multivalent poultry vaccine development using protein glycan coupling technology CORD-Papers-2022-06-02 (Version 1)

Title: Multivalent poultry vaccine development using Protein Glycan Coupling Technology
Abstract: BACKGROUND: Poultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen have a proven track record in human vaccinology but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this we use Protein Glycan Coupling Technology (PGCT) which enables the production of glycoconjugates in bacterial cells at considerably reduced costs to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry. RESULTS: We demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC 7122 serotype O78:H9. The integration caused mild attenuation of the 7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the 7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered 7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain. CONCLUSIONS: We generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni APEC C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless we present a strategy towards the production of low-cost live-attenuated multivalent vaccine factories with the ability to express glycoconjugates in poultry. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01682-4.
Published: 2021-10-02
Journal: Microb Cell Fact
DOI: 10.1186/s12934-021-01682-4
DOI_URL: http://doi.org/10.1186/s12934-021-01682-4
Author Name: Mauri Marta
Author link: https://covid19-data.nist.gov/pid/rest/local/author/mauri_marta
Author Name: Sannasiddappa Thippeswamy H
Author link: https://covid19-data.nist.gov/pid/rest/local/author/sannasiddappa_thippeswamy_h
Author Name: Vohra Prerna
Author link: https://covid19-data.nist.gov/pid/rest/local/author/vohra_prerna
Author Name: Corona Torres Ricardo
Author link: https://covid19-data.nist.gov/pid/rest/local/author/corona_torres_ricardo
Author Name: Smith Alexander A
Author link: https://covid19-data.nist.gov/pid/rest/local/author/smith_alexander_a
Author Name: Chintoan Uta Cosmin
Author link: https://covid19-data.nist.gov/pid/rest/local/author/chintoan_uta_cosmin
Author Name: Bremner Abi
Author link: https://covid19-data.nist.gov/pid/rest/local/author/bremner_abi
Author Name: Terra Vanessa S
Author link: https://covid19-data.nist.gov/pid/rest/local/author/terra_vanessa_s
Author Name: Abouelhadid Sherif
Author link: https://covid19-data.nist.gov/pid/rest/local/author/abouelhadid_sherif
Author Name: Stevens Mark P
Author link: https://covid19-data.nist.gov/pid/rest/local/author/stevens_mark_p
Author Name: Grant Andrew J
Author link: https://covid19-data.nist.gov/pid/rest/local/author/grant_andrew_j
Author Name: Cuccui Jon
Author link: https://covid19-data.nist.gov/pid/rest/local/author/cuccui_jon
Author Name: Wren Brendan W
Author link: https://covid19-data.nist.gov/pid/rest/local/author/wren_brendan_w
sha: 883da04890347324d10b6db26c09fa46238fd3b2
license: cc-by
license_url: https://creativecommons.org/licenses/by/4.0/
source_x: PMC
source_x_url: https://www.ncbi.nlm.nih.gov/pubmed/
pubmed_id: 34600535
pubmed_id_url: https://www.ncbi.nlm.nih.gov/pubmed/34600535
pmcid: PMC8487346
pmcid_url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487346
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487346/
has_full_text: TRUE
Keywords Extracted from Text Content: oral colibacillosis bacterial cells poultry Escherichia coli PGCT NetB toxoid C. perfringens glycoconjugates Poultry C. jejuni C. jejuni heptasaccharide glycan antigen human caecal chickens livestock Clostridium perfringens sac PBS-washed O-antigen intestinal Fig. 5b E. coli K-12 Salmonella enterica species His-purifications plasmid pSECpgl c cultures HRP G-/unG-NetB Clostridium perfringens NetB toxoid EcoRI-PstI flgG × 10 6 B-and T-cell PGCT NZ_HE962388_Pgl G-ExoA avian gut Uniprot ID χ7122 cells caeca 1 C. jejuni strain M1 O2 periplasmic side C. jejuni M1 O-antigen ligase WaaL Poultry meat NZ_HE962388 chicken flock N-acetylgalactosamine gastrointestinal inner membrane [53] Kan R Poultry Chickens Transformants BioLegend His-purified Campylobacter glycoconjugates goat anti-mouse IgG IRDye 680 (LI-COR Biosciences leaves Fig. 8d AhdI-SacII meat Salmonella NZ_HE962388 [1 1:50 serum C. coli poultry livers E. coli S17-1 λpir cells