Structure-based drug design studies of the interactions ofent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparumsarco/endoplasmic reticulum Ca2+-ATPase PfATP6

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosawere tested in silico against the Plasmodium falciparumCa2+-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.

CD1d-restricted NK T cells are dispensable for specific antibody responses and protective immunity against liver stage malaria infection in mice.

Immunization with a single dose of irradiated sporozoites is sufficient to induce protection against malaria in wild-type mice. Although this protection is classically attributed to conventional CD4+ and CD8+ T cells, several recent reports have suggested an important role for CD1-restricted NK T cells in immunity to malaria. In this study, we directly compared the ability of C57BL/6 wild-type and CD1-deficient mice to mount a protective immune response against Plasmodium berghei sporozoites. Our data indicate that CD1-restricted NK T cells are not required for protection in this model system. Moreover, specific IgG antibody responses to the P. berghei circumsporozoite repeat sequence were also unaffected by CD1 deficiency. Collectively, our data demonstrate that CD1-restricted NK T cells are dispensable for protective immunity to liver stage P. berghei infection.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium bergheistrain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Immunoregulation in human malaria: the challenge of understanding asymptomatic infection

Asymptomatic Plasmodium infection carriers represent a major threat to malaria control worldwide as they are silent natural reservoirs and do not seek medical care. There are no standard criteria for asymptomaticPlasmodium infection; therefore, its diagnosis relies on the presence of the parasite during a specific period of symptomless infection. The antiparasitic immune response can result in reducedPlasmodium sp. load with control of disease manifestations, which leads to asymptomatic infection. Both the innate and adaptive immune responses seem to play major roles in asymptomatic Plasmodiuminfection; T regulatory cell activity (through the production of interleukin-10 and transforming growth factor-β) and B-cells (with a broad antibody response) both play prominent roles. Furthermore, molecules involved in the haem detoxification pathway (such as haptoglobin and haeme oxygenase-1) and iron metabolism (ferritin and activated c-Jun N-terminal kinase) have emerged in recent years as potential biomarkers and thus are helping to unravel the immune response underlying asymptomatic Plasmodium infection. The acquisition of large data sets and the use of robust statistical tools, including network analysis, associated with well-designed malaria studies will likely help elucidate the immune mechanisms responsible for asymptomatic infection.

Do antibody responses to malaria vaccine candidates influenced by the level of malaria transmission protect from malaria?

OBJECTIVES: To examine whether the humoural response to malaria vaccine candidate antigens, Plasmodium falciparum [circumsporozoite repetitive sequence (NANP)(5) GLURP fragments (R0 and R2) and MSP3] varies with the level of malaria transmission and to determine whether the antibodies (IgG) present at the beginning of the malaria transmission season protect against clinical malaria. METHODS: Cross-sectional surveys were conducted to measure antibody response before, at the peak and at the end of the transmission season in children aged 6 months to 10 years in two villages with different levels of malaria transmission. A cohort study was performed to estimate the incidence of clinical malaria. RESULTS: Antibodies to these antigens showed different seasonal patterns. IgG concentrations to any of the four antigens were higher in the village with high entomological inoculation rate. Multivariate analysis of combined data from the two villages indicated that children who were classified as responders to the selected antigens were at lower risk of clinical malaria than children classified as non-responders [(NANP)(5) (incidence rate ratio (IRR) = 0.65, 95% CI: 0.46-0.92; P = 0.016), R0 (IRR = 0.69, 95% CI: 0.48-0.97; P = 0.032), R2 (IRR = 0.73, 95% CI: 0.50-1.06; P = 0.09), MSP3 (IRR = 0.52, 95% CI: 0.32-0.85; P = 0.009)]. Fitting a model with all four antibody responses showed that MSP3 looked the best malaria vaccine candidate (IRR = 0.63; 95% CI: 0.38-1.05; P = 0.08). CONCLUSION: Antibody levels to the four antigens are affected by the intensity of malaria transmission and associated with protection against clinical malaria. It is worthwhile investing in the development of these antigens as potential malaria vaccine candidates.

Malaria vaccines - The long synthetic peptide approach: Technical and conceptual advancements.

This review describes the advances in malaria antigen discovery and vaccine development using the long synthetic peptide platforms that have been made available during the past 5 years. The most recent technical developments regarding peptide synthesis with the optimized production of large synthetic fragments are discussed. Clinical trials of long synthetic peptides are also reviewed. These trials demonstrated that long synthetic peptides are safe and immunogenic when formulated with various adjuvants. In addition, long synthetic peptides can elicit an antibody response in humans and have demonstrated inhibitory activity against parasite growth in vitro. Finally, new approaches to exploit the abundance of genomic data and the flexibility and speed of peptide synthesis are proposed.

