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

Adam M Weiss,Aaron P Esser-Kahn,Aym Berges,Jainu Ajit,Tyler J Albin,Neeraj Kapoor,Lucy Pill,Jeff Fairman,Shilpa Maroju

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.

Highlights

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
Noting that a pioneering study of non-specific conjugation of CpG to OVA at a ≥ 2:1 ratio induced aggregation and reduced antigen p resentation[11], we introduced two site-specific conjugatable handles on one OVA protein to overcome this limitation of non-specific conjugation
We demonstrate that cell-free protein synthesis (CFPS) is a facile technique to synthesize large quantities of protein containing sitespecific non-native amino acids (nnAA) for antigen-adjuvant conjugation without disrupting protein folding or epitope recognition

Summary

Introduction

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. 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. Traditional attenuated vaccines are highly potent, and their administration has led to reductions in morbidity from many d iseases[1] Despite their potency, attenuated vaccines pose a safety risk resulting from the presence of live pathogens, limiting use in elderly or immunocompromised individuals. Subunit vaccines comprising a protein antigen do not contain live pathogenic material and serve as desirable alternatives to attenuated vaccines. Precise control over the amount of adjuvant administered with the antigen can prevent adverse responses and improve efficiency of subunit vaccines

Objectives

Methods

Results

Conclusion