The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity

The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.     

Differential effects of C3d on the immunogenicity of gene gun vaccines encoding Plasmodium falciparum and Plasmodium berghei MSP142

The complement fragment C3d mediates B-cell activation via simultaneous engagement of the B-cell receptor and CD21 by antigen/C3d conjugates. Several studies demonstrated the potential of C3d as a molecular adjuvant for vaccination. In this work, C3d exerted differential effects on humoral immune responses after gene gun immunization of mice with plasmids encoding the malaria blood stage antigen MSP1₄₂ depending on the nature of the protein (Plasmodium falciparum vs. Plasmodium berghei MSP), the localization of the C3d moiety (C-terminal vs. N-terminal), and the presence of putative N-glycosylation sites. No improvement of protective efficacy by C3d attachment or mutation of glycosylation sites could be demonstrated by in vitro parasite growth inhibition assays or in vivo blood stage parasite challenges. Our data underscore the controversial role of C3d as molecular adjuvant.     

No benefits from combining chloroquine with artesunate for three days for treatment of Plasmodium falciparum in Guinea-Bissau

The use of a combination of chloroquine and artesunate has been suggested for treatment of malaria in Africa. We used concomitant as well as sequential medication with these 2 drugs in relation to each drug separately for children infected with Plasmodium falciparum in Guinea-Bissau from March 2000 to November 2001. By block-randomization, 474 children with symptomatic malaria were divided into 4 groups and given either a total of 8 mg artesunate per kg bodyweight for 3 d, a total of 25 mg chloroquine base per kg bodyweight for 3 d, both drugs concomitantly for 3 d, or both drugs in sequence. All children were followed weekly for 5 weeks. On day 28, parasites had been detected in 40% of the children who were treated with artesunate only compared with 21% treated with chloroquine, 20% treated with artesunate in combination with chloroquine, and 16% treated with artesunate and chloroquine in sequence; on day 35 the corresponding percentages were 48%, 29%, 27%, and 24%, respectively. The outcome of the combination of chloroquine and artesunate in the doses studied was similar to the outcome of chloroquine monotherapy regardless of whether the 2 drugs are given concomitantly (relative risk [RR] = 0.93, 95% CI 0.56-1.53, P = 0.76) or in sequence (RR = 0.78, 95% CI 0.47-1.28, P = 0.32). Thus, neither an antagonistic, an additive, or a synergistic effect of the 2 drugs was indicated.     

Synthesis and in vitro antimalarial activity of tetraoxane-amine/amide conjugates

A series of tetraoxanes, tetraoxane-amine and tetraoxane-amide conjugates have been synthesized and screened for in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Most of the conjugates showed slightly better antimalarial activity than the parent tetraoxanes. Three of the conjugate compounds were potentially active with IC₅₀ values in the range of 0.38-0.80 μM. Cytotoxicity of four selected compounds was also evaluated in a panel of four cancer (SK-MEL, KB, BT-549, SK-OV-3) and two non-cancer (Vero and LLC-PK₁₁) cell lines up to a concentration of 25 μM and none of the compounds was found toxic to any of the cells.     

Antimalarial activity of imidazo[2,1-a]isoindol-5-ol derivatives and related compounds

The synthesis of several series of imidazo[2,1-a]isoindol-5-ol derivatives and the results of their evaluation against Plasmodium falciparum are presented and discussed. The effects of electron-withdrawing or-donating substituents on different parts of the molecule, as well as those produced by the incorporation of an additional fused ring, were analyzed. Several compounds showed significant antimalarial activity in vitro with IC₅₀ values as low as 60 nM and a certain efficacy in vivo by reducing parasitemia in Plasmodium berghei mouse models.     

In Vivo Antimalarial Effects of Iranian Flora Artemisia khorassanica against Plasmodium berghei and Pharmacochemistry of its Natural Components

Background

The aim of this study was to evaluate the antimalarial effects of Iranian flora Artemisia khorassanica against Plasmodium berghei in vivo and pharmacochemistry of its natural components.

