ABSTRACT
Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins , Bacterial Vaccines/administration & dosage , Heat-Shock Proteins/immunology , Mycobacterium leprae/immunology , Trehalose/chemistry , Acylation , Adjuvants, Immunologic , Animals , Antibodies, Bacterial/analysis , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/chemistry , Enzyme-Linked Immunosorbent Assay , Freeze Fracturing , Immunization , Immunoglobulin G/analysis , Immunoglobulin G/biosynthesis , Immunoglobulin M/analysis , Immunoglobulin M/biosynthesis , Liposomes , Membranes, Artificial , Mice , Pharmaceutical VehiclesABSTRACT
Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein fromMycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with largeunilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehaloseavoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrappedprotein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on theliposomal membrane integrity.
Subject(s)
Humans , Animals , Vaccines/administration & dosage , Vaccines/isolation & purification , Vaccines/supply & distribution , TrehaloseABSTRACT
A thrombin-like enzyme was isolated in 6% yield from the venom of Crotalus durissus terrificus by ammonium sulfate precipitation followed by gel filtration on Sephadex G-75 and finally affinity chromatography on Sepharose-1,4-butanediol-diglycyl-p-aminobenzamide eluted with 0.15 M benzamidine. The enzyme behaved like a single component on SDS-PAGE corresponding to a molecular weight of 34 kDa. The specific activity of the enzyme toward bovine fibrinogen was 71 NIH U/mg protein. The pH optimum for the coagulation of human fibrinogen was 8.0. The enzyme hydrolyzes the alpha-chain of fibrinogen, has amidase activity on L-arginine-p-nitroanilide and L-arginine-7-amido-4-methyl-coumarin amino terminal blocked peptides and presents esterolytic activity on N-alpha-tosyl-L-arginine-methylester.