Causal prophylactic efficacy of atovaquone-proguanil (Malarone) in a human challenge model.

Plasmodia infect the liver for about 7 days before subsequently infecting the blood. Present prophylaxis against Plasmodium falciparum malaria employs agents that primarily kill blood stages and must be continued for 28 days after the last exposure. Atovaquone-proguanil (Malarone) is a new antimalarial agent that is licensed in 35 countries as treatment against blood-stage infection, but its components (atovaquone and proguanil) have separately been shown to be active also against liver stages. To determine whether atovaquone-proguanil is sufficiently active against liver stages to be discontinued 7 days after exposure, we challenged 16 volunteers with P. falciparum via infected mosquitoes. Twelve volunteers received atovaquone-proguanil (1 tablet daily) on the day prior to challenge, on the day of challenge, and for the next 6 days; 4 volunteers received matching placebo. All placebo volunteers demonstrated parasitaemia and malarial symptoms beginning on days 11-12 after challenge. No atovaquone-proguanil volunteer acquired malaria. Atovaquone-proguanil is the first licensed antimalarial agent that kills P. falciparum in the liver and that may be discontinued 7 days after the last exposure.

Atovaquone-proguanil versus mefloquine for malaria prophylaxis in nonimmune travelers: results from a randomized, double-blind study.

Concerns about the tolerability of mefloquine highlight the need for new drugs to prevent malaria. Atovaquone-proguanil (Malarone; GlaxoSmithKline) was safe and effective for prevention of falciparum malaria in lifelong residents of malaria-endemic countries, but experience in nonimmune people is limited. In a randomized, double-blind study, nonimmune travelers received malaria prophylaxis with atovaquone-proguanil (493 subjects) or mefloquine (483 subjects). Information about adverse events (AEs) and potential episodes of malaria was obtained 7, 28, and 60 days after travel. AEs were reported by an equivalent proportion of subjects who had received atovaquone-proguanil or mefloquine (71.4% versus 67.3%; difference, 4.1%; 95% confidence interval, -1.71 to 9.9). Subjects who received atovaquone-proguanil had fewer treatment-related neuropsychiatric AEs (14% versus 29%; P=.001), fewer AEs of moderate or severe intensity (10% versus 19%; P=.001), and fewer AEs that caused prophylaxis to be discontinued (1.2% versus 5.0%; P=.001), compared with subjects who received melfoquine. No confirmed diagnoses of malaria occurred in either group. Atovaquone-proguanil was better tolerated than was mefloquine, and it was similarly effective for malaria prophylaxis in nonimmune travelers.

Atovaquone-proguanil versus chloroquine-proguanil for malaria prophylaxis in non-immune travellers: a randomised, double-blind study. Malarone International Study Team.

BACKGROUND: Chloroquine plus proguanil is widely used for malaria chemoprophylaxis despite low effectiveness in areas where multidrug-resistant malaria occurs. Studies have shown that atovaquone and proguanil hydrochloride is safe and effective for prevention of falciparum malaria in lifelong residents of malaria-endemic countries, but little is known about non-immune travellers. METHODS: In a double-blind equivalence trial, 1083 participants travelling to a malaria-endemic area were randomly assigned to two treatment groups: atovaquone-proguanil plus placebos for chloroquine and proguanil, or chloroquine, proguanil, and placebo for atovaquone-proguanil. Follow-up was by telephone 7 and 60 days after travel and at a clinic at 28 days. Serum samples were tested for antibodies to a malaria circumsporozoite protein. Blood and serum samples of participants with a potential malaria diagnosis were tested in a reference laboratory. FINDINGS: 7 days after travel, at least one adverse event was reported by 311 (61%) of 511 participants who received atovaquone-proguanil and 329 (64%) of 511 who received chloroquine-proguanil. People receiving atovaquone-proguanil had a lower frequency of treatment-related gastrointestinal adverse events (59 [12%] vs 100 [20%], p=0.001), and of treatment-related adverse events of moderate or severe intensity (37 [7%] vs 56 [11%], p=0.05). There were fewer treatment-related adverse events that caused prophylaxis to be discontinued in the atovaquone-proguanil group than in the chloroquine-proguanil group (one [0.2%] vs ten [2%], p=0.015). INTERPRETATION: Overall the two preparations were similarly tolerated. However, significantly fewer adverse gastrointestinal events were observed in the atovaquone-proguanil group in than in the chloroquine-proguanil group.

Atovaquone plus proguanil versus halofantrine for the treatment of imported acute uncomplicated Plasmodium falciparum malaria in non-immune adults: a randomized comparative trial.