Methods

The aerial parts of Iranian flora A. khorasanica were collected at flowering stage from Khorassan Province, northeastern Iran in 2008. They were air-dried at room temperature; powder was macerated in methanol and the extract defatted in refrigerator, filtered, diluted with water, then eluted with n-hexane and finally non-polar components were identified through Gas Chromatography and Mass Spectroscopy (GC-MS). Toxicity of herbal extracts was assessed on naïve NMRI mice, and its anti-malarial efficacy was investigated on infected Plasmodium berghei animals. This is the first application on A. khorssanica extract for treatment of murine malaria. The significance of differences was determined by Analysis of Variances (ANOVA) and Student''s t-test using Graph Pad Prism Software.

Results

The herbal extract was successfully tested in vivo for its anti-plasmodial activity through artemisin composition, which is widely used as a standard malaria treatment.

Conclusion

Although, this study confirmed less anti-malarial effects of A. khorssanica against murine malaria in vivo, however there are some evidences on reducing pathophysiology by this medication. In complementary assay, major components were detected by GC-MS analysis in herbal extract including chrysanthenone (7.8%), palmitic acid (7.4%) and cis-thujone (5.8%). The most retention indices of the component are given as n-eicosane, palmitic acid and n-octadecane.     

Epidemic of Plasmodium falciparum malaria in Central India, an area where chloroquine has been replaced by artemisinin-based combination therapy

India contributes greatly to the global incidence of malaria. The factors influencing malaria in India are highly diverse and vary greatly from the epidemiological setting of any other country. Central India is the most vulnerable area to malaria in India. This study was carried out in three community health centres in Dindori District, Madhya Pradesh (Central India). Dindori District is mesoendemic for malaria, with both Plasmodium falciparum and P. vivax being present in all age groups. Anopheles culicifacies and A. fluviatilis are highly efficient vectors of malaria. In this study, an epidemic of malaria among indigenous ethnic group Baigas was investigated to determine the causes of the epidemic and the population involved in order to aid in disease containment. The existence of sporozoite-positive A. culicifacies and A. fluviatilis indicates either that spraying had not been done properly or the presence of insecticide resistance. A combination of factors propagated the epidemic. Evidence suggests that the non-availability of artemisinin-based combination therapy and rapid diagnostic tests along with an immunogenically vulnerable population each played an important role. As the global prevalence of malaria decreases owing to initiatives to control or eliminate the disease, more areas will become mesoendemic or hypoendemic for malaria. Detection and control of epidemics requires greater attention, and mechanisms to ensure the quality of interventions are essential.     

Antimalarial efficacy of chloroquine, amodiaquine, sulfadoxine-pyrimethamine, and the combinations of amodiaquine + artesunate and sulfadoxine-pyrimethamine + artesunate in Huambo and Bie provinces, central Angola

We studied three antimalarial treatments in Caala and Kuito, Angola, in 2002 and 2003. We tested chloroquine (CQ), amodiaquine (AQ) and sulfadoxine-pyrimethamine (SP) in Caala, and AQ, SP and the combinations AQ+artesunate (AQ+AS) and SP+artesunate (SP+AS) in Kuito. A total of 619 children (240 in Caala, 379 in Kuito) with uncomplicated Plasmodium falciparum malaria were followed-up for 28 days, with PCR genotyping to distinguish recrudescence from reinfection. PCR-corrected failure proportions at day 28 were very high in the CQ group (83.5%, 95% CI 74.1-90.5), high in the SP groups (Caala: 25.3%, 95% CI 16.7-35.8; Kuito: 38.8%, 95% CI 28.4-50.0), around 20% in the AQ groups (Caala: 17.3%, 95% CI 10.0-27.2; Kuito: 21.6%, 95% CI 14.3-30.6) and very low in the artemisinin-based combination groups (1.2%, 95% CI 0.0-6.4 for each combination AQ+AS and SP+AS). These results show that CQ and SP are no longer efficacious in Caala and Kuito and that the moderate efficacy of AQ is likely to be compromised in the short term if used as monotherapy. We recommend the use of AQ with AS, though this combination might not have a long useful therapeutic life because of AQ resistance.     

Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination with different adjuvants

The Plasmodium falciparum antigen 332 (Pf332) is a conserved blood-stage antigen, which has been suggested to play a role in parasite invasion. In the present study, we have investigated the immunogenicity of the Duffy-binding like (DBL)-domain of the Pf332 molecule in combination with different adjuvants in four animal species. Three of the adjuvants are applicable for human use (Montanide ISA 720, alum and levamisole), whilst Freund's adjuvant served as a positive control adjuvant. Montanide ISA 720 was able to generate a significant and Th2-biased IgG response in BALB/c and C57BL/6 mice. Alum was a strong inducer of a Th2-type immune response only in BALB/c mice, whereas it was a poor adjuvant together with Pf332-DBL in C57BL/6 mice, rabbits and rats. Levamisole did not show any obvious adjuvant effect in any of the immunized animals. Thus in the case with Pf332-DBL, Montanide ISA 720 may be an adjuvant to further explore in the development of a vaccine against malaria.     

