Optimization of the Synthesis and Conjugation of the Methyl Rhamnan Tip of Pseudomonas aeruginosa A-Band Polysaccharide and Immunogenicity Evaluation for the Continued Development of a Potential Glycoconjugate Vaccine.

Pseudomonas aeruginosa is an antimicrobial-resistant bacterium that has no vaccine approved for human use. Additionally, it has been identified by the World Health Organization as a priority pathogen for novel vaccines and therapeutic development. We previously developed a synthetic mimic of the A-band polysaccharide tip that showed promise in terms of immunogenicity for use as a glycoconjugate vaccine. In this current manuscript, we improve upon the previous work to continue the development of this glycoconjugate vaccine. Herein, we report a higher-yielding synthesis of mimics containing a handle and a spacer that improved conjugation efficiency, resulting in better carbohydrate-to-protein ratios and also good immunogenicity of these conjugates in mice and rabbits. The data suggested that perhaps only a tetrasaccharide was required to induce an immune response capable of recognizing whole cells of P. aeruginosa.

Formation and immunological evaluation of Moraxella catarrhalis glycoconjugates based on synthetic oligosaccharides.

Published work has shown that glycoconjugate vaccines, based on truncated detoxified lipopolysaccharides from Moraxella catarrhalis attached through their reducing end to a carrier protein, gave good protection for all three serotypes A, B, and C in mice immunisation experiments. The (from the non-reducing end) truncated LPS structures were obtained from bacterial glycosyl transferase knock-out mutants and contained the de-esterified Lipid A, two Kdo residues and five glucose moieties. This work describes the chemical synthesis of the same outer Moraxella LPS structures, spacer-equipped and further truncated from the reducing end, i.e., without the Lipid A part and containing four or five glucose moieties or four glucose moieties and one Kdo residue, and their subsequent conjugation to a carrier protein via a five­carbon bifunctional spacer to form glycoconjugates. Immunisation experiments both in mice and rabbits of these gave a good antibody response, being 2-7 times that of pre-immune sera. However, the sera produced only recognized the immunizing glycan immunogens and failed to bind to native LPS or whole bacterial cells. Comparative molecular modelling of three alternative antigens shows that an additional (2 â†’ 4)-linked Kdo residue, not present in the synthetic structures, has a significant impact on the shape and volume of the molecule, with implications for antigen binding and cross-reactivity.

The structure of the LPS O-chain from five Fusobacterium nucleatum strains CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, containing alditol and phosphate in the main chain and development of mouse monoclonal antibodies specific to the O-antigens.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report four new structures of the lipopolysaccharide O-chain (OPS) from five strains of F. nucleatum CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, isolated from cancerous tissues. Three of the four structures have a common sequence of hexose-diaminofucose-hexitol-phosphate in the main chain.

Synthesis and Immunogenicity of a Methyl Rhamnan Pentasaccharide Conjugate from Pseudomonas aeruginosa A-Band Polysaccharide.

Pseudomonas aeruginosa was added to the World Health Organization's priority pathogen list for research and development of new antibiotics in 2017. Alongside the development of new antibiotics to fight antimicrobial-resistant P. aeruginosa, vaccines would be an appealing addition to the toolbox health professionals have against this bacteria, which causes life-threatening respiratory infections. Recently, the structure of a novel immunogenic terminal carbohydrate moiety on the cell surface of P. aeruginosa was elucidated, consisting of a 3-O-methyl (1→4)-α-d-rhamnan pentasaccharide. As isolating this oligosaccharide from P. aeruginosa in sufficient amounts for producing a conjugate vaccine is challenging, herein we describe the synthesis of 3-O-methyl d-rhamnose oligosaccharide. We also report the conjugation of the synthetic pentasaccharide to human serum albumin and its resulting immunogenicity.

Structural Characterization and Evaluation of an Epitope at the Tip of the A-Band Rhamnan Polysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Structure of the lipopolysaccharide O-antigens from Fusobacterium nucleatum strains SB-106CP and HM-992 and immunological comparison to the O-antigen of strain 12230.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structures of the lipopolysaccharide O-chain (OPS) of two strains of F. nucleatum, SB-106CP and HM-992, both isolated from cancerous tissues. These strains elaborate the same sugar chain, differing only by their N-acylation pattern: -6-α-D-GlcNAc-4-ß-D-GlcNHBu3NABuA-3-ß-D-QuiNAc4NABuAc- SB-106CP -6-α-D-GlcNAc-4-ß-D-GlcNHBu3NABuA-3-ß-D-QuiNAc4NAc- HM-992 ABu = (R)-3-amino-butyryl AbuAc = (R)-3-N-acetyl-3-aminobutyryl HBu = (R)-3-hydroxy-butyryl All monosaccharides are in the pyranose form. Previously we published the structure of the OPS from F. nucleatum 12230, a transtracheal isolate, which had similar sugar chain, differing by replacement of GlcNAc with Glc and a different acylation pattern: -6-α-d-Glc-4-ß-d-GlcNHBu3NHBuA-3-ß-d-QuiNAc4NABu- A mouse monoclonal antibody specific for the 12230 O-antigen did not cross react with the LPS of strains SB-106CP and HM-992 confirming the structural differentiation.

