Immunological evaluation of lipopeptide group A streptococcus (GAS) vaccine: structure-activity relationship

Abstract

Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.

Figure 1. The structure of lipoamino acid (Laa).

Lipoamino acids are described according to the total number of carbon atoms in the molecule. C12 Laa; n = 9. C14 Laa; n = 11. C16 Laa; n = 13.

Figure 2. Structure of the lipopeptide vaccine candidates grouped according to the orientation of epitopes and Laa in the current study.

* Lipopeptide 1 (C16 Laa), Lipopeptide 2 (C14 Laa), Lipopeptide 3 (C12 Laa).

Figure 3. J14-specific serum IgG titers (log10) at the final bleed (day 60) after primary immunization for each individual mouse.

Mean J14-specific IgG antibody titers are represented as a bar. Statistical analysis was performed using a one-way ANOVA followed by the Tukey post hoc test (ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001).

Figure 4. Isotypes of J14-specific serum IgG titers (log10) present in the final bleed (day 60) after primary immunization.

Bars represent the titer for pooled sera obtained from each group. Error bars represent standard error of mean.

Figure 5. The ability of the lipopeptides to signal through TLR2 was tested by using HEK293 cells stably expressing TLR2 transfected with an NFκB-luciferase reporter gene.

The several fold increase in NFκB levels in treated cells are shown relative to that of the TLR2-expressing cells treated with media, which was set to a value of 1. Bars are means ± SD of indicated number of experiments (ns, p>0.05; *, p<0.05; ** p<0.01; ***, p<0.001).