lipidA-linked heptasaccharide glycoprotein C.jejuni glycan C. jejuni pgl lectin Kanamycin Salmonella enterica serovar Pullorum flocks cervical IPTG-inducible pEXT20 caecum flock PglB OST E7645 IPTG-inducible PglB TMB Streptavidin-HRP pACYC pglΔpglB C. jejuni glycoproteins chickens cardiac puncture Biomasses surface inner membrane chicken gut [27] lanes 3 His-pulldown fastQ cytoplasmic DQNAT G-NetB(2 Kan [46] co-integrants Serum IgY SAMtools mouse b diphtheria toxin PglB lungs unG-NetB pEXT20-G-NetB periplasmic fractions carbonatebicarbonate buffer G-NetB/ unG-NetB kan R -marked pgl undecaprenyl-pyrophosphate oral G-ExoA(2 cells lactose Fig. 6c samples ampicillin A8ULG6 snpEff D/EXNXS/T lipid Post-mortem μl/well H 2 SO 4 capsular polysaccharides algorithm-mem CO2 lanes 3-4 heptasaccharide antigen pEXT20-G-NetB(10 chicken body μg/lane pFPV25.1-G-NetB(10 liquid cultures G-NetB(10) antigens C. jejuni heptasaccharide antigen sacB Fig. 5a LDS sample buffer tac WaaL tubes b, c gut 2 M H 2 SO 4 sac cell ELI-SAs [34] liver human χ7122 Fig. 2b oligosaccharyltransferase E. coli K-12 W3110 Plasmid Salmonella enterica serovar Paratyphi [30] GlcNAc eggs strain-appropriate mouse anti-His monoclonal antibody ( saline Figure S4a PD-10 LB broth Lennox birds E. coli W3110 IS1 rabbits let Fig. 4a 2-3 1:100 E. coli S17-1-λpir Clones 1 IntDN intraair sac 1:10,000 G/ unG-NetB pEXT20 homogenates children 19,932 O-antigens Figure S1 plasmid DNA E. coli K-12 strains Figure S3 Mycoplasma synoviae Figure S6 spleen chicken intestines amino acid AKTÄ tetramethylbenzidine E. coli χ7122 Invitrogen IIlumina glycan antigen Cell clones c, d) C. perfringens toxoid NetB C. jejuni strains M1 chickens [59 lanes 4-6 ELI-SAs FlpA glycoconjugates rpsM E. coli S17-1 λ pir cells h. lipid A sugar N-glycosylate † left homology arm FRT PCD70CB48 cell His-pulldowns NEB HiFi assembly kit WCL lanes 6-7 χ7122 pgl tissue pACYCpglΔpglB LLC human lectin C. perfringens cellular Figure S4b chicken gut GalNAc residues chicken anti-mouse IgG-HRP ST23 sucrose bioSBA lectin G-NetB C. jejuni strain M1 (Fig. 7c AU pEC415 Tween-20 pMAF10 NaH 2 PO 4 C. jejuni glycan antigen T ECA Oral E. coli pFPV25.1-unG-NetB Glycoconjugate chicken caeca °C caecal SDB1 glycoconjugates 1:5000 λpir + strains DNA lanes 4-5 SDB1 cells ~ 1-4 electrocompetent E. coli S17-1 λ pir serum Lysates gidB Cells c SDS-PAGE enteritis 4-12 UnG-NetB glycoconjugate serum IgY. glycoconjugate vaccines infants Biolabs Fast-Prep TM C. jejuni heptasaccharide glycan transferase WecA lipids W262A G-NetB(10 CLM24 cedA::pglB strain toxoid FeatureCounts Electrocompetent E. coli strains glutamine botulism IPTG-inducible G-NetB(10)/unG-NetB chicken meat colibacillosis serotypes Bacillus subtilis OST PglB Blood avian influenza viruses E. coli cells Salmonella enterica serovar Gallinarum C terminal 6xHis tag NetB nucleotide glucose On-cell broilers Figure S7 glycan/protein post-oral sucrose R streptavidin-HRP OST NetB antigens C. perfringens NetB Nal R antigen His-purified G-NetB(10 UK fowl cholera UltraII DNA Library Prep Kit for Illumina pFPV25.1-unGNetB transferase chromosomal green dotted line N-linked heptasaccharide glycan humans chicken intestine Fig. 5c b GalNAc E. coli O-antigen μl/ Fig. 6g Fig. 8d, ligase periplasmic O-antigen Cell resuspensions LB tissues O78 O-antigen G-NetB(10)/ unG-NetB × 10 8 unG-NetB. left C. jejuni heptasaccharide LiCOR PelB S. Typhimurium promoter P rpsM [63] glycoconjugate G-NetB(10 Inocula G-NetB. Lanes 1-3 pir-dependent R6K PglC G-/ unG-NetB CLM24 cedA::pglB antichicken IgY-horseradish peroxidase 1:1000 S17-1 λ pir Fig. 6b Rstudio O78-antigen nalidixic acid × IPTG pSEC-Pgl5 pFPV25 clones 1 Amp S colonies transformants OD-normalised immune Leghorn chickens pACYC pglΔpglB. IPTG IntUP glycans cell lysates L-arabinose membranes SodB Glc people Figure S2 atpI Haemophilus influenzae type b membrane three-ygeV Neisseria meningitidis human gastroenteritis glycoconjugate vaccine [12] vaccine antigens − Pgl1-3 NQ Mycoplasma gallisepticum Fig. 7b lanes 1-2 G-ExoA(10 lanes 1-4 caeca e intra-air sac vaccinated chickens pACYC pgl ycfC-demonstrated blood egg G-ExoA (10) women vaccine formulations Fig. 3a Figure S5 cell surface × 10 9 kanamycin IPTG-inducible S. Typhimurium C. jejuni host pgl1-3 lipid-linked glycan lung Cultures ena periplasmic extracts lectin Figure 3b G-NetB PCD70CB48
