Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum.
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 microg 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.
Hillier
Collette J
CJ
Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910-7500, USA.
Ware
Lisa A
LA
Barbosa
Arnoldo
A
Angov
Evelina
E
Lyon
Jeffrey A
JA
Heppner
D Gray
DG
Lanar
David E
DE
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007371:Interferon-gamma / Q000096:biosynthesis; D017780:Malaria Vaccines / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014614:Vaccines, Synthetic / Q000276:immunology*
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C067302:liver stage-specific antigen, Plasmodium; D007371:Interferon-gamma