Induction of T cell response to selected CTL epitopes following LVS or DNA-PolyEp immunization.

<p>^a CTL epitopes were derived from six proteins respectively: FTL_1916_400–410, FTL_0966_126–133, FTL_1708_13–23, FTL_0283_95–104, FTL_1101_64–74, and FTL_1673_308–318.</p><p>^b Stimulating antigens: 10 mM of the individual peptides or 10⁷ CFU/ml of formalin-inactivated LVS.</p><p>^c C57BL/6 mice were immunized with LVS or the DNA-PolyEp vaccine with CTL epitopes numbered (1)-(6) (see Materials and Methods).</p><p>^d Data represent the mean and SD of three experiments (at least two animals per experiment) that were carried out in duplicate.</p><p>^e The pCI vector was used to express a 186 bp DNA fragment encoding for the 1–6 epitopes (see Materials and Methods).</p><p>^f The peptide “ICYVSTNIM”, an identified CTL epitope in LVS not included in the DNA-PolyEp vaccine, was used as a positive control (see Materials and Methods).</p><p>^g Scrambled sequence of peptide (5).</p><p>^h LVS was inactivated by formalin (see Materials and Methods).</p

Survival after lethal inhalational <i>F. tularensis</i> LVS challenge.

<p>Two weeks after the completion of the immunization schedule (see Materials and Methods), 10 mice in each vaccine group were challenged <i>i.n.</i> with 10⁴ CFU LVS (equivalent to 10 LD₅₀) and monitored for survival for 28 days. squares, DNA-PolyEP immunization; circles, pCI immunization; triangles, non-immunized mice.</p

Selective T cell response of vaccinated mice.

<p>^a Number of IFNγ secreting cells/10⁶ splenocytes (splenocytes were removed 14 days following the last immunization (see Materials and Methods)). Data represents mean and SD from 3 individual animals (per group) derived from 3 independent experiments.</p><p>^b 10⁷ CFU/ml of formalin-inactivated LVS (see Materials and Methods).</p><p>^c 10 µg of anti-CD4 antibodies were added to 10⁶ splenocytes 1 hour before stimulation with formalin inactivated LVS.</p><p>^d 10 µg of anti-CD8 antibodies were added to 10⁶ splenocytes 1 hour before stimulation with formalin inactivated LVS.</p><p>^e No-Ag, samples were tested without any added antigen.</p><p>^f mice were immunized by gene gun. (See Materials and Methods).</p

Selective T cell response of vaccinated mice 3 and 5 days post LVS lethal challenge.

<p>^a Number of IFNγ secreting cells/10⁶ splenocytes were removed at the indicated day post challenge (see Materials and Methods). Data represents mean and SD from 3 individual animals (per group) derived from 2 independent experiments.</p><p>^b 10⁷ CFU/ml of formalin-inactivated LVS (see Materials and Methods).</p><p>^c 10 µg of anti-CD4 antibodies were added to 10⁶ splenocytes 1 hour before stimulation with formalin inactivated LVS.</p><p>^d 10 µg of anti-CD8 antibodies were added to 10⁶ splenocytes 1 hour before stimulation with formalin inactivated LVS.</p><p>^e Samples from harvested spleens were taken for the evaluation of bacterial load, which was determined by real time-PCR or CFU counts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085215#pone-0085215-g003" target="_blank">Fig. 3A</a>).</p><p>^f Survival data are derived from 3 groups of 10 mice each at 28 days post challenge (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085215#pone-0085215-g002" target="_blank">Fig. 2A</a>).</p><p>^g Mice were challenged <i>i.p.</i> (14 days following the last immunization) with 10³ CFU of LVS (equivalent to 10 LD₅₀)_.</p

Bacterial burden in vaccinated mice after <i>F. tularensis</i> LVS challenge.

<p>Mice were vaccinated and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085215#pone-0085215-g002" target="_blank">figure 2</a>. At two, three and five days post-challenge, 3 mice in each group were sacrificed, and their spleens (A) and livers (B) were removed for bacterial load inspection by CFU counts; similar results were obtained by quantitative real time-PCR. The data represent the means ± SD from 2 independent experiments. Black squares, DNA-PolyEp immunization; white circles, pCI immunization; grey triangles, non-immunized mice.</p

Proliferative responses of memory CD8+ and CD4+ T cells after <i>in vitro</i> stimulation.

<p>Splenocytes from DNA-PolyEp-immunized or naive mice were labeled with CFSE and stimulated for 48 hours with formalin-inactivated LVS. Stimulated cells were stained for CD4 and CD8 expression, and the proliferative responses were analyzed by flow cytometry. The percentage of splenocytes in each group represents the mean and SD of 3 individual animals from a single experiment. FACS proliferation plots are from a representative experiment.</p

Neutrophil frequencies in the spleens of <i>F. tularensis</i> LVS challenged mice.

<p>Neutrophil (CD11b⁺ Gr1⁺) counts in the splenocytes from naive or DNA-PolyEp-vaccinated mice were analyzed by flow cytometry on three consecutive days after <i>i.p.</i> challenge with 10³ LVS. Bars represent the mean and SD of 3 individual animals from a single experiment.</p