Method for the Synthesis of Multi-Epitopic Streptococcus pyogenes Lipopeptide Vaccines Using Native Chemical Ligation Peter M. Moyle, ² Colleen Olive, ‡ Mei-Fong Ho, ‡ Melinda Burgess, ‡ Levente Karpati, § Michael F. Good, ‡ and Istvan Toth* ,²,§ The School of Pharmacy and School of Molecular and Microbial Sciences (SMMS), The UniVersity of Queensland, St. Lucia, QLD, Australia, and the Queensland Institute of Medical Research (QIMR), Herston QLD, Australia i.toth@uq.edu.au ReceiVed May 8, 2006 The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes. Introduction Group A Streptococcus (GAS; Streptococcus pyogenes) is responsible for many diseases, including streptococcal toxic shock syndrome and necrotizing fasciitis, with streptococcal pharyngitis and impetigo being the most common GAS associ- ated conditions. In a small number of GAS infections, patients who have not been treated, or treated inadequately, may develop acute rheumatic fever. Acute rheumatic fever (ARF) is an autoimmune disease characterized by the production of antibod- ies and T-cells against GAS, which cross-react with human tissues in the heart, joints, and brain. 1 The associated heart valve and pericardial inflammation may lead to rheumatic heart disease (RHD) and eventually heart failure. As ARF and RHD only occur following GAS infection, a means to prevent GAS infection (e.g., a vaccine) would provide the best opportunity to prevent these diseases. Several prophylactic GAS vaccines are currently under development. 2-7 Many of these vaccines target the GAS M protein, an R-helical coiled-coil cell surface protein that is associated with resistance to phagocytosis. 1 The sequence of the GAS M protein amino (N)-terminus is highly variable between serotypes, with serotype-specific antibodies elicited to this region. 1 As over 100 GAS serotypes have been character- ized, vaccines based on N-terminal peptides need to be multi- epitopic, including epitopes selected to offer broad-strain coverage against circulating serotypes. In comparison, the M protein carboxyl (C)-terminus region is highly conserved ² School of Pharmacy, The University of Queensland. ‡ Queensland Institute of Medical Research. § School of Molecular and Microbial Sciences, The University of Queensland. (1) Batzloff, M. R.; Sriprakash, K. S.; Good, M. F. Curr. Drug Targets 2004, 5, 57. (2) Kotloff, K. L.; Corretti, M.; Palmer, K.; Campbell, J. D.; Reddish, M. A.; Hu, M. C.; Wasserman, S. S.; Dale, J. B. JAMA 2004, 292, 709. (3) Hruby, D. E.; Hodges, W. M.; Wilson, E. M.; Franke, C. A.; Fischetti, V. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5714. (4) Ji, Y. D.; Carlson, B.; Kondagunta, A.; Cleary, P. P. Infect. Immun. 1997, 65, 2080. (5) Brandt, E. R.; Sriprakash, K. S.; Hobb, R. I.; Hayman, W. A.; Zeng, W.; Batzloff, M. R.; Jackson, D. C.; Good, M. F. Nat. Med. 2000, 6, 455. (6) Olive, C.; Batzloff, M.; Horva ´th, A.; Clair, T.; Yarwood, P.; Toth, I.; Good, M. F. Infect. Immun. 2003, 71, 2373. (7) Horva ´th, A.; Olive, C.; Karpati, L.; Sun, H. K.; Good, M.; Toth, I. J. Med. Chem. 2004, 47, 4100. 6846 J. Org. Chem. 2006, 71, 6846-6850 10.1021/jo060960p CCC: $33.50 © 2006 American Chemical Society Published on Web 08/02/2006