In endemic zones, the atovaquone-proguanil (AP) combination is well tolerated and effective in treating acute, uncomplicated malaria. Trials involving non-immune patients are lacking, however. We conducted a randomized, multicenter open-label trial to determine the efficacy and tolerability of the AP combination (1,000 mg + 400 mg once daily for 3 days) in comparison with halofantrine (HF) (1,500 mg in 3 doses) in non-immune adults with imported uncomplicated Plasmodium falciparum malaria. Follow-up visits were programmed on Days 7, 14, 21, 28, and 35 after hospital discharge. Out of 48 patients enrolled in the study, 41 were assessable for the cure rate (21 in the AP group and 20 in the HF group). All the patients were cured. The mean parasite clearance time was longer (63+/-23 hours) in the AP group than in the HF group (48+/-15 hours) (P = 0.02). The frequency of gastrointestinal adverse events was higher in the AP group. No noteworthy electrocardiographic changes were observed, particularly in the QTc interval. The AP combination appears to be a valuable alternative treatment in non-immune adults.

Malarone (atovaquone and proguanil hydrochloride): a review of its clinical development for treatment of malaria. Malarone Clinical Trials Study Group.

The continuing spread of drug-resistant malaria emphasizes the need for new antimalarial drugs. Atovaquone is a broad-spectrum antiprotozoal drug with a novel mechanism of action, via inhibition of parasite mitochondrial electron transport, and a favorable safety profile. Early studies with atovaquone alone for treatment of malaria demonstrated good initial control of parasitemia but an unacceptable rate of recrudescent parasitemia. Parasites isolated during recrudescence after treatment with atovaquone alone were resistant to atovaquone in vitro. The combination of atovaquone and proguanil is synergistic in vitro, and clinical studies demonstrated enhanced efficacy of the combination compared to either drug alone for treatment of malaria. Malarone, a fixed-dose combination of 250 mg of atovaquone and 100 mg of proguanil hydrochloride, is available in many countries for treatment of acute, uncomplicated malaria caused by Plasmodium falciparum. At the recommended dose (in adults, four tablets once a day for three days), the overall cure rate was > 98% in more than 500 patients with falciparum malaria. In four randomized, controlled clinical trials, treatment with atovaquone and proguanil hydrochloride was significantly more effective than mefloquine (Thailand), amodiaquine (Gabon), chloroquine (Peru and the Philippines) or chloroquine plus pyrimethamine/sulfadoxine (Philippines). In clinical trials where the comparator drug was highly effective, treatment with atovaquone and proguanil hydrochloride was equally effective. Parasites isolated during recrudescence after treatment with the combination of atovaquone and proguanil were not resistant to atovaquone in vitro. The most commonly reported adverse events in clinical trials (abdominal pain, anorexia, nausea, vomiting, diarrhea and coughing) occurred with similar frequency in patients treated with a comparator drug. Malarone is a safe and effective new agent for treatment of malaria.

Atovaquone and proguanil hydrochloride followed by primaquine for treatment of Plasmodium vivax malaria in Thailand.

Chloroquine-resistant Plasmodium vivax malaria has been reported in several geographical areas. The P. vivax life-cycle includes dormant hepatic parasites (hypnozoites) that cause relapsing malaria weeks to years after initial infection. Curative therapy must therefore target both the erythrocytic and hepatic stages of infection. Between July 1997 and June 1998, we conducted an open-label study in Thailand to evaluate the efficacy and tolerability of a sequential regimen of combination atovaquone (1000 mg) and proguanil hydrochloride (400 mg), once daily for 3 days, followed by primaquine (30 mg daily for 14 days) for treatment of vivax malaria. All 46 patients who completed the 3-day course of atovaquone-proguanil cleared their parasitaemia within 2-6 days. During a 12-week follow-up period in 35 patients, recurrent parasitaemia occurred in 2. Both recurrent episodes occurred 8 weeks after the start of therapy, consistent with relapse from persistent hypnozoites rather than recrudescence of persistent blood-stage parasites. The dosing regimen was well tolerated. Results of this trial indicate that atovaquone-proguanil followed by primaquine is safe and effective for treatment of vivax malaria.

Atovaquone and proguanil hydrochloride for treatment of malaria.

