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.

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.

A historical perspective on malaria control in Brazil

Malaria has always been an important public health problem in Brazil. The early history of Brazilian malaria and its control was powered by colonisation by Europeans and the forced relocation of Africans as slaves. Internal migration brought malaria to many regions in Brazil where, given suitableAnopheles mosquito vectors, it thrived. Almost from the start, officials recognised the problem malaria presented to economic development, but early control efforts were hampered by still developing public health control and ignorance of the underlying biology and ecology of malaria. Multiple regional and national malaria control efforts have been attempted with varying success. At present, the Amazon Basin accounts for 99% of Brazil’s reported malaria cases with regional increases in incidence often associated with large scale public works or migration. Here, we provide an exhaustive summary of primary literature in English, Spanish and Portuguese regarding Brazilian malaria control. Our goal was not to interpret the history of Brazilian malaria control from a particular political or theoretical perspective, but rather to provide a straightforward, chronological narrative of the events that have transpired in Brazil over the past 200 years and identify common themes.

Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodium vivaxmalaria cases in China that are imported across its Southeast Asia border, especially in the China-Myanmar border area (CMB). To date, little is known about the genetic diversity of P. vivaxin this region. In this paper, we report the first genome sequencing of a P. vivaxisolate (CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto 96.43% of the P. vivaxSalvador I reference strain (Sal I) genome with 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Using the de novoassembly approach, we generated 8,541 scaffolds and assembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, we identified all 295 known virgenes, which is the largest subtelomeric multigene family in malaria parasites. These results provide an important foundation for further research onP. vivaxpopulation genetics.

Safety of falciparum malaria diagnostic strategy based on rapid diagnostic tests in returning travellers and migrants: a retrospective study.

BACKGROUND: Rapid diagnostic tests for malaria (RDTs) allow accurate diagnosis and prompt treatment. Validation of their usefulness in travellers with fever was needed. The safety of a strategy to diagnose falciparum malaria based on RDT followed by immediate or delayed microscopy reading at first attendance was evaluated in one referral hospital in Switzerland. METHODS: A retrospective study was conducted in the outpatient clinic and emergency ward of University Hospital, covering a period of eight years (1999-2007). The study was conducted in the outpatient clinic and emergency ward of University Hospital. All adults suspected of malaria with a diagnostic test performed were included. RDT and microscopy as immediate tests were performed during working hours, and RDT as immediate test and delayed microscopy reading out of laboratory working hours. The main outcome measure was occurrence of specific complications in RDT negative and RDT positive adults. RESULTS: 2,139 patients were recruited. 1987 had both initial RDT and blood smear (BS) result negative. Among those, 2/1987 (0.1%) developed uncomplicated malaria with both RDT and BS positive on day 1 and day 6 respectively. Among the 152 patients initially malaria positive, 137 had both RDT and BS positive, four only BS positive and five only RDT positive (PCR confirmed) (six had only one test performed). None of the four initially RDT negative/BS positive and none of the five initially BS negative/RDT positive developed severe malaria while 6/137 of both RDT and BS positive did so. The use of RDT allowed a reduction of a median of 2.1 hours to get a first malaria test result. CONCLUSIONS: A malaria diagnostic strategy based on RDTs and a delayed BS is safe in non-immune populations, and shortens the time to first malaria test result.

Antigenicity and immunogenicity of a novel Plasmodium vivax circumsporozoite derived synthetic vaccine construct.

