Subject(s)
Bioprosthesis , Heart Defects, Congenital/surgery , Heart Valve Diseases/surgery , Heart Valve Prosthesis , Hip/surgery , Aged , Aortic Valve/diagnostic imaging , Aortic Valve/surgery , Bicuspid Aortic Valve Disease , Bioprosthesis/adverse effects , Heart Defects, Congenital/diagnostic imaging , Heart Valve Diseases/diagnostic imaging , Heart Valve Prosthesis/adverse effects , Hip/diagnostic imaging , Hip/microbiology , Humans , Male , UltrasonographyABSTRACT
A synthetic multistage, multi-epitope Plasmodium falciparum malaria antigen (FALVAC-1A) was designed and evaluated in silico, and then the gene was constructed and expressed in Escherichia coli. The FALVAC-1A protein was purified by inclusion body isolation, followed by affinity and ion exchange chromatography. Although FALVAC-1A was a synthetic antigen, it folded to a specific, but as yet incompletely defined, molecular conformation that was stable and comparable from lot to lot. When formulated with four different adjuvants, FALVAC-1A was highly immunogenic in rabbits, inducing not only ELISA reactivity to the cognate antigen and most of its component epitopes, but also in vitro activity against P. falciparum parasites as demonstrated by inhibition of sporozoite invasion, antibody dependent cellular inhibition and the immunofluorescence assay.
Subject(s)
Antigens, Protozoan/immunology , Antigens, Protozoan/metabolism , Escherichia coli/metabolism , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Escherichia coli/genetics , Malaria, Falciparum/prevention & control , RabbitsABSTRACT
This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.
