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 malar ia blood-stage vaccine candidates that have induced protection in rodent an d nonhuman primate models of malaria. Correct conformation of the protein a ppears to be essential for the induction of parasite inhibitory responses, and these responses appear to be primarily antibody mediated. Here we descr ibe 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 methylotro phic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservat ively mutagenized PV66/AMA-1 gene (PV66 Delta glyc) lacking N-glycosylation sites was also developed. Expression of the PV66 Delta glyc ectodomain yie lded similar levels of a homogeneous product that was nonglycosylated and w as readily purified by ion-exchange and gel filtration chromatographies. Re combinant 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 immun oglobulin 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.