BackgroundThis Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems.Methodology/Principal FindingsAfter a preliminary safety evaluation of low dose AMA-1/AS01B (10 µg/0.5 mL) in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 µg/0.5 mL) of AMA-1/AS01B (n = 15) or AMA-1/AS02A (n = 15), followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs) with 95% Confidence Intervals (CIs) were high: low dose AMA-1/AS01B 196 µg/mL (103–371 µg/mL), full dose AMA-1/AS01B 279 µg/mL (210–369 µg/mL) and full dose AMA-1/AS02A 216 µg/mL (169–276 µg/mL) with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA), against homologous but not against heterologous (FVO) parasites as well as demonstrable interferon-gamma (IFN-γ) responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR). However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements.SignificanceAll three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali.Trial Registration www.clinicaltrials.gov NCT00385047

We report the first safety and immunogenicity trial of the Plasmodium falciparum vaccine candidate FMP2.1/AS02A, a recombinant E. coli-expressed protein based upon the apical membrane antigen-1 (AMA-1) of the 3D7 clone formulated with the AS02A adjuvant. We conducted an open-label, staggered-start, dose-escalating Phase I trial in 23 malaria-naïve volunteers who received 8, 20 or 40 g of FMP2.1 in a fixed volume of 0.5 mL of AS02A on a 0, 1, and 2 month schedule. Nineteen of 23 volunteers received all three scheduled immunizations. The most frequent solicited local and systemic adverse events associated with immunization were injection site pain (68%) and headache (29%). There were no significant laboratory abnormalities or vaccine-related serious adverse events. All volunteers seroconverted after second immunization as determined by ELISA. Immune sera recognized sporozoites and merozoites by immunofluorescence assay (IFA), and exhibited both growth inhibition and processing inhibition activity against homologous (3D7) asexual stage parasites. Post-immunization, peripheral blood mononuculear cells exhibited FMP2.1-specific lymphoproliferation and IFN-␥ and IL-5 ELISPOT assay responses. This is the first PfAMA-1-based vaccine shown to elicit both potent humoral and cellular immunity in humans. Encouraged by the potential of FMP1/AS02A to target host immunity against PfAMA-1 that is known to be expressed by sporozoite, hepatic and erythrocytic stages, we have initiated field trials of FMP2.1/AS02A in an endemic population in the Republic of Mali.

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The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83 Gly to 531 Glu ) of Plasmodium falciparum (3D7) produced in Escherichia coli. A synthetic gene containing an E. coli codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified E. coli strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the P. falciparum homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.Plasmodium falciparum causes more than three million deaths each year, mostly among children below the age of five (30). The spread of multi-drug-resistant strains of the parasite has underlined an urgent need for a malaria vaccine. Evidence exists from both animal models and human studies that antibodies to erythrocytic and exoerythrocytic parasite antigens can induce protection. Apical membrane antigen 1 (AMA1) is one of the most promising erythrocytic-stage vaccine targets under investigation. Present on the extracellular merozoite stage of the parasite, AMA1 is amenable to host immune intervention during the process of invasion. Indeed, immunization in animal models with affinity-purified or recombinant forms of AMA1 along with adjuvants permissible for human use can induce a protective response against homologous parasite challenge in vivo (1,5,7,23). Homologues of the AMA1 gene have been identified in all of the commonly studied species of Plasmodium (4,8,16,18,20,24,25,29), and knockout studies have revealed that the expression of AMA1 protein is vital for parasite survival (28).P. falciparum AMA1 is an integral membrane protein synthesized as a 72-kDa polypeptide (apparent molecular mass, 83 kDa) (24); it is localized in the apical rhoptries of the...

The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T-and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, D.G. Heppner Jr. et al. / Vaccine 23 (2005) [2243][2244][2245][2246][2247][2248][2249][2250] academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.