title: Improved formulation of a recombinant ricin A-chain vaccine increases its stability and effective antigenicity.
abstract: Ricin is a potent toxin associated with bioterrorism for which no vaccine or specific countermeasures are currently available. A stable, non-toxic and immunogenic recombinant ricin A-chain vaccine (RTA 1-33/44-198) has been developed by protein engineering. We identified optimal formulation conditions for this vaccine under which it remained stable and potent in storage for up to 18 months, and resisted multiple rounds of freeze-thawing without stabilizing co-solvents. Reformulation from phosphate buffer to succinate buffer increased adherence of the protein to aluminum hydroxide adjuvant from 15 to 91%, with a concomitant increase of nearly threefold in effective antigenicity in a mouse model. Using Fourier-transform infrared spectroscopy, we examined the secondary structure of the protein while it was adhered to aluminum hydroxide. Adjuvant adsorption produced only a small apparent change in secondary structure, while significantly stabilizing the protein to thermal denaturation. The vaccine therefore may be safely stored in the presence of adjuvant. Our results suggest that optimization of adherence of a protein antigen to aluminum adjuvant can be a useful route to increasing both stability and effectiveness, and support a role for a "depot effect" of adjuvant.
authors.affiliation: Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, United States.
authors.forename: John H
authors.initials: JH
authors.lastname: Carra
authors.forename: Robert W
authors.initials: RW
authors.lastname: Wannemacher
authors.forename: Ralph F
authors.initials: RF
authors.lastname: Tammariello
authors.forename: Changhong Y
authors.initials: CY
authors.lastname: Lindsey
authors.forename: Richard E
authors.initials: RE
authors.lastname: Dinterman
authors.forename: Rowena D
authors.initials: RD
authors.lastname: Schokman
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry; D000536:Aluminum Hydroxide / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000992:Antitoxins / Q000097:blood; D002626:Chemistry, Pharmaceutical; D004195:Disease Models, Animal; D004356:Drug Storage; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009500:Neutralization Tests; D011041:Poisoning / Q000517:prevention & control; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology*; D012276:Ricin / Q000506:poisoning*; D016019:Survival Analysis; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000276:Adjuvants, Immunologic; D000992:Antitoxins; D021122:Protein Subunits; D014614:Vaccines, Synthetic; D000536:Aluminum Hydroxide; D012276:Ricin