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

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.

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

BACKGROUND: Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. RESULTS: We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. CONCLUSIONS: We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.

Multivalent poultry vaccine development using Protein Glycan Coupling Technology.

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.

Construction of a pneumolysin deficient mutant in streptococcus pneumoniae serotype 1 strain 519/43 and phenotypic characterisation.

Streptococcus pneumoniae capsular serotype 1 continues to pose a huge infectious disease burden in low- and middle-income countries, particularly in West Africa. However, studies on this important serotype have been hampered by the inability to genetically modify these strains. In this study we have genetically modified a serotype 1 strain (519/43), the first time that this has been achieved for this serotype, providing the methodology for a deeper understanding of its biology and pathogenicity. As proof of principle we constructed a defined pneumolysin mutant and showed that it lost its ability to lyse red blood cells. We also showed that when mice were infected intranasally with the mutant 519/43Δply there was no significant difference between the load of bacteria in lungs and blood when compared to the wild type 519/43. When mice were infected intraperitoneally there were significantly fewer bacteria recovered from blood for the mutant 519/43Δply strain, although all mice still displayed signs of disease. Our study demonstrates S. pneumoniae serotype 1 strains can be genetically manipulated using our methodology and demonstrate that the ability to cause pneumonia in mice is independent of active pneumolysin for the 519/43 serotype 1 strain.

Evaluation of a FlpA Glycoconjugate Vaccine with Ten N-Heptasaccharide Glycan Moieties to reduce Campylobacter jejuni Colonisation in Chickens.

Campylobacter is a major cause of acute gastroenteritis in humans, and infections can be followed by inflammatory neuropathies and other sequelae. Handling or consumption of poultry meat is the primary risk factor for human campylobacteriosis, and C. jejuni remains highly prevalent in retail chicken in many countries. Control of Campylobacter in the avian reservoir is expected to limit the incidence of human disease. Toward this aim, we evaluated a glycoconjugate vaccine comprising the fibronectin-binding adhesin FlpA conjugated to up to ten moieties of the conserved N-linked heptasaccharide glycan of C. jejuni or with FlpA alone. The glycan dose significantly exceeded previous trials using FlpA with two N-glycan moieties. Vaccinated birds were challenged with C. jejuni orally or by exposure to seeder-birds colonised by C. jejuni to mimic natural transmission. No protection against caecal colonisation was observed with FlpA or the FlpA glycoconjugate vaccine. FlpA-specific antibody responses were significantly induced in vaccinated birds at the point of challenge relative to mock-vaccinated birds. A slight but significant antibody response to the N-glycan was detected after vaccination with FlpA-10×GT and challenge. As other laboratories have reported protection against Campylobacter with FlpA and glycoconjugate vaccines in chickens, our data indicate that vaccine-mediated immunity may be sensitive to host- or study-specific variables.

Production of Vaccines Using Biological Conjugation.

The production of conjugate vaccines within an E. coli (Escherichia coli) host provides an inexhaustible supply without the need for culture of pathogenic organisms. The machinery for expression of glycan and acceptor protein, as well as the coupling enzyme, are all housed within the E. coli chassis, meaning that there are no additional steps required for individual purification and chemical conjugation of components. In addition, there are far fewer purification steps necessary to obtain a purified glycoconjugate for use in vaccine testing. Here we describe production and purification of a HIS-tagged Campylobacter jejuni AcrA protein conjugated to Streptococcus pneumoniae serotype 4 capsule.

