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Protein self-assembling nanoparticles (NPs) can be used as carriers for antigen delivery to increase vaccine immunogenicity. NPs mimic the majority of invading pathogens, inducing a robust adaptive immune response and long-lasting protective immunity. In this context, we investigated the potential of NPs of different sizes and shapes-ring-, rod-like, and spherical particles-as carriers for bacterial oligosaccharides by evaluating in murine models the role of these parameters on the immune response. Oligosaccharides from Neisseria meningitidis type W capsular polysaccharide were conjugated to ring-shape or nanotubes of engineered Pseudomonas aeruginosa Hemolysin-corregulated protein 1 (Hcp1cc) and to spherical Helicobacter pylori ferritin. Glycoconjugated NPs were characterized using advanced technologies such as High-Performance Liquid Chromatography (HPLC), Asymmetric Flow-Field Flow fractionation (AF4), and Transmission electron microscopy (TEM) to verify their correct assembly, dimensions, and glycosylation degrees. Our results showed that spherical ferritin was able to induce the highest immune response in mice against the saccharide antigen compared to the other glycoconjugate NPs, with increased bactericidal activity compared to benchmark MenW-CRM 197 . We conclude that shape is a key attribute over size to be considered for glycoconjugate vaccine development.

Glycoconjugate vaccines so far licensed are generally composed of a native or size-reduced capsular polysaccharide conjugated to carrier proteins. Detailed information on the structural requirements necessary for CPS recognition is becoming the key to accelerating the development of next-generation improved glycoconjugate vaccines. Structural glycobiology studies using oligosaccharides (OS) complexed with functional monoclonal antibodies represent a powerful tool for gaining information on CPS immunological determinants at the atomic level. Herein, the minimal structural epitope of Haemophilus influenzae type b (Hib) CPS recognized by a functional human monoclonal antibody (hmAb) is reported. Short and well-defined Hib oligosaccharides originating from the depolymerization of the native CPS have been used to elucidate saccharide-mAb interactions by using a multidisciplinary approach combining surface plasmon resonance (SPR), saturation transfer difference-nanomagnetic resonance (STD-NMR), and X-ray crystallography. Our study demonstrates that the minimal structural epitope of Hib is comprised within two repeating units (RUs) where ribose and ribitol are directly engaged in the hmAb interaction, and the binding pocket fully accommodates two RUs without any additional involvement of a third one. Understanding saccharide antigen structural characteristics can provide the basis for the design of innovative glycoconjugate vaccines based on alternative technologies, such as synthetic or enzymatic approaches., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

QUIN database integrates and organizes structural-geological information from published and unpublished sources to constrain deformation in seismotectonic studies. The initial release, QUIN1.0, comprised 3,339 Fault Striation Pairs, mapped on 445 sites exposed along the Quaternary faults of central Italy. The present Data Descriptor introduces the QUIN 2.0 release, which includes 4,297 Fault Striation Pairs on 738 Structural Sites from southern Italy. The newly investigated faults span ~500 km along the Apennines chain, with strikes transitioning from ~SE to ~SW and comprehensively details Fault Striation Pairs' location, attitude, kinematics, and deformation axes. Additionally, it offers a shapefile of the fault traces hosting the data. The QUIN 2.0 release offers a significant geographic extension to the QUIN 1.0, with comprehensive description of local geometric-kinematic complexities of the regional pattern. The QUIN data may be especially relevant for constraining intra-Apennine potential seismogenic deformation patterns, where earthquake data only offer scattered or incomplete information. QUIN's data will support studies aimed at enhancing geological understanding, hazard assessment and comprehension of fault rupture propagation and barriers., (© 2024. The Author(s).)

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., (© 2023. Springer Nature Limited.)

Multivalent vaccines addressing an increasing number of Streptococcus pneumoniae types (7-, 10-, 13-, 15-, 20-valent) have been licensed over the last 22 years. The use of polysaccharide-protein conjugate vaccines has been pivotal in reducing the incidence of invasive pneumococcal disease despite the emergence of non-vaccine serotypes. Notwithstanding its undoubtable success, some weaknesses have called for continuous improvement of pneumococcal vaccination. For instance, despite their inclusion in pneumococcal conjugate vaccines, there are challenges associated with some serotypes. In particular, Streptococcus pneumoniae type 3 remains a major cause of invasive pneumococcal disease in several countries.Here a deep revision of the strengths and weaknesses of the licensed pneumococcal conjugate vaccines and other vaccine candidates currently in clinical development is reported., (© 2023. The Author(s).)

