High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris:: Strong immunogenicity in Macaca mulatta immunized with P-vivax AMA-1 and adjuvant SBAS2

The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66 Delta glyc) lacking N-glycosylation sites was also developed. Expression of the PV66 Delta glyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66 Delta glyc(43-487) was reactive with conformation-dependent monoclonal antibodies, With the SBAS2 adjuvant, Pichia-expressed PV66 Delta glyc(43-487) was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000, This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.