Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria.

Immunization of volunteers by the bite of mosquitoes carrying radiation-attenuated Plasmodium falciparum sporozoites protects greater than 90% of such volunteers against malaria, if adequate numbers of immunizing biting sessions and sporozoite-infected mosquitoes are used. Nonetheless, until recently it was considered impossible to develop, license and commercialize a live, whole parasite P. falciparum sporozoite (PfSPZ) vaccine. In 2003 Sanaria scientists reappraised the potential impact of a metabolically active, non-replicating PfSPZ vaccine, and outlined the challenges to producing such a vaccine. Six years later, significant progress has been made in overcoming these challenges. This progress has enabled the manufacture and release of multiple clinical lots of a 1(st) generation metabolically active, non-replicating PfSPZ vaccine, the Sanaria PfSPZ Vaccine, submission of a successful Investigational New Drug application to the US Food and Drug Administration, and initiation of safety, immunogenicity and protective efficacy studies in volunteers in MD, US. Efforts are now focused on how best to achieve submission of a successful Biologics License Application and introduce the vaccine to the primary target population of African children in the shortest possible period of time. This will require implementation of a systematic, efficient clinical development plan. Short term challenges include optimizing the (1) efficiency and scale up of the manufacturing process and quality control assays, (2) dosage regimen and method of administration, (3) potency of the vaccine, and (4) logistics of delivering the vaccine to those who need it most, and finalizing the methods for vaccine stabilization and attenuation. A medium term goal is to design and build a facility for manufacturing highly potent and stable vaccine for pivotal Phase 3 studies and commercial launch.

Association of serum transferrin receptor concentration with markers of inflammation in Zimbabwean children.

BACKGROUND: Clinical studies have shown that degree of erythropoiesis, the hypoxic response, and iron status each independently influences transferrin receptor concentration, but there is conflicting information regarding the effect of inflammation on transferrin receptor expression. SUBJECTS AND METHODS: Levels of hemoglobin, reticulocytes, serum ferritin, transferrin receptors and inflammatory markers (C-reactive protein, interleukin-6 and neutrophils) were determined in 208 Zimbabwean children 10 ng/mL that adjusted for erythropoiesis with log(10) reticulocyte count, the hypoxic response with hemoglobin concentration and iron status with log(10) ferritin concentration, positive correlations were found between log(10) transferrin receptor concentration and log(10) C-reactive protein concentration (P=0.012), log(10) interleukin-6 concentration (P=0.011) and log(10) neutrophil count (P=0.013). These models predict that, with a baseline transferrin receptor concentration in the upper normal range of 8.0 mg/L and holding hemoglobin concentration and reticulocyte count constant, an increase from 1 to 10 mg/L in C-reactive protein is associated with a rise of 1.6 mg/L in transferrin receptor (95% C.I. 0.3-3.0 mg/L), an increase from 0.5-to-5.0 pg/mL in interleukin-6 with a rise of 1.9 mg/L (0.4-3.7 mg/L), and an increase from 2000 to 20,000/microL in neutrophil count with a rise of 3.6 mg/L (0.7-7.5 mg/L). CONCLUSION: Our results suggest that inflammation leads to an increase in circulating transferrin receptor concentration that is independent of the degree of erythropoiesis, the hypoxic response and iron status.

Effect of ferroportin Q248H polymorphism on iron status in African children.

BACKGROUND: Iron deficiency is common in African children, but genetic variations affecting susceptibility have not been identified. The Q248H mutation in ferroportin, a cellular iron exporter regulated by iron status and inflammation, may be associated with high iron stores in African adults. OBJECTIVE: The study examined the prevalence of iron deficiency in African children in an area where malaria transmission is low to absent and investigated whether ferroportin Q248H provides protection from iron deficiency. DESIGN: Complete blood counts, serum markers of iron status and inflammation, and ferroportin Q247H were measured in 208 preschool children in Harare, Zimbabwe. Iron deficiency was defined by serum ferritin and C-reactive protein (CRP) concentrations (definition 1) or by ferritin and the ratio of transferrin receptor to log10 ferritin (definition 2). RESULTS: Q248H was present in 40 children (38 heterozygotes, 2 homozygotes), elevated CRP was present in 26 (12.5%), and iron deficiency was present in 50 (24.0%) (definition 1) or 55 (26.4%) (definition 2). The interaction between ferroportin Q248H and CRP was significant for ferritin concentrations (P = 0.027) in a 2-factor analysis of variance model. With elevated CRP, the estimated geometric mean (SE range) ferritin concentration was 74 (52-106) microg/L for Q248H heterozygotes but 24 (20-30) microg/L for wild-type subjects (P = 0.016). With normal CRP, the ferritin concentration was 16 (14-19) microg/L whether or not the mutation was present. After adjustment for age and weight-for-height z score, the odds ratio (OR) for iron deficiency in Q248H heterozygotes was not significant according to definition 1 (OR: 0.53; 95% CI: 0.18, 1.40; P = 0.222) or definition 2 (OR: 0.39; 95% CI: 0.14, 1.07; P = 0.068). CONCLUSIONS: Any effect of Q248H in protecting against iron deficiency may be observable in children exposed to repeated inflammatory conditions. Further studies of iron status and ferroportin Q248H in African children are needed.

