title: Expeditious screening of candidate proteins for microbial vaccines.
abstract: Advancements in high-throughput "omics" technologies have revolutionized the way vaccine candidates are identified. Now every surface expressed protein that an organism produces can be identified in silico and possibly made available for the rapid development of recombinant/subunit vaccines. However, evaluating the antigenicity of a large number of candidate proteins is an immense challenge, typically requiring cloning of several hundred candidates followed by immunogenicity screening. Here we report the development of a rapid, high-throughput method for screening candidate proteins for vaccines. This method involves utilizing a coupled, cell-free transcription-translation system to screen tagged proteins that are captured at the C-termini using appropriate ligand coated wells in 96 well ELISA plates. The template DNA for the cell-free expression is generated by two sequential PCRs and includes gene coding sequences, promoter, terminator, other necessary cis-acting elements and appropriate tag sequences. The process generates expressible candidate proteins containing two different peptide tags at the N- and the C-termini of the protein molecules. Proteins are screened in parallel for their quantity and immunoreactivity with N-terminal tag antibodies and antisera raised against the pathogen of interest, respectively. Normalization against the total detectable bound protein in the control wells allows for the identification of highly immunoreactive candidates. For this study we selected 30 representatives of >300 potential candidate proteins from Mannheimia haemolytica, a bacterial agent of pneumonia in feedlot cattle for expression with N-terminal Strep-II and C-terminal His(x6)-tag and evaluated their relative immunoreactivities using Strep-tactin-HRP and rabbit antisera generated against M. haemolytica. Using this system we were able to swiftly and quantitatively analyze and rank the suitability of proteins to identify potentially viable vaccine candidates, with the majority of the high ranking candidates being associated with virulence and pathogenicity. The system is adaptable to any bacterial target and presents an alternative to conventional laborious cloning, expression and screening procedures.
authors.affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1 Canada.
authors.forename: Rahat
authors.initials: R
authors.lastname: Zaheer
authors.affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1 Canada.
authors.forename: Cassidy L
authors.initials: CL
authors.lastname: Klima
authors.affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1 Canada. Electronic address: tim.mcallister@agr.gc.ca.
authors.forename: Tim A
authors.initials: TA
authors.lastname: McAllister
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification*; D001428:Bacterial Vaccines; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D057166:High-Throughput Screening Assays / Q000379:methods*; D016978:Mannheimia haemolytica / Q000737:chemistry* / Q000276:immunology* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction / Q000379:methods; D040901:Proteomics; D011817:Rabbits; D014774:Virulence
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines
keywords: Antigen; Cell-free protein expression; ELISA; Immunoreactivity; Vaccine