Carbohydrates are abundantly expressed on the surface of both eukaryotic and prokaryotic cells, often as post translational modifications of proteins. Glycoproteins are recognized by the immune system and can trigger both innate and humoral responses. This feature has been harnessed to generate vaccines against polysaccharide-encapsulated bacteria such as Streptococcus pneumoniae, Hemophilus influenzae type b and Neisseria meningitidis. In cancer, glycosylation plays a pivotal role in malignancy development and progression. Since glycans are specifically expressed on the surface of tumor cells, they have been targeted for the discovery of anticancer preventive and therapeutic treatments, such as vaccines and monoclonal antibodies. Despite the various efforts made over the last years, resulting in a series of clinical studies, attempts of vaccination with carbohydrate-based candidates have proven unsuccessful, primarily due to the immune tolerance often associated with these glycans. New strategies are thus deployed to enhance carbohydrate-based cancer vaccines. Moreover, lessons learned from glycan immunobiology paved the way to the development of new monoclonal antibodies specifically designed to recognize cancer-bound carbohydrates and induce tumor cell killing. Herein we provide an overview of the immunological principles behind the immune response towards glycans and glycoconjugates and the approaches exploited at both preclinical and clinical level to target cancer-associated glycans for the development of vaccines and therapeutic monoclonal antibodies. We also discuss gaps and opportunities to successfully advance glycan-directed cancer therapies, which could provide patients with innovative and effective treatments., Competing Interests: Declaration of Competing Interest C.S., F.P., F.C., S.P. and R.A. are employees of the GSK group of companies. The authors declare no other non-financial relationships and activities or conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Studying faults capable of releasing moderate-to-strong earthquakes is fundamental for seismic hazard studies, especially in a territory that was subject to the strongest peninsular Italy earthquake (1857, M w 7.1) and hosting the largest European oil field on-land. Fieldwork-based observations in the Campania-Lucania area highlight a SSW-dipping ~ 65 km-long normal-oblique-segmented fault, showing evidence of recent activity and possibly responsible for the 1857 earthquake. It crosses the Maddalena ridge, linking separate Quaternary basins. Two seismic reflection profiles cross the fault trace where it is buried beneath the Val d'Agri Quaternary deposits. Similarities between fault-controlled small basins in the highest portion of the massifs in the study area and the neighboring 1980 Irpinia area (1980 earthquake, M w 6.9) are interpreted as evidence of trans-ridge fault activity. Kinematic analyses and the stress field inversion provide a N032-trending near-horizontal s3-axis, the same computed in literature for the Irpinia area, highlighting a deviation from the ~N045-axis which characterizes most of the Apennines. This study demonstrates how detailed fieldwork, supported by geophysics and innovative data analysis techniques, can unravel unknown faults while giving a novel interpretation of the trans-ridge faults' style in controlling strong earthquakes, moving away from classical interpretations, and providing a helpful approach in similar contexts worldwide., (© 2022. The Author(s).)

Large magnitude earthquakes produce complex surface deformations, which are typically mapped by field geologists within the months following the mainshock. We present detailed maps of the surface deformation pattern produced by the M. Vettore Fault System during the October 2016 earthquakes in central Italy, derived from ALOS-2 SAR data, via DInSAR technique. On these maps, we trace a set of cross-sections to analyse the coseismic vertical displacement, essential to identify both surface fault ruptures and off-fault deformations. At a local scale, we identify a large number of surface ruptures, in agreement with those observed in the field. At a larger scale, the inferred coseismic deformation shows a typical long-wavelength convex curvature of the subsiding block, not directly recognizable in the field. The detection of deformation patterns from DInSAR technique can furnish important constraints on the activated fault segments, their spatial distribution and interaction soon after the seismic events. Thanks to the large availability of satellite SAR acquisitions, the proposed methodological approach can be potentially applied to worldwide earthquakes (according to the environmental characteristics of the sensed scene) to provide a wider and faster picture of surface ruptures. Thus, the derived information can be crucial for emergency management by civil protection and helpful to drive and support the geological field surveys during an ongoing seismic crisis., (© 2022. The Author(s).)

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity., Competing Interests: FC, DV, LDI, DO, EB, BB, FA, LDB, IM, MRR, and RA are employees of the GSK group of companies. AK was hosted in a secondment in GSK during her PhD programme. Prevnar and HibTITER are trademarks from Pfizer; Vaxem-Hib, Menveo and Menjugate are trademarks from GSK., (This journal is © The Royal Society of Chemistry.)

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.

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.)

Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) consists of (1 → 6)-2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, O-acetylated at position C3 or C4. Glycomimetics appear attractive to overcome the CPS intrinsic lability in physiological media, due to cleavage of the phosphodiester bridge, and to develop a stable vaccine with longer shelf life in liquid formulation. Here, we generate a series of non-acetylated carbaMenA oligomers which are proven more stable than the CPS. An octamer (DP8) inhibits the binding of a MenA specific bactericidal mAb and polyclonal serum to the CPS, and is selected for further in vivo testing. However, its CRM 197 conjugate raises murine antibodies towards the non-acetylated CPS backbone, but not the natural acetylated form. Accordingly, random O-acetylation of the DP8 is performed, resulting in a structure (Ac-carbaMenA) showing improved inhibition of anti-MenA CPS antibody binding and, after conjugation to CRM 197 , eliciting anti-MenA protective murine antibodies, comparably to the vaccine benchmark.

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., Competing Interests: Declaration of Competing Interest GB, IP, FC, MRR, DO, UD and RA are employees of the GSK group of companies. RA and DO are owners of GSK stocks. Other authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Invited for the cover of this issue is the group of Roberto Adamo at GlaxoSmithKline Research Center, Siena, and colleagues at The University of the Basque Country and Basque Research Technology Alliance. The image depicts a tactical plan with the different elements of the research as part of the team. Read the full text of the article at 10.1002/chem.202000284., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.)

The well-known Toll like receptor 9 (TLR9) agonist CpG ODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, however its in vivo stability and potential systemic toxicity remain a concern. In an effort to overcome these issues, different strategies have been explored including conjugation of CpG ODN with proteins or encapsulation/adsorption of CpG ODN into/onto liposomes. Although these methods have resulted in enhanced immunopotency compared to co-administration of free CpG ODN and antigen, we believe that this effect could be further improved. Here, we designed a novel delivery system of CpG ODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate model protein with the CpG ODN 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) in a very high degree. The novel cationic liposomes-protein conjugate complex shared similar vesicle characteristics (size and charge) compared to free liposomes. The conjugation of CpG ODN to protein in conjunction with adsorption on cationic liposomes, could promote co-delivery leading to the induction of immune response at low antigen and CpG ODN doses.•The CpG ODN Toll-like receptor (TLR) 9 agonist was conjugated to protein antigens via thiol-maleimide chemistry.•Due to their negative charge, protein conjugates readily electrostatically bound cationic liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA) resulting to the design of novel cationic liposomes-protein conjugate complexes.•The method is suited for the liposomal delivery of a variety of adjuvant-protein conjugates., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: FC, MRR, DO and RA are employees of the GSK group of companies. RA and DO are owners of GSK stocks. Other authors declare no conflict of interest., (© 2020 The Authors.)

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.)

Introduction : Over the last decades, glycoconjugate vaccines have been proven to be a successful strategy to prevent infectious diseases. Many diseases remain to be controlled, especially in developing countries, and emerging antibiotic-resistant bacteria present an alarming public-health threat. The increasing complexity of future vaccines, and the need to accelerate development processes have triggered the development of faster approaches to glycoconjugate vaccines design. Areas covered : This review provides an overview of recent progress in glycoconjugation technologies toward faster vaccine design. Expert opinion : Among the different emerging approaches, glycoengineering has the potential to combine glycan assembly and conjugation to carrier systems (such as proteins or outer membrane vesicles) in one step, resulting in a simplified manufacturing process and fewer analytical controls. Chemical and enzymatic strategies, and their automation can facilitate glycoepitope identification for vaccine design. Other approaches, such as the liposomal encapsulation of polysaccharides, potentially enable fast and easy combination of numerous antigens in the same formulation. Additional progress is envisaged in the near future, and some of these systems still need to be further validated in humans. In parallel, new strategies are needed to accelerate the vaccine development process, including the associated clinical trials, up to vaccine release onto the market.

Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of Enterococcus faecalis , and the secreted antigen A (SagA), an immunogenic protein from Enterococcus faecium , are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against E. faecalis type 2, and SagA.01 showed 40% killing against E. faecium 11231/6. In addition, both MAbs showed cross-reactivity toward other E. faecalis and E. faecium strains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections., (Copyright © 2019 Kalfopoulou et al.)