Rh1 high activity binding peptides inhibit high percentages of Plasmodium falciparum FVO strain invasion

Identifying the minimal functional regions of the proteins which the malaria parasite uses when invading its host cells constitutes the first and most important approach in an effective design for a chemically synthesised, multi-antigen, multi-stage, subunit-based vaccine. This work has been aimed at identifying the PfRh1 protein binding regions (residues 1–2580) belonging to the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) family implicated in the parasite's alternative target cell invasion routes. Eighteen peptide regions (called high activity binding peptides – HABPs) binding to red blood cells (RBC) were identified in peptides mapped in a highly robust, specific and sensitive receptor–ligand assay. These HABPs were saturable in the experimental conditions assayed here and most had an alpha helix structure. Polymorphism studies revealed that only six of the eighteen HABPs identified had changes at amino acid level amongst the seven P. falciparum strains evaluated. Most HABPs’ specific binding became altered when RBC were treated with neuraminidase, chymotrypsin and trypsin, suggesting differing sensitivity for RBC membrane receptors. After ascertaining that the Rh1 gene was transcribed and expressed in late-stage schizonts of the FCB-2 strain, invasion inhibition assays were carried out. When most of these HABPs were assayed in P. falciparum in vitro culture they were able to inhibit high percentages of FVO strain invasion compared to low inhibition percentages observed with the FCB-2 strain. This data shows small Rh1 regions’ participation during invasion and suggests that these units should be included in further immunological and structural studies.     

Malaria in France: Mainland and territories

Malaria, which was eliminated first from Metropolitan France (mainland and Corsica), then in the French West Indies and the Reunion Island during the 20^th century, remains endemic in two French territories: French Guiana and the Indian Ocean Mayotte island. Despite differences in the dominating plasmodial species and epidemiological patterns, these two territories have achieved marked quantitative improvements (in the reported number of cases and severe cases) thanks to efforts undertaken over the past decade. The situation, however, remains a concern from a qualitative standpoint with the emergence of resistance to antimalarial drugs and logistical and administrative issues which hinder access to treatment. Although malaria was eradicated in Metropolitan France half a century ago, competent vectors remain present in part or all of these territories and can give rise to limited outbreaks.     

Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys

In an effort to broaden the immune response induced by the RTS,S/AS02_A,vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1₄₂ and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1₄₂ and AMA1 vaccines formulated with the AS02_A Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1₄₂ or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1₄₂ and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-γ and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1₄₂ and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1₄₂ lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1₄₂ significantly reduced AMA1 IFN-γ and IL-5 responses. MSP1₄₂ suppression of AMA1 IFN-γ responses was not seen in animals receiving RTS,S + AMA1 + MSP1₄₂ suggesting that RTS,S restored IFN-γ responses. Conversely, AMA1 had no effect on MSP1₄₂ antibody and IFN-γ and IL-5 responses. Neither AMA1 alone or combined with MSP1₄₂ affected RTS,S antibody or IFN-γ and IL-5 responses. Immune interference by MSP1₄₂ on AMA1 antibody responses was also evident when AMA1, MSP1₄₂ and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.     

Sequence Diversity of pfmdr1 and Sequence Conserve of pldh in Plasmodium falciparum from Indonesia: Its implications on Designing a Novel Antimalarial Drug with Less Prone to Resistance

Background

pfmdr1 and its variants are molecular marker which are responsible for antibiotics resistance in Plasmodium falciparum, a parasitic carrier for malaria disease. A novel strategy to treat malaria disease is by disrupting parasite lactate dehydrogenase (pLDH), a crucial enzyme for Plasmodium survival during their erythrocytic stages. This research was aimed to investigate and characterize the pfmdr1 and pldh genes of P. falciparum isolated from Nusa Tenggara Indonesia.

