MVATG18377 induces multiple cytokines-producing T cells in C57BL/6 mice.

<p>Cells from MVATG18377-immunized C57BL/6 mice were stimulated with antigen peptide pools or an irrelevant E7 peptide (Irr). Results are presented as (<b>A</b>) the percentage of IFNγ⁺, IFNγ⁺TNFα⁺ or IFNγ⁺TNFα⁺IL2⁺ cell subsets among total CD4 T cells or (<b>B</b>) percentage of IFNγ⁺, TNFα⁺ or IFNγ⁺TNFα⁺ cell subsets among total CD8 T cells. Plain bars represent response from individual mice and hatched bars represent median response for each cell subset. Cut-off value (dotted line, 0.02%) is represented for both CD4⁺ and CD8⁺ T cell responses. Only antigens with median value above the cut-off value are represented. For these antigens, only cell subgroups with a percentage above the cut-off value are represented. No response was detected in MVATGN33.1-immunized mice (data not shown). For each cell population, background signal obtained in unstimulated cell condition was subtracted. Pie charts represent a more global analysis for each responder antigen. All analyzed single (IFNγ⁺, TNFα⁺ and IL2⁺), double (IFNγ⁺TNFα⁺, IFNγ⁺IL2⁺ and TNFα⁺IL2⁺) or triple (IFNγ⁺TNFα⁺IL2⁺) cytokine producer cells are included under the corresponding color codes. Results are representative of two independent experiments.</p

ELISpot analysis of IFNγ responses specific of MVATG18377-encoded Mtb antigens in different strains of mice.

<p>(<b>A</b>) BALB/c, (<b>B</b>) C57BL/6 and (<b>C</b>) C3H/HeN mice were immunized once with either MVATGN33.1 (light grey) or MVATG18377 (dark grey). Results are shown as the number of IFNγ-producing T cells (spots-forming cells) per 10⁶ splenocytes following stimulation with either peptide pools specific of each of the 14 antigens or the irrelevant GLL peptide (Irr). For long sequence antigens (Rv2029, Rv2626, Rv1733, Rv0111, RpfB-RpfD, Ag85B, Rv3478 and Rv1807), only results obtained with the peptide pool leading to the highest response are shown. Full bars represent individual mice and hatched bars represent median values of each group. The experimental cut-off value (dotted line) is represented for each mouse strain: 51 spots/10⁶ cells for BALB/c, 56 spots/10⁶ cells for C57BL/6 and 72 spots/10⁶ cells for C3H/HeN mice. Results are representative of two independent experiments.</p

MVATG18377 induces multiple cytokines-producing T cells in BALB/c mice.

<p>Cells from MVATG18377-immunized BALB/c mice were stimulated with antigen peptide pools or an irrelevant E7 peptide (Irr) and IFNγ, IL2 and TNFα intracellular cytokine staining was measured by flow cytometry. Results are presented as (<b>A</b>) the percentage of IFNγ⁺, IFNγ⁺TNFα⁺ or IFNγ⁺TNFα⁺IL2⁺ cell subsets among total CD4 T cells or (<b>B</b>) percentage of IFNγ⁺, TNFα⁺ or IFNγ⁺TNFα⁺ cell subsets among total CD8 T cells. Plain bars represent response from individual mice and hatched bars represent median response for each cell subset. Cut-off value (dotted line, 0.02%) is represented for both CD4⁺ and CD8⁺ T cell responses. Only antigens with median values above the cut-off value are represented. For these antigens, only cell subgroups with a percentage above the cut-off value are represented. No response was detected in MVATGN33.1-immunized mice (data not shown). For each cell population, background signal obtained in unstimulated cell condition was subtracted. Pie charts represent a more global analysis for each responder antigen. All analyzed single (IFNγ⁺, TNFα⁺ and IL2⁺), double (IFNγ⁺TNFα⁺, IFNγ⁺IL2⁺ and TNFα⁺IL2⁺) or triple (IFNγ⁺TNFα⁺IL2⁺) cytokine producer cells are included under the corresponding color codes. Results are representative of two independent experiments.</p

Schematic representation of the antigen fusions of MVATG18377.

<p>Three antigen fusions were inserted in the deletion III of MVA vector. The first fusion is constituted by the fusion of Rv2029, Rv2626, Rv1733 and Rv0111 proteins and is placed under the control of p7.5K promoter. The second fusion contains a fusion of RpfB-RpfD, Ag85B, TB10.4 and ESAT-6 proteins and its expression is driven by the pH5R promoter. The third fusion is constituted by the fusion of Rv0569, Rv1813, Rv3407, Rv3478 and Rv1807 proteins and is placed under the control of pB2R promoter. SF, signal peptide of the F protein of measles virus. SR, signal peptide of the glycoprotein precursor of rabies virus ERA strain. TMR, membrane-anchoring peptide derived from the rabies glycoprotein of PG strain.</p

ELISpot analysis of IFNγ responses specific of MVATG18377-encoded Mtb antigens in non-human primates.

<p>(<b>A</b>) Overview of primate experimental design. Three primates were immunized thrice with MVATG18377 at Weeks 0, 8 and 18 (arrowhead). IFNγ ELISpot assays were performed with PBMCs collected at indicated time points (red point). (<b>B</b>) Antigen-specific IFNγ responses. Results are shown as the number of IFNγ-producing cells (spots) per 10⁶ cells following stimulation with peptide pools specific of each of the 14 antigens. At each time point, cumulative results following stimulation with each of 14 antigens are represented. A color code is used for each antigen. †, data are not available due to cell culture issues.</p

<i>In vivo</i> CTL analysis in immunized BALB/c mice.

<p>Mice were immunized twice with either MVATGN33.1 or MVATG18377. Labelled target cell populations pulsed with individual antigen peptide pool were adoptively transferred into those 7 days after last immunization. Results are expressed as mean ± S.D. of the percentage of specific killing based on the relative ratio of target cells present in MVATG18377-vaccinated mice compared to those in the MVATGN33.1-vaccinated mice. For each time point and antigen peptide pool (P), significant lysis is indicated by * (p<0.05) using a permutation resampling test.</p