Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets

Authors

  • Michael D. Valentino,

    1. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY
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  • Zachary J. Maben,

    1. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY
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  • Lucinda L. Hensley,

    1. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • Matthew D. Woolard,

    1. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC
    2. Department of Microbiology and Immunology, Louisiana State University Health Science Center at Shreveport, Shreveport, LA
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  • Thomas H. Kawula,

    1. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC
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  • Jeffrey A. Frelinger,

    1. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC
    2. Department of Immunobiology, University of Arizona, Tucson, AZ, USA
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  • John G. Frelinger

    1. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY
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Dr M. D. Valentino, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue Box 672, Rochester, NY 14642, USA. Email: michael_valentino@urmc.rochester.edu
Senior author: John G. Frelinger, email: jfrelinger@gmail.com

Summary

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularaemia. Concerns regarding its use as a bioterrorism agent have led to a renewed interest in the biology of infection, host response and pathogenesis. A robust T-cell response is critical to confer protection against F. tularensis. However, characterization of the cellular immune response has been hindered by the paucity of tools to examine the anti-Francisella immune response at the molecular level. We set out to combine recent advances of genomics with solid-phase antigen delivery coupled with a T-cell functional assay to identify T-cell epitopes. A subset of clones, encoding serological targets, was selected from an F. tularensis SchuS4 ordered genomic library and subcloned into a bacterial expression vector to test the feasibility of this approach. Proteins were expressed and purified individually employing the BioRobot 3000 in a semi-automated purification method. The purified proteins were coupled to beads, delivered to antigen-presenting cells for processing, and screened with Francisella-specific T-cell hybridomas of unknown specificity. We identified cellular reactivity against the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK. Further analyses using genetic deletions and synthetic peptides were performed to identify the minimal peptide epitopes. Priming with the peptide epitopes before infection with F. tularensis LVS increased the frequency of antigen-specific CD4 T cells as assessed by intracellular interferon-γ staining. These results illustrate the feasibility of screening an arrayed protein library that should be applicable to a variety of pathogens.

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