Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum.
Infect Immun
; 73(4): 2109-15, 2005 Apr.
Article
em En
| MEDLINE
| ID: mdl-15784552
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Plasmodium falciparum
/
Vacinas Sintéticas
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Vacinas Antimaláricas
/
Antígenos de Protozoários
Limite:
Animals
Idioma:
En
Revista:
Infect Immun
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos