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

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.

Sweetening the Deal: Glycosylation and its Clinical Applications

“…Some success was seen in vitro with the Anti-spore serum recognizing the spore-specific glycan moiety. However, animal testing showed that the vaccine did not provide protection against the infection [18]. Despite the results, this study demonstrates the usefulness of glycosylation patterns in vaccine development.…”

Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores

“…For epidemic strain R20291, our results indicate that a deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore (Castro-Córdova et al, 2020). The gene bclA3 is the second element in a bicistronic operon, where the first cistron, sgtA, has been characterized as a glycosyltransferase enzyme that is required for the glycosylation of collagen-like fragments of BclA3 (Aubry et al, 2020, Strong et al, 2014. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer.…”

Non-Toxin-Based Clostridioides difficile Vaccination Approaches

“…Another group used a recombined BclA3 peptide and the corresponding glycopeptide conjugated to the KLH (keyhole limpet hemocyanin) carrier protein and administered it intranasally to mice. Again, although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease [ 15 ]. One of the reasons might be that spore coat proteins cover the exosporium proteins, therefore preventing antibody binding [ 16 ].…”