The tetrasaccharide (2- O -methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-α-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose) from the major exosporium protein (BclA) of Bacillus anthracis has been proposed as a target for development of diagnostics and immune therapy or prophylaxis. While the immunodominant character of the anthrose residue has been previously elucidated, the role of the stereochemical configuration of the downstream rhamnose is unknown. Because the linkage of this residue to the GlcNAc bridging the glycan and the protein is lost during isolation of the tetrasaccharide, its α- and β-glycoforms have been synthesized. Herein, we prepared neoglycoconjugates from a series of fragments of the tetrasaccharide, including the complete α- and β-tetrasaccharide glycoforms, a 2-demethoxylated version of the α-tetrasaccharide, and the α- and β-trirhamnosides and CRM 197 . By immunization of mice, we showed that the anti α- and β-tetrasaccharide serum equally recognized both glycoforms. In contrast the sera produced following immunization with the α- and β-trirhamnoside fragments exhibited higher recognition for their own antigens than for their anomeric counterparts. The anti α- and β-tetrasaccharide sera recognized Sterne spores in a comparable fashion. ΔBclA spores not expressing the major exosporium protein were also recognized by the same sera, while mutants that produced the carbohydrate antigen with deletion of either rhamnose or anthrose were not. The tetrasaccharide could, therefore, be expressed in proteins other than BlcA. This work proves that α- and β-tetrasaccharide are equally potent immunogens.

A four-membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site-selective bis-alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb-Aβ against the human Amyloid-β peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM 197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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.)

We have recently described a method for tyrosine-ligation of complex glycans that was proven efficient for the site selective coupling of GBS capsular polysaccharides (PSs). Herein, we explored the effect of conjugation of type V polysaccharide onto predetermined lysine or tyrosine residues of the GBS67 pilus protein with the dual role of T-cell carrier for the PS and antigen. For the preparation of a conjugate at predetermined lysine residues of the protein, we investigated a two-step procedure based on microbial Transglutaminase (mTGase) catalyzed insertion of a tag bearing an azide for following copper-free strain-promoted azide-alkyne [3 + 2] cycloaddition (SPAAC) with the polysaccharide. Two glycoconjugates were obtained by tyrosine-ligation through the known SPAAC and a novel thiol-maleimide addition based approach. Controls were prepared by random conjugation of PSV to GBS67 and CRM197, a carrier protein present in many commercial vaccines. Immunological evaluation in mice showed that all the site-directed constructs were able to induce good levels of anti-polysaccharide and anti-protein antibodies inducing osponophagocytic killing of strains expressing individually PSV or GBS67. GBS67 randomly conjugated to PSV showed carrier properties similar to CRM197. Among the tested site-directed conjugates, tyrosine-directed ligation and thiol-malemide addition was elected as the best combination to ensure production of anti-polysaccharide and anti-protein functional antibodies (in vitro opsonophagocytic killing titers) comparable to the controls made by random conjugation, while avoiding anti-linker antibodies. Our findings demonstrate that (i) mTGase based conjugation at lysine residues is an alternative approach for the synthesis of large capsular polysaccharide-protein conjugates; (ii) GBS67 can be used with the dual role of antigen and carrier for PSV; and (iii) thiol-maleimide addition in combination with tyrosine-ligation ensures the production of anti-polysaccharide and anti-protein functional antibodies while maintaining low levels of anti-linker antibodies. Site-specific conjugation methods aid in defining conjugation site and chemistry in carbohydrate-protein conjugates.

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.

Background: Outer membrane vesicles (OMVs) from Gram-negative bacteria are gaining increasing attention as vaccine platform for their built-in adjuvanticity and for their potential use as carriers of heterologous antigens. These 2 properties offer the opportunity to make highly effective, easy to produce multi-valent vaccines. OMVs can be loaded with foreign antigens by targeting protein expression either to the outer membrane or to the periplasm of the OMV-producing strain. Periplasmic expression is simple and relatively efficient but leads to the accumulation of recombinant antigens in the lumen of OMVs and the ability of OMVs carrying internalized antigens to induce antigen-specific antibody responses has been only marginally investigated and is considered to be sub-optimal., Methods: We have systematically analyzed in qualitative and quantitative terms antibody responses induced by OMVs carrying different heterologous antigens in their lumen. Group A Streptococcus (GAS) Slo, SpyCEP, Spy0269 and Group B Streptococcus (GBS) SAM_1372 were fused to the OmpA leader sequence for secretion and expressed in Escherichia coli. OMVs from the recombinant strains were purified and tested for immunogenicity and protective activity., Results: All proteins were incorporated into the OMVs lumen in their native conformation. Upon mice immunization, OMVs induced high functional antibody titers against the recombinant proteins. Furthermore, immunization with Slo-OMVs and SpyCEP-OMVs protected mice against GAS lethal challenge., Conclusions: The efficiency of antigen delivery to the vesicular lumen via periplasmic expression, and the surprisingly high immunogenicity and protective activity of OMVs carrying internalized recombinant antigens further strengthens the potential of OMVs as vaccine platform.

Class D β-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae. Carbapenemase activity is an unusual feature among class D β-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands β5 and β6 (the β5-β6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D β-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the β5-β6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10-derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted β5-β6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the β5-β6 loop in the carbapenemase activity of class D β-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.