BACKGROUND: The circumsporozoite (CS) protein is a major malaria sporozoite surface antigen currently being considered as vaccine candidate. Plasmodium vivax CS (PvCS) protein comprises a dimorphic central repeat fragment flanked by conserved regions that contain functional domains involved in parasite invasion of host cells. The protein amino (N-terminal) flank has a cleavage region (region I), essential for proteolytic processing prior to parasite invasion of liver cells. METHODS: We have developed a 131-mer long synthetic polypeptide (LSP) named PvNR1R2 that includes the N-terminal flank and the two natural repeat variant regions known as VK210 and VK247. We studied the natural immune response to this region in human sera from different malaria-endemic areas and its immunogenicity in mice. RESULTS: PvNR1R2 was more frequently recognized by sera from Papua New Guinea (PNG) (83%) than by samples from Colombia (24%) when tested by ELISA. The polypeptide formulated in Montanide ISA51 adjuvant elicited strong antibody responses in both C3H and CB6F1 mice strains. Antibodies from immunized mice as well as affinity-purified human IgG reacted with native protein by IFA test. Moreover, mouse immune sera induced strong (90%) in vitro inhibition of sporozoite invasion (ISI) of hepatoma cell lines. CONCLUSIONS: These results encourage further studies in non-human primates to confirm the elicitation of sporozoite invasion blocking antibodies, to assess cell mediated immune responses and the protective efficacy of this polypeptide.

Papel da Coagulação na Resposta do Mosquito Anopheles Gambiae ao Parasita da malária Plasmodium Berghei

Malária é uma doença parasitária infecciosa aguda ou crónica, causada pelo protozoário do género Plasmodium que é transmitido ao homem através de picada de mosquitos fêmeas do género Anopheles. Causa a morte a milhões de pessoas por ano, na sua maioria crianças até aos 5 anos de idade. A inexistência de estratégias eficazes, contra a transmissão da malária, deve-se sobretudo à falta de conhecimento de moléculas cruciais ao desenvolvimento do parasita no vector. Mecanismos de reconhecimento do parasita e a resposta imune do mosquito à infecção são claramente alvos de novas estratégias de controlo da malária. O ciclo natural de transmissão de Plasmodium requer a conclusão com sucesso, do ciclo esporogónico no intestino médio e nas glândulas salivares do mosquito Anopheles, um processo que demora cerca de duas semanas. Este processo de desenvolvimento pode ser bloqueado pelo sistema imune inato do mosquito, resultando assim na eliminação do parasita do vector. A resposta imunológica envolve vários mecanismos como a fagocitose, encapsulação, nodulação, síntese de péptidos antimicrobianos e coagulação, que são acompanhadas pela activação proteolítica da pró-fenoloxidase presente na hemolinfa.O sistema imune é um factor determinante da capacidade vectorial do mosquito, pelo que vários estudos têm sido feitos para melhor compreender as respostas do mosquito Anopheles, principal vector da malária, ao parasita Plasmodium. Uma das principais respostas desencadeadas pelo sistema imune do mosquito é a coagulação. Estudos recentes demonstram que a coagulação da hemolinfa de Anopheles requer actividade da fenoloxidase e difere de Drosophila s.p na formação, estrutura e composição. O objectivo deste trabalho é estudar o papel da coagulação na resposta do mosquito Anopheles gambiae ao parasita da malária Plasmodium berghei, através do estudo da transglutaminase. Para tal foi feita a caracterização dos genes que codificam para a transglutaminases em A. gambiae, através da sequenciação destes genes e dos seus transcritos, verificando-se alguns polimorfismos quando comparada com a sequência do genoma disponível na base de dados. Para caracterizar do papel desta enzima durante a infecção com P. berghei, foi feita a inibição da actividade enzimática dos genes AGAP009097 e AGAP009098 que codificam para a transglutaminases. Foi feita a descrição da dinâmica de transcrição durante a infecção e o silenciamento destes mesmos genes usando dsRNA. Observou-se um aumento da taxa de infecção e do número médio de oocistos por intestino médio nos mosquitos em que as transglutaminases foram inibidas quimicamente bem como naqueles que possuíam os genes silenciados, quando comparados com os grupos controlo. Com este trabalho espera-se ter contribuído para uma melhor compreensão do funcionamento da capacidade imunológica dos mosquitos na resposta ao parasita da malária e a possibilidade de manipular o sistema imune dos mesmos de modo a eliminar o parasita e contribuir para a diminuição/irradicação da malária, uma das principais doença e causa de morte a nível mundial.

