Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM 197 as
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ORIGINAL ARTICLE
Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM197 as carrier protein G. Stefanetti 1
&
M. Allan 2 & A. Usera 2 & F. Micoli 3
Received: 13 February 2020 / Revised: 15 April 2020 / Accepted: 30 May 2020 # The Author(s) 2020
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 wellcharacterized 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. Keywords Glycoconjugate vaccine . Click chemistry . Thiol chemistry . Conjugation chemistry . Salmonella Typhimurium . O-antigen . CRM197
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]. Traditional glycoconjugate approaches involve random linkage of the sugar
* G. Stefanetti [email protected] 1
Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
2
Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA
3
GSK Vaccines Institute For Global Health (GVGH) S.r.l, Via Fiorentina 1, Siena 53100, Italy
hapten to a carrier protein, or end-group modification of the saccharide chain to achieve better control and characterization of the resulting vaccine. More defined constructs where the covalent linking between sugar and the protein is limited to wellestablished attachment sites are highly desirable. They allow a better characterization of the product by physicochemical techniques facilitating the control of manufacturing consistency. Furthermore, the role of glycosylation site on the protein still has to be fully understood
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