Data sets for bacteriophage induction and effect of chitosan

<p>(Figure 1A): Induction of TOX:GFP strain by ciprofloxacin and chitosan effect<br>The induction of TOX:GFP, an E. coli strain carrying a lysogneic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding GFP, and the effect of chitosan in vitro are shown below. The strain was was induced with ciprofloxacin and the optical density was measured at 600 nm over time (0, 2, 4, 6 hours).</p> <p>(Figure 1B): Bacteriophage titers<br>Bacteriophage titers at different conditions are shown over time (hours). Bacteriophage titers were analyzed at 0, 2, 4 and 6 hours post-induction. Chitosan was added at 2 or 4 h post-induction.</p> <p>(Figure 3B): GFP expression in vivo by IVIS<br>GFP expression in vivo in mice infected with C600TOX:GFP was measured using In Vivo Imaging System (IVIS). Radiant efficiency in organs of different groups is shown. Four animals per group were analyzed and the fluorescence intensity was quantified using Living Imaging 4.3.1 in Calipter Life Sciences. Each number is an individual mouse. N=5 per group.</p> <p> </p

LT-IIb and LT-IIc enhance the formation of IL-7Rα⁺KLRG1⁺ effector memory CD8⁺ T cells.

<p>(A) Representative IL-7Rα and KLRG1 staining of OVA-specific CD8⁺ T cells 28 days after primary immunization with 1.0 µg HLT adjuvants. (B) Percentages of memory (IL-7Rα⁺KLRG1⁻), effector memory (IL-7Rα⁺KLRG1⁺), and effector (IL-7Rα⁻KLRG1⁺) OVA-specific CD8⁺ T cells 28 days post-immunization utilizing 1.0 µg and (C) 0.1 µg of HLT adjuvants. Data shown (n = 8) as a representative of two independent experiments. Statistical analysis: One-way ANOVA with Bonferroni post-test compared to OVA alone. *P≤0.05; **P≤0.01; ***P≤0.001. Results shown as the arithmetic mean with error bars denoting SEM.</p

Enhancement of dendritic cell migration and expression of costimulatory molecules in the cutaneous draining inguinal lymph node by LT-IIb, LT-IIc, and LT-I after ID vaccination.

<p>(A) Percentages of live CD11c⁺MHC-II⁺ dendritic cells in the total cell population of the inguinal lymph node, and (B) representative gating of cells 24 hr post-immunization with 50 µg of OVA and 1.0 µg of HLT adjuvants. (C) Percentages of positive staining DC, MFI, and representative histograms of (C–E) CD80, (F–H) CD86, and (I–K) CD40. Data shown (n = 8) with MFI denoting geometric means of fluorescence where applicable. Statistical Analysis: (A–K) One-way ANOVA with Bonferroni post-test compared to OVA alone unless otherwise noted. *P≤0.05; **P≤0.01; ***P≤0.001. Error bars denote SEM.</p

LT-IIb and LT-IIc enhance humoral responses.

<p>Total OVA-specific serum IgG levels on day 14 and 30 post-vaccination. Mice were immunized with 50 µg OVA +/−1.0 µg HLT. Data shown (n = 8) as a representative of two independent experiments. Unpooled serum was analyzed in duplicate by ELISA. Statistical analysis: One-way ANOVA with Bonferroni post-test compared to OVA. *P≤0.05; **P≤0.01; ***P≤0.001. Results shown as the arithmetic mean with error bars denoting SEM.</p

LT-IIb and LT-IIc induce less inflammatory neutrophil infiltrate than LT-I at the site of injection.

<p>(A) H&E stained sections of injection site cutaneous tissue after 48 hr with 0.5 µg of HLT adjuvant (100x magnification). (B) Visual quantification of infiltrating cells and neutrophils per high field (600x, 5 fields, n = 5) at 48 hr post-injection with 1 µg of HLT adjuvant. (C) Myeloperoxidase reaction in skin homogenates obtained after 48 hr post-injection with 1.0 µg of HLT adjuvant. Data shown (n = 2) as a representative of two independent experiments. Statistical Analysis: (B, C) One-way ANOVA with Bonferroni post-test. *P≤0.05; **P≤0.01; ***P≤0.001.</p

LT-IIb exhibits superior clearance of rLM-OVA and CD8⁺ T cell recall.

