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Infection and Immunity, January 1999, p. 43-49, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Clemens H. M. Kocken,1 Martin A. Dubbeld,1 Annemarie Van Der Wel,1 Jack T. Pronk,2 Andrew P. Waters,3 Jan A. M. Langermans,1 and Alan W. Thomas1,*

Department of Parasitology, Biomedical Primate Research Centre, 2280 GH Rijswijk,1 Kluyver Laboratory of Biotechnology, Industrial Biotechnology Section, Delft University of Technology, 2628 BC Delft,2 and Leiden University Medical Centre, 2300 RC Leiden,3 The Netherlands

Received 12 May 1998/Returned for modification 24 July 1998/Accepted 5 October 1998

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 (PV66Delta glyc) lacking N-glycosylation sites was also developed. Expression of the PV66Delta glyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Delta glyc43-487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant, Pichia-expressed PV66Delta glyc43-487 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.

* Corresponding author. Mailing address: BPRC, Dept. of Parasitology, Lange Kleiweg 157, 2288 GJ Rijswijk, The Netherlands. Phone: (31) 15-2842 538. Fax: (31) 15-2843 986. E-mail: thomas{at}bprc.nl.

Infection and Immunity, January 1999, p. 43-49, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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