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, † and Jeffrey J. Adamovicz † United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-1201 A 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. Introduction The United States Department of Homeland Security and Department of Defense require an effective vaccine to protect against human plague. The new vaccine will also benefit the Centers for Disease Control and Preven- tion, state and local emergency response and rescue teams, as well as some scientists and members of the World Heath Organization who currently have no U.S. licensed plague vaccine for prophylaxis during labora- tory and field work with Yersinia pestis. This paper describes the design, research level production, charac- terization, proof of concept testing, and comparative analysis of an improved fusion protein antigen that has been recommended by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) (1) as the active biological substance for the next plague vaccine. Background. Plague is a zoonotic disease caused by Y. pestis that has impacted society, medicine, and mili- tary history. An estimated 200 million human deaths are attributable to plague through three recorded global pandemics and innumerable regional and local outbreaks. Y. pestis apparently emerged 15,000-20,000 years ago from its ancestor, Yersinia pseudotuberculosis, by acquir- ing and adapting genes for survival and increased virulence in both the flea and mammalian host (2-5). Y. pestis bacilli are typically transmitted among wild ani- mals by fleas, thereby maintaining a natural reservoir as the sylvatic form of the disease. More than 200 mammalian species are known carriers or incidental hosts for Y. pestis, including humans and domestic and wild cats and dogs (6). Foci of epizootic disease continue to occur throughout the world today as plague is enzootic in semi-arid climates from the tropics to temperate zones. The World Health Organization tabulated a worldwide annual average of 2,547 human cases (15 in the U.S.) during a recent 10-year period (7). Contemporary out- breaks in India and East Africa and the increasing use of modern air travel have raised concern for possible global re-emergence of plague. Furthermore, the natural ability for gene transfer and documented occurrence of multi-drug-resistant strains (4, 8, 9) forewarns the ap- * To whom correspondence should be addressed. Ph: +1-301- 619-4933. Fax: +1-301-619-2152. E-mail: bradford.powell@ amedd.army.mil. † Division of Bacteriology, USAMRIID. | Division of Toxinology, USAMRIID. ‡ Biopharmaceutical Development Program, SAIC Frederick, National Cancer Institute at Frederick, Frederick, MD 21702-1201. ⊥ Vaccine and Prevention Research Program, Division of AIDS, NIAID/NIH/DHHS, 6700B Rockledge Dr., Bethesda, MD 20892- 7630. § Department of Veterinary Sciences, University of Wyoming, Laramie, WY 82070. 1490 Biotechnol. Prog. 2005, 21, 1490-1510 10.1021/bp050098r This article not subject to U.S. Copyright. Published 2005 by the American Chemical Society and American Institute of Chemical Engineers Published on Web 08/09/2005