Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen.
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.
['Thomas|Justin C|JC|', "O'Hara|Joanne M|JM|", 'Hu|Lei|L|', 'Gao|Fei P|FP|', 'Joshi|Sangeeta B|SB|', 'Volkin|David B|DB|', 'Brey|Robert N|RN|', 'Fang|Jianwen|J|', 'Karanicolas|John|J|', 'Mantis|Nicholas J|NJ|', 'Middaugh|C Russell|CR|']
D000818:Animals D057134:Antibodies, Neutralizing D000992:Antitoxins D001786:Blood Glucose D002151:Calorimetry D002942:Circular Dichroism D004195:Disease Models, Animal D004355:Drug Stability D051379:Mice D008807:Mice, Inbred BALB C D050505:Mutant Proteins D017354:Point Mutation D011041:Poisoning D011487:Protein Conformation D055550:Protein Stability D012276:Ricin D013050:Spectrometry, Fluorescence D016019:Survival Analysis D022223:Vaccines, Subunit D014614:Vaccines, Synthetic
immunogenicity ricin vaccine antigen computational protein design protein stability