key: cord-0296992-g6revx4l
authors: Abouelhadid, Sherif; Atkins, Elizabeth; Kay, Emily; Passmore, Ian; North, Simon J; Lehri, Burhan; Hitchen, Paul; Bakke, Eirik; Rahman, Mohammed; Bosse, Janine; Li, Yanwen; Terra, Vanessa S.; Langford, Paul; Dell, Anne; Wren, Brendan W; Cuccui, Jon
title: Development of a novel glycoengineering platform for the rapid production of conjugate vaccines
date: 2021-11-25
journal: bioRxiv
DOI: 10.1101/2021.11.25.470047
sha: c24f5b5f444b822a8a66772c0325883b989278cf
doc_id: 296992
cord_uid: g6revx4l

Antimicrobial resistance (AMR) is threatening the lives of millions worldwide. Antibiotics which once saved countless lives, are now failing, ushering in vaccines development as a current global imperative. Conjugate vaccines produced either by chemical synthesis or biologically in Escherichia coli cells, have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccines 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 method that overcomes the limitations of the current conjugate vaccine design method(s). We demonstrate at least 2-fold increase in glycoconjugate yield via MAGIC when compared to conventional bioconjugate method(s). Furthermore, the modularity of the MAGIC platform also allowed us to perform glycoengineering in genetically intractable bacterial species other than E. coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against bacteria, especially AMR pathogens, and could be applied for biopreparedness.

The alarming rise in antimicrobial resistance necessitates global efforts to prevent a future health crisis. For more than half a century, antibiotics were considered the first line of defence against bacterial pathogens (1) .

However, the spread of antibiotic resistance amongst pathogenic bacteria entails considerable efforts to look for antibiotic alternatives. Vaccines have been successful in curbing infectious diseases for decades, not only among adults but also among children and the elderly, thus saving millions of lives worldwide (2) . According to the Market Information for Access to Vaccines (MI4A), World Health Organization, the vaccines market is estimated to be worth approximately $33 billion in 2019 (3). Current biotechnological platforms however, might not be able to fulfil the vaccines supply demand.

To satisfy the market's demand for conjugate vaccines, to protect humanity from a foreseeable pandemic, and to be able to tailor novel efficacious vaccines at lower cost, significant biotechnological innovation is needed.

Glycoconjugate vaccines are considered to be one of the safest and most effective tools to combat serious infectious diseases including bacterial meningitis and pneumonia (4) . Conjugation is achieved by linking glycans (carbohydrate moiety), either chemically or enzymatically, to proteins via covalent bonds. This leads to a T-cell dependent immune response, offering excellent protection in people of all ages (5) . Traditionally chemical approaches to produce glycoconjugate vaccine involve the activation of functional groups on the glycan and protein that are linked chemically in a multi-step method that is expensive and laborious, requiring several rounds of purification after each step (6) . Additionally, chemical conjugation methods such as reductive amination can alter the polysaccharide epitope, affecting the immunogenicity of the glycoconjugate against the disease, besides its inherent batch-to-batch variation (7) . Biological conjugation (bioconjugation) offers an excellent alternative to chemical conjugation. It is based on using a bacterial cell, usually E. coli, as a chassis to express a pathway that encodes the desired bacterial polysaccharide, carrier protein, and an oligosaccharytransferase enzyme, OST, that catalyses the conjugation process (6) .

