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In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine

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 recap...

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Autores principales: Aubry, Annie, Zou, Wei, Vinogradov, Evguenii, Williams, Dean, Chen, Wangxue, Harris, Greg, Zhou, Hongyan, Schur, Melissa J., Gilbert, Michel, Douce, Gillian R., Logan, Susan M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157674/
https://www.ncbi.nlm.nih.gov/pubmed/32046000
http://dx.doi.org/10.3390/vaccines8010073
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author Aubry, Annie
Zou, Wei
Vinogradov, Evguenii
Williams, Dean
Chen, Wangxue
Harris, Greg
Zhou, Hongyan
Schur, Melissa J.
Gilbert, Michel
Douce, Gillian R.
Logan, Susan M.
author_facet Aubry, Annie
Zou, Wei
Vinogradov, Evguenii
Williams, Dean
Chen, Wangxue
Harris, Greg
Zhou, Hongyan
Schur, Melissa J.
Gilbert, Michel
Douce, Gillian R.
Logan, Susan M.
author_sort Aubry, Annie
collection PubMed
description 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.
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spelling pubmed-71576742020-05-01 In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine Aubry, Annie Zou, Wei Vinogradov, Evguenii Williams, Dean Chen, Wangxue Harris, Greg Zhou, Hongyan Schur, Melissa J. Gilbert, Michel Douce, Gillian R. Logan, Susan M. Vaccines (Basel) Article 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. MDPI 2020-02-07 /pmc/articles/PMC7157674/ /pubmed/32046000 http://dx.doi.org/10.3390/vaccines8010073 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Aubry, Annie
Zou, Wei
Vinogradov, Evguenii
Williams, Dean
Chen, Wangxue
Harris, Greg
Zhou, Hongyan
Schur, Melissa J.
Gilbert, Michel
Douce, Gillian R.
Logan, Susan M.
In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine
title In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine
title_full In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine
title_fullStr In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine
title_full_unstemmed In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine
title_short In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine
title_sort in vitro production and immunogenicity of a clostridium difficile spore-specific bcla3 glycopeptide conjugate vaccine
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157674/
https://www.ncbi.nlm.nih.gov/pubmed/32046000
http://dx.doi.org/10.3390/vaccines8010073
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