Protective Immunity against Lethal F. tularensis holarctica LVS Provided by Vaccination with Selected Novel CD8+ T Cell Epitopes

Shahar Rotem,Sharon Ehrlich,Erez Bar-Haim,Ofer Cohen,Liat Bar-On,Avigdor Shafferman,Dennis W Metzger

doi:10.1371/journal.pone.0085215

Abstract

Recently we described an unbiased bacterial whole-genome immunoinformatic analysis aimed at selection of potential CTL epitopes located in “hotspots” of predicted MHC-I binders. Applying this approach to the proteome of the facultative intra-cellular pathogen Francisella tularensis resulted in identification of 170 novel CTL epitopes, several of which were shown to elicit highly robust T cell responses. Here we demonstrate that by DNA immunization using a short DNA fragment expressing six of the most prominent identified CTL epitopes a potent and specific CD8+ T cell responses is being induced, to all encoded epitopes, a response not observed in control mice immunized with the DNA vector alone Moreover, this CTL-specific mediated immune response prevented disease development, allowed for a rapid clearance of the bacterial infection and provided complete protection against lethal challenge (10LD50) with F. tularensis holarctica Live Vaccine Strain (LVS) (a total to 30 of 30 immunized mice survived the challenge while all control DNA vector immunized mice succumbed). Furthermore, and in accordance with these results, CD8 deficient mice could not be protected from lethal challenge after immunization with the CTL-polyepitope. Vaccination with the DNA poly-epitope construct could even protect mice (8/10) against the more demanding pulmonary lethal challenge of LVS. Our approach provides a proof-of-principle for selecting and generating a multi-epitpoe CD8 T cell-stimulating vaccine against a model intracellular bacterium.

Highlights

Many virulent bacteria can grow intracellularly in infected hosts and exploit this ability as a key pathogenic and immune evasion strategy
We demonstrate that a DNA construct expressing a short poly-epitope composed of the six most prominent CTL epitopes can elicit in mice a specific CD8+ T cell response that is sufficient to provide effective protection against lethal systemic as well as a lethal pulmonary F. tularensis Live Vaccine Strain (LVS) challenge
Epitope Selection and DNA-PolyEp Design We previously described an unbiased F. tularensis whole genome analysis conducted in search for CD8+ T cell epitopes through a cluster based approach [26,27]

Summary

Introduction

Many virulent bacteria can grow intracellularly in infected hosts and exploit this ability as a key pathogenic and immune evasion strategy. The ability of DNA-PolyEp to elicit a T cell response was evaluated by measuring the numbers of IFNc secreting splenocytes after in vitro stimulation with one of the following (see Table 1): each of the individual six encoded peptides; a mix of all six peptides; an LVS-specific T cell responder peptide that was not included in the DNA-PolyEp construct (identified in the original screen [26]; ‘‘ICYVSTNIM’’ see Table 1); formalin inactivated LVS or a negative control peptide with a ‘‘scrambled’’ sequence.

Results

Conclusion