Volume 16, Issue 6, Pages 1065-1072 (June 2008) Therapeutic Effect of Recombinant Adenovirus Encoding Interferon-γ in a Murine Model of Progressive Pulmonary Tuberculosis  Dulce A Mata-Espinosa, Valentin Mendoza-Rodríguez, Diana Aguilar-León, Ricardo Rosales, Fernando López-Casillas, Rogelio Hernández-Pando  Molecular Therapy  Volume 16, Issue 6, Pages 1065-1072 (June 2008) DOI: 10.1038/mt.2008.69 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 In vitro analysis of adenovirus encoding interferon-γ (IFN-γ) (AdIFNγ), kinetics of IFN-γ, and tumor necrosis factor-α (TNF-α) mRNA expression, after intratracheal inoculation with AdIFNγ or control adenovirus expressing green fluorescent protein (AdGFP) in Balb/c mice. (a) Western blot of culture supernatants from Mv1Lu cells infected with AdIFNγ and AdGFP [multiplicity of infection (MOI) = 250]. The band of 16 kd corresponds to IFN-γ detected using an anti-mouse IFN-γ antibody. (b) Production of nitrites by peritoneal macrophages incubated with culture supernatants from Mv1Lu cells infected with AdIFNγ or AdGFP (MOI = 250); high concentration of nitrites was detected only in peritoneal macrophages incubated with supernatants from Mv1Lu cells infected with AdIFNγ. (c) Kinetics of IFN-γ and (d) TNF-α gene expression determined using real-time PCR in the lungs of healthy Balb/c mice infected with 1.7 × 109 plaque forming units/mouse of AdIFNγ (closed squares) or control AdGFP (open squares). Data are the mean values ± SD from two experiments, each carried out in triplicate. Asterisks represent statistical significance (P < 0.05). Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Representative lung histopathology findings and immunohistochemical data from healthy mice infected with recombinant adenoviruses by intratracheal cannulation. (a) Slight inflammatory infiltrate around small blood vessels and bronchi is seen 24 hours after adenovirus encoding interferon-γ (AdIFN-γ) administration. (b) At 21 days after AdIFNγ administration, there is mild chronic inflammation around blood vessels. (c) Small nodules of chronic inflammation, located around hepatic portal areas (arrows), as seen at 21 days after AdIFNγ administration. (d) When specific antibodies against adenoviral proteins are used, strong immunostaining is exhibited by bronchial epithelial cells (arrows) and macrophages (asterisk). Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Effect of a single administration of adenovirus encoding interferon-γ (AdIFNγ) on the number of colony forming units (CFUs), and morphometry results in mice lungs infected with Mycobacterium tuberculosis H37Rv strain. (a) Colony bacilli forming units from tuberculous mice treated with AdIFNγ (black bars), control adenovirus expressing green fluorescent protein (AdGFP) (white bars), AdIFNγ plus conventional chemotherapy (cross-hatched bars), and only with bacilli chemotherapy (dotted bars). The limits of detection of CFUs are 100–250 million numbers. (b) Percentage of the lung occupied by pneumonia. (c) Granuloma area. All treatments significantly reduced bacillary load in the lung when compared with results in control mice treated with AdGFP. The combination treatment significantly reduced the number of CFUs as compared to the results with chemotherapy alone, when measured on day 14 (P < 0.003). From days 90 to 180 there were no bacilli detected in the groups of mice that had received combination treatment and chemotherapy alone. On day 180 there were no surviving animals in the control group that had received AdGFP. The data are mean values ± SD from three different experiments. Asterisks on the AdIFNγ bar represent statistical significance relative to the control group (P < 0.05). Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Representative lung histopathology from mice treated with recombinant adenoviral vector 60 days after infection with Mycobacterium tuberculosis H37Rv strain or multidrug-resistant (MDR) clinical isolate. (a) Extensive pneumonia in mice infected with the H37Rv strain, as assessed at 2 months after treatment with control adenovirus expressing green fluorescent protein (AdGFP). (b) In contrast, very little pneumonia is apparent 2 months after adenovirus encoding interferon-γ (AdIFNγ) administration in mice infected with the same H37Rv strain. (c) Even less pneumonia is observed 2 months after a single dose of AdIFNγ plus conventional chemotherapy administered daily in animals infected with the same H37Rv strain. (d) Small granulomas were observed in mice infected with H37Rv and treated with control AdGFP. (e) Larger granulomas are observed in mice infected with H37Rv strain and treated with AdIFNγ. The inset represents immunohistochemical detection of adenovirus, showing strong positivity in macrophages (arrow) located in peribronchiolar granulomas, as well as in the bronchiolar epithelium. (f) Large granulomas are also seen in mice treated with the combination of AdIFNγ plus conventional chemotherapy. (g) Two months after administration of control vector AdGFP, mice infected with the MDR strain showed extensive pneumonia. In contrast, 2 months after AdIFNγ administration, mice that had been infected with the MDR strain showed (h) small pneumonic areas and (i) large granulomas. Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Effect of a single administration of adenovirus encoding interferon-γ (AdIFNγ) on the expression of cytokines and CCL2 in mice lungs infected with Mycobacterium tuberculosis H37Rv strain. Mice were treated with a single dose of AdIFNγ at 1.7 × 109 plaque forming units (pfu)/mouse (closed squares), or with control adenovirus expressing green fluorescent protein (AdGFP) at 1.7 × 109 pfu/mouse (open squares) on day 60 after infection with tuberculosis (TB), and the gene expression of the indicated cytokine or chemokine was determined using real-time reverse transcriptase-PCR. The results are given as mean values ± SD. Asterisks represent statistical significance (P < 0.05). iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-α. Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 6 Determination of CD4+ and CD8+ T cell subsets in lung homogenates from H37Rv-infected mice treated with recombinant adenoviruses. Adenovirus encoding interferon-γ treatment (black bars) induced a higher number of CD8+ T cells than control adenovirus expressing green fluorescent protein (AdGFP) treatment (white bars) at days 120 and 150, while similar numbers of CD4+ T cells were seen in both groups. Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 7 Effect of a single administration of adenovirus encoding interferon-γ (AdIFNγ) on the bacillary load and morphometry in mice lungs infected with multidrug resistant (MDR) Mycobacterium tuberculosis isolate. (a) Mice were treated with 1.7 × 109 plaque forming units (pfu)/mouse of AdIFNγ (black bars) or control adenovirus expressing green fluorescent protein (AdGFP) (white bars) on day 60 after infection with MDR bacilli. Bacillary loads in the lungs significantly decreased in mice treated with AdIFNγ after day 14 and up to day 180. (b) Percentage of the lung occupied by pneumonia. (c) Granuloma area. Results are mean values ± SD from four mice from three different experiments. Asterisks represent statistical significance (P < 0.05). There was no material available for morphometry after day 60 after treatment. Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 8 Effect of a single administration of adenovirus encoding interferon-γ (AdIFNγ) on the expression of cytokines and CCL2 in mice lungs infected with Mycobacterium tuberculosis multidrug-resistant (MDR) strain. Mice treated with 1.7 × 109 plaque forming units/mouse of AdIFNγ (closed squares) or control adenovirus expressing green fluorescent protein (AdGFP) (open squares) on day 60 after infection with MDR strain. Results are mean values ± SD. Asterisks represent statistical significance (P < 0.05). Molecular Therapy 2008 16, 1065-1072DOI: (10.1038/mt.2008.69) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions