followed-up on a daily basis with clinical examinations and thick blood smears to monitor P. falciparum parasitemia for 35 days. Malaria was defined as the presence of P. falciparum parasites in the blood associated with at least one symptom that can be associated to malaria over 35 days following DVI of PfSPZ Challenge. Soil-transmitted helminth (STH) infection was assessed by microscopy and by polymerase chain reaction (PCR) on stool, and Schistosoma infection was assessed by microscopy on urine. Participants were considered as infected if positive for any helminth either by PCR and/or microscopy at D0 and/or at D84 (Helm+) and were classified as mono-infection or co-infection. Total vaccine-specific IgG concentrations assessed on D84 were analysed as immunogenicity outcome. Main findings The helminth in mono-infection, particularly Schistosoma haematobium and STH were significantly associated with earlier malaria episodes following CHMI, while no association was found in case of coinfection. In further analyses, the anti-GMZ2 IgG concentration on D84 was significantly higher in the S. haematobium-infected and significantly lower in the Strongyloides stercoralis-infected groups, compared to helminth-negative volunteers. Interesting, in the absence of helminth infection, a high anti-GMZ2 IgG concentration on D84 was significantly associated with protection against malaria. Conclusions Our results suggest that helminth infection may reduce naturally acquired and vaccine-induced protection against malaria. Vaccine-specific antibody concentrations on D84 may be associated with protection in participants with no helminth infection. These results suggest that helminth infection affect malaria vaccine immunogenicity and efficacy in helminth endemic countries., Author summary Helminths, mainly because of their immune regulatory effects, are able to impact the response induced by vaccines. In the context of clinical trial designs that measure accrual of natural infections during follow up or outcome of controlled human malaria infection (CHMI), their effect on vaccine efficacy can be measured. Indeed, most of such clinical trials on malaria vaccine candidates conducted in Africa, especially where the prevalence of helminths is high, have shown a certain limit in their efficacy and immunogenicity, as compared to results observed in European and U.S volunteers. The present analysis assessed the effect of helminths on GMZ2, a malaria vaccine candidate. We found a high level of anti-GMZ2 antibodies among volunteers not infected with helminths and protected against CHMI, indicating efficacy of the candidate vaccine in this population. We found a species-dependent effect of helminths on the level of post-immunization GMZ2-specific IgG concentration, and an association of helminths with an early onset of malaria in CHMI. Our findings reveal that helminths are associated with immunogenicity and may decrease the protective effect of antibodies induced by vaccination. Helminth infection status shall be determined when measuring the immunogenicity and efficacy of malaria vaccine candidates in helminth endemic countries.

Helminth infections are common in sub-Saharan Africa. Besides direct clinical effects, a bias towards a T helper type 2 (Th2) cell immune response is observed. The consequences of parasite infection during pregnancy for the mother and particularly for the fetus and the newborn can be severe and may include impaired immune response during acute infection and vaccination. Here, we present data of immune responses to vaccines given within the expanded program on immunization (EPI) of infants born to helminth infected or non-infected mothers. The study was conducted in Lambar&eacute, n&eacute, and surroundings, Gabon. Maternal helminth infection was diagnosed microscopically using the Kato-Katz method for soil-transmitted helminths (STH), urine filtration for Schistosoma haematobium infections and the saponin-based method for filarial infections. Plasma antibody levels to different vaccine antigens were measured in mothers and their offspring by enzyme-linked immunosorbent assay (ELISA) at different timepoints. We found 42.3% of the mothers to be infected with at least one helminth species. Significantly lower anti-tetanus toxoid immunoglobulin (Ig) G was detected in the cord blood of infants born to helminth infected mothers. Following vaccination, immune responses of the infants to EPI vaccines were similar between the two groups at nine and 12 months. Even though infection with helminths is still common in pregnant women in Gabon, in our setting, there was no evidence seen for a substantial effect on infants&rsquo, immune responses to vaccines given as part of the EPI.

Background Despite appreciable immunogenicity in malaria-naive populations, many candidate malaria vaccines are considerably less immunogenic in malaria-exposed populations. This could reflect induction of immune regulatory mechanisms involving Human Leukocyte Antigen G (HLA-G), regulatory T (Treg), and regulatory B (Breg) cells. Here, we addressed the question whether there is correlation between these immune regulatory pathways and both plasmablast frequencies and vaccine-specific IgG concentrations. Methods Fifty Gabonese adults with lifelong exposure to Plasmodium spp were randomized to receive three doses of either 30 µg or 100 µg GMZ2-CAF01, or 100 µg GMZ2-alum, or control vaccine (rabies vaccine) at 4-week intervals. Only plasma and peripheral blood mononuclear cells isolated from blood samples collected before (D0) and 28 days after the third vaccination (D84) of 35 participants were used to measure sHLA-G levels and anti-GMZ2 IgG concentrations, and to quantify Treg, Breg and plasmablast cells. Vaccine efficacy was assessed using controlled human malaria infection (CHMI) by direct venous inoculation of Plasmodium falciparum sporozoites (PfSPZ Challenge). Results The sHLA-G concentration increased from D0 to D84 in all GMZ2 vaccinated participants and in the control group, whereas Treg frequencies increased only in those receiving 30 µg or 100 µg GMZ2-CAF01. The sHLA-G level on D84 was associated with a decrease of the anti-GMZ2 IgG concentration, whereas Treg frequencies on D0 or on D84, and Breg frequency on D84 were associated with lower plasmablast frequencies. Importantly, having a D84:D0 ratio of sHLA-G above the median was associated with an increased risk of P. falciparum infection after sporozoites injection. Conclusion Regulatory immune responses are induced following immunization. Stronger sHLA-G and Treg immune responses may suppress vaccine induced immune responses, and the magnitude of the sHLA-G response increased the risk of Plasmodium falciparum infection after CHMI. These findings could have implications for the design and testing of malaria vaccine candidates in semi-immune individuals.

