Purification and Protective Efficacy of Monomeric and Modified Yersina pestis Capsular F1-V Antigen Fusion Proteins for Vaccination Against Plague
Abstract
The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis to enable characterization of F1-V non-covalent multimer interactions and protective potency without participation by covalent disulfide-linkages. F1-V and F1-VC424S proteins were over-expressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, by using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2 mg per gram of cell paste of 95% pure, mono-disperse protein having ≤ 0.5 endotoxin units per mg by LAL. Both F1-V and F1-VC424S were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0, with partial precipitation occurring below pH 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible Alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-VC424S monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 g of F1-V, respectively, 100, 80, 80, and 70% of injected mice survived a subcutaneous lethal plague challenge with 108 LD50 Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.
Document Details
- Document Type
- Technical Report
- Publication Date
- Dec 31, 2006
- Accession Number
- ADA463927
Entities
People
- Bradford S. Powell
- David F. Nellis
- Dennis F. Michiel
- Jeffrey J. Adamovicz
- Jeffrey T. Enama
- Jeremy L. Goodin
- Lena C. Wang
- Patrick K. Clark
- Steven L. Giardina
- Vinay V. Vyas
Organizations
- United States Army Medical Research Institute of Infectious Diseases