Protective immunity against lethal F. tularensis holarctica LVS provided by vaccination with selected novel CD8+ T cell epitopes.

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

The RNA polymerase II subunit Rpb4p mediates decay of a specific class of mRNAs

It is commonly appreciated that the mRNA level is determined by the balance between its synthetic and decay kinetics. Yet, little is known about coordination between these distinct processes. A major pathway of the eukaryotic mRNA decay initiates with shortening of the mRNA poly(A) tail (deadenylation), followed by removal of the mRNA 5′ cap structure and its subsequent exonucleolytic degradation. Here we report that a subunit of RNA polymerase II, Rpb4p, is required for the decay of a class of mRNAs whose products are involved in protein synthesis. Cells lacking RPB4 are defective in the deadenylation and post-deadenylation steps of representatives of this class of mRNAs. Moreover, Rpb4p interacts with both the mRNP and with subunits of the mRNA decay complex Pat1/Lsm1–7 that enhances decapping. Consistently, a portion of Rpb4p is localized in P bodies, where mRNA decapping and degradation is executed, and mutations in RPB4 increase the number of P bodies per cell. We propose that Rpb4p has a dual function in mRNA decay. It promotes or enhances the deadenylation process of specific mRNAs and recruits Pat1/Lsm1–7 to these mRNAs, thus stimulating their decapping and further decay. In this way, Rpb4p might link the activity of the basal transcription apparatus with that of the mRNA decay machinery

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

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

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

Multiscale Elasticity of 3D Boron Carbonitride Foam for Tunable Mechanical Resisting Devices

Boron carbonitride (BCN) foam is a three-dimensional material with a hierarchical structure, which has promising potential due to its semiconducting properties and high surface area. However, the lack of understanding of its elastic properties impedes its large-scale integration into advanced applications. We grew BCN foam samples with different atomic compositions and studied their microscopic- and macroscopic-scale mechanics, which revealed that samples with high concentrations of carbon have lower elastic resistance across different scales (i.e., lower Young’s moduli). While the microscopic elasticity is dominated by interlayer interactions, the macroscopic elasticity is also strongly influenced by the buckling and fracturing of the three-dimensional structure of the BCN foam, and thus, the macroscopic Young’s moduli are lower than the microscopic ones. Our findings shed light on the mechanism that underlies the multiscale mechanics of BCN foam and pave the path toward its integration into tunable mechanical resisting devices such as flexible electronic devices and resonators

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