Whole-Genome Immunoinformatic Analysis of F. tularensis: Predicted CTL Epitopes Clustered in Hotspots Are Prone to Elicit a T-Cell Response

Abstract

The cellular arm of the immune response plays a central role in the defense against intracellular pathogens, such as F. tularensis. To date, whole genome immunoinformatic analyses were limited either to relatively small genomes (e.g. viral) or to preselected subsets of proteins in complex pathogens. Here we present, for the first time, an unbiased bacterial global immunoinformatic screen of the 1740 proteins of F. tularensis subs. holarctica (LVS), aiming at identification of immunogenic peptides eliciting a CTL response. The very large number of predicted MHC class I binders (about 100,000, IC50 of 1000 nM or less) required the design of a strategy for further down selection of CTL candidates. The approach developed focused on mapping clusters rich in overlapping predicted epitopes, and ranking these “hotspot” regions according to the density of putative binding epitopes. Limited by the experimental load, we selected to screen a library of 1240 putative MHC binders derived from 104 top-ranking highly dense clusters. Peptides were tested for their ability to stimulate IFNγ secretion from splenocytes isolated from LVS vaccinated C57BL/6 mice. The majority of the clusters contained one or more CTL responder peptides and altogether 127 novel epitopes were identified, of which 82 are non-redundant. Accordingly, the level of success in identification of positive CTL responders was 17–25 fold higher than that found for a randomly selected library of 500 predicted MHC binders (IC50 of 500 nM or less). Most proteins (ca. 2/3) harboring the highly dense hotspots are membrane-associated. The approach for enrichment of true positive CTL epitopes described in this study, which allowed for over 50% increase in the dataset of known T-cell epitopes of F. tularensis, could be applied in immunoinformatic analyses of many other complex pathogen genomes.

Clusters versus Affinity-Based Approaches in F. tularensis Whole Genome Search of CTL Epitopes

“…Moreover, there appears to be an enrichment of peptides with higher predicted affinities concomitant with increase in the density [14]. Overall, the data demonstrate that the two approaches – high affinity based and cluster based - are not mutually exclusive but are rather complementary yielding largely different populations of CTL epitopes.…”

“…We previously reported on a clear bias toward membranal proteins detected for the 51 parental proteins of Subset I (cluster densities 1.0–1.4) [14]. It was therefore interesting to determine if such a bias exists for the 28 proteins antigens of Subset III, which contain the clusters at lower densities of 0.8–1.0.…”

“…Data on Subset I and II is from [14]. Peptides which were shown to stimulate lymphocytes and induce IFNγ production are referred to as “responders".…”

“…Boxes shaded in light blue: Subsets I and II were described in a previous study [14] and are added for the sake of completeness. Boxes shaded in cyan: Subsets III–V of peptides selected and evaluated in this study.…”

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis

“…To further concentrate our identification on the appropriate T cell response, we elected to track antigen-specific CD4 + T cells, since these cells would directly recognize and respond to Francisella infected cells. Despite identified and predicted CD4 + and CD8 + T cell epitopes in Francisella vaccine strains (17–19), there has been no success in producing MHCI and MHCII tetramers to track Ftt antigen-specific cells in Francisella infected mice. To overcome this problem, we generated recombinant ATCC LVS, RML LVS, and SchuS4 strains ectopically expressing the well-characterized gp61-80 epitope from lymphocytic choriomenigitis virus (LCMV) fused to the Francisella protein, IglC.…”