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

<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

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

A multi-antigenic MVA vaccine increases efficacy of combination chemotherapy against <i>Mycobacterium tuberculosis</i>

<div><p>Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by <i>Mycobacterium tuberculosis</i> (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host’s immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. <i>In vitro</i> analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, <i>i</i>.<i>e</i>. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.</p></div

Statistical analyses of bacterial load of pooled post-exposure experiments performed in both BALB/c and C57BL/6 mouse models.

<p>Groups of mice co-treated with antibiotics (ATB) and MVATG18598 or MVATGN33.1 given thrice subcutaneously during antibiotic therapy were analyzed.</p

Schematic representation of antigenic expression cassettes in MVATG18598 and <i>in vitro</i> detection of antigen expression.

<p><b>(A)</b> MVATG18598 contains the fusion Rv2626/T2a/Ag85B under the control of pB2R promoter, the fusion CFP10/ESAT6 under the control of pH5R promoter, the fusion TB10.4/Rv0287 under the control of pSE/L promoter, the fusion RpfB-RpfD under the control of p7.5K promoter and the fusion Rv3407/E2a/Rv1813 under the control of pA35R promoter. <b>(B)</b> CEF cells were infected or not (Mock) with MVATG18598 or MVATGN33.1 and cell extracts analyzed by Western blot. Fusion Rv2626/T2a/Ag85B was detected using a mouse monoclonal anti-Rv2626 antibody (expected molecular weight of cleaved product: 17.4 kDa) and a rabbit polyclonal anti-Ag85B antibody (expected molecular weight of cleaved product: 31.3 kDa). CFP10/ESAT6 fusion (expected molecular weight: 21.8 kDa) was detected using a mouse monoclonal anti-ESAT6 antibody. TB10.4/Rv0287 fusion (expected molecular weight: 21.2 kDa) was detected using an anti-TB10.4 rabbit polyclonal antibody. Fusion RpfB-RpfD (expected molecular weight: 37.0 kDa) was detected using a rabbit polyclonal anti-RpfB antibody. Fusion Rv3407/E2a/Rv1813 was detected using a rabbit polyclonal anti-Rv3407 antibody (expected molecular weight of cleaved product: 13.2 kDa) and a rabbit polyclonal anti-Rv1813 antibody (expected molecular weight of cleaved product: 12.1 kDa). ND; not done.</p

Percentage of relapsing mice and bacterial burden in mice vaccinated SC or IN with MVATG18598 or empty vector, MVATGN33.1, during or after antibiotic regimen.

<p>Percentage of relapsing mice and bacterial burden in mice vaccinated SC or IN with MVATG18598 or empty vector, MVATGN33.1, during or after antibiotic regimen.</p