Immunology Letters 158 (2014) 88–94 Contents lists available at ScienceDirect Immunology Letters j our na l ho me page: www.elsevier.com/locate/immlet Immunotherapeutic potential of recombinant ESAT-6 protein in mouse model of experimental tuberculosis Shabir Ahmad Mir, Indu Verma, Sadhna Sharma ∗ Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India a r t i c l e i n f o Article history: Received 5 June 2013 Received in revised form 24 November 2013 Accepted 7 December 2013 Available online 15 December 2013 Keywords: Immunotherapy Chemotherapy ESAT-6 Histopathology Antituberculosis drugs a b s t r a c t Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunother- apy for the treatment of tuberculosis. Therapies that would upregulate the host antimycobacterial immune response and/or attenuate T-cell suppressive and macrophage-deactivating cytokines may prove to be useful in the treatment of tuberculosis. ESAT6, 6-kDa early secreted antigenic target, is a potent protective antigen and is considered as major target for long-lived memory cells. In the present study the immunotherapeutic potential of ESAT-6 has been evaluated in mouse model of experimental tuberculosis. In the present study the ESAT-6 protein was cloned in Escherichia coli using pET23a(+) plasmid and puri- fied by Ni 2+ -NTA chromatography. Further, the immunotherapeutic potential of the recombinant ESAT-6 (in terms of CFU enumeration in the target organs and histopathological analysis of lungs) was evaluated against experimental tuberculosis. The recombinant ESAT-6 with C-terminal histidine-tag and free N- terminus mimics the natural form of ESAT-6 has been successfully cloned and purified. The recombinant ESAT-6 protein adjuvanted with dimethyl dioctadecylammonium bromide (DDA) moderately reduced the bacterial load in the target organs of infected mice. Further, the formulation (ESAT-6-DDA) was able to act synergistically when given in combination with antituberculosis drugs. This recombinant ESAT- 6 showed good immunotherapeutic potential against experimental tuberculosis and can be used as an adjunct to the conventional antituberculosis chemotherapy. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Mycobacterium tuberculosis, the intracellular pathogen that causes tuberculosis is responsible for more human deaths than any other single infectious agent [1–3]. Currently, each year about 1.45 million people die of tuberculosis (TB), and 8.8 million develop tuberculosis [4]. The standard treatment of chemotherapeutic antibacterial drugs is either ineffective or suboptimal for sev- eral reasons including: (a) protracted drug treatment (at least 6 months after diagnosis) is apt to be interrupted; (b) large doses of antibacterial drug combinations may cause side-effects, such as liver damage; (c) the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis has further com- plicated the treatment; (d) the cost may be difficult to afford in some parts of the world. Therapeutic vaccines which aim at eliciting the host’s protective immune response to overcome the problem of drug resistance may improve the cure rates and shorten the treatment. Therefore, a combination of conventional chemotherapy ∗ Corresponding author. Tel.: +91 0172 2755180; fax: +91 0172 2745078/0172 2744401. E-mail addresses: mirshabir@gmail.com (S.A. Mir), sadhnabiochem@gmail.com (S. Sharma). and immunotherapeutics is a powerful weapon to defend against pathogen infection. There have been several studies on immunotherapeutic agents for TB control, such as Mycobacterium vaccae (M. vaccae) [5,6] and RUTI (a vaccine made of fragmented M. tuberculosis cells detoxified and liposomed) [7]. M. vaccae is a non- pathogenic, fast-growing bacterium isolated from soil, and RUTI is based on cell fragments of detoxified M. tuberculosis. They were reported as safe and useful adjuncts to conventional chemotherapy in the treatment of TB [5,8,9], although the efficacy of M. vaccae has been considered controversial most recently [10]. It is thought that, the benefit may be attributable to a type 1 immune response, cyto- toxic and interferon-gamma (IFN-) secreting CD8 + T cells, and/or to a new immunological response against antigens of M. tubercu- losis [7,11]. Moreover, therapeutic vaccinations with DNA vaccines have been reported and a few candidates such as Hsp65 and Ag85 antigens were considered to be protective [12–22]. Immunotherapy that modulates or enhances the host immune response to M. tuberculosis has proven to be an effective method for treatment of tuberculosis in mice [22,23]. One potential approach to immunotherapy is the direct use of cytokines. However, the admin- istration of cytokines or cytokine antagonists alters only one aspect of a complex immune response. Hence, an alternative approach is to provide a stimulus that generates a multifaceted response that favours bacillary elimination. Administration of the environmental 0165-2478/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2013.12.007