Glycoconjugate Vaccines Research Articles

What can rotavirus vaccines teach us about rotavirus?

Suspected and unexpected clinical features of pathogens might only become apparent during clinical trials to test vaccines or after implementation of vaccination programmes. For example, the role of Haemophilus influenzae type b (Hib) in early childhood pneumonia was not evident until findings of a clinical vaccine trial in The Gambia showed that—after 3 years of follow-up—Hib caused more than 20% of radiologically defined pneumonia in infants.1,2 Moreover, the ability of different pneumococcal serotypes, but not meningococcal serogroups, to replace competing strains in nasopharyngeal carriage and invasive disease was only noted after implementation of pneumococcal and group C meningococcal glycoconjugate vaccines.

ChemInform Abstract: Recent Advances in Synthesis of Bacterial Rare Sugar Building Blocks and Their Applications

AbstractReview: 79 refs.

Synthesis of antifungal vaccines by conjugation of β-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid

Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (β-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages.

Synthesis of Glycoconjugate Vaccines in Genetically Engineered Bacterial Cells

Conjugate vaccines have been the most successful way to prevent and control deadly bacterial infections; however their production is complex and tedious. The manufacturing process of these large molecules consists of a biological production and extraction of polysaccharide antigen and protein carrier and its chemical conjugation. Herein a simplified manufacturing process is described where the conjugate vaccine is completely produced in bacterial cells. The polysaccharide antigen and the protein carrier are expressed recombinantly in E. coli cells and linked covalently by an enzymatic process. This novel biological production system leads to improved products and opens up the possibility to develop a wide variety of novel vaccines. Keywords: Bacterial vaccines, bioconjugates, chemical conjugation, conjugate vaccines, glycosylation, production of glycoconjugates in E. coli.

Recombinant Clostridium difficile toxin fragments as carrier protein for PSII surface polysaccharide preserve their neutralizing activity.

Clostridium difficile is a Gram-positive bacterium and is the most commonly diagnosed cause of hospital-associated and antimicrobial-associated diarrhea. Despite the emergence of epidemic C. difficile strains having led to an increase in the incidence of the disease, a vaccine against this pathogen is not currently available. C. difficile strains produce two main toxins (TcdA and TcdB) and express three highly complex cell-surface polysaccharides (PSI, PSII and PSIII). PSII is the more abundantly expressed by most C. difficile ribotypes offering the opportunity of the development of a carbohydrate-based vaccine. In this paper, we evaluate the efficacy, in naive mice model, of PSII glycoconjugates where recombinant toxins A and B fragments (TcdA_B2 and TcdB_GT respectively) have been used as carriers. Both glycoconjugates elicited IgG titers anti-PSII although only the TcdB_GT conjugate induced a response comparable to that obtained with CRM197. Moreover, TcdA_B2 and TcdB_GT conjugated to PSII retained the ability to elicit IgG with neutralizing activity against the respective toxins. These results are a crucial proof of concept for the development of glycoconjugate vaccines against C. difficile infection (CDI) that combine different C. difficile antigens to potentially prevent bacterial colonization of the gut and neutralize toxin activity.

Engineering the product profile of a polysialyltransferase.

Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.

O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis

Bacterial lipopolysaccharide (LPS) is an essential cell envelope component for gram-negative bacteria. As the most variable region of LPS, O antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in Escherichia coli with an N-terminal His10-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemo-enzymatically synthesized the substrates of Wzy, the lipid-PP-linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that Wzy functions in a distributive manner.

Deciphering the structure–immunogenicity relationship of anti-Candidaglycoconjugate vaccines

Elucidation of the molecular immunity of glycoconjugate vaccines has focused on the carbohydrate moiety, herein the effect of the corresponding conjugation sites is studied.

Glycopolymers of microorganisms: achievements and future research (review)

This review presents the current literature data on the structure of peptidoglycans, lipopolysaccharides, teichoic acids, the mechanism of biological action of lipopolysaccharides, and the possibility of uising oligosaccharides for creation of glycoconjugate vaccines, as well as promising areas for further research of glycopolymers of microorganisms.

Structural analysis of O-polysaccharide chains extracted from different Salmonella Typhimurium strains

Salmonella Typhimurium is the major cause of invasive nontyphoidal Salmonella disease in Africa, with high mortality among children and HIV-infected individuals. Currently, no vaccine is available for use in humans. Antibodies directed against the O-polysaccharide of the lipopolysaccharide molecule of Salmonella mediate bacterial killing and are protective, and conjugation of the O-polysaccharide to a carrier protein represents a possible strategy for vaccine development.Here we have purified the O-polysaccharide from six different strains of S. Typhimurium and fully characterized them using analytical methods including HPLC–SEC, HPAEC-PAD, GC, GC–MS, 1D and 2D NMR spectroscopy. All the O-polysaccharide samples showed a similar bimodal molecular mass distribution, but differed with respect to the amount and position of O-acetylation and glucosylation. For some strains, O-acetyl groups were found not only on C-2 of abequose (factor 5 specificity), but also on C-2 and C-3 of rhamnose; glucose was found to be linked 1→4 or 1→6 to galactose in different amounts according to the strain of origin.This structural variability could have an impact on the immunogenicity of corresponding glycoconjugate vaccines and different strains need to be evaluated in order to identify the appropriate source of O-polysaccharide to use for the development of a candidate conjugate vaccine with broad coverage against S. Typhimurium.

Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X

Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM197 as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.

Novel Strategies for Development of Next-Generation Glycoconjugate Vaccines

Most bacterial pathogens are decorated with surface glycans called capsular polysaccharides (CPSs). Each CPS has a unique structure that is distinctively recognized by our immune cells. These polysaccharides are important vaccine candidates given that they are located on the surface of pathogens, are easily accessible by the immune system, and often result in formation of protective antibodies. To induce CPS specific adaptive immune response (i.e., T cell-mediated B cell response), CPSs are conjugated with carrier proteins, and the conjugation products are called glycoconjugate vaccines. Immunization with glycoconjugate vaccines has had significant health benefits in controlling infections caused by Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis. However, owing to insufficient understanding of their immune activation mechanisms, glycoconjugate vaccines have been designed and synthesized empirically. In recent years, we have witnessed important advancements in the glycoconjugate vaccine field: the discovery of the mechanism of action for glycoconjugate vaccines, a novel in vivo conjugation strategy, and progress in the use of novel carriers. These studies will be reviewed in detail herein.

Immunoactivity of Protein Conjugates of Carba Analogues from Neisseria meningitidis A Capsular Polysaccharide

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that is a major cause of epidemic meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of a liquid glycoconjugate vaccine against MenA are hampered by the poor hydrolytic stability of its capsular polysaccharide (CPS), consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to generate a carbocyclic analogue leads to enhancement of its chemical stability. Herein, we report conjugation of carbocyclic analogue monomer, dimer, and trimer to the protein carrier CRM197. After immunization in mice, only the conjugated trimer was able to induce specific anti-MenA polysaccharide IgG antibodies with in vitro bactericidal activity, although to a lesser extent than pentadecamer and hexamer oligomers obtained from mild acid hydrolysis of the native polysaccharide conjugated to the same protein carrier. This study represents the first proof-of-concept that hydrolytically stable structural analogues of saccharide antigens can be used for the development of efficacious antimicrobial preventative therapies. Conjugates with longer carbocyclic oligomers and/or precise acetylation patterns could further increase the induced immune response to a level comparable with those of commercially available anti-meningococcal glycoconjugate vaccines.

Simple polysaccharides and global responses in the era of vaccinomics

Simple polysaccharides and global responses in the era of vaccinomics

Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines

Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM197, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM197 showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM197 before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM197. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins.

Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates

Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP < 47 (median DP 25) and DP < 100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS(E) analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP < 47 and DP < 100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.

Long-term seroprotection after an adolescent booster meningococcal serogroup C vaccination

ObjectivesTo determine long-term seroprotection after serogroup C meningococcal (MenC) vaccination at the age of 9–12 years, with or without booster vaccination at the age of 13–15 years.DesignObservational cohort study; follow-on...

Management of secondary immune deficiencies

Management of secondary immune deficiencies

O19.6 Use of Surfactant Vesicles as a Potential Gonococcal Vaccine Delivery System to Generate Antibody Against Neisserial Lipooligosaccharide

BackgroundTo date, no one has been able to exploit the immunological potential of neisserial lipooligosaccharide (LOS) as a vaccine candidate.MethodsWe have developed a glycoconjugate vaccine (TRIAD) that contains native LOS...

Sustained Protection in Mice Immunized with Fractional Doses of Salmonella Enteritidis Core and O Polysaccharide-Flagellin Glycoconjugates

Non-typhoidal Salmonella (NTS) serovars S. Enteritidis and S. Typhimurium are a major cause of invasive bacterial disease (e.g., bacteremia, meningitis) in infants and young children in sub-Saharan Africa and also occasionally cause invasive disease in highly susceptible hosts (young infants, the elderly, and immunocompromised subjects) in industrialized countries. No licensed vaccines exist against human NTS infections. NTS core and O polysaccharide (COPS) and FliC (Phase 1 flagellin subunits) each constitute protective antigens in murine models. S. Enteritidis COPS conjugated to FliC represents a promising vaccine approach that elicits binding and opsonophagocytic antibodies and protects mice against lethal challenge with virulent S. Enteritidis. We examined the protective efficacy of fractional dosages of S. Enteritidis COPS:FliC conjugate vaccines in mice, and also established that protection can be passively transferred to naïve mice by administering sera from mice immunized with conjugate. Mice were immunized with three doses of either 10 µg, 2.5 µg (full dose), 0.25 µg, or 0.025 µg S. Enteritidis COPS:FliC conjugate at 28 day intervals. Antibody titers to COPS and FliC measured by ELISA fell consonant with progressively smaller vaccine dosage levels; anti-FliC IgG responses remained robust at fractional dosages for which anti-COPS serum IgG titers were decreased. Nevertheless, >90% protection against intraperitoneal challenge was observed in mice immunized with fractional dosages of conjugate that elicited diminished titers to both FliC and COPS. Passive transfer of immune sera from mice immunized with the highest dose of COPS:FliC to naïve mice was also protective, demonstrating the role of antibodies in mediating protection. These results provide important insights regarding the potency of Salmonella glycoconjugate vaccines that use flagellin as a carrier protein.