Abstract
Deciphering the cellular immunome of a bacterial pathogen is challenging due to the enormous number of putative peptidic determinants. State-of-the-art prediction methods developed in recent years enable to significantly reduce the number of peptides to be screened, yet the number of remaining candidates for experimental evaluation is still in the range of ten-thousands, even for a limited coverage of MHC alleles. We have recently established a resource-efficient approach for down selection of candidates and enrichment of true positives, based on selection of predicted MHC binders located in high density “hotspots" of putative epitopes. This cluster-based approach was applied to an unbiased, whole genome search of Francisella tularensis CTL epitopes and was shown to yield a 17–25 fold higher level of responders as compared to randomly selected predicted epitopes tested in Kb/Db C57BL/6 mice. In the present study, we further evaluate the cluster-based approach (down to a lower density range) and compare this approach to the classical affinity-based approach by testing putative CTL epitopes with predicted IC50 values of <10 nM. We demonstrate that while the percent of responders achieved by both approaches is similar, the profile of responders is different, and the predicted binding affinity of most responders in the cluster-based approach is relatively low (geometric mean of 170 nM), rendering the two approaches complimentary. The cluster-based approach is further validated in BALB/c F. tularensis immunized mice belonging to another allelic restriction (Kd/Dd) group. To date, the cluster-based approach yielded over 200 novel F. tularensis peptides eliciting a cellular response, all were verified as MHC class I binders, thereby substantially increasing the F. tularensis dataset of known CTL epitopes. The generality and power of the high density cluster-based approach suggest that it can be a valuable tool for identification of novel CTLs in proteomes of other bacterial pathogens.
Highlights
F. tularensis, the causative infective agent of the acute tularemia disease, is a facultative intracellular pathogen
Our previous study described in detail the screen conducted on 1740 putative CTL epitopes, preselected from a total of 90,879 predicted MHC binders [14]
Mapping the cellular immunome of a complex microbial genome, i.e. a bacterial pathogen, by identification of CTL epitopes, requires a rigorous filtering approach for down-selection of peptide candidates for experimental evaluation, and preferably an approach which enriches the extent of responders from the tested pool
Summary
F. tularensis, the causative infective agent of the acute tularemia disease, is a facultative intracellular pathogen. The bacterium has a broad range of host specificities and its clinical manifestation and disease severity depend on the route of infection and on the infecting strain. Inhalation of as few as 15 CFU of the more virulent strains (F. tularensis tularensis type A) is sufficient to infect humans, and without treatment the mortality rate for respiratory disease is 30–60% [1,2,3]. There is no available licensed vaccine against F. tularensis. Taken together with the well documented evidence on contribution of the cellular response to protection against the intracellular F. tularensis [11], identification of determinants which could elicit an effective cellular-mediated immune response is crucial. The knowledge on F. tularensis epitopes, and on cytotoxic T-cell (CTL)
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