Evaluation of a Campylobacter jejuni N-glycan-ExoA glycoconjugate vaccine to reduce C. jejuni colonisation in chickens.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and handling or consumption of contaminated poultry meat is the key source of infection. Glycoconjugate vaccines containing the C. jejuni N-glycan have been reported to be partially protective in chickens. However, our previous studies with subunit vaccines comprising the C. jejuni FlpA or SodB proteins with up to two or three C. jejuni N-glycans, respectively, failed to elicit significant protection. In this study, protein glycan coupling technology was used to add up to ten C. jejuni N-glycans onto a detoxified form of Pseudomonas aeruginosa exotoxin A (ExoA). The glycoprotein, G-ExoA, was evaluated for efficacy against intestinal colonisation of White Leghorn chickens by C. jejuni strains M1 and 11168H relative to unglycosylated ExoA. Chickens were challenged with the minimum dose required for reliable colonisation, which was 102 colony-forming units (CFU) for strain M1 and and 104 CFU for strain 11168H. Vaccine-specific serum IgY was detected in chickens vaccinated with both ExoA and G-ExoA. However, no reduction in caecal colonisation by C. jejuni was observed. While the glycan dose achieved with G-ExoA was higher than FlpA- or SodB-based glycoconjugates that were previously evaluated, it was lower than that of glycoconjugates where protection against C. jejuni has been reported, indicating that protection may be highly sensitive to the amount of glycan presented and/or study-specific variables.

Characterization of novel beta-galactosidase activity that contributes to glycoprotein degradation and virulence in Streptococcus pneumoniae.

The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore, efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterized the Streptococcus pneumoniae strain D39 protein encoded by SPD_0065 and found that this gene encodes a beta-galactosidase. The SPD_0065 recombinant protein released galactose from desialylated fetuin, which was used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD_0065 showed diminished beta-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium, and cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal beta-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total beta-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but beta-galactosidase activity in the double mutant, although significantly less than that in either of the single mutants, was not completely abolished. The expression of SPD_0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly upregulated compared to that in pneumococci from glucose-containing medium. The SPD_0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43.

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus' ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Evaluation of Glycosylated FlpA and SodB as Subunit Vaccines Against Campylobacter jejuni Colonisation in Chickens.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and the handling or consumption of contaminated poultry meat is the key source of infection. C. jejuni proteins FlpA and SodB and glycoconjugates containing the C. jejuni N-glycan have been separately reported to be partially protective vaccines in chickens. In this study, two novel glycoproteins generated by protein glycan coupling technology-G-FlpA and G-SodB (with two and three N-glycosylation sites, respectively)-were evaluated for efficacy against intestinal colonisation of chickens by C. jejuni strain M1 relative to their unglycosylated variants. Two independent trials of the same design were performed with either a high challenge dose of 107 colony-forming units (CFU) or a minimum challenge dose of 102 CFU of C. jejuni M1. While antigen-specific serum IgY was detected in both trials, no reduction in caecal colonisation by C. jejuni M1 was observed and glycosylation of vaccine antigens had no effect on the outcome. Our data highlight inconsistencies in the outcome of C. jejuni vaccination trials that may reflect antigen-, challenge strain-, vaccine administration-, adjuvant- and chicken line-specific differences from previously published studies. Refinement of glycoconjugate vaccines by increasing glycosylation levels or using highly immunogenic protein carriers could improve their efficacy.

CSF Levels of Elongation Factor Tu Is Associated With Increased Mortality in Malawian Adults With Streptococcus pneumoniae Meningitis.