Methods

Genomic DNA of P.falciparum was isolated from malaria patients in Nusa Tenggara Indonesia. pfmdr1 was amplified using nested PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). pldh was amplified, sequenced, and analyzed using NCBI public domain databases and alignment using Clustal W ver. 1.83.

Results

Genotyping of the pfmdr1 revealed that sequence diversity was extremely high among isolates. However, a sequence analysis of pldh indicated that open reading frame of 316 amino acids of the gene showing 100% homology to the P. falciparum 3D7 reference pldh (GeneBank: {"type":"entrez-nucleotide","attrs":{"text":"XM_001349953.1","term_id":"124513265","term_text":"XM_001349953.1"}}XM_001349953.1).

Conclusion

This is the first report which confirms the heterologous of pfmdr1 and the homologous sequences of P.falciparum pldh isolated from Nusa Tenggara Islands of Indonesia, indicating that the chloroquine could not be used effectively as antimalarial target in the region and the pLDH-targeted antimalarial compound would have higher chance to be successful than using chloroquine for curbing malaria worldwide.     

Efficacy and safety of artemether-lumefantrine (Coartem) tablets (six-dose regimen) in African infants and children with acute, uncomplicated falciparum malaria

Approximately one million children die from malaria each year. A recently approved artemisinin-based tablet, Coartem (co-artemether), comprising artemether 120 mg plus lumefantrine 20 mg, given in four doses, provides effective antimalarial treatment for children in many sub-Saharan countries. However, this regimen is considered insufficient for non-immune infants and in areas where multidrug-resistant Plasmodium falciparum predominates. This open-label study assessed the efficacy and safety of co-artemether administered to 310 African children weighing 5-25 kg, with acute, uncomplicated falciparum malaria. Six doses of co-artemether were given over 3 days, with follow-up at 7, 14 and 28 days. Treatment rapidly cleared parasitemia and fever. The overall 28-day cure rate was 86.5%, and 93.9% when corrected by PCR for reinfection. Cure rates at 7 and 14 days exceeded 97.0% (uncorrected) and, on day 28, were similar in infants (5-<10 kg) previously exposed to malaria infection (partially immune: 88.6% uncorrected; 93.3% corrected), and in those who were non-immune (82.5% uncorrected; 95.0% corrected). Adverse events were mostly mild. There was no electrocardiographic evidence of cardiotoxicity. The co-artemether six-dose regimen, treating acute uncomplicated falciparum malaria in African children, achieved rapid parasite clearance and a high cure rate. Treatment was generally safe and well tolerated.     

Molecular method for the diagnosis of imported pediatric malaria

Malaria is a polymorphous disease; it can be life threatening especially for children. We report a case of imported malaria in a boy, illustrating the epidemiological and clinical aspects of severe pediatric malaria. In this case real-time PCR was used to quantify Plasmodium falciparum DNA levels, to monitor the evolution under treatment, and to determine genetic mutations involved in chloroquine resistance. The major epidemiological features of imported malaria, and the difficulty to diagnose childhood severe malaria are described. The contribution of molecular methods for the diagnosis of imported malaria is discussed.     

Clinical pattern of severe Plasmodium falciparum malaria in Sudan in an area characterized by seasonal and unstable malaria transmission

A hospital-based study was carried out in Gedarif town, eastern Sudan, an area of markedly unstable malaria transmission. Among the 2488 diagnosed malaria patients, 4.4% fulfilled the WHO criteria for severe malaria, and seven died of cerebral malaria. The predominant complication was severe malarial anemia (45.4%), followed by convulsions (21%), cerebral malaria (16. 4%) and hypotension (11.8%). Severe malaria was recognized in all age groups, but 44.5% of patients were aged 2 to 4 years. The mean ages of patients with severe anemia (5.6 years) and convulsions (5.9 years) were significantly lower than the mean ages of patients with cerebral malaria (14.1 years) or hypotension (35.2 years). Patients with convulsions and cerebral malaria had significantly higher mean parasite count (69972 and 56110 parasites/microL, respectively) than patients with severe anemia (24637 parasites/microL) or hypotension (13667 parasites/microL). The mean blood glucose level was higher in patients with cerebral malaria than in patients with anemia, and higher in patients who died than in patients who survived. In this setting, the clinico-epidemiological pattern of severe malaria varies considerably from that of hyperendemic regions in sub-Saharan Africa, and there is considerable variation between the individual complications of severe malaria.