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Infection and Immunity, April 2005, p. 2109-2115, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2109-2115.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Process Development and Analysis of Liver-Stage Antigen 1, a Preerythrocyte-Stage Protein-Based Vaccine for Plasmodium falciparum

Collette J. Hillier, Lisa A. Ware, Arnoldo Barbosa, Evelina Angov, Jeffrey A. Lyon, D. Gray Heppner, and David E. Lanar*

Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland

Received 28 September 2004/ Returned for modification 4 November 2004/ Accepted 26 November 2004

Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 µg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.

* Corresponding author. Mailing address: Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910-7500. Phone: (301) 319-9003. Fax: (202) 318-7594. E-mail: david.lanar{at}na.amedd.army.mil.

Editor: W. A. Petri, Jr.

Infection and Immunity, April 2005, p. 2109-2115, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2109-2115.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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