Adaptive immune response induced in mice immunized with the LACK vectors.

<p>A. Analysis of the antigen-specific IFNγ secreting cells by splenocytes measured by ELISPOT 11 days after boost. B. Analysis of the total magnitude of CD4⁺ and CD8⁺ T cell responses in splenocytes re-stimulated with LACK protein. Among the lymphocyte population, T cells were gated and analyzed for IFNγ, TNFα and/or IL-2 production. MVA-wt background was subtracted before representation. Cytokine production by LACK-specific CD8⁺ T cells (C), LACK-specific CD4⁺ T cells (D) or LACK_157–173 peptide-specific CD4⁺ T cells (E). The different combinations of cytokines are indicated on the <i>x</i>-axis; percentages of T cells producing any cytokine are indicated on the <i>y</i>-axis. The different pies show the quality of the response measured as the relative quantity of single, double or triple cytokine producing cells (F). Analysis of the Geometric Mean of Fluorescence Intensity of IFNγ, TNFα or IL-2; produced by different populations of LACK-specific CD4⁺ T cells isolated from animals that received heterologous vaccination with DNA-LACK/MVA-LACK. Data is representative of two independent experiments.</p

Analysis of humoral and cellular responses triggered by the MVA vector.

<p>A. IgG total antibodies at different points post-immunization where measured by ELISA using extracts of BSC40 cells infected with VACV. B. Analysis of the E3-specific IFNγ secreting cells by splenocytes measured by ELISPOT 11 days after the booster (prechallenge), 10 days after experimental challenge (postchallenge) or 53 days after the booster (memory). Among the lymphocyte population, T cells were gated and analyzed for IFNγ, TNFα and/or IL-2 production. C. Cytokine production by E3-specific CD8⁺ T cells 11 or 53 days after the booster.</p

Deletion of <i>A46R</i> gene from NYVAC-C enhances innate immune responses.

<p>Human macrophages were mock-infected (0) or infected with NYVAC-WT, NYVAC-C or NYVAC-C-Δ46R (1 or 5 PFU/cell). 24 hours later, cell-free supernatants were collected to quantify the concentrations of TNF and IL-6 by bioassay and of IL-8 by ELISA. Data are means ± SD of duplicates and are representative of three independent experiments. * <i>p</i><0.05, ** p<0.005.</p

Adaptive HIV-specific T cell immune responses elicited by <i>A46R</i> deletion mutant in the spleen of BALB/c mice in heterologous prime/boost immunization protocol.

<p>(A) Magnitude of the vaccine-specific CD4 or CD8 T cell response. The HIV-specific CD4 or CD8 T cells were measured 10 days after the last immunization by ICS assay following stimulation of splenocytes derived from immunized animals (n=4) with the different HIV peptide pools. The total value in each group represents the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting IFN-γ and/or IL-2 and/or TNF-α (CD4) or CD107a and/or IFN-γ and/or IL-2 and/or TNF-α (CD8) against all HIV peptide pools. All data are background-subtracted. *** <i>p</i><0.001. <i>p</i> value indicates significantly higher responses compared to parental group or between DNA-C/NYVAC-C-ΔA46R and DNA-C/NYVAC-C immunization groups. (B) Flow cytometry profiles of vaccine-induced CD4 or CD8 T cell responses against Env pool. (C) Functional profile of the adaptive HIV-specific CD4 or CD8 T cell response in the different immunization groups. The possible combinations of the responses are shown on the <i>x</i> axis, whereas the percentages of the functionally distinct cell populations within the total CD4 or CD8 T cell population are shown on the <i>y</i> axis. Combinations that did not contribute significantly to the functional profile are not shown. Responses are grouped and colour-coded on the basis of the number of functions. The non-specific responses obtained in the control group DNA-ϕ/NYVAC-WT were subtracted in all populations. ** p<0.005, *** <i>p</i><0.001. <i>p</i> values indicate significantly higher responses compared to DNA-C/NYVAC-C immunization group.</p

Memory HIV-specific T cell immune responses elicited by <i>A46R</i> deletion mutant in the spleen of BALB/c mice after prime/boost immunization.

