Intranasal vaccination with a lipopeptide containing a minimal, conserved M-protein derived peptide and a self-adjuvanting lipid induces both systemic and mucosal immune responses in mice
Introduction
A primary route of GAS infection in humans is via colonization of the mucosal epithelium of the pharynx. Colonization followed by tissue invasion can lead to local suppurative complications or systematic infections [1]. On mucosal surfaces such as the mucosal epithelium production of immunoglobulin A (IgA) is one of the primary defense mechanisms in the host to prevent bacterial attachment to these cells and subsequent colonization [2].
We have previously defined a conformationally constrained minimal conserved peptide (J14) not containing a potentially deleterious T-cell epitope from the M-protein of GAS [3]. It has previously been demonstrated that passively acquired IgA directed to the M-protein is able to significantly inhibit streptococcal infection in mice when administered mucosally [4]. Furthermore, mice immunised intranasally with synthetic peptides from the conserved region of the M-protein in the presence of the mucosal adjuvant CTB showed a significant decrease in the incidence of pharyngeal GAS colonization [4]. Taken together, these data suggest that intranasal immunisation is the preferred route of administration for a GAS vaccine.
Currently, there is no mucosal adjuvant available that is suitable for human use for the administration of peptides. However, a variety of lipid moieties have been studied for the purpose of vaccine design. Pam2Cys, a synthetic version of the lipid moiety from the 2-kDa macrophage-activating lipopeptide 2 derived from Mycoplasma fermentans, has been found to have a very potent adjuvanting activity [5] and target the Toll-like receptors on the dendritic cells [6]. We made a lipopeptide in which the conserved peptide (J14), a universal T cell epitope and the lipid moiety Pam2Cys were assembled together in a branched geometry (Fig. 1) as described previously [6] and assessed its ability to induce systemic and mucosal immune responses in mice.
Section snippets
Peptides synthesis and intranasal immunisation of mice
All peptide constructs were synthesized using standard F-moc chemistry. Lipidation of the peptides was carried out as previously described [6]. Purification and characterization of the peptide and lipopeptide products was carried out using the methods as described in detail elsewhere [6]. Outbred Quackenbush mice (Animal Resources Centre, Western Australia) were administered 60 μg of the P25-P2C-J14 formulation or its derivates in 30 μl of PBS (15 μl/nare). Mice received two boosts 21 days
Results
We observed that intranasal immunisation of outbred Quackenbush mice with the lipopeptide construct (P25-P2C-J14) induced antibodies in all outbred mice tested (Fig. 2A). Mice immunised with the P25-P2C-J14 had significantly higher (p < 0.05) J14-specific serum IgG titres than in the control groups (at day 60 post-primary immunisation) including P25-P2C-LHRH.
One of the main reasons for the use of an intranasal vaccine is the ability to induce a local immune response at the primary point of GAS
Discussion
A lipopeptide-based vaccine that combines the advantages of synthetic peptide-vaccines (consistency and purity) with the self-adjuvanting properties of lipids such as Pam2Cys and Pam3Cys have enormous potential as mucosal and systemic vaccines [8]. We have expanded on these findings by assembling a lipopeptide construct in a branched geometry [6] that incorporates a minimal B-cell epitope, namely, the peptide (J14), a universal T-cell epitope peptide (P25) and the Toll-like receptor-targeting
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