Novel Generation Mycobacterial Adjuvant Based on Liposome-Encapsulated Monomycoloyl Glycerol from Mycobacterium bovis Bacillus Calmette-Gue ´rin 1 Claire A. Swetman Andersen,* Ida Rosenkrands,* Anja W. Olsen,* Pernille Nordly,* Dennis Christensen,* Roland Lang, ‡ Carsten Kirschning, § Jessica M. Gomes, Veemal Bhowruth, † David E. Minnikin, † Gurdyal S. Besra, † Frank Follmann,* Peter Andersen,* and Else Marie Agger 2 * The immunostimulatory activity of lipids associated with the mycobacterial cell wall has been recognized for several decades and exploited in a large variety of different adjuvant preparations. Previously, we have shown that a mycobacterial lipid extract from Mycobacterium bovis bacillus Calmette-Gue ´rin delivered in cationic liposomes was a particular efficient Th1-inducing adjuvant formulation effective against tuberculosis. Herein, we have dissected the adjuvant activity of the bacillus Calmette-Gue ´rin lipid extract showing that the majority of the activity was attributable to the apolar lipids and more specifically to a single lipid, monomycoloyl glycerol (MMG), previously also shown to stimulate human dendritic cells. Delivered in cationic liposomes, MMG induced the most prominent Th1-biased immune response that provided significant protection against tuberculosis. Importantly, a simple synthetic analog of MMG, based on a 32 carbon mycolic acid, was found to give rise to comparable high Th1-biased responses with a major representation of polyfunctional CD4 T cells coexpressing IFN-, TNF-, and IL-2. Furthermore, com- parable activity was shown by an even simpler monoacyl glycerol analog, based on octadecanoic acid. The use of these synthetic analogs of MMG represents a promising new strategy for exploiting the immunostimulatory activity and adjuvant potential of components from the mycobacterial cell wall without the associated toxicity issues observed with complex mycobacterial preparations. The Journal of Immunology, 2009, 183: 2294 –2302. M ycobacterium species, including one of the world’s most successful pathogens, Mycobacterium tuberculo- sis, constitute some of the most immunostimulatory organisms known to date. This is illustrated by infection with tu- berculosis (TB), 3 which gives rise to granulomatous inflammation at infection site and a powerful induction of T cell responses. The existence of mycobacterial components with substantial immuno- stimulatory activity is furthermore reflected in the prominent ac- tivity of Freund’s complete adjuvant (FCA) based on mineral oil formulated with heat-killed mycobacteria. Because FCA causes acute inflammation, granulomas, and chronic toxicity, it is consid- ered far too reactogenic for use in human vaccines; however, in- dividual purified bioactive molecules from mycobacteria may prove useful in adjuvant formulations without the associated ad- verse effects. Several compounds from the mycobacterial cell wall have been implicated in mediating host cell immune activation, in- cluding lipids that constitute up to 40% of the dry weight of the cell envelope (1, 2). Sprott et al. (3) demonstrated that lipo- somes formed of different polar lipids extracted from Mycobac- terium bovis bacillus Calmette-Gue ´rin (BCG) and in particular purified phosphatidylinositol mannosides (PIM) were effective in stimulating murine bone marrow dendritic cells (BMDC). In addition, mice immunized with these liposomes admixed with soluble OVA generated OVA-specific Abs and CTL responses. In a recent study (4), comparing seven cell wall lipids, the tre- halose mycolates were identified as the principal biologically active lipid from mycobacteria. In particular, trehalose dimy- colate (TDM) composed of trehalose covalently linked to two mycolic acid chains induced the highest levels of the proin- flammatory cytokines TNF-, IL-6, and IL-12 when used for stimulation of BMDC (5). This immunostimulatory activity has been exploited in different adjuvant preparations, e.g., in the Ribi adjuvant systems where the TDM analog trehalose-dico- rynomycolate is one of the key constituents. Recent studies in our laboratory have shown that a mycobacterial lipid extract from M. bovis BCG, comprising both apolar and polar lipids, is particularly effective when delivered in cationic liposomes composed of dimethyldioctadecylammonium (DDA). This ad- juvant formulation, designated mycosomes, combined with the *Department of Infectious Disease Immunology, Adjuvant Research, Statens Serum Institut, Copenhagen, Denmark; † School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom; ‡ Mikrobiologisches Institut, Universita ¨- tsklinikum Erlangen, Erlangen, Germany; and § Institut fu ¨r Medizinische Mikrobiolo- gie, Immunologie und Hygiene, Munchen, Germany Received for publication December 8, 2008. Accepted for publication June 10, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the European Commission (contract no. LSHP-CT- 2003-503367). G.S.B. has a James Bardrick Research Chair and a Royal Society Wolfson Research Merit Award, and D.E.M. was a Leverhulme Emeritus Fellow; both acknowledge support from the Medical Research Council (U.K.) and the Well- come Trust. 2 Address correspondence and reprint requests to Dr. Else Marie Agger, Department of Infectious Disease Immunology, Adjuvant Research, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark. E-mail address: eag@ssi.dk 3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Gue ´rin; BMDC, bone marrow dendritic cell; C 18 MAG, C 18 mono-acyl glycerol analog; DC, dendritic cell; DDA, dimethyldioctadecylammonium; FCA, Freund’s complete adju- vant; MMG, monomycoloyl glycerol; NMR, nuclear magnetic resonance; PDIM, ph- thiocerol dimycocerosate; PGL, phenolic glycolipid; PIM, phosphatidylinositol man- noside; TAG, triacylglycerol; TDM, trehalose dimycolate. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 The Journal of Immunology www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804091