Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

详细信息    查看全文

• 作者：Jeremy L. Goodin ; David F. Nellis ; Bradford S. Powell ; Vinay V. Vyas ; Jeffrey T. Enama ; Lena C. Wang ; Patrick K. Clark ; Steven L. Giardina ; Jeffery. J. Adamovicz ; Dennis F. Michiel
• 关键词：F1-V ; Fusion protein ; Vaccine ; Development ; Yersinia pestis ; Monodisperse ; Alhydrogel ; Protective efficacy
• 刊名：Protein Expression and Purification
• 出版年：2007
• 出版时间：May 2007
• 年：2007
• 卷：53
• 期：1
• 页码：63-79
• 全文大小：1394 K

文摘

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 for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V<sub>C424Ssub> proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2 mg/g of cell paste of 95 % pure, mono-disperse protein having 0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V<sub>C424Ssub> 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. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel<sup>®sup> adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V<sub>C424Ssub> 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 × 20 μg of F1-V, respectively, 100 % , 80 % , 80 % , and 70 % of injected mice survived a subcutaneous lethal plague challenge with 10<sup>8sup> LD<sub>50sub> Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.