Cross-reactivity of Haemophilus influenzae type a and b polysaccharides: molecular modeling and conjugate immunogenicity studies.

Haemophilus influenzae is a leading cause of meningitis disease and mortality, particularly in young children. Since the introduction of a licensed conjugate vaccine (targeting the outer capsular polysaccharide) against the most prevalent serotype, Haemophilus influenzae serotype b, the epidemiology of the disease has changed and Haemophilus influenzae serotype a is on the rise, especially in Indigenous North American populations. Here we apply molecular modeling to explore the preferred conformations of the serotype a and b capsular polysaccharides as well as a modified hydrolysis resistant serotype b polysaccharide. Although both serotype b and the modified serotype b have similar random coil behavior, our simulations reveal some differences in the polysaccharide conformations and surfaces which may impact antibody cross-reactivity between these two antigens. Importantly, we find significant conformational differences between the serotype a and b polysaccharides, indicating a potential lack of cross-reactivity that is corroborated by immunological data showing little recognition or killing between heterologous serotypes. These findings support the current development of a serotype a conjugate vaccine.

The capsular polysaccharides of Pasteurella multocida serotypes B and E: Structural, genetic and serological comparisons.

We describe the structural characterization of the capsular polysaccharides (CPSs) of Pasteurella multocida serotypes B and E. CPS was isolated following organic solvent precipitation of the supernatant from flask grown cells. Structural analysis utilizing nuclear magnetic resonance spectroscopy enabled the determination of the CPS structures and revealed significant structural similarities between the two serotypes, but also provided an explanation for the serological distinction. This observation was extended by the development of polyclonal sera to the glycoconjugate of serotype B CPS that corroborated the structural likenesses and differences. Finally, identification of these structures enabled a more comprehensive interrogation of the genetic loci and prediction of roles for some of the encoded proteins in repeat unit biosynthesis.

Structural analysis of the lipopolysaccharide O-antigen from Fusobacterium nucleatum strain CC 7/3 JVN3 C1 and development of a mouse monoclonal antibody specific to the O-antigen.

Fusobacterium nucleatum is becoming increasingly recognised as an emerging pathogen, gaining attention as a potential factor for exacerbating colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism; thus, we have initiated studies to examine the bacterium's surface glycochemistry. In an effort to characterise the surface carbohydrates of F. nucleatum, the aims of this study were to investigate the structure of the lipopolysaccharide (LPS) O-antigen of the cancer-associated isolate F. nucleatum strain CC 7/3 JVN3 C1 (hereafter C1) and to develop monoclonal antibodies (mAbs) to the LPS O-antigen that may be beneficial to the growing field of F. nucleatum research. In this study, we combined several technologies, including nuclear magnetic resonance (NMR) spectroscopy, to elucidate the structure of the LPS O-antigen repeat unit as -[-4-ß-Gal-3-α-FucNAc4N-4-α-NeuNAc-]-. We have previously identified this structure as the LPS O-antigen repeat unit from strain 10953. In this present study, we developed a mAb to the C1 LPS O-antigen and confirmed the mAbs cross-reactivity to the 10953 strain, thus confirming the structural identity.

Development and Characterization of Mouse Monoclonal Antibodies Specific for Clostridiodes (Clostridium) difficile Lipoteichoic Acid.

Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA)of C. difficile. The mAbs were characterized in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen, and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerized with human constant region domains and was found to recognize the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (standalone, in conjunction with known antitoxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.

Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen Streptococcus mutans.

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.

Investigating the candidacy of a capsular polysaccharide-based glycoconjugate as a vaccine to combat Haemophilus influenzae type a disease: A solution for an unmet public health need.

After the introduction of the glycoconjugate vaccine based upon the capsular polysaccharide ofHaemophilus influenzaetype b in the mid 1980s there was a remarkable decrease in the number of invasive cases reported for this organism. Since the 1990s several groups have observed the emergence ofHaemophilus influenzaetype a (Hia), especially in indigenous communities in the northern regions of Canada and Alaska, to a stage where a solution is warranted to prevent further unnecessary deaths due to this pathogen. A glycoconjugate vaccine solution based upon the type a capsular polysaccharide (CPS) was investigated pre-clinically in an effort to illustrate the proof of concept for this approach. In this study we describe the growth of Hia and the isolation, purification and conjugation of the CPS to several carrier proteins. The resulting glycoconjugates were immunised in mice and rabbits provoking sera that facilitated bactericidal killing against all type a strains that we tested. This study has illustrated the pre-clinical proof of concept of a glycoconjugate vaccine based on the CPS of Hia asa solution to this emerging disease.

Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection.

