title: Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen.
abstract: There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.
authors.affiliation: Macromolecule and Vaccine Stabilization Center; Department of Pharmaceutical Chemistry; University of Kansas; Lawrence, KS USA.
authors.forename: Justin C
authors.initials: JC
authors.lastname: Thomas
authors.forename: Joanne M
authors.initials: JM
authors.lastname: O'Hara
authors.forename: Lei
authors.initials: L
authors.lastname: Hu
authors.forename: Fei P
authors.initials: FP
authors.lastname: Gao
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
authors.forename: Robert N
authors.initials: RN
authors.lastname: Brey
authors.forename: Jianwen
authors.initials: J
authors.lastname: Fang
authors.forename: John
authors.initials: J
authors.lastname: Karanicolas
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Mantis
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000992:Antitoxins / Q000097:blood; D001786:Blood Glucose / Q000032:analysis; D002151:Calorimetry; D002942:Circular Dichroism; D004195:Disease Models, Animal; D004355:Drug Stability; D051379:Mice; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017354:Point Mutation; D011041:Poisoning / Q000517:prevention & control; D011487:Protein Conformation; D055550:Protein Stability; D012276:Ricin / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D013050:Spectrometry, Fluorescence; D016019:Survival Analysis; D022223:Vaccines, Subunit / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D000992:Antitoxins; D001786:Blood Glucose; D050505:Mutant Proteins; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D012276:Ricin
keywords: antigen; computational protein design; immunogenicity; protein stability; ricin; vaccine
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