Site-specific antigen-adjuvant conjugation using cell-free protein synthesis enhances antigen presentation and CD8+ T-cell response

Sci Rep. 2021 Mar 18;11(1):6267. doi: 10.1038/s41598-021-85709-1.

Abstract

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-L-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adjuvants, Immunologic / metabolism*
  • Animals
  • Antigen Presentation*
  • Antigen-Presenting Cells / immunology
  • Antigens / genetics
  • Antigens / immunology*
  • CD8-Positive T-Lymphocytes / immunology*
  • Cell-Free System
  • Click Chemistry / methods
  • HEK293 Cells
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Models, Animal
  • Mutant Proteins / immunology*
  • Oligodeoxyribonucleotides / immunology*
  • Oligodeoxyribonucleotides / metabolism
  • Oligodeoxyribonucleotides / pharmacology
  • Ovalbumin / genetics
  • Ovalbumin / immunology*
  • Toll-Like Receptor 9 / agonists
  • Toll-Like Receptor 9 / genetics
  • Toll-Like Receptor 9 / metabolism
  • Transfection
  • Vaccination / methods
  • Vaccines, Conjugate / administration & dosage
  • Vaccines, Conjugate / immunology
  • Vaccines, Subunit / administration & dosage
  • Vaccines, Subunit / immunology

Substances

  • Adjuvants, Immunologic
  • Antigens
  • CPG-oligonucleotide
  • Mutant Proteins
  • Oligodeoxyribonucleotides
  • Tlr9 protein, mouse
  • Toll-Like Receptor 9
  • Vaccines, Conjugate
  • Vaccines, Subunit
  • 1018 oligonucleotide
  • Ovalbumin