More than 200 million malaria clinical cases are reported each year due to Plasmodium vivax, the most widespread Plasmodium species in the world. This species has been neglected and understudied for a long time, due to its lower mortality in comparison with Plasmodium falciparum. A renewed interest has emerged in the past decade with the discovery of antimalarial drug resistance and of severe and even fatal human cases. Nonetheless, today there are still significant gaps in our understanding of the population genetics and evolutionary history of P. vivax, particularly because of a lack of genetic data from Africa. To address these gaps, we genotyped 14 microsatellite loci in 834 samples obtained from 28 locations in 20 countries from around the world. We discuss the worldwide population genetic structure and diversity and the evolutionary origin of P. vivax in the world and its introduction into the Americas. This study demonstrates the importance of conducting genome-wide analyses of P. vivax in order to unravel its complex evolutionary history., Author summary Among the five Plasmodium species infecting humans, P. vivax is the most prevalent parasite outside Africa. To date, there has been less research on this species than for Plasmodium falciparum, a more lethal species, principally because of the lack of an in vitro culture system and also because P. vivax is considered relatively benign. Nevertheless, P. vivax is responsible for severe and incapacitating clinical symptoms with significant effects on human health. The emergence of new drug resistance and the discovery of severe and even fatal cases due to P. vivax question the benign status of P. vivax malaria. In recent years, there has been increased interest in characterizing the distribution of genetic variation in P. vivax. However, these studies either generated genetic information from a regional geographic scale or combine genetic datasets generated in different molecular platforms, which is known to generate biased results. In this study, we used a single genotyping platform to genotype 14 microsatellite markers in 834 samples of P. vivax obtained from 28 locations in 20 countries from around the world, including several populations from East and West Africa. We discuss the worldwide population genetic structure and the evolutionary origins of P. vivax, as well as its introduction into the Americas.

Summary Background Loiasis is a highly prevalent helminth infection found in distinct regions of sub-Saharan Africa. The disease has been considered to be of minor clinical significance, but this belief is being increasingly challenged by recent evidence. We aimed to prospectively quantify the overall burden of disease caused by loiasis in an endemic region of Gabon, using disability-adjusted life years (DALYs). Methods We did a cross-sectional survey during 2017 and 2018 in rural Gabon. Volunteers underwent diagnostic tests for loiasis and were given a standardised questionnaire on symptoms. Participants reporting eye worm migration or harbouring Loa loa microfilariae were defined as loiasis positive. Morbidity-based DALYs associated with loiasis were estimated for the rural population of Gabon. Findings Between Sept 1, 2017 and May 31, 2018, 1235 participants residing in 38 villages in the Gabonese departments of Tsamba-Magotsi and Ogooue et des Lacs were screened. 626 (50·8%) of 1232 eligible participants had loiasis. 520 (42·2%) of 1232 participants reported eye worm migration. 478 (93·9%) of 509 individuals with eye worm migration also reported associated pain, and 397 (78·6%) of 505 reported vision disturbances. After correcting for age and sex, loiasis was significantly associated with a variety of symptoms, including transient painful oedema (adjusted odds ratio 1·76 [95% CI 1·37–2·26]) and arthralgia (1·30 [1·01–1·69]). Application of attributable fractions of correlating symptoms resulted in 412·9 (95% CI 273·9–567·7) morbidity-based DALYs per 100 000 people in rural Gabon. Interpretation Loiasis, with the pathognomonic sign of eye worm migration, appears to not be benign, but severely impeding to affected individuals. Furthermore, loiasis is associated with substantial morbidity, comparable to that of other neglected tropical parasitic diseases. These findings call for reconsideration of L loa as a relevant pathogen in affected populations, with a need for more concerted research and control of these infections. Funding Federal Ministry of Science, Research and Economy of Austria, and the European Union.