The advent of the bacterial bioconjugation method allowed several protein glycan vaccine combinations to be successfully developed, emphasizing its immense potential to become the preferred method to develop glycoconjugate vaccines in the future (4, (8) (9) (10) (11) (12) . However, several challenges remain. Firstly, the process places significant metabolic stress on the E. coli cell, vaccine micro-factory, due to the expression of orthogonal pathways (13) . This process requires the prior genetic and structural information of the polysaccharide structure of choice. Secondly, the use of three independent replicons has limitations due to incompatibility of plasmid origins of replication and antibiotic selection markers which may lead to the plasmid loss that results in reduction in glycoconjugate yield (8, 10, 14) . Thirdly, reports have demonstrated that the expression of the OST PglB, that catalyses the linking of glycans to carrier proteins, has a detrimental effect on bacterial growth, thus decreasing cellular fitness to produce glycoconjugates (13, 15) . All this together results in a low biomass which often translates to a reduction in the vaccine yield. Consequently, this leads to an increase in the production cost of a glycoconjugate vaccine, making it unaffordable in low-income countries where they are most needed, putting millions of lives at risk as a result of vaccines inequity (6) .

Previous attempts to engineer robust glycoengineering host strains using homologous recombination have had limited success (15) . Although this technology managed to moderately boost glycoprotein production and reduce the dependence on plasmids, it suffers from major drawbacks.

Firstly, the method is slow and requires the successful expression of recombinase systems from plasmids to allow chromosomal integration of glycoengineering components. Secondly, it cannot be applied to other Gram-negative bacteria since prior knowledge of the genome sequence is required to allow for the design of homologous arms for homologous recombination to occur. Thirdly, the scarcity of genetic manipulation tools, which are available for few bacteria, impede the wide use of homologous recombination platforms.

Here, we present a novel platform to overcome some of the limitations of bioconjugation by creating a modular system to rapidly develop conjugate vaccine candidates. This platform could be biologically tailored in a "plugand-play" manner to allow the integration and stable expression of the glycoengineering component(s) not only in E. coli but also in other Gramnegative bacteria. We term this technique, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome (MAGIC). We demonstrate how this platform enables the rapid assembly of stable glycoconjugate combinations. We also report that once a bacterial cell has undergone the MAGIC process, it can be used as a chassis strain to achieve superior glycoconjugate yields and higher glycosylation efficiency when compared to the traditional three plasmid-based bioconjugation methods, and cell free glycosylation method. Furthermore, integration of the OST into host E. coli was shown to alleviate much of the metabolic burden from the bacterium that was translated into increase of biomass and glycoconjugate yield. In addition, we demonstrate the versatility and robustness of MAGIC in not only glycoengineering E. coli strains but also in other genetically intractable host bacteria such as Citrobacter species. As an exemplar, we demonstrate how MAGIC could provide a first line of defence in an E. coli O157 outbreak scenario by developing a candidate glycoconjugate vaccine in less than a week. The modular nature of MAGIC highlights its applicability as a tool for biopreparedness especially against emerging multi-drug resistant bacteria.

To demonstrate the proof-of-principle of MAGIC application in glycoconjugate production, we first constructed E. coli MAGIC v.1, based on inducible pglB under a P tac promoter system (16) Fig 1, (21) . Previously, we demonstrated that a biologically conjugated vaccine against S. pneumoniae confers protection and increased survival rate in laboratory animals (14) . We sought to apply MAGIC v.2 in enhancing the production of a S. pneumoniae vaccine. As a control to this experiment, E. coli W3110 CmeA-Sp4 was assembled. This strain expresses the orthogonal pathway of S. pneumoniae serotype 4 (Sp4), 

One of the most challenging steps in bioconjugation and cell free glycosylation methods is the successful expression of the glycan orthogonal pathway in E. coli. This problem is further complicated when ORFs of a certain glycan are scattered on the genome and/or when a certain degree of acetylation is necessary for a carbohydrate to be immunogenic (24) coli strain. Additionally, we demonstrate that the biotechnology is compatible with health and safety procedures that minimize any biohazard risk.