BACKGROUND: Safe and effective new drugs are needed for treatment of malaria. Atovaquone and proguanil hydrochloride is a new antimalarial combination that has recently become available in many countries. METHODS: Data from clinical trials evaluating atovaquone/proguanil for treatment of malaria were reviewed. RESULTS: In 10 open-label clinical trials, treatment of uncomplicated falciparum malaria with 1000 mg atovaquone and 400 mg proguanil hydrochloride (or the equivalent based on body weight in patients < or = 40 kg) once daily for 3 days achieved cure in 514 of 521 (99%) evaluable patients. Treatment-limiting adverse events occurred in < 1% of patients (vomiting in four, anaphylaxis in one). Atovaquone/proguanil has been used to provide radical cure of asymptomatic Plasmodium falciparum infections prior to initiation of placebo-controlled trials of malaria prophylaxis. Recurrent parasitemia occurred within 28 days in 0 of 99 subjects who subsequently received prophylaxis with atovaquone/proguanil and 1 of 81 subjects who subsequently received placebo. Atovaquone/proguanil is also effective for treatment of malaria caused by the other three Plasmodium species that cause malaria in humans. For treatment of vivax malaria, therapy with primaquine in addition to atovaquone/proguanil is needed to prevent relapse from latent hepatic hypnozoites. CONCLUSION: Atovaquone and proguanil hydrochloride is a safe and effective combination for treatment of malaria.

Atovaquone and proguanil hydrochloride for prophylaxis of malaria.

BACKGROUND: The spread of drug-resistant malaria and appreciation of side effects associated with existing antimalarial drugs emphasize the need for new drugs to prevent malaria. The combination of atovaquone and proguanil hydrochloride was previously shown to be safe and highly effective for treatment of malaria, including multi-drug-resistant Plasmodium falciparum. METHODS: We reviewed results of clinical trials that evaluated either a fixed-dose combination of atovaquone and proguanil hydrochloride for malaria prophylaxis or atovaquone alone for causal prophylactic activity against P. falciparum. RESULTS: In three placebo-controlled trials, 331 subjects received 250 mg atovaquone and 100 mg proguanil hydrochloride (or an equivalent dose based on body weight in children) once daily for 10 to 12 weeks. The overall efficacy for preventing parasitemia was 98%. Among 175 nonimmune volunteers taking the same dose of atovaquone/proguanil once daily for 10 weeks while temporarily residing in a malaria-endemic area, malaria developed in one patient who was noncompliant with therapy. Results of volunteer challenge studies indicate that both atovaquone and proguanil have causal prophylactic activity directed against the liver stages of P. falciparum. Adverse events occurred with similar or lower frequencies in subjects treated with atovaquone/proguanil compared to placebo. Less than 1% of patients discontinued from these studies due to a treatment-related adverse event. CONCLUSION: A fixed-dose combination of atovaquone and proguanil hydrocloride is a promising new alternative for malaria prophylaxis.

Clinical manifestations of Plasmodium falciparum malaria experimentally induced by mosquito challenge.

To determine the characteristics of clinical illness accompanying Plasmodium falciparum infection induced by controlled exposure to infected mosquitoes, records of 118 volunteers participating in studies conducted between 1985 and 1992 were reviewed. One hundred fourteen volunteers (97%) reported at least one symptom attributable to malaria, with fatigue, myalgias or arthralgias, headache, and chills most commonly reported. The median duration of symptoms was 3 days. Fever was recorded in 61% of volunteers; 4 volunteers had temperatures >40 degrees C. Neutropenia and thrombocytopenia were present in 9% and 12% of volunteers, respectively. Despite counts as low as 658/microL (neutrophils) or 73,000/microL (platelets), no secondary infectious or hemorrhagic complications occurred. In all cases, volunteers recovered completely and laboratory values returned to baseline after specific antimalarial therapy. Recrudescence did not occur in any volunteer. In this model, mosquito inoculation of P. falciparum is a reliable, safe, and well-tolerated method of experimental challenge.

Merozoite surface protein-1 epitopes recognized by antibodies that inhibit Plasmodium falciparum merozoite dispersal.

