Design and Testing for a Nontagged F1-V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague

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• 作者：Bradford S. Powell ; Gerard P. Andrews ; Jeffrey T. Enama ; Scott Jendrek ; Chris Bolt ; Patricia Worsham ; Jeffrey K. Pullen ; Wilson Ribot ; Harry Hines ; Leonard Smith ; David G. Heath ; and Jeffrey J. Adamov
• 刊名：Biotechnology Progress
• 出版年：2005
• 出版时间：October 2005
• 年：2005
• 卷：21
• 期：5
• 页码：1490 - 1510
• 全文大小：474K
• 年卷期：v.21,no.5(October 2005)
• ISSN：1520-6033

文摘

A two-component recombinant fusion protein antigen was re-engineered and testedas a medical counter measure against the possible biological threat of aerosolizedYersinia pestis. The active component of the proposed subunit vaccine combines theF1 capsular protein and V virulence antigen of Y. pestis and improves upon the designof an earlier histidine-tagged fusion protein. In the current study, different productionstrains were screened for suitable expression and a purification process was optimizedto isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-Vprotein was isolated to 99% purity by sequential liquid chromatography includingcapture and refolding of urea-denatured protein via anion exchange, followed byhydrophobic interaction, concentration, and then transfer into buffered saline for directuse after frozen storage. Protein identity and primary structure were verified by massspectrometry and Edman sequencing, confirming a purified product of 477 amino acidsand removal of the N-terminal methionine. Purity, quality, and higher-order structurewere compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy,and multi-angle light scattering spectroscopy, all of which indicated a consistent andproperly folded product. As formulated with aluminum hydroxide adjuvant andadministered in a single subcutaneous dose, this new F1-V protein also protected micefrom wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxisagainst pneumonic and bubonic plague. These findings confirm that the fusion proteinarchitecture provides superior protection over the former licensed product, establisha foundation from which to create a robust production process, and set forth assaysfor the development of F1-V as the active pharmaceutical ingredient of the next plaguevaccine.