In this work we have established MAGIC, as a versatile and robust, "plug- provided superior protection than low molecular size glycoconjugate against intranasal F. tularensis challenge in a mouse model of tularemia (28) . Previous attempts to produce highly polymerized glycoconjugates using bioconjugation methods were hampered by expressing several glycoengineering components in the cell from plasmids, which consequently, led to lower cellular biomass and glycoconjugate yield. By alleviating the metabolic stress in the cell when applying MAGIC to one or more glycoengineering components, cellular biomass could increase leading to a higher glycoconjugate yield. Indeed, cellular biomass increased by 41% when MAGIC was applied, compared to traditional bioconjugation three plasmids method. This increase in biomass was accompanied by improved glycosylation efficiency, seen as an increase in the polymer length in the glycoconjugate, and a significant increase in the glycoconjugate yield.

In-depth analysis of the vaccine market shows that pneumococcal conjugate vaccine (PCV) is the most likely to see a high value growth by 2030, requiring billions of doses to be manufactured (2) . The instrumental role played by MAGIC in developing another efficacious and inexpensive vaccine is exemplified by the S. pneumoniae glycoconjugate generated.

When compared to the three plasmid bioconjugation method, the MAGIC assembled strain showed a significant 3-fold increase in glycoconjugate yield with glycosylation efficiency reaching 90.4 % ± 2.9. Additionally, glycoconjugates produced using the MAGIC platform showed a higher degree of polymerization of SP4 glycan when compared to traditional bioconjugation method. When tested in outbred mice these glycoconjugates had significant immunogenicity and protection resulting in higher survival rate than animals immunized with PCV13 (low dose) following challenge with S. pneumoniae (14) . Taken together, we set out a novel benchmark in biological conjugation that allows for enhancement of glycoconjugates production with key advantages over the current biological conjugation technologies.

In contrast to the recently published cell free glycosylation method, MAGIC can provide an inexhaustible and renewable source of glycoconjugates (29) .

This main difference stems from the fact that MAGIC is based on converting the bacterial cell, either E. coli or any other Gram-negative bacterium, into a factory for glycoconjugate vaccine production, contrary to the limited reaction volumes in cell free glycosylation (30) . One appealing feature of bioconjugation is the reduction of the batch-to-batch variation bottleneck, since no component mixing is required with specific quantities that increase the probability of a human error (29) . Key steps are also eliminated when compared to cell free methods such as, ultracentrifugation, protein and LLO quantification (30) . In summary, we present a novel glycoengineering platform that will 

The gene coding for C. jejuni NCTC11168 PglB was loaded into a Mini-Tn5Km2 transposon within a pUT backbone targeting the NotI site.

However, in order to assemble a construct that could be induced, we first cloned C. jejuni pglB into pEXT20. This is a vector that enables IPTG 

In gel reduction, alkylation, and digestion with trypsin or chymotrypsin was performed on the gel sample prior to subsequent analysis by mass spectrometry. Cysteine residues were reduced with dithiothreitol and derivatized by treatment with iodoacetamide to form stable carbamidomethyl derivatives. Trypsin digestion was carried out overnight at room temperature after initial incubation at 37°C for 2 h. Sample digests, 

Cell free glycosylation was conducted in S30 buffer with 0.1% n-dodecylβ-d-maltopyranoside (DDM; Thermo Scientific) and 10 mM MnCl 2 (Across Organics The plasmid was then digested with SfiI and ligated to pUTminiTn5 to create the plasmid pELLA1 (pglBx4, pELLA2 (pglBx10), pELLA3

(pglBx15). Conjugation to the E. coli strain CROW was carried out to deliver the constitutive pglB copy. Table 1 Strains and plasmids used in this study

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We would like to thank Steven Lynham, mass spectrometry centre of excellence, King's college London, UK for his help with mass spectrometry. We thank the Biotechnology and Biological Sciences Research Council, including grants BB/M01925X/1, BBSRC BB/H017437/1 for helping to fund this research.

Description Reference or source E. coli DH5α F-φ80lacZΔM15 Δ(lacZYA-argF) U169 deoRrecA1 endA1 hsdR17 (rk-, mk+) gal-phoAsupE44 λ-thi-1 gyrA96 relA1 This study