In vitro production and immunogenicity of a Clostridium difficile spore-specific BclA3 glycopeptide conjugate vaccine

Aubry, A. et al. (2020) In vitro production and immunogenicity of a Clostridium difficile spore-specific BclA3 glycopeptide conjugate vaccine. Vaccines, 8(1), 73. (doi: 10.3390/vaccines8010073) (PMID:32046000) (PMCID:PMC7157674)

[img]
Preview
Text
207650.pdf - Published Version
Available under License Creative Commons Attribution.

1MB

Abstract

The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of β-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Douce, Dr Gillian
Creator Roles:
Douce, G. R.Methodology, Formal analysis, Data curation, Writing – review and editing, Visualization
Authors: Aubry, A., Zou, W., Vinogradov, E., Williams, D., Chen, W., Harris, G., Zhou, H., Schur, M. J., Gilbert, M., Douce, G. R., and Logan, S. M.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Journal Name:Vaccines
Publisher:MDPI
ISSN:2076-393X
ISSN (Online):2076-393X
Published Online:07 February 2020
Copyright Holders:Copyright © 2020 The Authors
First Published:First published in Vaccines 8(1): 73
Publisher Policy:Reproduced under a Creative Commons License

University Staff: Request a correction | Enlighten Editors: Update this record

Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
174080Unravelling the link between S-layer morphogenesis and sprulation in Clostridium difficileGillian DouceWellcome Trust (WELLCOTR)204877/Z/16/ZIII - Bacteriology