Prophylactic and therapeutic tumor immunity against a peripheral solid tumor.

<p>(A) B6 mice were i.v. immunized with 10⁵ pfu of the different vectors or received PBS as control. Seven days later, 5×10⁵ gp-recombinant Lewis lung carcinoma cells were injected s.c. on the left flank. (B) Assessment of therapeutic tumor immunity induced by 10⁵ pfu of the different vectors applied on day 4 post s.c. inoculation with 5×10⁵ gp-recombinant Lewis lung carcinoma cells. Tumor growth was monitored on the indicated days. Values indicate mean tumor volume ±SEM (n=9 mice). Values in parentheses indicate number of growing tumors of inoculated tumors. Statistical analysis in (B) was performed using one way ANOVA with Bonferroni multiple comparison test (**, p< 0.01; ***, p< 0.001; ns, non-significant).</p

Generation and in vitro characterization of cytokine-encoding murine coronavirus vectors.

<p>(A) Schematic representation of MHV A59 genome and construction of cytokine-encoding vectors. (B, C) Growth kinetics of the indicated MHV vectors in 17ECl20 packaging cells (B) and macrophages (Mph) (C). Cells were infected at an MOI of 1 and titres in supernatants were determined at the indicated time points. (D) Transduction in bone marrow-derived DCs with murine coronavirus vectors. DCs from B6 mice were transduced with the indicated vectors at a multiplicity of infection (MOI) of 1. Cells were harvested 24 h later and EGFP expression on CD11c⁺ cells was assessed. Pooled data from three independent experiments with values indicating mean percentage ±SEM of EGFP⁺CD11c⁺ cells. (E) Cytokine production induced by Flt3L and IL-2 encoding vectors. DCs or macrophages were transduced at a MOI of 1 and concentration of cytokines in the supernatants was determined by ELISA. Representative data from one out of three independent experiments.</p

In vivo maturation of DCs following vaccination with coronavirus vectors.

<p>B6 mice were i.v. immunized with 10⁶ pfu of the indicated viral vectors or left untreated (mock). (A, B) Transduction of DCs as assessed by EGFP expression. Spleens were collected after 24 h, digested with collagenase and low-density cells were analyzed by flow cytometry. Values in dot plots (A) and bar graph (B) show the mean percentage ± SEM of EGFP⁺IA^bhigh cells gated on CD11c⁺ cells. Data from two independent experiments with three mice per group (n=6). (C, D) Cytokine concentration in serum and spleen homogenates at 24 h post immunization with Flt3L (C) and IL-2 (D) vectors. Pooled data from three independent experiments with three mice per group (mean ±SEM, n=9). (E) Representative histograms showing expression of the DC maturation markers CD40 and CD86 on EGFP⁺CD11c⁺ cells transduced with the indicated vectors. (F) Mean fluorescence values ±SEM (n=9) of CD40 and CD86 expression in transduced EGFP⁺CD11c⁺ cells (+) and non-transduced EGFP⁺CD11c^- cells (-) cells (*, p< 0.05; **, p< 0.01).</p

Coronavirus vector-induced immunity against a metastasizing tumor.

<p>(A, B) B6 mice were i.v. immunized with the indicated doses of the different vectors or infected with 200 pfu LCMV WE. PBS was administered as negative control. Seven days later mice were challenged with 5×10⁵ B16F10-gp melanoma cells or the parental B16F10 cells. (A) Representative microphotographs of lungs on day 12 post tumor inoculation. (B) Number of metastatic foci per lung was determined 12 days after challenge (pooled data from three independent experiments, mean ±SEM, n=6-9 mice per time point). (C, D) Assessment of therapeutic tumor immunity. B6 mice received 5×10⁵ B16F10-gp melanoma cells i.v. at day 0 and were vaccinated 10 days later with 10⁵ pfu of the indicated vectors or left untreated. (C) Representative microphotographs of affected lungs on day 20 after tumor inoculation. (D) Disease severity was quantified by black pixel counting on lung surfaces on day 20. Values represent mean percentage ±SEM (n=6-8 mice) of affected lung surface. Statistical analysis in (D) was performed using one way ANOVA with Tukey’s post analysis (**, p< 0.01; ns, non-significant).</p

Evaluation of CD8⁺ T cell response induced by cytokine-encoding coronavirus vectors.

