High-level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical membrane antigen 1 and induction of antibodies that inhibit erythrocyte invasion

Abstract

Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum.

FIG. 1.

Analysis of expression of recombinant PfAMA-1 ectodomain in P. pastoris. Cultures were methanol induced for 48 h. (A) Secretion of Pf4mH was analyzed by Western blotting with the conformation-dependent MAb 4G2. One milliliter of the culture was centrifuged to pellet the yeast cells. The cell pellet was disrupted in a total of 1 ml of lysis buffer, and equal amounts of the culture supernatant (lane S) and cell pellet extract (lane P) were subjected to SDS-PAGE under nonreducing conditions and analyzed by Western blotting with MAb 4G2. The majority of the 4G2-reactive protein was secreted into the culture supernatant under these conditions. (B) Ni-agarose-purified Pf4mH from culture supernatant was analyzed by SDS-PAGE. Samples containing 1 μg of purified protein were applied to an SDS-polyacrylamide gel and stained with Coomassie blue. Lane 1, Pf4mH under reducing conditions; lane 2, Pf4mH under nonreducing conditions. Relative molecular masses are indicated in kilodaltons. Note the small increase in relative molecular mass upon reduction, indicative of formation of disulfide bond structures in the recombinant material.

FIG. 2.

Antigenicity of Pf4mH was evaluated by ELISA with MAbs 28G2 (reactive with the extreme C terminus of PfAMA-1, not present in Pf4mH), 58F8 (reactive with the N terminus of PfAMA-1), and 4G2 (reactive with a conformational AMA-1 epitope), European control human serum (Hu −ve), and human serum from Guinea-Bissau, where malaria is endemic (Hu +ve). MAbs were incubated at 10 ng ml⁻¹, and sera were diluted 1:1,000. OD, optical density.

FIG. 3.

Quantitative recognition of Pf4mH by MAb 4G2. Purified Pf4mH in PBS/T (lane 1) was incubated with MAb 4G2-Sepharose for 1 h. A sample of the protein remaining in solution after this period was taken (lane 2), and then the 4G2 Sepharose was washed extensively and a sample of the final wash was taken (lane 3), prior to elution of bound protein (lane 4). Samples were analyzed by SDS-PAGE and Coomassie blue staining. Essentially all the Pf4mH could be bound to 4G2-Sepharose. The minor additional bands present in lane 4 compared to lane 1 are probably due to antibody leaching from the 4G2-Sepharose under the low-pH conditions used for elution.