Inhalation of recombinant adenovirus expressing granulysin protects mice infected with Mycobacterium tuberculosis

Abstract

Granulysin is a cytolytic molecule with perforin and granzymes that is expressed by activated human CTLs, NK and γδ T cells, and it has broad antimicrobial activity, including to drug-sensitive and drug-resistant Mycobacterium tuberculosis. We hypothesized that approaches facilitating the expression of granulysin in M. tuberculosis-infected host cells in the lung may provide a novel treatment strategy for pulmonary TB. In this study, a recombinant replication-deficient adenovirus serotype 5-based rAdhGLi was constructed that expressed human granulysin in the cytosol of the U937 and RAW264.7 macrophage-like cell lines as confirmed by western blotting and co-localization technology using indirect immunofluorescence staining. Ninety-six hours after both cell lines were infected with M. tuberculosis, acid-fast staining and enumeration demonstrated that rAdhGLi-treated cells had a lower colony-forming units (CFU) of intracellular bacteria than culture medium or AdNull controls. Granulysin was only expressed in the lung and not in other organs following inhalation of rAdhGLi. In particular, immunocompetent BALB/c mice or SCID mice intranasally infected with ~200 CFU of virulent M. tuberculosis H37Rv intranasally were treated with rAdhGLi, and they showed decreased bacterial loads in the lung when compared with phosphate-buffered saline or AdNull controls. Importantly, a clear dose-dependent rAdhGLi treatment efficacy was found in infected BALB/c mice, with the most significant reduction in lung bacteria obtained in BALB/c mice treated with 10(9) plaque-forming units of rAdhGLi without any pathological changes. Our study indicates that rAdhGLi may be used as a novel and efficient treatment strategy with the capability to directly kill intracellular M. tuberculosis.

A Multistage Subunit Vaccine Effectively Protects Mice Against Primary Progressive Tuberculosis, Latency and Reactivation

“…with virulent M. tuberculosis strain H37Rv. The next day, three mice in the PBS control group were sacrificed and the whole lung was removed aseptically from each mouse to determine the actual infectious dose by enumeration of bacterial load ( Ma et al, 2015 ). Four weeks post-challenge, differential protective efficacy between three subunit vaccines and BCG was determined by comparing the bacterial load in the spleen and lung (n = 6), and by observing the lung histopathological changes as previously described (n = 3) ( Wang et al, 2015 ).…”

“…The negative control mice were treated with DMT or PBS. At week 15, protective efficacy between groups (n = 6) was compared as previously described ( Ma et al, 2015 ). The remaining mice in each group were injected s.c. with dexamethasone (DXM; Cat#50022; Sigma) at 6 mg per kg body weight every 2 days, for a total of three injections ( Lowrie et al, 1999 ).…”

Lipoarabinomannan-Responsive Polycytotoxic T Cells Are Associated with Protection in Human Tuberculosis

“…The plasma levels of granulysin correlate with successful treatment of tuberculosis (26,27), and the expression of granulysin at the site of mycobacterial infection correlates with protection (28,29) and vaccine efficacy (30). Strikingly, the local delivery of granulysin by a recombinant adenovirus reduced the proliferation of Mtb in mice, highlighting the therapeutic potential of cytotoxic molecules (31).…”

Lipoarabinomannan-responsive polycytotoxic T cells are associated with protection in human tuberculosis

“…The plasma levels of granulysin correlate with successful treatment of tuberculosis (26,27), and the expression of granulysin at the site of mycobacterial infection correlates with protection (28,29) and vaccine efficacy (30). Strikingly, the local delivery of granulysin by a recombinant adenovirus reduced the proliferation of Mtb in mice, highlighting the therapeutic potential of cytotoxic molecules (31).…”