Background Malaria remains a major public health problem, affecting mainly low-and middle-income countries. The management of this parasitic disease is challenged by ever increasing drug resistance. This study, investigated the therapeutic efficacy, tolerability and safety of artemether–lumefantrine (AL) and artesunate–amodiaquine (AS–AQ), used as first-line drugs to treat uncomplicated malaria in Lambaréné, Gabon. Methods A non-randomized clinical trial was conducted between October 2017 and March 2018 to assess safety, clinical and parasitological efficacy of fixed-doses of AL and AS–AQ administered to treat uncomplicated Plasmodium falciparum malaria in children aged from 6 months to 12 years. After 50 children were treated with AL, another 50 children received ASAQ. The 2009 World Health Organization protocol for monitoring of the efficacy of anti‑malarial drugs was followed. Molecular markers msp1 and msp2 were used to differentiate recrudescence and reinfection. For the investigation of artemisinin resistant markers, gene mutations in Pfk13 were screened. Results Per-protocol analysis on day 28 showed a PCR corrected cure rate of 97% (95% CI 86–100) and 95% (95% CI 84–99) for AL and AS–AQ, respectively. The most frequent adverse event in both groups was asthenia. No mutations in the kelch-13 gene associated with artemisinin resistance were identified. All participants had completed microscopic parasite clearance by day 3 post-treatment. Conclusion This study showed that AL and AS–AQ remain efficacious, well-tolerated, and are safe to treat uncomplicated malaria in children from Lambaréné. However, a regular monitoring of efficacy and a study of molecular markers of drug resistance to artemisinin in field isolates is essential. Trial registration ANZCTR, ACTRN12616001600437. Registered 18 November, http://www.anzctr.org.au/TrialSearch.aspx?searchTxt=ACTRN12616001600437p&isBasic=True

Background Sub-Saharan Africa carries most of the global burden of schistosomiasis. To optimize disease control and reduce morbidity, precise data are needed for control measures adapted to the local epidemiological situation. The objective of this study is to provide baseline information on schistosomiasis dynamics, including praziquantel (PZQ) treatment outcome in children and young adults living in the vicinity of Lambaréné, Gabon. Methods Eligible volunteers were included into a prospective longitudinal study. Urine filtration technique was used to detect eggs in urine for schistosomiasis diagnosis. Subjects were treated with 60 mg of PZQ once per month for three consecutive months, and the outcome was assessed by cure rate (CR) and egg reduction rate (ERR). Results A total of 328 volunteers were enrolled in the study with a mean (± SD) age of 12.2 ± 4.7 years-old. The female-to-male ratio was 0.99. Out of 258 participants in total, 45% had schistosomiasis during the survey and 43% presented with heavy infections. The incidences of haematuria and schistosomiasis were 0.11 and 0.17 person-years, respectively. After the first and third dose of PZQ, overall ERR of 93% and 95% were found, respectively; while the CR were 78% and 88%, respectively. Both ERR (100 vs 88%) and CR (90 vs 68%) were higher among females than males after the first dose. The CR increased for both groups after the third dose to 95% and 80%, respectively. After the first PZQ dose, ERR was higher for heavy compared to light infections (94 vs 89%), while the CR was higher for light than for heavy infections (87 vs 59%). After the third PZQ dose, ERR increased only for light infections to 99%, while CR increased to 98% and 75% for light and for heavy infections, respectively. The reinfection rate assessed at a mean of 44.6 weeks post-treatment was 25%. Conclusions The prevalence of schistosomiasis is moderate in communities living in the vicinity of Lambaréné, where a subpopulation with a high risk of reinfection bears most of the burden of the disease. To improve schistosomiasis control in this scenario, we suggest education of these high-risk groups to seek themselves a one-year PZQ treatment. Trial registration clinicaltrials.gov Identifier NCT 02769103. Registered 11 May 2016, retrospectively registered. https://clinicaltrials.gov/ct2/show/NCT02769013

Fosmidomycin–piperaquine is being developed as nonartemisinin-based combination therapy to meet the challenge of emerging artemisinin resistance. The combination appeared to have high efficacy and be safe and well tolerated despite observed transient changes in electrocardiogram with prolongation of the QT interval., Background Fosmidomycin–piperaquine is being developed as nonartemisinin-based combination therapy to meet the challenge of emerging artemisinin resistance. Methods The study was a phase 2, single-arm, proof-of-concept study of the efficacy, tolerability, and safety of fosmidomycin–piperaquine for the treatment of uncomplicated Plasmodium falciparum monoinfection in Gabon. Adults and children of both sexes with initial parasite counts between 1000 and 150000/µL received oral treatment with fosmidomycin (twice daily doses of 30 mg/kg) and piperaquine (once daily dose of 16 mg/kg) for 3 days and followed-up for 63 days. The primary efficacy endpoint was the per-protocol polymerase chain reaction (PCR)–corrected day 28 adequate clinical and parasitological response (ACPR). Results One hundred patients were enrolled. The PCR-corrected day 28 ACPR rate was 83/83, or 100% (95% confidence interval, 96–100). Fourteen patients had asexual parasitaemia between day 28 and day 63; all were typed by PCR as new infections. Fosmidomycin–piperaquine therapy led to rapid parasite clearance (median, 36 hours; interquartile range [IQR], 6–60) and fever clearance time (median, 12 hours; IQR, 6–48). The electrocardiogram assessments showed 2 patients with prolonged QT interval >500 msec following study drug administration. The majority of adverse events affected the gastrointestinal and respiratory tracts and were transient and mild to moderate in severity. Conclusions This is the first report of the use of the combination fosmidomycin–piperaquine. The combination appeared to have high efficacy and be safe and well tolerated despite observed transient changes in electrocardiogram with prolongation of the QT interval. Clinical Trials Registration. NCT02198807.

Scally et al. show the molecular, structural, and functional characterization of human antibodies against the C-terminal domain of Plasmodium falciparum (Pf) circumsporozoite (CSP [C-PfCSP]) and reveal that its arrangement on the Pf sporozoite surface and epitope polymorphism contribute to poor C-PfCSP immunogenicity and ineffective humoral responses in volunteers protected against Pf malaria., Antibodies against the central repeat of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) inhibit parasite activity and correlate with protection from malaria. However, the humoral response to the PfCSP C terminus (C-PfCSP) is less well characterized. Here, we describe B cell responses to C-PfCSP from European donors who underwent immunization with live Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), and were protected against controlled human malaria infection. Out of 215 PfCSP-reactive monoclonal antibodies, only two unique antibodies were specific for C-PfCSP, highlighting the rare occurrence of C-PfCSP–reactive B cells in PfSPZ-CVac–induced protective immunity. These two antibodies showed poor sporozoite binding and weak inhibition of parasite traversal and development, and did not protect mice from infection with PfCSP transgenic Plasmodium berghei sporozoites. Structural analyses demonstrated that one antibody interacts with a polymorphic region overlapping two T cell epitopes, suggesting that variability in C-PfCSP may benefit parasite escape from humoral and cellular immunity. Our data identify important features underlying C-PfCSP shortcomings as a vaccine target.

Background The RTS,S/AS01E malaria vaccine has moderate efficacy, lower in infants than children. Current efforts to enhance RTS,S/AS01E efficacy would benefit from learning about the vaccine-induced immunity and identifying correlates of malaria protection, which could, for instance, inform the choice of adjuvants. Here, we sought cellular immunity-based correlates of malaria protection and risk associated with RTS,S/AS01E vaccination. Methods We performed a matched case-control study nested within the multicenter African RTS,S/AS01E phase 3 trial. Children and infant samples from 57 clinical malaria cases (32 RTS,S/25 comparator vaccinees) and 152 controls without malaria (106 RTS,S/46 comparator vaccinees) were analyzed. We measured 30 markers by Luminex following RTS,S/AS01E antigen stimulation of cells 1 month postimmunization. Crude concentrations and ratios of antigen to background control were analyzed. Results Interleukin (IL) 2 and IL-5 ratios were associated with RTS,S/AS01E vaccination (adjusted P ≤ .01). IL-5 circumsporozoite protein (CSP) ratios, a helper T cell type 2 cytokine, correlated with higher odds of malaria in RTS,S/AS01E vaccinees (odds ratio, 1.17 per 10% increases of CSP ratios; P value adjusted for multiple testing = .03). In multimarker analysis, the helper T cell type 1 (TH1)–related markers interferon-γ, IL-15, and granulocyte-macrophage colony-stimulating factor protected from subsequent malaria, in contrast to IL-5 and RANTES, which increased the odds of malaria. Conclusions RTS,S/AS01E-induced IL-5 may be a surrogate of lack of protection, whereas TH1-related responses may be involved in protective mechanisms. Efforts to develop second-generation vaccine candidates may concentrate on adjuvants that modulate the immune system to support enhanced TH1 responses and decreased IL-5 responses.

Background Malaria remains one of the most important infectious diseases. Treatment options for severe malaria are limited and the choline analogue SAR97276A is a novel chemical entity that was developed primarily as treatment for severe malaria. Before starting clinical investigations in severely ill malaria patients, safety and efficacy of SAR97276A was studied in patients with uncomplicated malaria. Here, we summarize two open-label, multi-center phase 2 trials assessing safety and efficacy of parenterally administered SAR97276A in African adults and children with falciparum malaria. Results Study 1 was conducted in Burkina Faso, Gabon, Benin and Tanzania between August 2008 and July 2009 in malaria patients in an age de-escalating design (adults, children). A total of 113 malaria patients received SAR97276A. Adults were randomized to receive a single dose SAR97296A given either intramuscularly (IM) (0.18 mg/kg) or intravenously (IV) (0.14 mg/kg). If a single dose was not efficacious a second adult group was planned to test a three dose regimen administered IM once daily for 3 days. Single dose SAR97276A showed insufficient efficacy in adults (IM: 20 of 34 cured, 59%; and IV: 23/30 cured, 77%). The 3-day IM regimen showed acceptable efficacy in adults (27/30, 90%) but not in children (13/19, 68%). SAR97276A was well tolerated but no further groups were recruited due lack of efficacy. Study 2 was conducted between October 2011 and January 2012 in Burkina Faso, Gabon and Kenya. SAR97276A administered at a higher dose given IM was compared to artemether–lumefantrine. The study population was restricted to underage malaria patients to be subsequently enrolled in two age cohorts (teenagers, children). Rescue therapy was required in all teenaged malaria patients (8/8) receiving SAR97276A once daily (0.5 mg/kg) for 3 days and in 5 out of 8 teenaged patients treated twice daily (0.25 mg/kg) for 3 days. All patients (4/4) in the control group were cured. The study was stopped, before enrollment of children, due to lack of efficacy but the overall safety profile was good. Conclusions Monotherapy with SAR97276A up to twice daily for 3 days is not an efficacious treatment for falciparum malaria. SAR97276A will not be further developed for the treatment of malaria. Trial registration at clinicaltrials.gov: NCT00739206, retrospectively registered August 20, 2008 for Study 1 and NCT01445938 registered September 26, 2011 for Study 2. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1832-x) contains supplementary material, which is available to authorized users.

Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambarene, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

Ivermectin is the drug of choice for many parasitic infections, with more than one billion doses being distributed in onchocerciasis programs. The drug has been put into focus recently by the malaria community because of its potential to kill blood-sucking mosquitoes, thereby reducing malaria transmission. However, the activity of ivermectin against the malaria parasite itself has been only partly investigated. This study aimed to investigate the in vitro activity of ivermectin against asexual and sexual stages of Plasmodium falciparum. Both asexual and late-stage gametocytes were incubated with ivermectin and control drugs in vitro. The growth-inhibiting effects were assessed for asexual stages of different Plasmodium falciparum laboratory strains and culture-adapted clinical isolates using the histidine-rich protein 2 enzyme-linked immunosorbent assay technique. The effect against stage IV/V gametocytes was evaluated based on ATP quantification. Ivermectin showed activities at nanomolar concentrations against asexual stages (50% inhibitory concentration of ∼100 nM) and stage IV/V gametocytes (500 nM) of P. falciparum. Stage-specific assays suggested that ivermectin arrests the parasite cycle at the trophozoite stage. Ivermectin might add a feature to its “wonder drug” properties with activity against asexual stages of the malaria parasite Plasmodium falciparum. The observed activities might be difficult to reach with current regimens but will be more relevant with future high-dose regimens under investigation. Further studies should be performed to confirm these results in vitro and in vivo.

Background: The transcriptome of Plasmodium falciparum clinical isolates varies according to strain, mosquito bites, disease severity and clinical history. Therefore, it remains a challenge to directly interpret the parasite's transcriptomic information into a more general biological signature in a natural human malaria infection. These confounding variations can be potentially overcome with parasites derived from controlled-human malaria infection (CHMI) studies. Methods: We performed CHMI studies in healthy and immunologically naïve volunteers receiving the same P. falciparum strain ((Sanaria® PfSPZ Challenge (NF54)), but with different sporozoite dosage and route of infection. Parasites isolated from these volunteers at the day of patency were subjected to in vitro culture for several generations and synchronized ring-stage parasites were subjected to transcriptome profiling. Findings: We observed clear deviations between CHMI-derived parasites from volunteer groups receiving different PfSPZ dose and route. CHMI-derived parasites and the pre-mosquito strain used for PfSPZ generation showed significant transcriptional variability for gene clusters associated with malaria pathogenesis, immune evasion and transmission. These transcriptional variation signature clusters were also observed in the transcriptome of P. falciparum isolates from acute clinical infections. Interpretation: Our work identifies a previously unrecognized transcriptional pattern in malaria infections in a non-immune background. Significant transcriptome heterogeneity exits between parasites derived from human infections and the pre-mosquito strain, implying that the malaria parasites undergo a change in functional state to adapt to its host environment. Our work also highlights the potential use of transcriptomics data from CHMI study advance our understanding of malaria parasite adaptation and transmission in humans. Ministry of Education (MOE) National Medical Research Council (NMRC) Published version This work is supported by German Israeli Foundation, German ministry for education and research, MOE Tier 1 from the Singapore Ministry of Education Academic Research Fund, Singapore Ministry of Health's National Medical Research Council, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA and the German Centre for Infection Research (Deutsches Zentrum für Infektionsforschung-DZIF).

Background: Ivermectin is safe and widely used for treating helminth infections. It also kills arthropods feeding on treated subjects, including malaria vectors. Thus, ivermectin mass drug administration as an additional tool for malaria control is being evaluated by the World Health Organization: As in vitro data, animal experiments and epidemiological observations suggest that ivermectin has a direct effect on the liver stages of the malaria parasite, this study was designed to assess the prophylactic effect of ivermectin on Plasmodium falciparum controlled human malaria infection. Methods: 4 volunteers were randomized to placebo, and 8 volunteers were randomized to receive ivermectin 0,4 mg/kg, orally, once 2 hours before being experimentally infected intravenously with 3200 P. falciparum sporozoites. The primary endpoint was time to parasitaemia detected by positive thick blood smear; RT-qPCR was performed in parallel. Results: All but one volunteers became thick blood smear positive between day 11 and day 12 after infection and there was no significant effect of ivermectin on parasitaemia. Conclusions: Ivermectin - at the dose used - has no clinically relevant activity against the preerythrocytic stages of P. falciparum. Clinical Trial Registration Number: The study was registered with the European Clinical Trials Register (EudraCT-Nr. 2017-002723-16). Funding Statement: This study was carried out at the clinical trial platform at the Institute of Tropical Medicine at the University of Tubingen, Germany, and was partly funded by the Else Kroner-Fresenius-Stiftung/Bad Homburg, Germany. Declaration of Interests: Dr. Metzger has nothing to disclose. All other authors have nothing to disclose. Ethics Approval Statement: The study complied with the International Council for Harmonisation Good Clinical Practice guidelines and the German Medicines Law and was approved by the ethics committee of the Eberhard Karls Universitat and the Universitatsklinikum Tubingen and the competent regulatory authority Bundesinstitut fur Arzneimittel und Medizinprodukte and the Regierungsprasidium Tubingen. A Safety Monitoring Committee reviewed the study to evaluate the safety of individual volunteers.