An iron regulatory-like protein expressed in Plasmodium falciparum displays aconitase activity.

Plasmodium falciparum iron regulatory-like protein (PfIRPa) has homology to both mammalian iron regulatory proteins and aconitases and is capable of binding RNA iron response elements. We examined the subcellular localization of PfIRPa and its enzymatic properties at low oxygen tension. Differential digitonin permeabilization of isolated trophozoites with subsequent Western blot analysis suggests that the localization of PfIRPa is predominantly in the membranous compartments of the parasite, such as the mitochondrion. Immunofluorescence analysis showed that PfIRPa colocalizes with heat shock protein 60 (Hsp60), a mitochondrial marker, and is also present in the parasitic cytosol/food vacuole. Under conditions favoring the formation of an iron-sulfur cluster, recombinant PfIRPa (rPfIRPa) had aconitase activity as detected by a colorimetric NADPH-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. As assessed by the hydration of cis-aconitate spectrophotometrically at 240 nm, rPfIRPa had high affinity for cis-aconitate (Km=3.5 microM) but a low turnover number (Kcat= 3.3 s(-1)). The overall catalytic efficiency (Kcat/Km) of rPfIRPa was similar in magnitude to human cytosolic IRP1/aconitase and human mitochondrial aconitase. PfIRPa immunoprecipitated from parasite lysates also had aconitase activity, as assessed by an MTT-based assay. Our results provide evidence that PfIRPa localizes in the mitochondrion and in the cytosol/food vacuole and is able to demonstrate aconitase activity. Further understanding of the role of PfIRPa/aconitase in the regulation of P. falciparum homeostasis may contribute towards the development of novel antimalarial strategies against plasmodial species.

Novel aroylhydrazone and thiosemicarbazone iron chelators with anti-malarial activity against chloroquine-resistant and -sensitive parasites.

Iron (Fe) is crucial for cellular proliferation, and Fe chelators have shown activity at preventing the growth of the malarial parasite in cell culture and in animal and human studies. We investigated the anti-malarial activity of novel aroylhydrazone and thiosemicarbazone Fe chelators that show high activity at inhibiting the growth of tumour cells in cell culture [Blood 100 (2002) 666]. Experiments with the chelators were performed using the chloroquine-sensitive, 3D7, and chloroquine-resistant, 7G8, strains of Plasmodium falciparum in vitro. The new ligands were significantly more active in both strains than the Fe chelator in widespread clinical use, desferrioxamine (DFO). The most effective chelators examined were 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone and 2-hydroxy-1-naphthylaldehyde-4-phenyl-3-thiosemicarbazone. The anti-malarial activity correlates with anti-proliferative activity against neoplastic cells demonstrated in a previous study. Our studies suggest that this class of lipophilic chelators may have potential as useful agents for the treatment of malaria.

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs.

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.

Plasmodium falciparum malaria challenge by the bite of aseptic Anopheles stephensi mosquitoes: results of a randomized infectivity trial.

BACKGROUND: Experimental infection of malaria-naïve volunteers by the bite of Plasmodium falciparum-infected mosquitoes is a preferred means to test the protective effect of malaria vaccines and drugs. The standard model relies on the bite of five infected mosquitoes to induce malaria. We examined the efficacy of malaria transmission using mosquitoes raised aseptically in compliance with current Good Manufacturing Practices (cGMPs). METHODS AND FINDINGS: Eighteen adults aged 18-40 years were randomized to receive 1, 3 or 5 bites of Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of P. falciparum. Seventeen participants developed malaria; fourteen occurring on Day 11. The mean prepatent period was 10.9 days (9-12 days). The geometric mean parasitemia was 15.7 parasites/µL (range: 4-70) by microscopy. Polymerase chain reaction (PCR) detected parasites 3.1 (range: 0-4) days prior to microscopy. The geometric mean sporozoite load was 16,753 sporozoites per infected mosquito (range: 1,000-57,500). A 1-bite participant withdrew from the study on Day 13 post-challenge and was PCR and smear negative. CONCLUSIONS: The use of aseptic, cGMP-compliant P. falciparum-infected mosquitoes is safe, is associated with a precise prepatent period compared to the standard model and appears more efficient than the standard approach, as it led to infection in 100% (6/6) of volunteers exposed to three mosquito bites and 83% (5/6) of volunteers exposed to one mosquito bite. TRIAL REGISTRATION: ClinicalTrials.gov NCT00744133.

Inhibition of in-vitro growth of Plasmodium falciparum by Pseudocedrela kotschyi extract alone and in combination with Fagara zanthoxyloides extract.