Extracted Text Content in Record: First 5000 Characters:Background: Poultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry. We demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain. We generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost "live-attenuated multivalent vaccine factories" with the ability to express glycoconjugates in poultry. Healthily maintained livestock are essential for economic and societal prosperity [1] . Poultry are the main source of meat and eggs worldwide. The world's chicken flock is now estimated to be around 66 billion broilers and 21 billion layers [2] . Poultry meat production has grown 12-fold in the past 50 years [2] . Additionally, poultry accounts for egg production, which globally has increased threefold in the last three decades with c. 87 billion eggs estimated to be produced per annum [3] . However, infectious diseases remain a significant impediment to poultry welfare and productivity. Consequently, there is a growing demand to identify successful strategies to prevent the spread of diseases within and from flocks, to avert significant economic losses and to mitigate the healthcare burden resulting from zoonoses arising from poultry products. The most common bacterial infections encountered in poultry are colibacillosis, mycoplasmosis, and salmonellosis, caused by avian pathogenic E. coli (APEC), Mycoplasma gallisepticum (and less frequently by Mycoplasma synoviae and Mycoplasma meleagridis), and Salmonella enterica species (mostly Salmonella enterica serovar Pullorum and Salmonella enterica serovar Gallinarum), respectively [4] . Other less common, but possibly severe bacterial infections include fowl cholera, necrotic enteritis, botulism and tuberculosis [4] . Aside from contracting infections, poultry can also transmit zoonotic diseases of public health concern to humans, such as campylobacteriosis, salmonellosis, and avian influenza viruses causing gastroenteritis, diarrhoea and fever [5] . In Europe and the UK, for instance, bacterial species of Campylobacter and Salmonella are the top two reported bacterial gastrointestinal pathogens in humans. These are WHOlisted high priority pathogens given the rise of antibiotic resistant species, and undercooked chicken meat and eggs are key sources of human infection [6] [7] [8] . The diseases they cause are usually self-limiting in people, but in severe cases they require hospitalisation, and can result in death, generally posing a higher threat for children younger than 5 years, people with weakened immune systems, pregnant women and the elderly [5] . Worse outcomes, particularly in young children, are more common in poor settings lacking safe water, effective sanitation, standard hygiene, and hospital access, emphasising the global health concerns around zoonotic enteritis and the importance of the One Health approach (health for people, animals and the environment) to tackle them [6, 9, 10] . General recommendations to reduce the spread of infectious diseases are the implementation of generic hygiene measures, together with safe cooking and food handling practices to avoid
Keywords Extracted from PMC Text: ST23 E. coli cells rpsM clones 1 flocks strain-appropriate egg lipid Figure S1 atpI tetramethylbenzidine c C. jejuni pgl Mycoplasma synoviae membranes botulism O2 anti-chicken IgY-horseradish peroxidase G-ExoA(2 membrane T plasmid DNA rabbits Pgl1-3 O78 O-antigen fowl cholera O-antigens pFPV25.1-unGNetB transferase WecA pEXT20 S17-1 λ pir glycan/protein Salmonella cell surface Salmonella enterica serovar Pullorum Neisseria meningitidis 1:100 Figure S2 LB Cell resuspensions UnG-NetB chicken intestines E. coli S17-1-λpir C. jejuni heptasaccharide glycan chicken E. coli S17-1 λpir cells humans OD600nm-matched cultures Figure S4a IntDN " capsular polysaccharides G-ExoA undecaprenyl-pyrophosphate blood glycan antigen caeca Fig. 8d, ycfC-demonstrated UltraII DNA Library Prep Kit for Illumina C terminal 6xHis tag Qiazol Lysates Glycoconjugate CO2 pACYCpglΔpglB gut Fig. 5c nucleotide Biomasses E. coli S17-1 λ pir cells PGCT Figure S5 NetB antigens C. jejuni PglB AhdI-SacII Salmonella