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 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. 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.

PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome

“…To achieve chromosomal integration of the pgl locus, we used the suicide vector, pSEC pgl [ 13 ] based on the pCVD442 plasmid [ 20 ] containing a pir -dependent R6K replicon and the sacB gene of Bacillus subtilis conferring sucrose sensitivity, which enables positive selection of integrants for the loss of backbone sequence. pSEC pgl contains the pgl locus from C. jejuni 81116 C-terminally tagged with a kanamycin resistance (KanR) cassette and surrounded by homology arms for insertion between E. coli gidB and atpI genes (Fig.…”

“…It would mean the determination of a minimum amount of glycoconjugate that confers protection and ensuring this could be achieved per bacterial cell. Indeed, it may explain the lack of protection observed with an APEC pgl integrant expressing G-NetB in our recent work [ 13 ]. Alternatively, LAVS could be produced at 30 °C, and repeatedly introduced to chickens over time.…”

“…Introduction of large polysaccharide coding regions in the chromosome of bacteria has been shown as enabling stable and metabolically efficient glycan production [ 14 , 15 , 32 ]. Furthermore, having multiple copies of two or three plasmids performing complex metabolic functions can elevate the metabolic burden arresting cell growth [ 13 , 33 ], having a deleterious effect on glycoconjugate production.…”

“…In this work, we generated a versatile glycoengineering strain ( E. coli SDB1 pgl ) for the rapid production of glycoconjugates to be used recombinantly as well as to investigate the usefulness of such strains as part of a live attenuated vaccine strategy. The E. coli SDB1 pgl was used to investigate glycosylation in vivo in a bid to understand the lack of protection observed in our previous study where poultry was vaccinated using avian pathogenic Escherichia coli (APEC) strain Chi7122 pgl [ 13 ]. By creating a strain that can chromosomally express the conserved Campylobacter heptasaccharide glycan we introduced greater flexibility to the PGCT system, enabling the rapid testing of preferred carrier proteins, obviating the need for antibiotics for plasmid maintenance, minimising the possible spread of plasmids and increasing stability of the polysaccharide coding region as well as contributing to greater efficiency by decreasing the metabolic burden to the E. coli host [ 14 – 17 ].…”

“…Due to results we have observed previously, where no protection was seen against C. jejuni when using Chi7122 pgl, we set out to determine factors that affect glycosylation when the pgl locus is inserted in the chromosome. As the live attenuated vaccines (LAVs) created in our previous study [ 13 ] were intended to be used in poultry which have a higher body temperature than humans or other farm animals [ 19 ], we investigated the effect of temperature on protein expression and glycosylation. An unequivocal relationship between temperature and glycosylation efficiency was demonstrated within bacterial cells as well as when carrying out the reaction using an in vitro glycosylation assay.…”