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Microbial surface polysaccharides are important virulence factors and targets for vaccine development. Glycoconjugate vaccines, obtained by covalently linking carbohydrates and proteins, are well established tools for prevention of bacterial infections. Elucidation of the minimal portion involved in the interactions with functional antibodies is of utmost importance for the understanding of their mechanism of induction of protective immune responses and the design of synthetic glycan based vaccines. Typically, this is achieved by combination of different techniques, which include ELISA, glycoarray, Surface Plasmon Resonance in conjunction with approaches for mapping at atomic level the position involved in binding, such as Saturation Transfer NMR and X-ray crystallography. This review provides an overview of the structural studies performed to map glycan epitopes (glycotopes), with focus on the highly complex structure of Group B Streptococcus type III (GBSIII) capsular polysaccharide. Furthermore, it describes the rational process followed to translate the obtained information into the design of a protective glycoconjugate vaccine based on a well-defined synthetic glycan epitope., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment-mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in-silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM 197 , a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X-ray crystallography-guided design of a synthetic carbohydrate-based conjugate vaccine., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Recent structural studies demonstrated that the epitope recognized by a monoclonal antibody representative of the protective response against the type III group B Streptococcus polysaccharide was comprised within 2 of the repeating units that constitute the full-length native structure. In the current study, we took advantage of this discovery to design a novel vaccine based on multivalent presentation of the identified minimal epitope on a carrier protein. We show that highly glycosylated short oligosaccharide conjugates elicit functional immune responses comparable to those of the full-length native polysaccharide. The obtained results pave the way to the design of well-defined glycoconjugate vaccines based on short synthetic oligosaccharides., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines., Competing Interests: Conflict of interest statement: All authors are employees of GSK Vaccines (known as Novartis Vaccines at the time of the study).

Background: Group B Streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. A vaccine targeting pregnant women could protect infants through placentally transferred antibodies. The association between GBS maternal antibody concentrations and the risk of neonatal infection has been investigated in US and African populations. Here we studied naturally acquired immunoglobulin G (IgG) responses to GBS capsular polysaccharides (CPS) and pilus proteins in European pregnant women., Methods: Maternal sera were prospectively collected in 8 EU countries from 473 GBS non-colonized and 984 colonized pregnant women who delivered healthy neonates and from 153 mothers of infants with GBS disease. GBS strains from these colonized women and infected infants were obtained in parallel and their capsular and pilus types were identified by serological and molecular methods. Maternal serum concentrations of IgG anti- Ia, -Ib, -III and -V polysaccharides and anti-BP-1, -AP1-2a and -BP-2b pilus proteins were determined by enzyme-linked immunosorbent assay. Antibody functional activity was quantified by Opsonophagocytic Killing Assay., Results: Antibody levels against CPS and pilus proteins were significantly higher in GBS colonized women delivering healthy babies than in mothers of neonates with GBS disease or non-colonized women. Moreover, maternal anti-capsular IgG concentrations showed a significant correlation with functional titers measured by Opsonophagocytic Killing Assay., Conclusions: Maternal anti-capsular IgG concentrations above 1 µg/mL mediated GBS killing in vitro and were predicted to respectively reduce by 81% (95% confidence interval, 40%-100%) and 78% (45%-100%) the risk of GBS Ia and III early-onset disease in Europe., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates.

A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Proof of concept for a single-dose Group B Streptococcus vaccine based on capsular polysaccharide conjugated to Qβ virus-like particles" published in NPJ Vaccines. The correction states that the author Roberto Adamo was mistakenly omitted from the original article and has now been included in the corrected versions. The document provides the names of all the authors involved in the article. [Extracted from the article]

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full‐length conjugated polysaccharide. Here, starting from the X‐ray crystallographic structure of the polysaccharide fragment–mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in‐silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197, a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X‐ray crystallography‐guided design of a synthetic carbohydrate‐based conjugate vaccine. [ABSTRACT FROM AUTHOR]

Keywords: carbohydrates; glycoconjugates; immunology; streptococcus; structural glycobiology; vaccines EN carbohydrates glycoconjugates immunology streptococcus structural glycobiology vaccines 6940 6940 1 06/04/20 20200602 NES 200602 B Group B Streptococcus III b has been considered a model of highly complex glycan epitope. This study underlines the role of structural glycobiology in simplifying the design of vaccines based on complex bacterial polysaccharides. Carbohydrates, glycoconjugates, immunology, streptococcus, structural glycobiology, vaccines. [Extracted from the article]

Although the well-known Toll like receptor 9 (TLR9) agonist CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity remain a concern. In an effort to circumvent these issues, different strategies have been employed to increase its stability, localise action and reduce dosage. These include conjugation of CpGODN with proteins or encapsulation/adsorption of CpGODN into/onto liposomes, and have resulted in enhanced immunopotency compared to co-administration of free CpGODN and antigen. Here, we designed a novel delivery system of CpGODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate the Group B Streptococcus (GBS) GBS67 protein antigen with the CpGODN TLR9 agonist. This treatment did not alter protein's ability to be recognised by specific antibodies or the CpGODN to function as a TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The novel cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following intramuscular immunisation, GBS67-CpGODN+L formed a vaccine depot at the injection site and induced a remarkable increase of functional immune responses against GBS compared to the simple co-administration of GBS67, CpGODN and liposomes. This work demonstrates that the conjugation of CpGODN to GBS67 in conjunction with adsorption on cationic liposomes, can promote co-delivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. Our findings highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms. Unlabelled Image • Delivery system combines synergistically protein-adjuvant conjugate and liposomes. • Immune response was dramatically improved compared to previously published data. • Pathogen Group B Streptococcus was used as model for which there is no vaccine yet. • The methodology could be extended to other agonists/adjuvant liposome formulations. [ABSTRACT FROM AUTHOR]