Co-Administration of IL-1+IL-6+TNF-a with Mycobacterium tuberculosis Infected Macrophages Vaccine Induces Better Protective T Cell Memory than BCG Vijender Singh 1 , Shweta Jain1, Uthaman Gowthaman 1 , Pankaj Parihar1, Pushpa Gupta2, Umesh D. Gupta2, Javed N. Agrewala1* 1 Immunology Laboratory, Institute of Microbial Technology, Chandigarh, India, 2 National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, India Abstract BCG has been administered globally for more than 75 years, yet tuberculosis (TB) continues to kill more than 2 million people annually. Further, BCG protects childhood TB but is quite inefficient in adults. This indicates that BCG fails to induce long-term protection. Hence there is a need to explore alternative vaccination strategies that can stimulate enduring T cell memory response. Dendritic cell based vaccination has attained extensive popularity following their success in various malignancies. In our previous study, we have established a novel and unique vaccination strategy against Mycobacterium tuberculosis (M. tb) and Salmonella typhimurium by utilizing infected macrophages (IM). In short-term experiments (30 days), substantial degree of protection was observed. However, remarkable difference was not observed in long-term studies (240 days) due to failure of the vaccine to generate long-lasting memory T cells. Hence, in the present study we employed T cell memory augmenting cytokines IL-1+IL-6+TNF-a and IL-7+IL-15 for the induction of the enhancement of long-term protection by the vaccine. We co-administered the M. tb infected macrophages vaccine with IL-1+IL-6+TNF-a (IM-1.6.a) and IL-7+IL-15 (IM-7.15). The mice were then rested for a reasonably large period (240 days) to study the bona fide T cell memory response before exposing them to aerosolized M. tb. IM-1.6.a but not IM-7.15 significantly improved memory T cell response against M. tb, as evidenced by recall responses of memory T cells, expansion of both central as well as effector memory CD4 and CD8 T cell pools, elicitation of mainly Th1 memory response, reduction in the mycobacterial load and alleviated lung pathology. Importantly, the protection induced by IM-1.6.a was significantly better than BCG. Thus, this study demonstrates that not only antigen-pulsed DCs can be successfully employed as vaccines against cancer and infectious diseases but also macrophages infected with M. tb can be utilized with great efficacy especially in protection against TB. Citation: Singh V, Jain S, Gowthaman U, Parihar P, Gupta P, et al. (2011) Co-Administration of IL-1+IL-6+TNF-a with Mycobacterium tuberculosis Infected Macrophages Vaccine Induces Better Protective T Cell Memory than BCG. PLoS ONE 6(1): e16097. doi:10.1371/journal.pone.0016097 Editor: Leonardo A. Sechi, Universita di Sassari, Italy Received November 30, 2010; Accepted December 10, 2010; Published January 19, 2011 Copyright: 2011 Singh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study is supported by the Indian Council of Medical Research (Grant Reference No. 5/8/5/6/2005-ECD-I) and Department of Biotechnology (Grant Reference No. BT/PR6219/Med/14/762/2005), New Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: javed@imtech.res.in Introduction In 1992, nearly 100 million children received BCG [1]. Despite the reality that more people have been immunized with BCG than any other vaccine, TB continues to kill some 2 million people annually and 2 billion people worldwide are infected with M. tb [2]. Hence, the protective efficacy of the BCG vaccine remains doubtful. The wide spread of TB has been further aggravated by the emergence of multidrug-resistant (MDR) strains of mycobac- teria and the AIDS-pandemic [3]. Therefore, there is a serious need and challenge for scientific community to develop alternative vaccines for the control of the disease. Approximately one-third of the world population is infected with M. tb, but only 5%–10% contract active disease while remaining develop effective immunity [2]. Further, mice cleared of TB infection respond vigorously upon re-exposure to mycobacteria and remain protected [4]. This suggests that infection with viable mycobacteria develop effective and long-lasting protective immu- nity. There may exist a possibility that growing bacilli express discrete molecules inside the macrophage [5], that may act as protective antigens necessary for the generation of effector and memory T cells responsible for protective immunity. Whether such protective antigens of M. tb that can be exploited as vaccines, still remains to be identified. [4,5]. Incidentally, the antigens isolated from in vitro cultures have failed to generate considerable protective immunity, corroborating to the above mentioned hypothesis [6,7]. In the past few decades, a number of new vaccine approaches like naked DNA vaccines, live attenuated vaccines and subunit vaccines have been elucidated. Unfortunately, none of them worked successfully against TB since they failed to generate long- lasting memory cells [8]. This indicates that novel and unique vaccination strategies still need to be explored for TB. Recently, many studies have highlighted the role of cell-based vaccines viz. antigen-loaded DCs to evoke protective T cell PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16097