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Role of hepatocyte wounding on " Plasmodium " liver-stage development and survival

RESUME La première étape primordiale au cycle de vie du Plasmodium dans un hôte mammifère est l'invasion des hepatocytes par des sporozoites. L'infection finale des hepatocytes est précédée de la traversée de plusieurs cellules hôtes, rompant les membranes plasmiques et ayant comme résultat la sécrétion des facteurs cytotoliques dans le micro-environnement. Ce matériel endogène libéré est fortement stimulant/immunogène et peut servir de signal de danger initiant des réponses distinctes dans diverses cellules. De nos jours, le caractère essentiel et salutaire de la migration des sporozoites comme étape d'infection du Plasmodium est vivement controversée. Ainsi, notre étude a visé à caractériser l'effet de l'interaction du parasite avec ses cellules hôtes d'un point de vue immunologique. En particulier, nous avons voulu évaluer l'effet de la perte de matériel cellulaire pendant l'infection de Plasmodium sur les hepatocytes primaires de souris et sur des cultures cellulaires HepG2. Nous avons observé que les facteurs cytotoxiques dérivés des cellules endommagés activent NF-κB - un important régulateur de réponse inflammatoires -dans des cellules voisines des cellules endommagés, qui sont des cellules hôtes potentielles pour l'infection finale du parasite. Cette activation de NF-κB s'est produite peu de temps après l'infection et a mené in vitro et in vivo à une réduction d'infection de façon dépendante du temps, un effet qui a pu être compensé par l'addition de BAY11-7082, un inhibiteur spécifique de NF-κB. De plus, aucune activation de NF-κB avec des parasites SPECT-/-, incapables de traverser les hepatocytes, n'a été observée. Nous avons montré parla suite que l'activation de NF-κB induit l'expression de l'enzyme iNOS dans les hepatocytes, qui est responsable d'une diminution des hepatocytes infectés. En outre, les hepatocytes primaires des souris MyD88-/- n'ont montré ni activation de NF-κB, ni expression d'iNOS lors de l'infection, ce qui suggère la participation des membres de famille du Toll/IL-1 récepteur dans la reconnaissance des facteurs cytosoxiques. En effet, le manque de MyD88 a augmenté significativement l'infection in vitro et in vivo. D'autre part, un rôle bénéfique pour l'activation de NF-κB a été évalué. Les cellules infectées étaient plus résistantes contre l'apoptose induite par Fas (CD95/Apo-1) que les cellules non infectées ou les cellules infectées dans lesquelles NF-κB a été bloqué par BAY11-7082 in vitro. Paradoxalement, l'expression d'iNOS contribue à la protection des cellules infectées contre l'apoptose pax Fas, puisque le traitement avec l'inhibiteur spécifique SMT (S-methylisothiourea) a rendu les cellules infectées plus susceptibles à l'apoptose. Un effet bénéfique additionnel pour le parasite est que la plupart des cellules hôtes traversées présentent des peptides du parasite aux cellules T cytotoxiques spécifiques et peuvent donc réorienter la réaction immune spécifique sur les cellules non infectées. Nous montrons que les cellules hôtes endommagés par la migration du parasite induit l'inflammation, qui limite l'ampleur de l'infection. D'autre part, nos données soutiennent que la survie du parasite Plasmodium dans le foie est assurée par une augmentation de la résistance des hepatocytes contre l'apoptose. SUMMARY The first obligatory step of the Plasmodium life cycle in the mammalian host is the invasion of hepatocytes by sporozoites. Final hepatocyte infection involves the penetration of several host cells, whose plasma membranes are ruptured in the process, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory / immunogenic and can serve as a danger signal initiating distinct responses in various cells. To date, it is highly controversial whether sporozoite migration through hepatocytes is an essential and beneficial step for Plasmodium infection. Thus, our study aimed at characterizing the effect of the interaction of the parasite with its host cells from an immunological point of view In particular, we wanted to evaluate the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB - a main regulator of host inflammatory responses - in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time dependent manner in vitro and in viva, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. In addition, no NF-κB activation was observed when SPECT-/- parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible nitric oxide synthase (iNOS) expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88-/- mice showed no NF-κB activation and iNOS expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. In a further complementary series of experiments, we assessed a possible beneficial role for the activation of NF-κB. Infected cells were more resistant to Fas (CD95/Apo-1)-mediated apoptosis than uninfected cells or infected cells in which NF-κB was blocked by BAYl1-7082 in vitro. Paradoxically, iNOS expression contributes to the protection of infected cells from Fas-induced apoptosis, since treatment with the specific iNOS inhibitor SMT (S-Methylisothiourea Sulfate) rendered the infected cells more susceptible to apoptosis. An additional beneficial effect of host cell traversal for the parasite is the fact that mainly traversed cells present parasite-derived peptides to specific cytotoxic T cells and therefore may redirect the specific immune response to uninfected cells. In summary, we have shown that host cells wounded by parasite migration induce inflammation, which limits the extent of parasite infection. In addition, our data support the notion that survival of Plasmodium parasites in the liver is mediated by increasing the resistance of hepatocytes to Fas-induced apoptosis.

Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge.

Needle-free procedures are very attractive ways to deliver vaccines because they diminish the risk of contamination and may reduce local reactions, pain or pain fear especially in young children with a consequence of increasing the vaccination coverage for the whole population. For this purpose, the possible development of a mucosal malaria vaccine was investigated. Intranasal immunization was performed in BALB/c mice using a well-studied Plasmodium berghei model antigen derived from the circumsporozoite protein with the modified heat-labile toxin of Escherichia coli (LTK63), which is devoid of any enzymatic activity compared to the wild type form. Here, we show that intranasal administration of the two compounds activates the T and B cell immune response locally and systemically. In addition, a total protection of mice is obtained upon a challenge with live sporozoites.

Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

Antibody-mediated and cellular immune responses induced in naive volunteers by vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.

Sporozoite-mediated hepatocyte wounding limits Plasmodium parasite development via MyD88-mediated NF-kappa B activation and inducible NO synthase expression

Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-kappaB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-kappaB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-kappaB inhibitor BAY11-7082. Furthermore, no NF-kappaB activation was observed when Spect(-/-) parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-kappaB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88(-/-) mice showed no NF-kappaB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection

CSP-A Model for In Vivo Presentation of Plasmodium berghei Sporozoite Antigens by Hepatocytes.

One target of protective immunity against the Plasmodium liver stage in BALB/c mice is represented by the circumsporozoite protein (CSP), and mainly involves its recognition by IFN-γ producing specific CD8+T-cells. In a previous in vitro study we showed that primary hepatocytes from BALB/c mice process Plasmodium berghei (Pb) CSP (PbCSP) and present CSP-derived peptides to specific H-2k(d) restricted CD8+T-cells with subsequent killing of the presenting cells. We now extend these observations to an in vivo infection model in which infected hepatocytes and antigen specific T-cell clones are transferred into recipient mice inducing protection from sporozoite (SPZ) challenge. In addition, using a similar protocol, we suggest the capacity of hepatocytes in priming of naïve T-cells to provide protection, as further confirmed by induction of protection after depletion of cross-presenting dendritic cells (DCs) by cytochrome c (cyt c) treatment or using traversal deficient parasites. Our results clearly show that hepatocytes present Plasmodium CSP to specific-primed CD8+T-cells, and could also prime naïve T-cells, leading to protection from infection. These results could contribute to a better understanding of liver stage immune response and design of malaria vaccines.