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

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; Jeffrey J. Adamovicz

doi:10.1021/bp050098r

journal article Jan 01, 2005

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

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 Jeffrey J. Adamovicz

Biotechnology Progress Vol. 21 No. 5 pp. 1490-1510 · Wiley

View at Publisher Save 10.1021/bp050098r

Abstract

AbstractA two‐component recombinant fusion protein antigen was re‐engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine‐tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1‐V fusion protein absent extraneous coding sequences. Soluble F1‐V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea‐denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N‐terminal methionine. Purity, quality, and higher‐order structure were compared between lots using RP‐HPLC, intrinsic fluorescence, CD spectroscopy, and multi‐angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1‐V protein also protected mice from wild‐type and non‐encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1‐V as the active pharmaceutical ingredient of the next plague vaccine.

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Abbreviations used: Ab antibody; AEX anion exchange chromatography; CEX cation exchange chromatography; CIEF capillary isoelectric focusing; des‐Met without the N‐terminal translation initiator methionine; DO2 dissolved oxygen; DPBS Dulbecco's phosphate‐buffered saline without Ca2+or Mg2+; F1‐V fusion protein comprisingY. pestisfraction 1 capsule joined to V antigen; FDA Food and Drug Administration; GMP good manufacturing practices; GMT geometric mean titer; GSD geometric standard deviation; HPLC high‐pressure liquid chromatography; IEP isoelectric point; IPTG isopropyl‐β‐d‐thiogalactoside; KWC killed whole cell; LD50 50% lethal dose; MALDI‐TOF‐MS matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry; Met methionine;Mr relative molecular weight; MST mean survival time as measured in days;m/z mass‐to‐charge; OD405 optical density at 405 nm wavelength; PCR polymerase chain reaction; RFLP restriction fragment length polymorphism; RP‐HPLC reversed‐phase high‐pressure liquid chromatography; SC subcutaneous; SE standard error; SEC size exclusion chromatography; SEC‐MALS size exclusion chromatography coupled to multi‐angle light scattering spectroscopy; TetR tetracycline resistance; USAMRIID United States Army Medical Research Institute of Infectious Diseases; YOP Yersiniaouter protein.

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Published: Jan 01, 2005
Vol/Issue: 21(5)
Pages: 1490-1510
License: View

Authors

Cite This Article

Bradford S. Powell, Gerard P. Andrews, Jeffrey T. Enama, et al. (2005). Design and Testing for a Nontagged F1‐V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague. Biotechnology Progress, 21(5), 1490-1510. https://doi.org/10.1021/bp050098r

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

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