A lipoteichoic acid has recently been shown to be conserved in the majority of strains from Clostridium difficile and as such is being considered as a possible vaccine antigen. In this study we examine the candidacy of the conserved lipoteichoic acid by demonstrating that it is possible to elicit antibodies against C. difficile strains following immunisation of rabbits and mice with glycoconjugates elaborating the conserved lipoteichoic acid antigen. The present study describes a conjugation strategy that utilises an amino functionality, present at approximately 33 % substitution of the N-acetyl-glucosamine residues within the LTA polymer repeating unit, as the attachment point for conjugation. A maleimide-thiol linker strategy with the maleimide linker on the carboxyl residues of the carrier protein and the thiol linker on the carbohydrate was employed. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, despite an immune response to the linkers also being observed. These sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has illustrated that the LTA polymer is a highly conserved surface polymer of C. difficile that is easily accessible to the immune system and as such merits consideration as a vaccine antigen to combat C. difficile infection.

Investigating the candidacy of lipopolysaccharide-based glycoconjugates as vaccines to combat Mannheimia haemolytica.

Inner core lipopolysaccharide (LPS) has been shown to be conserved in the majority of veterinary strains from the species Mannheimia haemolytica, Actinobacillus pleuropneumoniae and Pasteurella multocida and as such is being considered as a possible vaccine antigen. The proof-in-principle that a LPS-based antigen could be considered as a vaccine candidate has been demonstrated from studies with monoclonal antibodies raised to the inner core LPS of Mannheimia haemolytica, which were shown to be both bactericidal and protective in a mouse model of disease. In this study we confirm and extend the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against Mannheimia haemolytica wild-type strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes a conjugation strategy that uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule. To protect the amino functionality on the phosphoethanolamine (PEtn) residue of the inner core, we developed a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy with the thiol linker on the carboxyl residues of the carrier protein and the maleimide linker on the carbohydrate resulted in a high loading of carbohydrates per carrier protein. Immunisation derived antisera from rabbits recognised fully extended Mannheimia haemolytica LPS and whole cells from serotypes 1 and 2, despite a somewhat immunodominant response to the linkers also being observed. Moreover, bactericidal activity was demonstrated to a strain elaborating the immunising carbohydrate antigen and crucially to wild-type cells of serotypes 1 and 2, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by Mannheimia haemolytica.

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera.

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading.

We investigated the immune responses of rabbits that were immunised with lipopolysaccharide (LPS)-based glycoconjugates by measuring the reactivity of the derived sera to a panel of selected wild-type and mutant strains of Neisseria meningitidis. In all cases, high titers of antibodies capable of recognising LPS elaborating the identical structure as presented on the immunising glycoconjugate were obtained, and in most cases the derived sera also recognised heterologous strains including wild-type, but at lower titers. However, although serum bactericidal antibodies were consistently obtained against strains elaborating the same LPS structure as the immunising antigen, this functional response was not observed against wild-type strains. We identified several potentially competing neo-epitopes that had been introduced via our conjugation strategies, which might compete with the conserved inner core oligosaccharide target region, thus reducing the antibody titers to epitopes which could facilitate bactericidal killing. This study has therefore identified key factors that are crucial to control in order to increase the likelihood of obtaining bactericidal antibodies to wild-type meningococcal cells with LPS-derived glycoconjugates. Glycoconjugates utilised in this study, have been found to contain epitopes that do not contribute to the derivation of antibodies that may facilitate bactericidal killing of wild-type strains and must be avoided in future LPS-based glycoconjugate preparations.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading.

In previous studies protective antibodies that could facilitate bactericidal killing of Neisseria meningitidis (Nm) serogroup B strains were derived from immunisation with glycoconjugates prepared from O-deacylated lipopolysaccharide (LPS-OH) via direct reductive amination between the reducing end of the oligosaccharide molecule, created by treatment with alkaline phosphatase, and amino functionalities on the CRM(197) carrier protein. These glycoconjugates proved difficult to prepare because the presence of amide linked fatty-acyl groups results in glycolipids that are relatively insoluble and aggregate. Therefore, we have examined several strategies to prepare glycoconjugates in order to identify a robust, consistently reproducible strategy that produces glycoconjugates with a high loading of LPS derived oligosaccharides. Initially we used completely deacylated LPS molecules, but lacking phosphoethanolamine (PEtn) from the core OS as the strong basic conditions required to completely deacylate the LPS would modify the PEtn residue. We utilised a squarate linker and conjugated via the reducing end of the carbohydrate antigen following removal of the glycosidic phosphate to amino groups on CRM(197), however carbohydrate loading on the carrier protein was low. Glycoconjugates were then produced utilising amidases produced by Dictyostelium discoideum (Dd), which partially remove N-linked fatty acids from the lipid A region of the Nm LPS molecule, which enabled the retention of the PEtn residue. LPS-OH was treated with Dd amidase, the reducing glycosidic phosphate removed, and using a cystamine linker strategy, conjugated to the carrier protein. Carbohydrate loading was somewhat improved but still not high. Finally, we have developed a novel conjugation strategy that targets the amino functionality created by the amidase activity as the attachment point. The amino functionality on the PEtn residue of the inner core was protected via a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy, targeting lysine residues on the carrier protein did not result in high loading of the carbohydrate molecules, however when we targeted the carboxyl residues we have consistently obtained a high loading of carbohydrate antigens per CRM(197), which can be controlled by variation in the amount of activated carbohydrate utilised in the conjugation reaction.