Immunization with a Tetraepitopic Lipid Core Peptide Vaccine Construct Induces Broadly Protective Immune Responses against Group A Streptococcus

MAJOR ARTICLE Immunization with a Tetraepitopic Lipid Core Peptide Vaccine Construct Induces Broadly Protective Immune Responses against Group A Streptococcus Colleen Olive,1 Mei-Fong Ho,1 Joanne Dyer,1 Douglas Lincoln,1 Nadia Barozzi,2 Istvan Toth,2 and Michael F. Good1 1 Cooperative Research Centre for Vaccine Technology, Queensland Institute of Medical Research, and 2School of Molecular and Microbial Sciences and School of Pharmacy, University of Queensland, Brisbane, Australia Infection with group A streptococcus (GAS) can lead to a plethora of illnesses and diseases, including pharyngitis, pyoderma, invasive diseases, rheumatic fever (RF), and rheumatic heart disease (RHD). It has been estimated that, collectively, GAS infections can cause 1500,000 deaths each year, the majority of which are related to RF and RHD [1]. The burden of GAS diseases is greater in less-developed countries and in indigenous populations of developed countries. Of note, the in- Received 22 November 2005; accepted 20 January 2006; electronically published 10 May 2006. Potential conflicts of interest: none reported. Financial support: National Health and Medical Research Council of Australia; National Heart Foundation of Australia; Prince Charles Hospital Foundation; Australian Government’s Cooperative Research Centres Program. Reprints or correspondence: Dr. Colleen Olive, Cooperative Research Centre for Vaccine Technology, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia (). The Journal of Infectious Diseases 2006; 193:1666–76  2006 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2006/19312-0009$15.00 1666 • JID 2006:193 (15 June) • Olive et al. digenous population of Australia has been reported to have the highest incidence of RF in the world [2]. To prevent GAS infection, research has focused on the development of a vaccine based on the M protein, the antiphagocytic bacterial surface protein that is the primary virulence factor [3]. Previous studies have indicated that protective immunity to GAS can be evoked by opsonic antibodies against serotypic epitopes at the amino-terminal region of the M protein that are type specific [4, 5]. The M protein, however, is highly variable, with 1150 different types having been identified to date. With the aim of developing a vaccine that has broad strain coverage, several strategies have been used to overcome the variability of the M protein, including designing recombinant multivalent vaccines that contain multiple serotypic epitopes [6, 7], vaccines that are based on the conserved carboxy-terminal C-repeat region [8–12, 13–15], and vaccines that incorporate serotypic and conserved-region determinants [16–18]; the last may be more advantageous in providing better protective immunity. Background. The development of a vaccine to prevent infection with group A streptococcus (GAS) is hampered by the widespread diversity of circulating GAS strains and M protein types, and it is widely believed that a multivalent vaccine would provide better protective immunity. Methods. We investigated the efficacy of incorporating 3 M protein serotypic amino-terminal epitopes from GAS isolates that are common in Australian Aboriginal communities and a conformational epitope from the conserved carboxy-terminal C-repeat region into a single synthetic lipid core peptide (LCP) vaccine construct in inducing broadly protective immune responses against GAS after parenteral delivery to mice. Results. Immunization with the tetraepitopic LCP vaccine construct led to high titers of systemic, antigenspecific IgG responses and the induction of broadly protective immune responses, as was demonstrated by the ability of immune serum to opsonize multiple GAS strains. Systemic challenge of mice with a lethal dose of GAS given 60 or 300 days after primary immunization showed that, compared with the control mice, the vaccinated mice were significantly protected against GAS infection, demonstrating that the vaccination stimulated long-lasting protective immunity. Conclusions. These data support the efficacy of the LCP vaccine delivery system in the development of a synthetic, multiepitopic vaccine for the prevention of GAS infection. We have previously investigated the lipid core peptide (LCP) vaccine delivery system [19] for the delivery of a GAS lipopeptide vaccine based on the conserved C-repeat region of the M protein [10]. Although epitopes of M proteins exist within the carboxyterminus—including the C-repeat region [20–22]—that can induce antibodies and T cells that are cross-reactive with human tissues, a conformationally constrained, minimally conserved peptide, J8, that lacks a T cell autoepitope [22] from the M protein has been identified [23, 24]. Our experiments demonstrated that an LCP vaccine formulation [10] containing multiple copies of the J8 epitope elicited opsonic antibodies in mice. Furthermore, parenteral delivery of an LCP vaccine formulation containing J8 and a serotypic epitope evoked complete protection after homologous GAS challenge [17]. The present study investigated the LCP system for the incorporation of 4 different nonhost cross-reactive peptide epitopes of the GAS M protein into a single immunogen. Mice were immunized with a tetraepitopic LCP vaccine construct, and the immunogenicity of the vaccine candidate was evaluated by measuring antigen-specific antibody responses in serum. In addition, we evaluated the induction of serum opsonic anti- bodies, bacterial surface binding, and protection against GAS infection after challenge. MATERIALS AND METHODS Design of a tetraepitopic LCP vaccine construct. We designed a GAS vaccine candidate, LCP-J8-8830-NS1-PL1, containing 3 amino-terminal serotypic epitopes—8830 (DNGKAIYERARERALQELGP), NS1 (RVTTRSQAQDAAGLKEKAD), and PL1 (EVLTRRQSQDPKYVTQRIS)—from GAS isolates that are common in Australian Aboriginal communities in the Northern Territory [16], a region highly endemic for GAS. To generate a vaccine construct with broader strain coverage, we also included a conformational epitope, J8 (QAEDKVKQSREAKKQVEKALKQLEDKVQ), from the conserved carboxy-terminal C-repeat region [23, 24]. One triepitopic LCP construct, LCP-J8-8830NS1, and 2 diepitopic LCP constructs containing either the J8 and 8830 or the NS1 and PL1 GAS peptide epitopes—LCP-J88830 and LCP-NS1-PL1, respectively—were also designed, for use in comparative experiments with the tetraepitopic LCP construct. Vaccine-Mediated Protection against GAS • JID 2006:193 (15 June) • 1667 Figure 1. Chemical structure of the lipid core peptide (LCP) vaccine constructs. The LCP constructs were synthesized to contain three 2-aminododecanoic acid residues, 2 glycine (Gly) spacers, and 2 copies each of 4 (A), 3 (B), or 2 (C) group A streptococcus peptide epitopes. The peptide epitopes are attached to the amino groups of the lysine (Lys) residues of a polylysine core. The tetraepi (...truncated)