title: Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.
abstract: 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(C424S) 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 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) 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 adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) 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 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.
authors.affiliation: Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA.
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Goodin
authors.forename: David F
authors.initials: DF
authors.lastname: Nellis
authors.forename: Bradford S
authors.initials: BS
authors.lastname: Powell
authors.forename: Vinay V
authors.initials: VV
authors.lastname: Vyas
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Enama
authors.forename: Lena C
authors.initials: LC
authors.lastname: Wang
authors.forename: Patrick K
authors.initials: PK
authors.lastname: Clark
authors.forename: Steven L
authors.initials: SL
authors.lastname: Giardina
authors.forename: Jeffery J
authors.initials: JJ
authors.lastname: Adamovicz
authors.forename: Dennis F
authors.initials: DF
authors.lastname: Michiel
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D021461:Animals, Outbred Strains; D000942:Antigens, Bacterial / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D001428:Bacterial Vaccines / Q000276:immunology; D002021:Buffers; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects; D008027:Light; D008033:Limulus Test; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010930:Plague / Q000276:immunology / Q000517:prevention & control*; D010931:Plague Vaccine / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D012542:Scattering, Radiation; D012694:Serine / Q000378:metabolism; D012995:Solubility; D015996:Survival Rate; D016896:Treatment Outcome; D014508:Urea / Q000494:pharmacology; D014611:Vaccination; D014614:Vaccines, Synthetic / Q000008:administration & dosage; D015010:Yersinia pestis / Q000276:immunology* / Q000472:pathogenicity
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D002021:Buffers; D010931:Plague Vaccine; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C086725:caf1 protein, Yersinia pestis; D012694:Serine; D014508:Urea