A Model Vaccine Exploiting Fungal Mannosylation to Increase Antigen Immunogenicity 1 Jennifer S. Lam,* † Michael K. Mansour,* † Charles A. Specht, † and Stuart M. Levitz 2 * † Ag mannosylation represents a promising strategy to augment vaccine immunogenicity by targeting Ag to mannose receptors (MRs) on dendritic cells. Because fungi naturally mannosylate proteins, we hypothesized that Ags engineered in fungi would have an enhanced capacity to stimulate T cell responses. Using the model Ag OVA, we generated proteins that differentially expressed N- and O-linked mannosylation in the yeast Pichia pastoris and compared them to their unglycosylated counterparts produced in Escherichia coli. We found that yeast-derived OVA proteins containing N-linkages, extensive O-linkages, or both were more potent than the unmannosylated Ags at inducing OVA-specific CD4  T cell proliferation. This elevated response to fungal Ags was inhibited by mannan, suggesting involvement of MRs. However, the macrophage MR (CD206) was not essential, because mac- rophage MR-deficient dendritic cells were fully competent in presenting yeast-derived OVA Ags. Thus, the use of fungal glyco- sylation to provide N-linked and/or extensive O-linked mannosylation increased the capacity of the model Ag OVA to stimulate Ag-specific T cell responses in an MR-dependent manner. These data have implications for vaccine design by providing proof of principle that yeast-derived mannosylation can enhance immunogenicity. The Journal of Immunology, 2005, 175: 7496 –7503. C urrently, the majority of the vaccines licensed for human use induce protective Ab responses (1, 2). However, vac- cine-mediated protection against many tumors and infec- tions is thought to require the induction of T cell-mediated immu- nity (1, 2). Mannosylation of Ags has been reported to enhance MHC class I- and MHC class II-restricted Ag presentation and T cell stimulation by up to 200-fold compared with nonmannosy- lated proteins (3–7). Furthermore, in vivo induction of Th1 cyto- kines and Ag-specific CTL responses were observed in mice upon immunization with mannosylated vaccines (6, 8, 9). Thus, Ag mannosylation is an attractive strategy for boosting cell-mediated immune responses. The effectiveness of mannosylated proteins is probably attrib- utable to their ability to target mannose receptors (MRs), 3 such as the macrophage MR (MMR; CD206) and dendritic cell (DC)-spe- cific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209), found on DCs. MMR binds oligosaccharides containing terminal mannose residues and traffics Ag into early endosomes (10), whereas DC- SIGN recognizes high mannose oligosaccharides and directs Ag into late endosomes or lysosomes for degradation (11, 12). Both the MMR and DC-SIGN have the capacity to direct internalized Ag into endocytic pathways that result in MHC presentation and subsequent T cell activation. Tumor Ags fused to a mAb specific for the MMR stimulated MHC class I- and II-restricted T cell responses (13), whereas IgG1 isotype Abs specific for DC-SIGN induced IgG1-specific CD4 + T cell proliferation (12). Many studies on MR-targeted vaccines involved the use of chemical conjugation to provide mannosylation of Ags (6) and therefore were unable to test the effects of N- and O-linked man- nosylation. Because fungi preferentially glycosylate their glycans with mannose residues, we wanted to examine the immunogenic potential of mannosylated proteins produced in a fungal system. We have previously established that mannosylation is essential to the capacity of mannoproteins (MP) from the pathogenic fungus, Cryptococcus neoformans, to stimulate a cell-mediated immune response (14). Furthermore, MRs on DCs bind to exposed man- nose residues on the MP, resulting in efficient Ag uptake, process- ing, and presentation. 4 Molecular characterization of cryptococcal MPs revealed serine/threonine (S/T)-rich C-terminal regions that serve as sites for extensive O-linked glycosylation and asparagine- X-S/T (N-X-S/T) sequences as potential N-glycosylation sites (15, 16). Given the above findings, we hypothesized that the natural abil- ity of fungi to mannosylate Ags could be exploited to make re- combinant mannosylated vaccines that efficiently stimulate CD4 + T cell responses. To test this hypothesis, mannosylated and ung- lycosylated Ags were engineered in the yeast Pichia pastoris and the bacterium Escherichia coli, respectively. The model Ag was a portion of OVA that includes epitopes recognized by OVA-spe- cific T cells and two potential N-linked glycosylation sites. Site- directed mutagenesis of the N-X-S/T sequences was performed to study the contribution of N-linked mannosylation, whereas the S/T-rich region from cryptococcal MP98 (15) was fused to OVA to study the effects of extensive O-linked mannosylation. We found that mannosylated OVA Ags were significantly more potent *Department of Microbiology and Immunology Training Program, Boston University School of Medicine, and † Evans Memorial Department of Medicine, Section of In- fectious Diseases, Boston Medical Center, Boston, MA 02118 Received for publication June 24, 2005. Accepted for publication September 25, 2005. 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 in part by National Institutes of Health Grants RO1AI25780, RO1AI066087, RO1AI37532, and T32AI07642. 2 Address correspondence and reprint requests to Dr. Stuart M. Levitz, Evans Me- morial Department of Medicine, Section of Infectious Diseases, Room X626, Boston Medical Center, 650 Albany Street, Boston, MA 02118. E-mail address: slevitz@bu.edu 3 Abbreviations used in this paper: MR, mannose receptor; BMDC, bone marrow- derived DC; DC, dendritic cell; DC-SIGN, DC-specific ICAM-3-grabbing noninte- grin (CD209); ec, E. coli derived construct; KO, knockout; LN, lymph node; MMR, macrophage MR (CD206); MP, mannoprotein; N, asparagine; PNGase F, peptide:N- glycosidase F; ppOVA, P. pastoris-derived protein containing the OVA portion; ppOVANQ, P. pastoris-derived counterpart of ppOVA lacking the N-linked glycan site; ppOVAST, protein consisting of the OVA portion fused to the S/T-rich region produced in P. pastoris; ppOVASTNQ, P. pastoris-derived counterpart of ppOVAST lacking the N-linked glycan site; S/T, serine/threonine; Tg, transgenic; WT, wild type. 4 M. K. Mansour, E. Latz, and S. M. Levitz. Cryptococcus neoformans glycoantigens are captured by multiple lectin receptors and exclusively presented by dendritic cells. Submitted for publication. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00