title: 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.
abstract: 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 (PV66Deltaglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Deltaglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Deltaglyc43-487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant, Pichia-expressed PV66Deltaglyc43-487 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.
authors.affiliation: Department of Parasitology, Biomedical Primate Research Centre, Rijswijk, The Netherlands.
authors.forename: C H
authors.initials: CH
authors.lastname: Kocken
authors.forename: M A
authors.initials: MA
authors.lastname: Dubbeld
authors.forename: A
authors.initials: A
authors.lastname: Van Der Wel
authors.forename: J T
authors.initials: JT
authors.lastname: Pronk
authors.forename: A P
authors.initials: AP
authors.lastname: Waters
authors.forename: J A
authors.initials: JA
authors.lastname: Langermans
authors.forename: A W
authors.initials: AW
authors.lastname: Thomas
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage*; D000818:Animals; D000913:Antibodies, Protozoan / Q000096:biosynthesis; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors / Q000276:immunology / Q000378:metabolism; D007117:Immunization, Secondary; D008253:Macaca mulatta; D017780:Malaria Vaccines / Q000276:immunology*; D016780:Malaria, Vivax / Q000276:immunology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics* / Q000276:immunology; D016789:Plasmodium cynomolgi / Q000276:immunology; D010966:Plasmodium vivax / Q000235:genetics / Q000276:immunology*; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D000276:Adjuvants, Immunologic; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D008565:Membrane Proteins; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic; C090077:apical membrane antigen I, Plasmodium