GMZ2 is a recombinant protein malaria vaccine, comprising two blood-stage antigens of Plasmodium falciparum, glutamate-rich protein and merozoite surface protein 3. We assessed efficacy of GMZ2 in children in Burkina Faso, Gabon, Ghana and Uganda. : Children 12-60months old were randomized to receive three injections of either 100μg GMZ2 adjuvanted with aluminum hydroxide or a control vaccine (rabies) four weeks apart and were followed up for six months to measure the incidence of malaria defined as fever or history of fever and a parasite density ⩾5000/μL. : A cohort of 1849 children were randomized, 1735 received three doses of vaccine (868 GMZ2, 867 control-vaccine). There were 641 malaria episodes in the GMZ2/Alum group and 720 in the control group. In the ATP analysis, vaccine efficacy (VE), adjusted for age and site was 14% (95% confidence interval [CI]: 3.6%, 23%, p-value=0.009). In the ITT analysis, age-adjusted VE was 11.3% (95% CI 2.5%, 19%, p-value=0.013). VE was higher in older children. In GMZ2-vaccinated children, the incidence of malaria decreased with increasing vaccine-induced anti-GMZ2 IgG concentration. There were 32 cases of severe malaria (18 in the rabies vaccine group and 14 in the GMZ2 group), VE 27% (95% CI -44%, 63%). : GMZ2 is the first blood-stage malaria vaccine to be evaluated in a large multicenter trial. GMZ2 was well tolerated and immunogenic, and reduced the incidence of malaria, but efficacy would need to be substantially improved, using a more immunogenic formulation, for the vaccine to have a public health role.

Background. Antigenic variation in Plasmodium falciparum is mediated by the multicopy var gene family. Each parasite possesses about 60 var genes, and switching between active var loci results in antigenic variation. In the current study, the effect of mosquito and host passage on in vitro var gene transcription was investigated. Methods. Thirty malaria-naive individuals were inoculated by intradermal or intravenous injection with cryopreserved, isogenic NF54 P. falciparum sporozoites (PfSPZ) generated from 1 premosquito culture. Microscopic parasitemia developed in 22 individuals, and 21 in vitro cultures were established. The var gene transcript levels were determined in early and late postpatient cultures and in the premosquito culture. Results. At the early time point, all cultures preferentially transcribed 8 subtelomeric var genes. Intradermal infections had higher var gene transcript levels than intravenous infections and a significantly longer intrahost replication time (P =.03). At the late time point, 9 subtelomeric and 8 central var genes were transcribed at the same levels in almost all cultures. Premosquito and late postpatient cultures transcribed the same subtelomeric and central var genes, except for var2csa. Conclusions. The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth.

Summary Background Artemisinin-based combination therapies (ACTs) are the recommended first-line treatment for uncomplicated Plasmodium falciparum malaria. Ferroquine is a new combination partner for fast-acting ACTs such as artesunate. We aimed to assess different doses of ferroquine in combination with artesunate against uncomplicated P falciparum malaria in a heterogeneous population in Africa. Methods We did a phase 2, multicentre, parallel-group, double-blind, randomised, dose-ranging non-inferiority trial at eight African hospitals (two in Gabon, three in Burkina Faso, one in Benin, and two in Kenya). We recruited patients presenting with acute P falciparum monoinfection (1000–200 000 parasites per μL), and a central body temperature of at least 37·5°C or history of fever in the past 24 h. We assessed patients in two sequential cohorts: cohort 1 contained adults (bodyweight >50 kg) and adolescents (aged ≥14 years, >30 kg), and cohort 2 contained children (aged 2–13 years, 15–30 kg). We randomly assigned patients (1:1:1:1) to receive artesunate 4 mg/kg per day plus ferroquine 2 mg/kg, 4 mg/kg, or 6 mg/kg, given double-blind once per day for 3 days, or ferroquine monotherapy 4 mg/kg per day given single-blind (ie, allocation was only masked from the patient) once per day for 3 days. We did 14 patient visits (screening, 3 treatment days and 48 h post-treatment surveillance, a visit on day 7, then one follow-up visit per week until day 63). The primary endpoint was non-inferiority of treatment in terms of PCR-corrected cure rate against a reference value of 90%, with a 10% non-inferiority margin, assessed in patients treated without major protocol deviations for parasitologically confirmed malaria. We assessed safety in all treated patients. This study is registered with ClinicalTrials.gov, number NCT00988507, and is closed. Findings Between Oct 16, 2009, and Sept 22, 2010, we randomly assigned 326 eligible patients to treatment groups, with last follow-up visit on Dec 1, 2010. 284 patients (87%) were available for per-protocol analyses. At day 28, PCR-confirmed cure was noted in 68 (97%, 95% CI 90–100) of 70 patients treated with ferroquine 2 mg/kg plus artesunate, 73 (99%, 93–100) of 74 with ferroquine 4 mg/kg plus artesunate, 71 (99%, 93–100) of 72 with ferroquine 6 mg/kg plus artesunate, and 54 (79%, 68–88) of 68 with ferroquine 4 mg/kg monotherapy. The three dose groups of ferroquine plus artesunate met the non-inferiority hypothesis. The most common adverse events were headache in cohort 1 (30 [19%] of 162 patients) and worsening malaria in cohort 2 (23 [14%] of 164 patients); occurrences were similar between treatment groups. Interpretation Ferroquine combined with artesunate was associated with high cure rates and was safe at all doses tested, and could be a promising new drug combination for the treatment of P falciparum malaria. Ferroquine could also partner other drugs to establish a new generation of antimalarial combinations, especially in regions that have developed resistance to ACTs. Funding Sanofi.