<p>(A) Magnitude of the vaccine-specific CD4 or CD8 T cell responses. The HIV-specific CD4 or CD8 T cells were measured 53 days after the last immunization by ICS assay following stimulation of splenocytes derived from immunized animals (n=4) with the different HIV peptide pools. The total value in each group represents the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting IFN-γ and/or IL-2 and/or TNF-α (CD4) or CD107a and/or IFN-γ and/or IL-2 and/or TNF-α (CD8) against all HIV peptide pools. All data are background-subtracted. *** <i>p</i><0.001. <i>p</i> value indicates significantly higher responses compared to parental group or between DNA-C/NYVAC-C and DNA-C/NYVAC-C-ΔA46R immunization groups. (B) Functional profile of the memory HIV-specific CD8 T cell response in the different immunization groups. The possible combinations of the responses are shown on the <i>x</i> axis, whereas the percentages of the functionally distinct cell populations within the total CD8 T cell population are shown on the <i>y</i> axis. Combinations that did not contribute significantly to the functional profile are not shown. Responses are grouped and colour-coded on the basis of the number of functions. *** <i>p</i><0.001. <i>p</i> values indicate significantly higher responses compared to DNA-C/NYVAC-C immunization group. (C) Phenotypic profile of memory HIV-specific CD8 T cells. Representative FACS plots showing the percentage of Env-specific CD8 T cells with central memory (TCM; CD127⁺CD62L⁺), effector memory (TEM; CD127⁺CD62L^-) or effector (TE; CD127^-CD62L^-) phenotype.</p

Cytotoxic activity of CD8⁺ T lymphocytes activated by MVA-B-infected MDDC.

<p>Effector cells were autologous monocyte-depleted PBMC co-cultured for 6 days with MDDC mock-treated or infected with MVA or MVA-B. After this co-culture period, CFSE staining of effector cells was performed and then were cultured for 4 h with target cells at a 10∶1 ratio. Target cells were autologous CD8-depleted T cells infected with a primary HIV-1 isolate and their corresponded to the phenotype CFSE-negative, CD3-positive CD8-negative. In (A) the flow cytometry analysis is shown. In (B), the mean (±SEM) count of target CD4⁺ T cells is a representative result of three independent experiments (performed in triplicates) is depicted. (*) p<0.05.</p

Antigen presentation study.

<p>(A) Phagocytosis assay. Immature MDDC were labeled with CFSE and infected with MVA at a MOI of 10 PFU/MDDC for 1 hour. They were then extensively washed and mixed with uninfected immature MDDC (ratio 1∶2) and maturation was induced for 48 h. Phagocytosis of apoptotic bodies resulting from MVA-infected MDDC by uninfected MDDC were detected by flow cytometry. Density plots showing infected MDDC, detected as CFSE⁺ CD80⁻ (<i>left box</i>), uninfected MDDC, detected as CFSE⁻ CD80⁺ (<i>middle box</i>) and CD80⁺ MDDC that ingested apoptotic CFSE-labeled MDDC during the incubation period, detected as CFSE⁺ CD80⁺ (<i>right box</i>). These data are representative of two individual experiments. (B) Phagocytosis assessed by fluorescence microscopy. Phagocytosis of apoptotic bodies resulting from MVA-infected MDDC by uninfected MDDC were visualized by fluorescence microscopy. MVA-B infected MDDC were CFSE⁺ CD80⁻ (green colour), uninfected matured MDDC were stained CFSE⁻ CD80⁺ antibody (red colour), nucleus was visualized with Hoechst staining (blue colour) and phagocytosis was observed as CFSE stained vacuoles (green) inside the MDDC CD80 positive cells (red). As negative controls of phagocytosis, uninfected MDDC and infected MDDC cultured separately were used. These data are representative of 2 independent experiments. (C) Cross-presentation induced proliferation of CD8⁺ T cells. Representative dot plots showing the proliferation rate of CD8⁺ T cells after co-culture between infected MDDC with autologous lymphocytes. The white plots correspond to a MVA-B infection model at 0.3 PFU/MDDC. The black plots correspond to infection of immature MDDC (two thirds of the initial culture; cultured during 72 hours) with apoptotic bodies (obtained after infection of MDDC –one third of the initial culture- with MVA-B at a dose of 10 PFU/MDDC and during a period of 72 hours). The grey plots correspond to infection between immature MDDC and apoptotic bodies obtained as described above and subsequently heat-inactivated at 55°C for one hour. Subsequently, infected MDDC were collected and co-cultured with CFSE labeled autologous lymphocytes in triplicates. Six days after, proliferation was tested by flow cytometry. These data are representative of 3 individual experiments.</p

MDDC infected with MVA-B expressed intracellular Gag protein in a dose-dependent manner.

