Structure–activity relationship of lipopeptide Group A streptococcus (GAS) vaccine candidates on toll-like receptor 2

Incorporation of lipoamino acids (LAAs) into peptide structures effectively imparts self-adjuvanting activity onto otherwise ineffective immunogens. Our fully synthetic lipopeptide vaccine candidates against group A streptococcus (GAS) were composed of J14 as a target GAS B-cell epitope alongside a universal helper T-cell epitope (P25) and a LAA-based lipid moiety. In the current study, we investigated the ability of our lipopeptides to activate nuclear factor-κB (NF-κB) in a toll-like receptor-2 (TLR2)-dependent manner as the possible mode of action and reported the structure–function requirements for novel TLR2 targeting lipopeptides based on LAAs. The NF-κB activation was dependent on the dose and the length of the alkyl chains of the incorporated lipid moieties with the hierarchy LAA 3 (16 carbons) > LAA 2 (14 carbons) > LAA 1 (12 carbons). The position of the lipid moiety (C-terminus vs. Nɛ-terminus of the central lysine residue) does not significantly affect NF-κB activation. Lipopeptides containing different copies of LAA 3 were synthesized and the di-lipidated analogue was the most effective in NFκB activation.