Background: Mortality from bacterial meningitis, predominately caused by Streptococcus pneumoniae, exceeds 50% in sub-Saharan African countries with high HIV prevalence. Underlying causes of high mortality are poorly understood. We examined the host and pathogen proteome in the CSF of adults with proven pneumococcal meningitis (PM), testing if there was an association between differentially expressed proteins and outcome. Materials/Methods: CSF proteomes were analyzed by quantitative Mass-Spectrometry. Spectra were identified using the Swissprot human and TIGR4 pneumococcal protein libraries. Proteins were quantitated and analyzed against mortality. Unique proteins in PM were identified against published normal CSF proteome. Random-Forest models were used to test for protein signatures discriminating outcome. Proteins of interest were tested for their effects on growth and neutrophil opsonophagocytic killing of S. pneumoniae. Results: CSF proteomes were available for 57 Adults with PM (median age 32 years, 60% male, 70% HIV-1 co-infected, mortality 63%). Three hundred sixty individual human and 23 pneumococcal proteins were identified. Of the human protein hits, 30% were not expressed in normal CSF, and these were strongly associated with inflammation and primarily related to neutrophil activity. No human protein signature predicted outcome. However, expression of the essential S. pneumoniae protein Elongation Factor Tu (EF-Tu) was significantly increased in CSF of non-survivors [False Discovery Rate (q) <0.001]. Expression of EF-Tu was negatively co-correlated against expression of Neutrophil defensin (r 0.4 p p < 0.002), but not against complement proteins C3 or Factor H. In vitro, addition of EF-Tu protein impaired S. pneumoniae neutrophil killing in CSF. Conclusions: Excessive S. pneumoniae EF-Tu protein in CSF was associated with reduced survival in meningitis in a high HIV prevalence population. We show EF-Tu may inhibit neutrophil mediated killing of S. pneumoniae in CSF. Further mechanistic work is required to better understand how S. pneumoniae avoids essential innate immune responses during PM through production of excess EF-Tu.

Pyruvate formate lyase is required for pneumococcal fermentative metabolism and virulence.

Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

The pneumococcal response to oxidative stress includes a role for Rgg.

Streptococcus pneumoniae resides in the oxygen-rich environment of the upper respiratory tract, and therefore the ability to survive in the presence of oxygen is an important aspect of its in vivo survival. To investigate how S. pneumoniae adapts to oxygen, we determined the global gene expression profile of the micro-organism in aerobiosis and anaerobiosis. It was found that exposure to aerobiosis elevated the expression of 54 genes, while the expression of 15 genes was downregulated. Notably there were significant changes in putative genome plasticity and hypothetical genes. In addition, increased expression of rgg, a putative transcriptional regulator, was detected. To test the role of Rgg in the pneumococcal oxidative stress response, an isogenic mutant was constructed. It was found that the mutant was sensitive to oxygen and paraquat, but not to H(2)O(2). In addition, the absence of Rgg strongly reduced the biofilm-forming ability of an unencapsulated pneumococcus. Virulence studies showed that the median survival time of mice infected intranasally with the rgg mutant was significantly longer than that of the wild-type-infected group, and the animals infected with the mutant developed septicaemia later than those infected intranasally with the wild-type.

The ability to utilize mucin affects the regulation of virulence gene expression in Streptococcus pneumoniae.

Streptococcus pneumoniae colonizes the mucin-rich environment of the nasopharynx. As colonization may be the first stage of infection, investigation was carried out as to whether the pneumococcus could utilize mucin as a source of nutrient and whether its virulence gene expression is influenced by this glycoprotein. It was found that when grown in Sicard's defined medium supplemented with 1% w/v mucin, the organism grew at a rate similar to that in rich medium. The presence of sialate O-acetylesterase activity, an enzyme implicated in glycoprotein degradation, in pneumococcal cell extracts was also reported. The authors hypothesized that neuraminidase A, which is linked to pneumococcal virulence, plays an important role in mucin utilization. Growth in mucin resulted in an increase in nanA transcription and a DeltananA-deficient strain of pneumococcus could not grow when mucin was used as the sole carbon source.

The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering.

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.

Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli.

Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology.

Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design.

The discovery of the Campylobacter jejuni N-linked glycosylation system combined with its functional expression in Escherichia coli marked the dawn of a new era in glycoengineering. The process, termed protein glycan coupling technology (PGCT), has, in particular, been applied to the development of glycoconjugate vaccines. In this review, we highlight recent technical developments in this area, including the first structural determination of the coupling enzyme PglB, the use of glycotags for optimal glycan attachment and the possible applications of other glycosylation systems and how these may improve and extend PGCT.