<p>Mice immunized with OVA +/− HLT adjuvants were challenged with 5×10⁶ CFU of recombinant OVA-expressing <i>Listeria monocytogenes</i> (rLM-OVA) 30 days post-vaccination. (A) CFU enumerated from homogenized spleens 3 days post rLM-OVA challenge of mice immunized with OVA and adjuvanted with 1.0 µg of HLT and (B) OVA-specific CD8⁺ T cell secondary recall 3 days post challenge. Cells gated on live OVA dextramer⁺TCRβ⁺CD8⁺ cells. (C) Splenic CFU and (D) OVA-specific CD8⁺ T cell recall as before in mice immunized OVA and 0.1 µg of HLT adjuvant. Data shown (n = 8) as a representative of two independent experiments. Statistical analysis: One-way ANOVA with Bonferroni post-test compared to OVA alone unless otherwise noted. *P≤0.05; **P≤0.01; ***P≤0.001. Results shown as the geometric (A, C) or arithmetic (B, D) mean with error bars denoting SEM.</p

LT-IIb and LT-IIc enhance antigen-specific CD8⁺ T cell expansion after immunization, but with different kinetics.

<p>(A) Representative flow analysis of OVA-specific CD8⁺ T cells identified by MHC-dextramers 7 days after 1.0 µg HLT adjuvanted immunization. Cells gated on live TCRβ⁺CD8⁺ cells. (B) OVA-specific CD8⁺ T cells from PBMCs after 7, 14, and 28 days post-immunization utilizing 1.0 µg and (C) 0.1 µg of HLT adjuvants. Data shown (n = 8) as a representative of two independent experiments. Statistical analysis: One-way ANOVA with Bonferroni post-test compared to OVA, unless otherwise noted. *P≤0.05; **P≤0.01; ***P≤0.001. Results shown as the arithmetic mean with error bars denoting SEM.</p

LT-IIb and LT-IIc induce less swelling than LT-I at the site of injection.

<p>Kinetics of swelling volume measured to day 10 after primary immunization with 50 µg of OVA and (A) 1.0 µg or (B) 0.1 µg of HLT adjuvants. Swelling at day 0 represents the size of the induration immediately after injection (open symbols). (C) Side profile of injection-associated swelling 24 hr after ID administration of 50 µg OVA +/−0.5 µg HLT adjuvant. Injection induration margins (black) and swelling margins (red). Data shown (n = 8) as a representative of two independent experiments. <i>Statistical Analysis:</i> (A, B) Two-way ANOVA with Bonferroni post-test compared to LT-IIb (*) or LT-IIc (#). *P≤0.05; **P≤0.01; ***P≤0.001 or respective symbol. Results shown as the arithmetic mean with error bars denoting SEM.</p

Activation of antigen-specific IFN-γ-producing CD8+ T cell precursors in mice immunized with pIRES I or pIRES II.

<p>(A-B) Spleen cells from BALB/c mice spleen cells were stimulated with the MHC-I-restricted p24-specific peptide, and the p24-specific IFN-γ-producing CD8⁺ T cells were detected by intracellular cytokine staining (A) or ELISPOT assay (B). (C–D) Spleen cells from C57BL/6 mice were stimulated with the MHC-I-restricted E7-specific peptide, and the E7-specific IFN-γ-producing CD8⁺ T cells were detected by IFN-γ intracellular staining (C) or ELISPOT assay (D). Mice were i.m. immunized with three doses of the DNA vaccines with one week intervals between doses (100 µg/dose). The CD8⁺ T-cell responses were analyzed two weeks after the last dose. *p<0.05. Data represent the compilation of two independent experiments with four mice per immunization group (n = 8) and results expressed by each animal analyzed. pIRES is the empty vector used as immunization control.</p

Construction of bicistronic DNA vaccines encoding HPV, HIV and HSV antigens for expression in mammalian cells.

<p>(A) Schematic linear representation of the trivalent DNA vaccines. pIRES I and pIRES II contain gDp24 and gDE7 chimeric gene fusions, which are inverted with regard to the CMV promoter and IRES sequence. The empty vector pIRES Ø was used as a control. The nucleotide numbers corresponding to the IRES sequence and the cloned chimeric genes are indicated. (B) In vitro expression of the chimeric proteins encoded by pIRES I (left panels) and pIRES II (right panels). Non-permeabilized pIRES I- or pIRES II-transfected COS-7 cells were labeled with antigen-specific antibodies for the simultaneous detection of the HSV-1 protein gD and the HIV-1 protein p24 or the HPV-16 oncoprotein E7. Green, gD; red, p24 or E7; yellow, co-localization of gD with p24 or E7; blue, DAPI nuclear staining.</p