Roots of Pseudocedrela kotschyi are commonly used as chewing sticks in West Africa. This study examined the effects of the plant extract on the in-vitro growth of Plasmodium falciparum. Ring-stage synchronised cultures of the malaria parasite were exposed to 30 and 60 microg/ml of P. kotschyi extract for 51 h. Aliquots were taken from the cultures every 3 h for preparation of Giemsa-stained thin films, which were evaluated by light microscopy for degree of parasitaemia and stage distribution of parasite development. The extracts did not show any inhibitory effects on the emergence of trophozoites in treated cultures. However, the results indicate that 80% of inhibition of the parasite transformation into schizont was obtained for both tested concentrations (30 and 60 microg/ml). Experiments with (3)H-hypoxanthine incorporation showed an IC(50) of 16 microg/ml for the Pseudocedrela extract. Pseudocedrela was combined with extract of Fagara zanthoxyloides in various concentrations to determine their interactive effects on the in-vitro cultures. Isobologram analysis of the results indicated a synergistic interaction between the two extracts at low concentrations, while interactions at higher concentrations showed antagonistic effects.

Effects of root extracts of Fagara zanthoxyloides on the in vitro growth and stage distribution of Plasmodium falciparum.

The development of resistance by Plasmodium falciparum to conventional drugs poses a threat to malaria control. There is therefore a need to find new, effective, and affordable remedies for malaria, including those derived from plants. This study demonstrates that crude, reverse-phase high-pressure liquid chromatography (RP-HPLC)-semipurified, and RP-HPLC-purified root extracts of Fagara zanthoxyloides inhibit the growth of P. falciparum in vitro, with 50% inhibitory concentrations (IC(50)s) of 4.90, 1.00, and 0.13 microg/ml, respectively. Roots of F. zanthoxyloides, known as chewing sticks, are widely used for tooth cleaning in West Africa. Microscopic examination of Giemsa-stained slides showed a virtual absence of schizonts in ring-stage synchronized cultures treated with crude extracts at concentrations of 30 to 60 microg/ml during 36 to 48 h of incubation. These observations suggest that the active constituent in the extract may be cytotoxic for P. falciparum trophozoites, thereby inhibiting their development to the schizont stage. A pure bioreactive fraction was subsequently obtained from the chromatographic separations. When this fraction was mixed with pure fagaronine, the mixture coeluted as a single peak on the analytical RP-HPLC column, suggesting that fagaronine may be the active antimalarial constituent of Fagara root extracts. Additional experiments showed that fagaronine also inhibited P. falciparum growth, with an IC(50) of 0.018 microg/ml. The results of this study suggest that the antimalarial activity of fagaronine deserves further investigation.

Targeting malaria with polyamines.

During the asexual cycle of Plasmodium falciparum within the host erythrocyte, the parasite induces a stage-dependent elevation in the levels of polyamines by increased metabolism and uptake of extracellular pools. Polyamine amides of N-methylanthranilic acid have been synthesized which have embedded within them putrescine, spermidine, or spermine and from a charge perspective mimic natural polyamines. The interaction of these polyamine conjugates with human erythrocytes infected with malaria is described using fluorescent microscopy. The fluorescent spermine mimic was the only probe to show measurable intracellular accumulation. This was observed in late stage development but not in the ring stages or in uninfected erythrocytes.

Fluorescence measurements of the labile iron pool of sickle erythrocytes.

Sickle erythrocytes have increased ferritin and increased molecular iron on the inner membrane leaflet, and we postulated that cytosolic labile iron is also elevated. We used the fluorescent metallosensor, calcein, and a permeant Fe2+ chelator to estimate labile cytoslic Fe2+, and calcein plus an Fe3+ chelator to estimate total cytosolic labile iron (Fe2+ + Fe3+). We measured membrane nonheme iron by its reactivity with ferrozine. As estimated by calcein and Fe2+ chelator, the mean +/- SD labile Fe2+ concentration was significantly lower in hemoglobin (Hb) SS (n = 29) than hemoglobin AA (n = 17) erythrocytes (0.56 +/- 0.35 microM versus 1.25 +/- 0.65 microM; P <.001). In contrast, as estimated by calcein and Fe3+ chelator, total erythrocyte labile iron was similar in hemoglobin SS (n = 12) and hemoglobin AA (n = 10) participants (1.75 +/- 0.41 microM versus 2.14 +/- 0.93 microM; P =.2). Mean membrane nonheme iron levels were higher in hemoglobin SS cells than hemoglobin AA cells (0.0016 x 10-4 versus 0.0004 x 10-4 fmol/cell; P =.01), but much lower than the mean amounts of total labile iron (1.6-1.8 x 10-4 fmol/cell) or hemoglobin iron (18 000-19 000 x 10-4 fmol/cell). Both membrane iron and total labile iron were much less than the mean amount of iron potentially present in erythrocyte ferritin as calculated from results of other investigators (15 x 10-4 versus 34 x 10-4 fmol/cell in HbAA versus HbSS erythrocytes). We conclude that cytosolic labile iron is not elevated in hemoglobin SS erythrocytes and that elemental membrane iron is present in only trace amounts.