Development of a novel glycoengineering platform for the rapid production of conjugate vaccines

Abstract

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Keywords: Bacterial vaccines; Bioconjugation; Conjugate vaccines; Glycoengineering.

Fig. 1

Glycoconjugate production in E. coli MAGIC strains compared to conventional bioconjugation method. A Schematic diagram of developing of constructing E. coli MAGIC; B Western blot of 5 µg His-tagged CmeA protein purified by nickel affinity chromatography. Biological samples were separated on a Bolt 4–12% bis–tris gel (Invitrogen) with MOPS buffer and transferred to nitrocellulose membrane with an iBlot 2 dry blotting system. The membrane was probed with anti-His (Invitrogen) and anti- Ft-O antigen monoclonal antibody (Abcam) and detected with fluorescently labelled secondary antisera (green-His, red-Ft-O-antigen) on a LiCor Odyssey scanner.; C densitometry analysis of glycoconjugate production in E. coli MAGIC v.1 Ft-O compared to E. coli bioconjugation Ft-O. Densitometry analysis of glycoconjugate was done from three biological replicates. Statistical analysis is from three biological replicates using Student’s t-test ns, p > 0.05; *,p < 0.05, **,p < 0.01, ***, p < 0.001

Fig. 2

Cell free glycosylation (CFG). A schematic diagram of cell free glycosylation analysis; B western blot analysis of cell free glycosylation upon gradual increase of cell debris containing glycan donor denoted by the grey triangle. Western blot of 5 µg His-tagged CmeA protein purified by nickel affinity chromatography Protein samples were separated by SDS-PAGE 4–12% bis–tris gel (Invitrogen) with MOPS buffer and transferred to nitrocellulose membrane with an iBlot 2 dry blotting system. The membrane was probed with anti-His (Invitrogen) and anti- Ft-O antigen monoclonal antibody (Abcam) and detected with fluorescently labelled secondary antisera (red-His, green-Ft-O-antigen) on a LI-COR Odyssey scanner

Fig. 3

Designing and testing of MAGIC v.2; A schematic diagram of I and O end of MAGIC v.2; B Western blot of 5 µg His-tagged CmeA protein purified by nickel affinity chromatography. Biological samples were separated on a Bolt 4–12% bis–tris gel (Invitrogen) with MOPS buffer and transferred to nitrocellulose membrane with an iBlot 2 dry blotting system. The membrane was probed with anti-His (Invitrogen) and anti-SP4 (Statens, Serum Institute) and detected with fluorescently labelled secondary antisera (red-His, green- anti-SP4) on a LI-COR Odyssey scanner.; C densitometry analysis of glycoconjugate production in E. coli MAGIC v.2 SP4 compared to E. coli bioconjugation SP4.; Densitometry analysis of glycoconjugate was done from three biological replicates. Statistical analysis is from three biological replicates using Student’s t-test ns, p > 0.05; *,p < 0.05, **,p < 0.01, ***, p < 0.001

Fig. 4

A, Designing and testing MAGIC v.3. A nucleotide sequence of Biobricks promoters used in constructing MAGIC v.3 and their corresponding promoter strength [19]; B Western blot of 5 µg His-tagged CmeA protein purified by nickel affinity chromatography. Biological samples were separated on a Bolt 4–12% bis–tris gel (Invitrogen) with MOPS buffer and transferred to nitrocellulose membrane with an iBlot 2 dry blotting system. The membrane was probed with anti-His (Invitrogen) and anti- Ft-O antigen monoclonal antibody (Abcam) and detected with fluorescently labelled secondary antisera (green-His, red-Ft-O-antigen) on a LI-COR Odyssey scanner; H, densitometry analysis of glycoconjugate production in E. coli MAGIC v.3 Ft-O compared to E. coli bioconjugation Ft-O. Densitometry analysis of glycoconjugate was done from three biological replicates. Statistical analysis is from three biological replicates using Student’s t-test ns, p > 0.05; *,p < 0.05, **,p < 0.01, ***, p < 0.001

Fig. 5

Developing of E. coli O157 candidate conjugate in C. sedlakii MAGIC v.1. A schematic diagram of construction of C. sedlakii MAGIC v.1; B Coomassie stain of His-tagged CmeA protein purified from C.sedlakii and C. sedlakii MAGIC v.1 by nickel affinity chromatography. Biological samples were separated on a Bolt 4–12% bis–tris gel (Invitrogen) with MOPS buffer; C western blot analysis of CmeA purified from C. sedlakii and C. sedlakii MAGIC v.1, Biological samples were separated on a Bolt 4–12% bis–tris gel (Invitrogen) with MOPS buffer transferred to nitrocellulose membrane with an iBlot 2 dry blotting system. The membrane was probed with anti-His (Invitrogen) and anti-O157 (Abcam) antibody and detected with fluorescently labelled secondary antisera (green-His, red-O157) on a LI-COR Odyssey scanner. D glycosylation of CmeA in C. sedlakii MAGIC v.1 in broth and plate of His-tagged CmeA

Fig. 6

Mass spectrometry analysis of CmeA glycopeptides A ²⁶⁸AVFDNNNSTLLPGAFATITSEGFIQK²⁹³; and B E¹²¹DFNR¹²⁴; purified CmeA was reduced, alkenylated, and treated with sequencing grade trypsin overnight, peptides were then run on LC–MS/MS (Waters). Precursor ion fragmentation shows the loss of HexNAc, Hex, deoxyHex, and deoxyHexNAc which corresponds to one repeating unit of O157. Glycan fragmentation is shown in blue lines and peptide fragmentation is shown in red