title: In silico prediction of Gallibacterium anatis pan-immunogens.
abstract: The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870, Frederiksberg C, Denmark. rjb@sund.ku.dk.
authors.forename: Ragnhild J
authors.initials: RJ
authors.lastname: Bager
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870, Frederiksberg C, Denmark. egle@sund.ku.dk.
authors.forename: Egle
authors.initials: E
authors.lastname: Kudirkiene
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870, Frederiksberg C, Denmark. idapiedade@sund.ku.dk.
authors.forename: Isabelle
authors.initials: I
authors.lastname: da Piedade
authors.affiliation: Victorian Bioinformatics Consortium, Monash University, 3800, Clayton, Melbourne, Australia. torsten.seemann@monash.edu.
authors.forename: Torsten
authors.initials: T
authors.lastname: Seemann
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200, Copenhagen N, Denmark. tine.nielsen@cpr.ku.dk.
authors.forename: Tine K
authors.initials: TK
authors.lastname: Nielsen
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870, Frederiksberg C, Denmark. sup@sund.ku.dk.
authors.forename: Susanne E
authors.initials: SE
authors.lastname: Pors
authors.affiliation: Center for Biological Sequence Analysis, Technical University of Denmark, 2800, Lyngby, Denmark. andreas.holm.mattsson@gmail.com.|Evaxion Biotech North America LLC, Wilmington, USA. andreas.holm.mattsson@gmail.com.
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Mattsson
authors.affiliation: Department of Microbiology, Monash University, 3800, Clayton, Melbourne, Australia. john.boyce@monash.edu.
authors.forename: John D
authors.initials: JD
authors.lastname: Boyce
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, 3800, Clayton, Melbourne, Australia. ben.adler@monash.edu.
authors.forename: Ben
authors.initials: B
authors.lastname: Adler
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870, Frederiksberg C, Denmark. miki@sund.ku.dk.
authors.forename: Anders M
authors.initials: AM
authors.lastname: Bojesen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001428:Bacterial Vaccines / Q000235:genetics* / Q000276:immunology; D002645:Chickens; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D015661:Pasteurellaceae / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism / Q000472:pathogenicity; D016871:Pasteurellaceae Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control / Q000662:veterinary*; D011201:Poultry Diseases / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D037521:Virulence Factors
chemical_list: D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D037521:Virulence Factors