The malaria vaccine Combination B comprises recombinant Plasmodium falciparum ring-infected erythrocyte surface antigen and 2 merozoite surface proteins (MSP1 and MSP2) formulated in oil-based adjuvant. A phase 1-2b double-blind, randomized, placebo-controlled trial in 120 children (5-9 years old) in Papua New Guinea demonstrated a 62% (95% confidence limits: 13%, 84%) reduction in parasite density in children not pretreated with sulfadoxine-pyrimethamine. Vaccinees had a lower prevalence of parasites carrying the MSP2-3D7 allelic form (corresponding to that in the vaccine) and a higher incidence of morbid episodes associated with FC27-type parasites. These results demonstrate functional activity of Combination B against P. falciparum in individuals with previous malaria exposure. The specific effects on parasites with particular msp2 genotypes suggest that the MSP2 component, at least in part, accounted for the activity. The vaccine-induced selection pressure exerted on the parasites and its consequences for morbidity strongly argue for developing vaccines comprising conserved antigens and/or multiple components covering all important allelic types.

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BackgroundPfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion.Methodology/Principal FindingsThe trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005–April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 µg of Pfs25/ISA 51, 5 µg of Pvs25/ISA 51, or 20 µg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51). Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity.Conclusion/SignificanceIt is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum.Trial RegistrationClinicalTrials.gov NCT00295581

Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYT bc 1 ) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25-to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.The widespread resistance of malaria parasites to standard antimalarial drugs is a serious global health problem. The urgent need for new antimalarial drugs has led to the development of atovaquone (566C80) which, combined with proguanil, has been licensed as Malarone. There is some concern that parasites may develop resistance to Malarone. In one study, 33% of patients treated with atovaquone alone experienced a recrudescence of parasitemia after treatment. These parasites tolerated up to 1,000-fold higher concentrations of atovaquone than did the pretreated parasites (16). Atovaquone-resistant parasites have been readily selected in vitro. Up to 1 in 10 5 parasites became resistant to the drug after having been cultured in the presence of 10 Ϫ8 M atovaquone for 5 weeks (21, 23).Atovaquone has potent blood schizonticidal activity and is also effective against the preerythrocytic (2, 4, 5) and sexual stages (8, 9) of the malaria parasite. It acts by inhibiting mitochondrial electron transport (10) and collapsing mitochondrial membrane potential (25). From these observations and on the basis of its structural similarity to ubiquinol, it has been postulated that atovaquone binds to parasite cytochrome b (CYT b) (31). The inhibitors stigmatellin and 5-n-undecyl-4,7-dioxobenzoxythiazol (UHDBT), which are structurally similar to atovaquone, have been shown to bind at the ubihydroquinone (Q o ) site of CYT b and inhibit electron transport. Single point mutations within the Q o site confer resistance to these inhibitors in a variety of microorganisms (7). Two mutations in close proximity to the Q o site in Pneumocystis carinii are associated with atovaquone prophylaxis failure (33). Atovaquone-resistant Plasmodium yoelii lines have been derived from infected mice treated with suboptimal doses of atovaquone. All resistant lines ...

Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.