The Tuberculosis Vaccine Candidate Bacillus Calmette-Guérin ΔureC::hly Coexpressing Human Interleukin-7 or -18 Enhances Antigen-Specific T Cell Responses in Mice

et al. (2013) The Tuberculosis Vaccine Candidate Bacillus Calmette-Guerin DureC::hly Coexpressing Human Interleukin-7 or -18 Enhances Antigen-Specific T Cell Responses in Mice. PLoS ONE 8(11): e78966. doi:10.1371/journal.pone.0078966 The Tuberculosis Vaccine Candidate Bacillus Calmette- Gue rin DureC ::hly Coexpressing Human Interleukin-7 or -18 Enhances Antigen-Specific T Cell Responses in Mice Martin Rao 0 Alexis Vogelzang 0 Peggy Kaiser 0 Stefanie Schuerer 0 Stefan H. E. Kaufmann 0 Martin Gengenbacher 0 0 Max Planck Institute for Infection Biology, Department of Immunology , Berlin , Germany Bacillus Calmette-Gue rin (BCG), the only approved tuberculosis vaccine, provides only limited protection. Previously, we generated a recombinant derivative (BCG DureC::hly), which secretes the pore-forming toxin listeriolysin O (LLO) of Listeria monocytogenes. This vaccine shows superior protection against tuberculosis in preclinical models and is safe in humans. Here we describe two new vaccine strains which express human interleukin-7 (hIL)-7 or hIL-18 in the genetic background of BCG DureC::hly to modulate specific T cell immunity. Both strains exhibited an uncompromised in vitro growth pattern, while inducing a proinflammatory cytokine profile in human dendritic cells (DCs). Human DCs harbouring either strain efficiently promoted secretion of IL-2 by autologous T cells in a coculture system, suggesting superior immunogenicity. BALB/c mice vaccinated with BCG DureC::hly, BCG DureC::hly_hIL7 or BCG DureC::hly_hIL18 developed a more robust Th1 response than after vaccination with parental BCG. Both strains provided significantly better protection than BCG in a murine Mycobacterium tuberculosis challenge model but efficacy remained comparable to that afforded by BCG DureC::hly. We conclude that expression of hIL-7 or hIL-18 enhanced specific T cell responses but failed to improve protection over BCG DureC::hly in mice. - Funding: This work received financial support from the European 7th Framework Program NEWTBVAC (HEALTH-F3-2009-241745). 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. An estimated 30% of the worlds population is latently infected with Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB) [1]. Of an estimated 8.7 million new TB cases worldwide in 2011, 1.4 million people died of whom 95% were from low- to middle-income countries [1]. In line with this, 1.1 million clinical TB cases have been reported to account for human immunodeficiency virus (HIV) coinfected individuals, with approximately 500,000 TB-related deaths globally [2,3]. Additionally, the advent of drug-resistant M. tuberculosis strains complicates treatment while limiting chances of survival [4]. Vaccines remain the most cost-effective means to counteract the global challenges related to infectious diseases including TB [5]. Bacillus CalmetteGuerin (BCG) is the only licensed vaccine for TB, and protects children but leaves adults unprotected from the most prevalent form of the disease, pulmonary TB [6]. This calls for better vaccines against TB. Two main strategies are pursued in TB vaccine research subunit and live vaccines. Subunit vaccines are generally aimed at boosting cellular immunity initially raised by BCG administered as prime vaccination [7]. Live vaccines are developed to replace BCG itself. Principally, a robust CD4+ T helper 1 (Th1) response represented by interferon gamma (IFN-c) and tumour necrosis factor-alpha (TNF-a) expression should be induced by the prime vaccine to eventually form a pool of memory T cells which control TB disease [8,9]. We have previously reported the superior protective efficacy of BCG DureC::hly (VPM1002), a recombinant BCG vaccine candidate that is urease-deficient and heterologously expresses listeriolysin O (LLO) [10,11]. One of the main features of this strain is its ability to perforate the host cell phagosomal membrane, thus releasing antigens into the cytosol of the host macrophage and promoting crosspriming [7,10,12]. Interleukin (IL)-7 and IL-18 have been implicated in immunity to M. tuberculosis infection [13,14,15,16]. More specifically, IL-7 is involved in homeostatic regulation of T- and B-cell proliferation in humans and mice [17]. Administration of purified recombinant IL-7 has been shown to influence recall T cell responses to M. tuberculosis infection with and without prior BCG vaccination [15,16]. IL-18, induces IFN-c secretion jointly with IL-12 as well as expression of TNF-a [18]. Both IFN-c and TNF-a are proinflammatory cytokines critical in shaping Th1-mediated immune responses in TB [19]. Mice lacking expression of IL-18 are susceptible to M. tuberculosis infection [13,20]. Moreover, high levels of IL-18 were detected in sera of patients with advanced pulmonary TB [21]. Expression of recombinant cytokines by BCG has been shown to promote better immunogenicity [22,23,24]. We therefore hypothesized that incorporating the expression of human (h)IL-7 or IL-18 into BCG DureC::hly could improve its immunogenicity. In this study, we describe two newly-derived candidate derivatives of BCG DureC::hly coexpressing either IL-7 or IL-18, namely BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18, respectively. Both strains were evaluated for their intracellular fitness in primary human cells, and their immunomodulatory properties as well as protective efficacies against aerosol challenge with M. tuberculosis. Materials and Methods Ethics Statement All experimental procedures involving mice were performed in accordance with requirements of, and approval by, the State Office for Health and Social Services (Landesamt fu r Gesundheit und Soziales), Berlin, Germany under permission number G0307/ 11. Mice were sacrificed by cervical dislocation, and all efforts were made to minimize suffering and pain. Bacterial Strains and Growth Conditions BCG SSI 1331 (American Type Culture Collection, #35733), BCG DureC::hly (VPM1002; [10,11]) and M. tuberculosis H37Rv (American Type Culture Collection, #27294) were grown in Middlebook 7H9 borth (Becton Dickinson) supplemented with 0.2% w/v glycerol, 0.05% w/v Tween 80, 10% v/v albumindextrose-catalase supplement (Becton Dickinson) (7H9-ADC) or on Middlebrook 7H11 agar (Becton Dickinson) containing 10% v/ v oleic acid-albumin-dextrose-catalase enrichment (Becton Dickinson) and 0.2% w/v glycerol. Mycobacterial cultures were grown to the mid-log phase in 1 L roller bottles (450 cm2) at 37uC and 2 rpm. For vaccine stock preparations, bacilli were collected by centrifugation [3200 rpm, room temperature (RT)], washed with phosphate-buffered saline (PBS) and stored at 80uC as PBS suspension with additional 10% glycerol. Prior to vaccination, vials were thawed, and cells harvested and resuspended in an appropriate volume of PBS. For CF (...truncated)