Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets
Article first published online: 7 JAN 2011
© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd
Volume 132, Issue 3, pages 348–360, March 2011
How to Cite
Valentino, M. D., Maben, Z. J., Hensley, L. L., Woolard, M. D., Kawula, T. H., Frelinger, J. A. and Frelinger, J. G. (2011), Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets. Immunology, 132: 348–360. doi: 10.1111/j.1365-2567.2010.03387.x
- Issue published online: 1 FEB 2011
- Article first published online: 7 JAN 2011
- Received 17 September 2010; revised 11 November 2010; accepted 12 November 2010.
- epitope discovery;
- Francisella tularensis;
- gateway genomic array;
- proteomic array;
- T lymphocyte
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.