enterica serovar Gallinarum electrocompetent E. coli S17-1 λ pir On-cell avian gut Oral Fig. 6c post-oral oligosaccharyltransferase sugar G-ExoA(10 E. coli OD600nm-matched periplasmic extracts Uniprot ID SodB Glc [34] O-antigen glycoproteins G-/unG-NetB − oral human °C bis–tris gels cytoplasmic KanR GlcNAc kanamycin transferase Blood LDS sample buffer periplasmic side NZ_HE962388 [1 Serum IgY cardiac puncture N-acetylgalactosamine post-hatch × IPTG-inducible PglB gidB sacB G-NetB(10)/unG-NetB vaccine formulations cellular anti-mouse IgG-HRP caecum homogenates UK OD600nm-matched plasmid pSECpgl EcoRI-PstI Salmonella enterica species T-cell C.jejuni glycan bioSBA lectin streptavidin-HRP infants C. perfringens IIlumina pFPV25.1-unG-NetB Chickens Figure S6 FlpA glycoconjugates women 19,932 heptasaccharide antigen WCL cell lysates gidA amino acid 1:50 serum Poultry C. jejuni glycan antigen IPTG His-purifications Fig. 2b pEC415 flock Figure S7 G-NetB(2 BioLegend TMB chicken flock NalR mouse anti-His monoclonal antibody ( poultry enteritis let chromosomal G-NetB(10 birds children Inocula glucose C. jejuni strain M1 ECA pSEC-Pgl5 sac Fig. 5b glycoprotein pir-dependent R6K serum E. coli W3110 E. coli K-12 W3110 spleen chicken gut Electrocompetent E. coli strains S. Typhimurium pMAF10 sucrose AmpS C. jejuni host HRP goat anti-mouse IgG IRDye 680 (LI-COR Biosciences Poultry meat tubes chicken caeca Clostridium perfringens NetB toxoid Rstudio C. jejuni strains M1 IPTG-inducible G-NetB(10)/unG-NetB χ7122 cells inner membrane [53] chicken meat Mycoplasma gallisepticum 1:5000 Figure S3 SDB1 lungs mouse snpEff pEXT20-G-NetB(10 GalNAc Fig. 6g b Salmonella enterica serovar Paratyphi [30] FRT E. coli K-12 strains NZ_HE962388 serum IgY. IPTG-inducible pEXT20 serotypes unG-NetB ligase E. coli O-antigen periplasmic fractions C. jejuni M1 chickens [59 OD450/620 PglB OST DNA E. coli χ7122 lanes 3 co-integrants Biolabs carbonate-bicarbonate buffer pFPV25.1-G-NetB(10 chickens CLM24 cedA::pglB strain eggs caecal periplasmic IntUP L-arabinose surface C. jejuni strain M1 (Fig. 7c [12] intestinal 1:1000 Post-mortem PglC Kanamycin 1:10,000 livers N-glycosylate liquid cultures Fig. 8d people Haemophilus influenzae type b C. coli C. perfringens toxoid NetB glycoconjugates transformants glycans glycoconjugate vaccines Fig. 5a tissue lipid A Bacillus subtilis C. jejuni heptasaccharide GalNAc residues G-NetB avian influenza viruses leaves cultures vaccine antigens meat clones human gastroenteritis lactose Fig. 6b lipid-linked glycan gastrointestinal h. antigen Fig. 3a Transformants liver DQNAT D/EXNXS/T OST PglB human lectin saline × 106 glutamine SAMtools LLC inner membrane His-pulldown LiCOR flgG C. perfringens NetB algorithm-mem A8ULG6 chicken body diphtheria toxin PrpsM [63 W262A Mueller–Hinton sucroseR WaaL Tween-20 CLM24 cedA::pglB Invitrogen His-pulldowns OD-normalised chicken gut [27] E. coli K-12 cell lectin E7645 chicken intestine Streptavidin-HRP Cells His-purified G-NetB(10 LB broth Lennox λpir + strains lipidA-linked heptasaccharide N-linked heptasaccharide glycan NEB HiFi assembly kit G-NetB(10) antigens FeatureCounts tissues C. jejuni heptasaccharide antigen Figure S4b PelB ampicillin lipids Cultures Cell PBS-washed O-antigen fastQ nalidixic acid cells O78-antigen OST NetB NZ_HE962388_Pgl Fig. 4a χ7122 colibacillosis broilers kanR -marked pgl 's Ptac O-antigen ligase WaaL His-purified Campylobacter glycoconjugates glycoconjugate
Extracted PMC Text Content in Record: First 5000 Characters:Healthily maintained livestock are essential for economic and societal prosperity [1]. Poultry are the main source of meat and eggs worldwide. The world's chicken flock is now estimated to be around 66 billion broilers and 21 billion layers [2]. Poultry meat production has grown 12-fold in the past 50 years [2]. Additionally, poultry accounts for egg production, which globally has increased threefold in the last three decades with c. 87 billion eggs estimated to be produced per annum [3]. However, infectious diseases remain a significant impediment to poultry welfare and productivity. Consequently, there is a growing demand to identify successful strategies to prevent the spread of diseases within and from flocks, to avert significant