Proteins were considered identified with the confidence when GPS protein score confidence interval was 100% and at least two peptides per protein were identified. == STATISTICS Amsilarotene (TAC-101) == Differences in cytokine levels were compared by two-way ANOVA followed by Tukeys multiple-comparison post-test as appropriate, using GraphPad Prizm 5 software. This work is focused on characterization of immune response duringin vivoinfection of two attenuatedFrancisella tularensismutant (dsbAandiglH) strains; importantly, the dsbAmutant, but not the iglHmutant, induced an early innate inflammatory response leading to strong Th1-like antibody response. == Graphical Abstract Physique. == This work is focused on characterization of immune response duringin vivoinfection of two attenuatedFrancisella tularensismutant (dsbAandiglH) strains; importantly, the dsbAmutant, but not the iglHmutant, induced an early innate inflammatory response leading to strong Th1-like antibody response. == INTRODUCTION == Tularemia is usually a severe disease caused by the intracellular pathogenic bacteriumFrancisella tularensis(F. tularensis). Human infections are most commonly acquired through direct contact with infected material (usually animals) or through vector-borne transmission, such as bites by infected insects. By contamination through skin, the ulceroglandular tularemia form evolves, which represents approximately 90% of all tularemia cases (Tarnvik and Berglund2003). A more severe form of tularemia may be caused by respiratory infections after inhalation of aerosols made up of as little as 10 bacteria of subsp.tularensis, which may result in 3060% mortality if untreated (Evanset al.1985). The potential risk ofF. tularensisto be misused as a biological weapon led to this bacterium being classified as a category A agent by Centers for Disease Control and Prevention, USA (Oyston, Sjostedt and Titball2004). In general, tularemia is usually treated with antibiotics where streptomycin is recommended as the drug of first choice with tetracyclines providing as potential alternatives (Russellet al.1998; Denniset al.2001; Johanssonet al.2001). However, the successful antibiotic therapy requires prompt diagnosis which is still a serious problem in some countries, and therefore, the development of a safe vaccine is usually urgently needed. Currently, tularemia vaccine development focuses on improvement of existing attenuatedFrancisellalive vaccine strain (LVS) or on construction of new attenuated mutant strains for genes that are involved in pathogenic mechanisms of tularemic microbe (Marohn and Barry2013). Compared to these two methods, designing a subunit vaccine represents much more difficult task because of the current lack of knowledge of suitable immunodominant antigens. Up to now, immunoproteomics exploiting immune sera Rabbit Polyclonal to Mouse IgG for identification of new immunoreactive antigens has been the easiest way to acquire information about candidates for protective antigens (Kilmury and Twine2010). Previously, we constructed two attenuated type BF. tularensisstrains, one with deletion in gene encoding a homolog to the protein family of disulfide oxidoreductases DsbA (FTS_1067) and the second one with deletion in gene encoding the FPI protein IglH (FTS_0106/FTS_1134) (Straskovaet al.2009,2012). Both mutants showed attenuated phenotype and protective potential against subsequent subcutaneous challenge with parental European clinical isolate of subsp.holarcticastrain, denoted as FSC200 strain. While immunization with dsbA/FSC200 led to complete protection of BALB/c mice against the FSC200 strain challenge, administration of the iglH/FSC200 mutant provided only partial dose-dependent protection with maximal protective effect when a dose of more than 3 107CFUs was applied (Straskovaet al.2009,2012). In this study, we investigated the immunological parameters which might be responsible for differential protection capacity of the dsbA/FSC200 Amsilarotene (TAC-101) and the iglH/FSC200 mutant strains. We found that the ability ofin vivoinduction of early innate inflammatory response and the Th1-like antibody response clearly differ between both mutants. Furthermore, we exhibited that immune response induced by the dsbA/FSC200 mutant is also sufficient for protection against challenge withFrancisellatype A strain SCHU S4. Finally, using an immunoproteomic approach, we defined the profile ofFrancisellamembrane Amsilarotene (TAC-101) proteins recognized by post-vaccination and post-challenge sera and their comparison enabled the determination of novel immunoreactive SCHU S4 antigens. == MATERIALS AND METHODS == == Animals == Female BALB/c mice were purchased from Velaz, s.r.o. (Unetice, Czech Republic) and joined experiments at 68 weeks of age. All procedures using mice were performed in accordance with guidelines of Animal Care and Use Ethical Committee.