<p>MDDC were infected with serial three-fold dilutions of a stock of MVA-B, from 0.003 to 10 PFU/MDDC. After MVA-B infection of MDDC, Gag and CD86 expression was measured by flow cytometry. (A) Intracellular viral protein expression in MDDC observed by fluorescence microscopy. MDDC at 16 h post-infection not infected (Mock) or infected with MVA-B (at 10 PFU/MDDC) were fixed-permeabilized and viral proteins were stained indirectly with FITC (green) and nucleus was visualized using Hoechst (blue). These data are representative of two individual experiments. (B) Density plots showing the percentage of Gag and CD86 double positive MDDC from 0.003 to 3.3 doses (PFU/DC). (C) Linear regression of percentage of double positive CD86 Gag cells versus viral dose (PFU/MDDC). A representative result of 3 independent experiments is shown. (D) Intracellular Gag expression in MDDC observed by fluorescence microscopy. MDDC were infected with MVA-B and MVA (at 10 PFU/MDDC) and at 16 h post-infection were stained with PE-CD86 (red) and fixed-permeabilized and stained with FITC-anti-HIV-Gag (green) antibody and nucleus was stained with Hoechst (blue). These data are representative of two individual experiments.</p

Correlation between CD86 expression on MVA-B-infected MDDC and HIV-1-specific CD8^<b>+</b> T-cell proliferation and cytokine secretion.

<p>MDDC were infected with MVA-B at serial three-fold dilutions (from 0.003 to 3.3 PFU/MDDC) and expression of the maturation surface marker of MDDC, CD86, measured by flow cytometry at 16 h post-infection in the absence of maturation cocktail was assessed. Subsequently, MVA-B-infected MDDC matured with cytokine cocktail were co-cultured for 6 days with autologous lymphocytes and HIV-1-specific CD8⁺ T proliferation and cytokine secretion were measured. A representative result of 2 independent experiments is shown.</p

Chemotaxis assay.

<p>Chemokine-induced migration of infected mature MDDC was tested 48 h and 72 h after infection in a chemotaxis assay (n = 6). Entire populations that migrated toward CCL9 and CCL21 were collected after incubation and counted during one minute by flow cytometry. The number of migrated DC is depicted in percent of the initial input. Subsequently, infected (MVA and MVA-B) and uninfected MDDC that migrated in the chemotaxis assay were collected and co-cultured with CFSE labeled autologous lymphocytes at a 1∶40 ratio (5×10³ MDDC: 2×10⁵ lymphocytes/well). After a period of 6 days, proliferation was tested by flow cytometry. (A) Percentage of CCL19 specific migration of MVA-B-infected MDDC 48 hours after infection. Error bars represent the mean (±SEM) from 6 independent experiments with 6 different HIV-1 patients (n = 6) performed in triplicates. (B) Percentage of CCL19 specific migration of MVA and MVA-B-infected and uninfected MDDC (Mock) 48 and 72 h post-infection and maturation. Error bars represent the mean (±SEM) from 3 independent experiments with 3 different HIV-1 patients (n = 3) performed in triplicates. (C) Representative flow cytometric plots showing CFSE dilution in gated CD3⁺ CD8⁺ lymphocytes after co-culture with MVA and MVA-B infected MDDC that were able to migrate in the chemotaxis assay. One representative result of 2 independent experiments is shown. Error bars represent the mean (±SEM) (n = 3). (D) Percentage of CFSE^low cells in the CD3⁺ CD8⁺ gate, observed in one sample. Error bars represent the SEM from the mean triplicates (***) p = 0.005.</p