Synthesis of a Highly Pure Lipid Core Peptide Based Self-Adjuvanting Triepitopic Group A Streptococcal Vaccine, and Subsequent Immunological Evaluation Peter M. Moyle, ² Colleen Olive, ‡ Mei-Fong Ho, ‡ Michael F. Good, ‡ and Istvan Toth ²,§, * School of Pharmacy, The UniVersity of Queensland, St. Lucia 4072, Queensland, Australia, School of Molecular and Microbial Sciences (SMMS), The UniVersity of Queensland, St. Lucia 4072, Queensland, Australia, and The Queensland Institute of Medical Research (QIMR), Herston QLD 4029, Australia ReceiVed April 23, 2006 We have developed a highly pure, self-adjuvanting, triepitopic Group A Streptococcal vaccine based on the lipid core peptide system, a vaccine delivery system incorporating lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity. Vaccine synthesis was performed using native chemical ligation. Due to the attachment of a highly lipophilic adjuvant, addition of 1% (w/v) sodium dodecyl sulfate was necessary to enhance peptide solubility in order to enable ligation. The vaccine was synthesized in three steps to yield a highly pure product (97.7% purity) with an excellent overall yield. Subcutaneous immunization of B10.BR (H-2 k ) mice with the synthesized vaccine, with or without the addition of complete Freund’s adjuvant, elicited high serum IgG antibody titers against each of the incorporated peptide epitopes. Introduction Streptococcus pyogenes (group A streptococcus; GAS) is a common human pathogen, associated with a broad spectrum of diseases including impetigo (an infection of the skin) and pharyngitis (strep throat). 1 Following GAS infection, particularly where patients are not treated adequately, immune-mediated post-streptococcal sequelae (e.g. glomerulonephritis or rheumatic fever) may develop. Rheumatic fever (RF) is associated with the production of antibodies and T cells against GAS, which cross-react with human tissues of the heart, joints, and brain. 1 The associated inflammation of the heart valves and pericardium may lead to rheumatic heart disease (RHD) and eventually heart failure. Because RF and RHD only occur following GAS infection, the development of a prophylactic GAS vaccine holds the potential to greatly reduce the incidence of these diseases. Numerous research groups 2-4 are working toward the devel- opment of prophylactic GAS vaccines, with the most advanced vaccine development strategies focusing on the GAS M protein. The M protein is an R-helical coiled-coil cell surface protein, which is associated with resistance to complement-mediated phagocytosis. 5 The M protein amino (N)-terminal domain is highly variable between GAS strains. 1 Antibodies elicited to N-terminal epitopes are type-specific, opsonic, and form the basis for GAS serotyping. 6 As over 100 different GAS serotypes have been characterized, vaccines based upon GAS M protein N-terminal peptides need to be multivalent, incorporating appropriate N-terminal epitopes to prevent infection with common circulating strains. 1 In comparison, the carboxyl (C)- terminal M protein (C-repeat) region is highly conserved among GAS strains, 2 offering the potential to develop vaccines capable of protecting against infection caused by many different GAS serotypes. However, the C-terminal region has been demon- strated to contain epitopes which are capable of eliciting auto- reactive antibodies and T cells. 7,8 Thus, the use of whole M protein or M protein components for vaccine development may result in RF. Investigations have therefore focused on identifying C-terminal sequences devoid of potentially deleterious T and B cell epitopes. An example is the J8 peptide 9 (QAEDKVKQS- REAKKQVEKALKQLEDKVQ), which contains an M protein C-terminal minimal B cell epitope (in bold) enclosed within peptide sequences designed to maintain the R-helical conforma- tion of the native M protein. Previously we have described the development of experi- mental M protein based GAS vaccines utilizing the J8 peptide, and N-terminal epitopes from GAS strains common to the Australian aboriginal populations of the Northern Territory and northern Queensland. 10-17 These vaccines were developed using the lipid core peptide (LCP) system, 18 a vaccine delivery system incorporating peptide epitopes, a lipidic adjuvant (the LCP lipid core; Figure 1), and a carrier into a single molecular entity. These systems were synthesized using solid-phase peptide synthesis (SPPS) and incorporated in up to four different GAS M protein epitopes. Subcutaneous immunization of mice in general elicited high-titer systemic IgG antibodies against each of the incorporated epitopes without the need for additional adjuvants. 10-16 Furthermore, the elicited antibodies have been demonstrated to protect mice against intraperitoneal challenge with GAS serotypes for which N-terminal M protein epitopes were included in the vaccine. 13 Despite these promising results, LCP systems synthesized using SPPS are generally not suitable for use in human clinical trials due to difficulties obtaining highly pure LCP systems. Attempts have therefore been made to synthesize analogues of the LCP system using native chemical ligation in order to provide highly pure LCP analogues suitable for use in humans. Previously we have published the synthesis, using native chemical ligation, of a branched peptide containing three GAS M protein derived peptides. 10 Two of these peptides, 88/30 (DNGKAIYERARERALQELGP) and PL1 (EVLTRRQSQD- PKYVTQRIS), are N-terminal peptides from GAS strains common to the Australian aboriginal populations of the Northern Territory and northern Queensland. 19 The other peptide was the previously described J8 peptide. 9 As the vaccine did not feature an inbuilt adjuvant, immunization of B10.BR mice failed to elicit systemic IgG antibodies unless the vaccine was administered with an adjuvant (e.g. complete Freund’s adjuvant (CFA)). Methods for incorporating the LCP lipid core into this vaccine using native chemical ligation were therefore investigated. This * To whom correspondence should be addressed. Phone: +61 (7) 3346 9892. Fax: +61 (7) 3365 1688. E-mail: i.toth@uq.edu.au. ² School of Pharmacy, The University of Queensland. ‡ The Queensland Institute of Medical Research. § SMMS, The University of Queensland. 6364 J. Med. Chem. 2006, 49, 6364-6370 10.1021/jm060475m CCC: $33.50 © 2006 American Chemical Society Published on Web 09/26/2006