Background The RTS,S/AS01E vaccine provides partial protection against malaria in African children, but immune responses have only been partially characterized and do not reliably predict protective efficacy. We aimed to evaluate comprehensively the immunogenicity of the vaccine at peak response, the factors affecting it, and the antibodies associated with protection against clinical malaria in young African children participating in the multicenter phase 3 trial for licensure. Methods We measured total IgM, IgG, and IgG1–4 subclass antibodies to three constructs of the Plasmodium falciparum circumsporozoite protein (CSP) and hepatitis B surface antigen (HBsAg) that are part of the RTS,S vaccine, by quantitative suspension array technology. Plasma and serum samples were analyzed in 195 infants and children from two sites in Ghana (Kintampo) and Mozambique (Manhiça) with different transmission intensities using a case-control study design. We applied regression models and machine learning techniques to analyze immunogenicity, correlates of protection, and factors affecting them. Results RTS,S/AS01E induced IgM and IgG, predominantly IgG1 and IgG3, but also IgG2 and IgG4, subclass responses. Age, site, previous malaria episodes, and baseline characteristics including antibodies to CSP and other antigens reflecting malaria exposure and maternal IgGs, nutritional status, and hemoglobin concentration, significantly affected vaccine immunogenicity. We identified distinct signatures of malaria protection and risk in RTS,S/AS01E but not in comparator vaccinees. IgG2 and IgG4 responses to RTS,S antigens post-vaccination, and anti-CSP and anti-P. falciparum antibody levels pre-vaccination, were associated with malaria risk over 1-year follow-up. In contrast, antibody responses to HBsAg (all isotypes, subclasses, and timepoints) and post-vaccination IgG1 and IgG3 to CSP C-terminus and NANP were associated with protection. Age and site affected the relative contribution of responses in the correlates identified. Conclusions Cytophilic IgG responses to the C-terminal and NANP repeat regions of CSP and anti-HBsAg antibodies induced by RTS,S/AS01E vaccination were associated with malaria protection. In contrast, higher malaria exposure at baseline and non-cytophilic IgG responses to CSP were associated with disease risk. Data provide new correlates of vaccine success and failure in African children and reveal key insights into the mode of action that can guide development of more efficacious next-generation vaccines. Electronic supplementary material The online version of this article (10.1186/s12916-018-1186-4) contains supplementary material, which is available to authorized users.

BackgroundGMZ2 is a recombinant malaria vaccine inducing immune responses against Plasmodium falciparum (Pf) merozoite surface protein-3 and glutamate-rich protein. We used standardized controlled human malaria infection (CHMI) to assess the efficacy of this asexual blood-stage vaccine.MethodsWe vaccinated 50 healthy, adult volunteers with lifelong exposure to Pf 3 times, at 4-week intervals, with 30 or 100 µg GMZ2 formulated in CAF01, a liposome-based adjuvant; 100 µg GMZ2, formulated in Alhydrogel; or a control vaccine (Verorab). Approximately 13 weeks after the last vaccination, 35/50 volunteers underwent CHMI by direct venous inoculation of 3200 Pf sporozoites (Sanaria® PfSPZ Challenge).ResultsAdverse events were similarly distributed between GMZ2 and control vaccinees. Baseline-corrected anti-GMZ2 antibody concentrations 4 weeks after the last vaccination were higher in all 3 GMZ2-vaccinated arms, compared to the control group. All GMZ2 formulations induced similar antibody levels. CHMI resulted in 29/34 (85%) volunteers with Pf parasitemia and 15/34 (44%) with malaria (parasitemia and symptoms). The proportion of participants with malaria (2/5 control, 6/10 GMZ2-Alhydrogel, 2/8 30 µg GMZ2-CAF01, and 5/11 100 µg GMZ2-CAF01) and the time it took them to develop malaria were similar in all groups. Baseline, vaccine-specific antibody concentrations were associated with protection against malaria.ConclusionsGMZ2 is well tolerated and immunogenic in lifelong–Pf-exposed adults from Gabon, with similar antibody responses regardless of formulation. CHMI showed no protective effect of prior vaccination with GMZ2, although baseline, vaccine-specific antibody concentrations were associated with protection. CHMI with the PfSPZ Challenge is a potent new tool to validate asexual, blood-stage malaria vaccines in Africa.Clinical Trials RegistrationPan-African Clinical Trials: PACTR201503001038304

