Solid-Phase Synthesis and Immunological Evaluation of Peptide Conjugates Containing Stereochemically Defined UPam-Based TLR2 Ligands and Epitopes from SARS-CoV-2 Virus

Abstract

The design of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on eliciting robust cellular and humoral immune responses. In this study, we report the synthesis and immunological evaluation of synthetic long peptide (SLP) vaccines incorporating immunodominant CD4⁺ and CD8⁺ T cell and B cell epitopes from the SARS-CoV-2 proteome, covalently conjugated to stereochemically defined UPam ligands, which serve as synthetic Toll-like receptor 2 (TLR2) agonists. Using solid-phase peptide synthesis, we generated a panel of R- and S-glycerol-based UPam conjugates with varied amino acids adjacent to the palmitoylated cysteine. These conjugates were evaluated for their ability to induce dendritic cell maturation, TLR2 activation, and antigen-specific immune responses. In vitro assays demonstrated that R-UPam ligands, particularly those containing Dab and Abu residues, were superior in stimulating IL-12p40 production and TLR2 activation compared with their S-epimer counterparts. In vivo immunization studies in mice revealed that covalent conjugation of UPam to CD8⁺ T cell epitopes (VNF and REL) significantly enhanced antigen-specific T cell responses compared to unconjugated mixtures. Furthermore, a UPam-conjugated B cell epitope fused to a universal helper T cell peptide (PADRE) induced strong dendritic cell activation and enhanced PADRE-specific CD4⁺ T and B cell responses, as evidenced by elevated IgG production. These findings underscore the potential of chirally pure UPam-conjugated peptide vaccines to induce balanced, durable immunity against SARS-CoV-2 by combining TLR2-mediated adjuvanticity with precise epitope targeting.