Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM197 as carrier protein

G Stefanetti,A Usera,F Micoli,M Allan

doi:10.1007/s10719-020-09930-2

Abstract

Conjugation chemistry is one of the main parameters affecting immunogenicity of glycoconjugate vaccines and a rational approach toward a deeper understanding of their mechanism of action will greatly benefit from highly-defined and well-characterized structures. Herein, different conjugation methods were investigated with the aim of controlling glycosylation site and glycosylation density on the carrier protein. S. Typhimurium lipopolysaccharide O-Antigen and CRM197 carrier protein were used as models. In particular, thiol and click chemistry were examined, both involving the linkage of the terminal reducing sugar unit of the O-Antigen chain to different amino acids on the carrier protein. Thiol chemistry allowed O-Antigen conjugation only when the carrier protein was activated on the lysines and with a relative high number of linkers, while click chemistry allowed conjugate generation even when just one position on the protein was activated and to both lysine and tyrosine sites. The study highlights click chemistry as a leading approach for the synthesis of well-defined glycoconjugates, useful to investigate the relationship between conjugate design and immune response.

Highlights

Glycoconjugate vaccines are important therapeutics for the prevention of infectious disease from severe pathogens like Neisseria meningitidis, Haemophilus influenza and Streptococcus pneumoniae [1, 2]
We have recently synthesized well-defined O-antigen (OAg)-based glycoconjugate vaccines to protect against nontyphoidal Salmonella (NTS) serovar Typhimurium, with CRM197 as carrier protein [6]
The following chemicals were used in this study: cystamine dihydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), (+)-sodium L-ascorbate, 2-(Nmorpholino)ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP), propargylamine, copper(II) sulfate pentahydrate (CuSO4·5H2O), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), phosphate buffer solution (PBS), adipic acid dihydrazide (ADH), sodium cyanoborohydride (NaBH3CN), dimethyl sulfoxide (DMSO), sodium phosphate monobasic (NaH2PO4) [Sigma]; triplex III (EDTA) [Merck]; N-[βmaleimidopropionic acid] hydrazide trifluoroacetic acid salt (BMPH) [Thermo]; N-[ε-maleimidocaproyloxy]succinimide ester (EMCS), succinimidyl 3-(bromoacetamido)propionate (SBAP), CRM197-BMPH via EDAC/NHS chemistry

Summary

Introduction

Glycoconjugate vaccines are important therapeutics for the prevention of infectious disease from severe pathogens like Neisseria meningitidis, Haemophilus influenza and Streptococcus pneumoniae [1, 2]. They require the covalent linking of a sugar antigen to a carrier protein, which can be obtained by different strategies influencing both the efficiency of conjugation and the structure of the glycoconjugate, with an important impact on immunogenicity [3]. In this study we compared four different orthogonal conjugation chemistries for the generation of selective OAg-based glycoconjugate vaccines against NTS, with the aim to identify efficient strategies to link a different number of sugar chains to defined amino acid sites on the protein. CRM197 is a 58 kDa nontoxic mutant of diphtheria toxin and was selected as carrier protein for this investigation because of its defined structure and extensive use for licensed glycoconjugate vaccines and other vaccines in development [16, 17]

Methods

Results

Conclusion