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

Abstract

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.

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

“…F1-V was originally purified using a polyhistidine tag and the his(10)-F1-V vaccine protected experimental mice against pneumonic as well as bubonic plague produced by either F1 + or F1 − strains of Y. pestis [5]. By a statistical comparison of potency [6], the recombinant fusion-protein vaccine provided far better protection against the wild-type (F1 + ) strain than did the former Plague Vaccine USP, and it also showed a significant improvement in protection over a cocktail vaccine composed of the separate F1 and V antigens, as was first indicated after its creation [4,6].…”

“…Based on the success of animal protection studies and with the intent to improve the fusion protein for product development, F1-V was subsequently re-engineered to remove the polyhistidine tag and placed under transcriptional control of the IPTG-inducible pET-24a expression system (plasmid pPW731) in Escherichia coli strain BL21 (DE3), and then purified from soft inclusion bodies using 6M urea and a two-column procedure including anionexchange and hydrophobic interaction chromatography [6]. The untagged fusion protein showed equivalent immunogenicity and protective efficacy, and was less polydisperse in molecular structure than the individual F1 subcomponent, but still showed a tendency to aggregate under certain conditions.…”

“…The untagged fusion protein showed equivalent immunogenicity and protective efficacy, and was less polydisperse in molecular structure than the individual F1 subcomponent, but still showed a tendency to aggregate under certain conditions. The tendency of F1-V to self-associate was revealed by analytical size exclusion chromatography (HPLC-SEC) coupled to multiple angle laser light scattering (called SEC-MALLS in combination), which clearly showed mixtures of monomer, dimer, and multimeric species of higher mass in all standard preparations of F1-V [6]. The prior study did not eliminate a possible contribution to aggregation or stability by the single cysteine in F1-V, which provided an un-paired, solution-accessible free-thiol.…”

Assessment of a Recombinant F1-v Fusion Protein Vaccine Intended to Protect Canada Lynx (Lynx Canadensis) From Plague

“…Our interest in potentially incorporating plague vaccination into the Colorado Division of Wildlife's lynx reintroduction protocol was motivated by these findings. Effective vaccines for preventing plague in mammalian species, including felids, have been developed only recently (Heath et al, 1998, Gasper andWatson, 2001;Rocke et al, 2004;Powell et al, 2005). Of these, a recombinant capsular F1-V fusion protein vaccine (Powell et al, 2005) has shown a promising combination of safety and efficacy in black-footed ferret recovery (Rocke et al, 2004(Rocke et al, , 2008 and appeared potentially useful in lynx restoration as well.…”

“…Effective vaccines for preventing plague in mammalian species, including felids, have been developed only recently (Heath et al, 1998, Gasper andWatson, 2001;Rocke et al, 2004;Powell et al, 2005). Of these, a recombinant capsular F1-V fusion protein vaccine (Powell et al, 2005) has shown a promising combination of safety and efficacy in black-footed ferret recovery (Rocke et al, 2004(Rocke et al, , 2008 and appeared potentially useful in lynx restoration as well. We report on assessment of responses to a recombinant F1-V fusion protein vaccine (US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland, USA) in captive lynx being held in southwestern Colorado prior to release as part of a reintroduction program.…”