Serum antibodies from malaria immune donors can inhibit merozoite dispersal by forming immune complexes through surface-accessible regions of membrane associated antigens. Such merozoite forms are referred to as immune clusters of merozoites (ICM). Antibodies dissociated from ICM of Plasmodium falciparum identify a restricted subset of antigens, including merozoite surface protein-1 (MSP-1). We performed epitope mapping by comparing the reactivity of whole immune sera and ICM-derived antibodies in immunoblotting assays, using fourteen overlapping recombinant MSP-1 fragments, and by ELISA, using each of the 1720 octapeptides encoded within MSP-1. Antibodies in immune sera reacted with thirteen recombinant fragments and hundreds of octapeptides, but antibodies derived from ICM reacted with only six recombinant fragments and twenty octapeptides. Recombinant fragment recognition by ICM-derived antibodies was delimited to three regions 150-200 residues long, with seven of the octapeptide epitopes also mapping to these regions. The octapeptides recognized most strongly by antibodies in whole serum corresponded to the degenerate repeats near the N-terminus of MSP-1, however, neither recombinant fragments, nor octapeptides containing these degenerate repeats, were recognized by ICM-derived antibodies. Compared to reactions with recombinant fragments, the reactions observed with octapeptides were weak and may represent low-affinity mimetopes or cross-reactions. Alternatively, they may represent reactions with a portion of an epitope assembled from more than one non-contiguous peptide. These results suggest that ICM-derived antibodies can be used to map surface-accessible epitopes on MSP-1 and that the recombinant fragments with which they react are appropriate candidates for further evaluation as components of a malaria vaccine.

Plasmodium falciparum: chymotryptic-like proteolysis associated with a 101-kDa acidic-basic repeat antigen.

Malaria proteinases appear to function in the release of merozoites from infected erythrocytes and the invasion of merozoites into erythrocytes. Chymostatin, an inhibitor of chymotrypsin-like proteinases, inhibits malaria invasion and also inhibits apparent autoproteolysis of a 101-kDa acidic-basic repeat antigen (p101-ABRA) that is found in the vacuolar space surrounding merozoites in Plasmodium falciparum-infected erythrocytes. After purification by a monoclonal antibody (MAb 3D5), p101-ABRA degrades into smaller fragments in the absence of chymostatin. In this study fluorogenic proteinase substrates of the type peptidyl-7-amino-4-trifluoromethyl coumarin with phenylalanine or tyrosine linked to AFC were used to characterize chymotryptic-like activity associated with p101-ABRA. When p101-ABRA from the cell extract of P. falciparum-schizont-infected erythrocytes was affinity purified on MAb 3D5 beads, chymotryptic-like activity bound to the beads. Seventy-four percent to 96% of the activity detected using MeOSuc-KLF-AFC, Suc-LLVY-AFC, or SY-AFC at a pH optimum of 7.0 was removed from the extract and 6 to 33% was detected on the washed beads. Attempts to recover active enzyme eluted from the beads were not successful. Enzymes cleaving two other substrates (MeOSuc-AAPM-AFC and F-AFC) did not significantly bind to mAB 3D5 beads. Chymotryptic-like activity was also associated with p101-ABRA in fractions from sequential DEAE-Sephacel chromatography, Sephacryl S-200 chromatography, and nondenaturing polyacrylamide gel electrophoresis.

Serum concentrations of penicillin, doxycycline, and ciprofloxacin during prolonged therapy in rhesus monkeys.

Concentrations of penicillin, doxycycline, and ciprofloxacin were measured by bioassay in sera of rhesus monkeys treated with these drugs for inhalation anthrax. Antibiotic doses were determined on the basis of published serum concentration data from humans and comparative body surface area calculations for humans and rhesus monkeys. The antibiotics were well tolerated. Serum peak and trough concentrations of penicillin, doxycycline, and ciprofloxacin, respectively, averaged 2.7 and 0.8, 1.31 and 0.26, and 1.22 and 0.14 microgram/mL. These were within the range usually observed with standard oral doses in humans, and peak concentrations in all monkeys exceeded the MICs for 90% of Bacillus anthracis strains.

Immunization of owl monkeys with a recombinant protein containing repeated epitopes of a Plasmodium falciparum glycophorin-binding protein.

A Plasmodium falciparum glycophorin binding protein (GBP-130) has been implicated in protective immunity to malaria. The gene for GBP-130 encodes a protein containing 11 tandemly repetitive 50 amino acid units. We report an immunization trial in Aotus monkeys using a recombinant DNA protein containing three of these 50 amino acid repeats. When administered with aluminum hydroxide, this antigen induced low levels of antibodies that reacted with the recombinant protein by ELISA and with parasite antigens in immunoblot and immunofluorescence assays, but not by immunoprecipitation. When administered with Freund's complete adjuvant, this antigen induced high levels of antibodies that reacted in ELISA, immunoblot, immunofluorescence, and immunoprecipitation assays. Serum from immunized monkeys did not inhibit parasite growth, and protection from intravenous challenge with P. falciparum-infected erythrocytes was not observed in any experimental group. These results suggest that the repetitive region of GBP-130 is not a useful vaccine candidate.

Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1.

The CD36 and ICAM-1 glycoproteins on vascular endothelial cells have been implicated as cytoadherence receptors for Plasmodium falciparum-infected erythrocytes (IRBC). Adhesion of IRBC from Thai patients with uncomplicated and severe falciparum malaria to purified CD36 or ICAM-1 and to C32 melanoma cells was compared. All malaria isolates bound to solid phase-adsorbed CD36 and to fluid-phase 125I-labeled CD36. IRBC adhesion to purified ICAM-1 varied widely, and no correlation with clinical severity of disease was observed. The cytoadherent phenotype of IRBC was modulated by selective panning on plates coated with purified CD36 or ICAM-1. IRBC selected by panning on CD36+, ICAM-1+ melanoma cells bound to cells that express surface CD36 but not to CD36-deficient cells, indicating that CD36 exerts a strong selective pressure on the IRBC cytoadherent phenotype. IRBC adhesion to CD36 and ICAM-1 suggests that P. falciparum parasites may use these receptors in vivo to promote parasite survival and immune evasion.

Use of adjuvant containing mycobacterial cell-wall skeleton, monophosphoryl lipid A, and squalane in malaria circumsporozoite protein vaccine.

Human immune responses to modern synthetic and recombinant peptide vaccines administered with the standard adjuvant, aluminum hydroxide, tend to be poor, hence the search for better adjuvants. Antibody responses to a Plasmodium falciparum circumsporozoite (CS) protein vaccine, R32NS1(81), administered with an adjuvant containing cell-wall skeleton of mycobacteria and monophosphoryl lipid A in squalane (MPL/CWS) have been compared to responses to the same immunogen administered with aluminum hydroxide. 2 weeks after the third dose the following indices were greater in the 5 patients who received MPL/CWS than in controls (p less than 0.05): the geometric mean concentration (2.0 vs 25.4 microgram/ml) and avidity index of antibodies to the P falciparum CS protein by ELISA, the geometric mean titre to P falciparum sporozoites by IFAT (1/115 vs 1/1600), and the geometric mean inhibition of sporozoite invasion of hepatoma cells in vitro (37.6 vs 90.3%). For R32NS1(81) MPL/CWS is superior to aluminum hydroxide as an adjuvant, and the data support the evaluation of this complex as an adjuvant for other vaccines.

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.

Host receptors for malaria-infected erythrocytes.

Mature trophozoites and schizonts of Plasmodium falciparum induce changes in their host erythrocyte membranes that cause infected erythrocytes to sequester by binding to capillary endothelial cells. Sequestration protects infected erythrocytes from destruction in the spleen and contributes to the pathogenesis of severe and complicated malaria. The use of in vitro parasite culture and cytoadherence assays that measure the binding of infected erythrocytes to target cells has enabled the identification of host proteins that are putative receptors to which infected erythrocytes bind. Three such receptors have been identified: the extracellular matrix protein thrombospondin, the leukocyte differentiation antigen CD36, and the intercellular adhesion molecule ICAM-1. Infected erythrocytes can bind in vitro to each of these proteins. Thrombospondin and CD36 bind to one another, but each also binds to infected erythrocytes independently. Triggering of the CD36 receptor on platelets and monocytes activates these cells in vitro, and stimulation of endothelial cells with cytokines that upregulate ICAM-1 expression can increase the binding of infected erythrocytes to endothelial cells. Studies of these and perhaps other host receptors to which infected erythrocytes bind may contribute to our understanding of pathophysiologic mechanisms in malaria.

Safety and immunogenicity of a Plasmodium vivax sporozoite vaccine.

A recombinant DNA Plasmodium vivax sporozoite vaccine containing the repeating region of the Salvador I strain circumsporozoite (CS) protein was produced in Escherichia coli. This vaccine was tested in 13 naive volunteers at doses of 10-1,000 micrograms. No serious adverse reactions were noted. None of 4 volunteers receiving the 10 micrograms dose developed antibodies measurable by ELISA. Six of 9 volunteers in the other dose groups developed measurable antibodies: 5 of 5 volunteers receiving 100 micrograms and 1 of 4 receiving 1,000 micrograms. Antibody responses measured by immunofluorescence assays paralleled those seen by ELISA. None of the volunteers developed antisera that inhibited sporozoite invasion of human hepatoma cells in vitro. Lack of a classical anamnestic response and lack of a typical dose response to increasing amounts of antigen suggests the possible presence of an immunosuppressive epitope in the repetitive region of the CS protein.