<p>(A) Induction of gp34-specific CD8⁺ T cells. B6 mice were immunized i.v. with 10⁵ pfu of the indicated vectors. At day 7 post immunization, splenocytes were analyzed for expression of CD8 and reactivity with H2-K^b/gp34-tetramers, and were tested for gp34-specific IFN-γ production. Values indicate mean percentages of tet⁺ cells ± SEM (upper row) or mean percentages of IFN-γ⁺ cells ± SEM (lower row) in the CD8⁺ T-cell compartment (pooled data of 3 independent experiments, n=12 mice). (B and C) Duration of vector-induced CD8⁺ T cell responses. B6 mice were immunized i.v. with 10⁵ pfu of the indicated vectors or infected with 200 pfu of LCMV WE. Frequencies (B) and total numbers (C) of splenic CD8⁺ tet-gp34-binding T cells were determined at the indicated time points (mean percentages or total numbers of tet-gp34⁺ cells ±SEM, n=6-12 mice per time point); nd, not detectable. (D) Induction of gp33-specific CD8⁺ T cells as determined by tetramer analysis on day 7 post i.v. immunization with the indicated vectors (mean percentages of tet-gp33⁺ cells ± SEM, n=8 mice). (E) Expansion of gp33-specific P14 TCR transgenic CD8⁺ T cells. One day before immunization with 10⁵ pfu of the indicated vectors, CD45.2⁺ B6 mice had received 10⁵ CD45.1⁺ P14 splenocytes. Expansion of CD45.1⁺CD8⁺ T cells in spleens was assessed on day 7 post immunization (mean percentages of CD45.1⁺CD8⁺ T cells ±SEM, n=8 mice) (*, p< 0.05; **, p< 0.01; ***, p< 0.001; comparison with gp control vector).</p

Activation profile of OmpC/F-specific CD4⁺ T cells after Ty21a vaccination.

<p>PBMCs were collected from vaccinated or control (Ctrl) subjects at the indicated time points. Activation of CD4⁺ T cells was assessed by flow cytometry after 24 h incubation with the indicated antigen. (A) Gating strategy for FACS analysis. (B) Representative dot plots showing the proportions of CD4⁺ CD40L⁺ T cells of Ctrl (upper panels) or vaccinated (lower panels) subjects after stimulation with <i>S</i>. Typhi OmpC/F porins or tetanus toxoid (TT). (C and D) Proportions of CD4⁺ CD40L⁺ T cells stimulated with TT (C) or OmpC/F (D) at the indicated time points. Dots represent individual values; bars represent mean ± SEM. (E) Representative dot plot showing IFN- γ and TNF-α production out of CD4⁺ CD40L⁺ cells after stimulation using medium or OmpC/F porins. (F) Proportions of OmpC/F-specific polyfunctional cells (IFN- γ ⁺ and/or TNF- α⁺) out of CD4⁺ CD40L⁺ T cells at indicated time points after vaccination in responder individuals. Statistical analysis in panels C and D was performed using one way ANOVA with Dunnett’s multiple comparison test for comparisons between individuals of the same group at different time points (pre- versus post-vaccination) or unpaired Student’s <i>t</i> test with Welch’s correction for comparison between Ctrl and vaccinated groups. Statistical analysis in panel F was performed using paired Student’s <i>t</i> test for comparison between individuals of the same group (Day 11 versus Day 60) (*, P< 0.05; **, P< 0.01).</p

Identification of activated B cells and antibody secreting cells in peripheral blood following Ty21a vaccination.

<p>PBMCs were collected from vaccinated or control (Ctrl) subjects at the indicated time points and analysis of different B cell populations was performed by flow cytometry. (A) Gating strategy for FACS analysis of activated B cells (ABC, blue) or antibody secreting cells (ASC, red). (B) Representative dot plots showing the proportions of ABC (CD20^hi CD71⁺) and ASC (CD20^- CD71⁺) in vaccinated and Ctrl individuals at the indicated time points. (C and D). Proportions of ABC and ASC in vaccinated and Ctrl individuals at the indicated time points. Dots represent individual values; bars indicate mean ± SEM. Statistical analysis was performed using one way ANOVA with Dunnett’s multiple comparison test for comparisons between individuals of the same group at different time points (pre- versus post-vaccination) (*, P< 0.05; **, P< 0.01).</p

Induction of anti-OmpC/F-specific porin antibodies in serum and stool after Ty21a vaccination.

<p>(A) IgM and (B) IgG, OmpC/F-specific antibody titers in serum samples from vaccinated subjects were analyzed by ELISA at the indicated time points after vaccination; participant numbers assigned according to increasing IgG reactivity. (C and D) Kinetics of anti-OmpC/F IgM (C) and IgG (D) antibody titers in vaccinated or control (Ctrl) individuals. Dots represent individual values; bars indicate mean ± SEM. (E) <i>S</i>. Typhi OmpC/F-specific IgA was determined from stool samples from vaccinated or Ctrl subjects by ELISA. Dots represent individual values; bars represent mean ± SEM. Statistical analysis was performed using one way ANOVA with Dunnett’s multiple comparison test for comparisons between individuals of the same group at different time points (pre- versus post-vaccination) (*, P< 0.05; **, P< 0.01 ***, P<0.001).</p

Study design.

<p>CONSORT flow diagram indicating the number of participants that have been included and have completed the study.</p

Study demographic details.

<p>Study demographic details.</p