economic losses and to mitigate the healthcare burden resulting from zoonoses arising from poultry products. The most common bacterial infections encountered in poultry are colibacillosis, mycoplasmosis, and salmonellosis, caused by avian pathogenic E. coli (APEC), Mycoplasma gallisepticum (and less frequently by Mycoplasma synoviae and Mycoplasma meleagridis), and Salmonella enterica species (mostly Salmonella enterica serovar Pullorum and Salmonella enterica serovar Gallinarum), respectively [4]. Other less common, but possibly severe bacterial infections include fowl cholera, necrotic enteritis, botulism and tuberculosis [4]. Aside from contracting infections, poultry can also transmit zoonotic diseases of public health concern to humans, such as campylobacteriosis, salmonellosis, and avian influenza viruses causing gastroenteritis, diarrhoea and fever [5]. In Europe and the UK, for instance, bacterial species of Campylobacter and Salmonella are the top two reported bacterial gastrointestinal pathogens in humans. These are WHO-listed high priority pathogens given the rise of antibiotic resistant species, and undercooked chicken meat and eggs are key sources of human infection [6–8]. The diseases they cause are usually self-limiting in people, but in severe cases they require hospitalisation, and can result in death, generally posing a higher threat for children younger than 5 years, people with weakened immune systems, pregnant women and the elderly [5]. Worse outcomes, particularly in young children, are more common in poor settings lacking safe water, effective sanitation, standard hygiene, and hospital access, emphasising the global health concerns around zoonotic enteritis and the importance of the One Health approach (health for people, animals and the environment) to tackle them [6, 9, 10]. General recommendations to reduce the spread of infectious diseases are the implementation of generic hygiene measures, together with safe cooking and food handling practices to avoid consumption of raw or undercooked animal products [11]. Alongside these practices, the introduction of vaccines has been one of the most impactful and cost-effective public health measures to prevent the spread of diseases (reviewed in [12]), saving an estimated 2–3 million lives each year [13]. Amongst the different types of vaccines that exist to date, glycoconjugates, consisting of a glycan antigen from the surface of the target pathogen covalently coupled to a protein carrier with strong immunogenic properties, have a proven track record in human vaccinology for safety and efficacy [14]. In fact, since the introduction of licensed glycoconjugate vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis from the 1980s, the incidence of pneumonia and meningitis has dramatically decreased [12, 15]. The key element of their effectiveness in inducing durable B- and T-cell responses is the coupling of the glycan and protein moieties, a crucial observation that stemmed from pioneering work of Avery and Goebbels in the 1920s [16]. Linking a carrier protein to a glycan antigen overcomes the limitations of polysaccharide-only vaccines, which fail to induce protection in infants younger than 18 months, are T-cell independent antigens and fail to induce immunological memory [17–19]. The traditional production of glycoconjugates, however, is a convoluted multi-step process that requires large scale cultures of pathogenic microorganisms as a source of the glycan antigen or chemical synthesis of the glycan, the separate production of a recombinant carrier protein, and the chemical or enzymatic coupling of the two. The complexity of the production process is reflected in high costs that has so far hindered their use in the veterinary field. To circumvent cost and biosafety issues, we employ Protein Glycan Coupling Technology (PGCT), which utilises modified bacterial strains to produce glycoconjugates recombinantly in vivo (reviewed in [20, 21]). This biotechnology stemmed from the discovery of an N-linked protein glycosylation locus (pgl) in the bacterium C. jejuni [22, 23] and its functional transfer to E. coli [24]. The subsequent observations that the oligosaccharyltr
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