Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the Plasmodium falciparum Apical Membrane Antigen 1 Expressed in Escherichia coli

Abstract

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...

Phase I dose escalation safety and immunogenicity trial of Plasmodium falciparum apical membrane protein (AMA-1) FMP2.1, adjuvanted with AS02A, in malaria-naïve adults at the Walter Reed Army Institute of Research

“…Fig. 3A shows the typical pattern of AMA-1 distribution, namely apical fluorescence of individual merozoites within a schizont [22]. …”

“…FMP2.1 antigen (Lot 0971), representing amino acids #83-531 of the P. falciparum (clone 3D7) AMA-1 protein, was manufactured according to current good manufacturing practices (cGMP) at the Walter Reed Army Institute of Research Bioproduction Facility at Forest Glen, Maryland [22]. The final FMP2.1 product was a single dose vial containing approximately 43 g of lyophilized protein.…”

“…Blood stage late schizonts were fixed with methanol and immunofluorescence assays (IFA) were performed as described previously [22]. Briefly, 2% fetal bovine serum containing PBS was used as a diluent and serial dilutions starting from 1:2 to 1:25,600 were tested.…”

“…At the Walter Reed Army Institute of Research (WRAIR) Pilot Bioproduction Facility, we produced a recombinant protein, designated FMP2.1 [22], representing a major portion of the ecto-domain of PfAMA-1 from the 3D7 clone of P. falciparum. FMP2.1 was formulated in the GlaxoSmithKline proprietary adjuvant system, AS02A, which had been shown to enhance the immunogenicity and efficacy of the circumsporozoite-based malaria antigen, RTS,S [23][24][25][26][27][28].…”

Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research

“…AMA-1, like MSP-1, is the target of protective immunity, as supported by epidemiological observations [58], in vitro demonstrations [55], and by protection studies in which mice [59] or monkeys [60] immunized with recombinant forms of AMA-1 were protected from lethal challenge with homologous parasites. Scientists at the WRAIR have produced a correctly folded GMP recombinant protein in E. coli based upon the P. falciparum 3D7 strain AMA-1 ectodomain [61]. This antigen was vialed as a single-dose lyophilized cake intended for formulation with AS02A just prior to administration.…”