The availability of an effective and appropriately implemented malaria vaccine would form a crucial cornerstone of public health efforts to fight this disease. Despite many decades of research, however, no malaria vaccine has yet shown satisfactory protective efficacy or been rolled-out. Validated immunological substitute endpoints have the potential to accelerate clinical vaccine development by reducing the required complexity, size, duration and cost of clinical trials. Besides facilitating clinical development of existing vaccine candidates, understanding immunological mechanisms of protection may drive the development of fundamentally new vaccination approaches. In this review we focus on correlates of protection in malaria vaccine development: Does immunogenicity predict malaria vaccine efficacy and why is this question particularly difficult? Have immunological correlates accelerated malaria vaccine development in the past and will they facilitate it in the future? Does Controlled Human Malaria Infection represent a valid model for identifying such immunological correlates, or a correlate of protection against naturally-acquired malaria in itself?

Background Diagnosis of malaria is usually based on samples of peripheral blood. However, it is unclear whether capillary (CAP) or venous (VEN) blood samples provide better diagnostic performance. Quantitative differences of parasitemia between CAP and VEN blood and diagnostic performance characteristics were investigated. Methods Patients were recruited between September 2015 and February 2016 in Gabon. Light microscopy and quantitative polymerase chain reaction (qPCR) measured parasitemia of paired CAP and VEN samples. CAP and VEN performance characteristics using microscopy were evaluated against a qPCR gold standard. Results Microscopy revealed a median parasitemia of 495/μL in CAP and 429/μL in VEN samples, manifesting in a 16.6% (P = .04) higher CAP parasitemia compared with VEN parasitemia. Concordantly, in qPCR -0.278 (P = .006) cycles were required for signal detection in CAP samples. CAP sensitivity of microscopy relative to the gold standard was 81.5% vs VEN sensitivity of 73.4%, while specificities were 91%. CAP and VEN sensitivities dropped to 63.3% and 45.9%, respectively, for a subpopulation of low-level parasitemias, whereas specificities were 92%. Conclusions CAP sampling leads to higher parasitemias compared to VEN sampling and improves diagnostic sensitivity. These findings may have important implications for routine diagnostics, research, and elimination campaigns of malaria.

Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here we cloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development in vivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines.

The deployment of a safe and effective malaria vaccine will be an important tool for the control of malaria and the reduction in malaria deaths. With the launch of the 2030 Malaria Vaccine Technology Roadmap, the malaria community has updated the goals and priorities for the development of such a vaccine and is now paving the way for a second phase of malaria vaccine development. During a workshop in Brussels in November 2014, hosted by the European Vaccine Initiative, key players from the European, North American and African malaria vaccine community discussed European strategies for future malaria vaccine development in the global context. The recommendations of the European malaria community should guide researchers, policy makers and funders of global health research and development in fulfilling the ambitious goals set in the updated Malaria Vaccine Technology Roadmap.

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drugs pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.

Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive in vivo retinal imaging. We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.

Treatment recommendations for Plasmodium malariae and Plasmodium ovale malaria are largely based on anecdotal evidence. The aim of this prospective study, conducted in Gabon, was to systematically assess the efficacy and safety of artemether-lumefantrine for the treatment of patients with uncomplicated P. malariae or P. ovale species monoinfections or mixed Plasmodium infections. Patients with microscopically confirmed P. malariae , P. ovale , or mixed-species malaria with at least one of these two Plasmodium species were treated with an oral, fixed-dose combination of artemether-lumefantrine for 3 consecutive days. The primary endpoints were per-protocol PCR-corrected adequate clinical and parasitological response (ACPR) on days 28 and 42. Tolerability and safety were recorded throughout the follow-up period. Seventy-two participants (42 male and 30 female) were enrolled; 62.5% of them had PCR-corrected mixed Plasmodium infections. Per protocol, PCR-corrected ACPR rates were 96.6% (95% confidence interval [CI], 91.9 to 100) on day 28 and 94.2% (95% CI, 87.7 to 100) on day 42. Considering Plasmodium species independently from their coinfecting species, day 42 ACPR rates were 95.5% (95% CI, 89.0 to 100) for P. falciparum , 100% (exact CI, 84.6 to 100) for P. malariae , 100% (exact CI, 76.8 to 100) for P. ovale curtisi , and 90.9% (95% CI, 70.7 to 100) for P. ovale wallikeri . Study drug-related adverse events were generally mild or moderate. In conclusion, this clinical trial demonstrated satisfying antimalarial activity of artemether-lumefantrine against P. ovale wallikeri , P. ovale curtisi , P. malariae , and mixed Plasmodium infections, with per-protocol efficacies of 90% to 100% and without evident tolerability or safety concerns. (This trial was registered in the clinical study database ClinicalTrials.gov under the identifier NCT02528279.)