Investigation of physical mixtures versus disulfide crosslinking self-adjuvanting peptide-based subunit vaccines...

Group A Streptococcus (GAS) is a Gram-positive pathogen responsible for a broad spectrum of diseases and significant global morbidity and mortality, yet no commercial vaccine currently exists. Peptide-based subunit vaccines offer a safer alternative to protein- and whole-pathogen-based approaches, minimising the risk of allergic and autoimmune responses while inducing adaptive immunity. In this study, we designed and evaluated a series of self-adjuvanting peptide-based subunit vaccine candidates incorporating the GAS B-cell epitope J8 and the universal helper T-cell epitope PADRE (P-J8). These were delivered using either a lipopeptide (C16) or polyleucine (L₁₅K₆) as self-adjuvanting domains. Constructs were developed by direct conjugation or simple physical mixing, and compared to disulfide-crosslinked formulations containing one, two, or three cysteine residues. Immunisation studies in mice revealed that single-disulfide crosslinked constructs elicited stronger humoral responses than those with multiple crosslinks. However, physical mixtures of P-J8 with either lipopeptide or polyleucine induced significantly higher IgG titres, outperforming all crosslinked counterparts. These formulations also promoted enhanced dendritic cell maturation, with increased expression of MHC II and CD40, suggesting superior antigen presentation and T-cell activation. Furthermore, sera from immunised mice demonstrated potent opsonisation and bactericidal activity against clinical GAS isolates. Our findings highlight the promise of physical mixture formulations as simple, effective, and self-adjuvanting peptide vaccine platforms for GAS prevention.