Assessement of Malaria Transmission in an Area with Very Low Mosquito Density

The increase in world travel in recent years, especially to and from areas where vector-borne diseases are endemic, has resulted in a substantial rise in imported cases of those diseases. In particular, malaria is a cause of concern. In those countries at the edge of its distribution, it can be difficult to distinguish between autochthonous and imported cases. However, distinguishing between the two is important because of the different allocation of resources to combat the disease that each requires. In general, observation of the various stages of parasite development in wild-caught female mosquitoes is considered evidence of autochthonous transmission. Observation of oocysts in the mosquito mid-gut testifies that mosquitoes are susceptible to infection but conclusions cannot be reached about their ability to complete the transmission cycle. Perhaps the best indication of autochthonous transmission is microscopic observation of sporozoites in mosquito salivary glands, since this detects parasites ready to be inoculated (BELER et al., 1990). Detection of circumsporozoite protein (CSP)(BURKOT, WILLIAMS & SCHNEIDER, 1984) in dry mosquito thoraxes, by Enzyme Linked Immunosorbent Assay (ELISA) is also widely used to determine transmission, especially when large numbers of mosquitoes need to be processed. Such assays provide information about the parasite species infecting the mosquito (BURKOT & WIRTZ, 1986; WIRTZ et al., 1987; BELER et al., 1990).

Vaccine potentials of an intrinsically unstructured fragment derived from the blood stage-associated Plasmodium falciparum protein PFF0165c.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

Treatment of acute uncomplicated falciparum malaria with artemether-lumefantrine in nonimmune populations: a safety, efficacy, and pharmacokinetic study.

The efficacy and safety of artemether-lumefantrine for the treatment of malaria in nonimmune populations are not well defined. In this study, 165 nonimmune patients from Europe and non-malarious areas of Colombia with acute, uncomplicated falciparum malaria or mixed infection including P. falciparum were treated with the six-dose regimen of artemether-lumefantrine. The parasitologic cure rate at 28 days was 96.0% for the per protocol population (119/124 patients). Median times to parasite clearance and fever clearance were 41.5 and 36.8 hours, respectively. No patient had gametocytes after Day 7. Treatment was well tolerated; most adverse events were mild to moderate and seemed to be related to malaria. There were few serious adverse events, none of which were considered to be drug-related. No significant effects on ECG or laboratory parameters were observed. In conclusion, the six-dose regimen of artemether-lumefantrine was effective and well tolerated in the treatment of acute uncomplicated falciparum malaria in nonimmune patients.

Malaria importée: des médicaments toujours plus efficaces et plus sûrs [Imported malaria: safer and more efficacious drugs?].

In response to the spread of parasite resistance to old antimalarial drugs, the large-scale implementation of artemisinine-based combinations has allowed to improving patient survival and reducing parasite transmission. Even though decreased susceptibility of parasites to artemisinine has been observed in South-East Asia, this phenomenon has no practical implications for travelers with uncomplicated malaria. The combination of artemether-lumefantrine is still very effective and safe, be it for P. falciparum or vivax. Intravenous administration of artesunate has allowed to significantly reducing case fatality rate of severe malaria patients when compared to quinine treatment in endemic areas. Artesunate is also recommended in travelers, but with close monitoring, especially for hematological parameters, in order to confirm its superiority.

Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif.

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling.

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.

The course of malaria in mice: major histocompatibility complex (MHC) effects, but no general MHC heterozygote advantage in single-strain infections.

A general MHC-heterozygote advantage in parasite-infected organisms is often assumed, although there is little experimental evidence for this. We tested the response of MHC-congenic mice (F2 segregants) to malaria and found the course of infection to be significantly influenced by MHC haplotype, parasite strain, and host gender. However, the MHC heterozygotes did worse than expected from the average response of the homozygotes.

The evolutionary host switches of Polychromophilus: a multi-gene phylogeny of the bat malaria genus suggests a second invasion of mammals by a haemosporidian parasite.

BACKGROUND: The majority of Haemosporida species infect birds or reptiles, but many important genera, including Plasmodium, infect mammals. Dipteran vectors shared by avian, reptilian and mammalian Haemosporida, suggest multiple invasions of Mammalia during haemosporidian evolution; yet, phylogenetic analyses have detected only a single invasion event. Until now, several important mammal-infecting genera have been absent in these analyses. This study focuses on the evolutionary origin of Polychromophilus, a unique malaria genus that only infects bats (Microchiroptera) and is transmitted by bat flies (Nycteribiidae). METHODS: Two species of Polychromophilus were obtained from wild bats caught in Switzerland. These were molecularly characterized using four genes (asl, clpc, coI, cytb) from the three different genomes (nucleus, apicoplast, mitochondrion). These data were then combined with data of 60 taxa of Haemosporida available in GenBank. Bayesian inference, maximum likelihood and a range of rooting methods were used to test specific hypotheses concerning the phylogenetic relationships between Polychromophilus and the other haemosporidian genera. RESULTS: The Polychromophilus melanipherus and Polychromophilus murinus samples show genetically distinct patterns and group according to species. The Bayesian tree topology suggests that the monophyletic clade of Polychromophilus falls within the avian/saurian clade of Plasmodium and directed hypothesis testing confirms the Plasmodium origin. CONCLUSION: Polychromophilus' ancestor was most likely a bird- or reptile-infecting Plasmodium before it switched to bats. The invasion of mammals as hosts has, therefore, not been a unique event in the evolutionary history of Haemosporida, despite the suspected costs of adapting to a new host. This was, moreover, accompanied by a switch in dipteran host.