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

Bioconjugate Chemistry, vol. 37, pp. 1–10

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.

Authors 8

1. Giulia Castello Aachen Department of Immunology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Immunology, Leiden University Medical Center, Albinusdreef 2, Leiden 2333 ZA, The Netherlands

2. Marjolein M. E. Isendoorn Aachen

   Leiden University

   Affiliation as printed

   Leiden University, Einsteinweg 55, Leiden 2333 CC, The Netherlands

3. Nico J. Meeuwenoord Aachen

   Leiden University

   Affiliation as printed

   Leiden University, Einsteinweg 55, Leiden 2333 CC, The Netherlands

4. Vincent V. F. Verhoeks Aachen

   Leiden University

   Affiliation as printed

   Leiden University, Einsteinweg 55, Leiden 2333 CC, The Netherlands

5. Thomas C. van den Ende Aachen

   Leiden University

   Affiliation as printed

   Leiden University, Einsteinweg 55, Leiden 2333 CC, The Netherlands

6. Jeroen D. C. Codée Aachen

   Leiden University

   Affiliation as printed

   Leiden University, Einsteinweg 55, Leiden 2333 CC, The Netherlands

7. FERRY A. OSSENDORP corresponding Aachen Department of Immunology, Leiden University Medical Center

   Leiden University Medical Center

   Affiliation as printed

   Department of Immunology, Leiden University Medical Center, Albinusdreef 2, Leiden 2333 ZA, The Netherlands

8. Dmitri V. Filippov corresponding Aachen

   Leiden University

   Affiliation as printed

   Leiden University, Einsteinweg 55, Leiden 2333 CC, The Netherlands