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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-CRM197. We conclude that shape is a key attribute over size to be considered for glycoconjugate vaccine development. [ABSTRACT FROM AUTHOR]

Differential SAR Interferometry is a largely exploited technique to study ground deformations. A key application is the detection of the effects promoted by earthquakes, including detailed variations in ground deformations at different scales. In this work, an implemented Python script (Snap2DQuake) based on the "snappy" module by SNAP software 9.0.8 (ESA) for the processing of satellite imagery is proposed. Snap2DQuake is aimed at producing detailed coseismic deformation maps using Sentinel-1 C-band data by the DInSAR technique. With this alternative approach, the processing is simplified, and several issues that may occur using the software are solved. The proposed tool has been tested on two case studies: the Mw 6.4 Petrinja earthquake (Croatia, December 2020) and the Mw 5.7 to Mw 6.3 earthquakes, which occurred near Tyrnavós (Greece, March 2021). The earthquakes, which occurred in two different tectonic contexts, are used to test and verify the validity of Snap2DQuake. Snap2DQuake allows us to provide detailed deformation maps along the vertical and E-W directions in perfect agreement with observations reported in previous works. These maps offer new insights into the deformation pattern linked to earthquakes, demonstrating the reliability of Snap2DQuake as an alternative tool for users working on different applications, even with basic coding skills. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

The presence of a set of well-known turbidite successions, deposited in progressively E-migrating foredeep basins and subsequently piled up with east vergence, makes the Northern Apennines of Italy paradigmatic of the evolution of deepwater fold-and-thrust belts. This study focuses on the early Apenninic collisional stage, early Miocene in age, which led to the accretion of the turbidites of the Trasimeno Tectonic Wedge (TTW), in the central part of the Northern Apennines. Based on the interpretation of previously unpublished seismic reflection profiles with new surface geology data and tectonic balancing, we present a detailed tectonic reconstruction of the TTW. In the study area, the TTW is characterized by a W-dipping shaly basal décollement located at a depth of 1–5 km. The tectonic wedge is c. 5 km thick at its central-western part and tapers progressively eastwards to c. 1 km. The total shortening, balanced along a 33 km long cross-section, is c. 60 km, including 20 km (40%) of internal imbrication, c. 23 km of horizontal ENE-wards translation along the basal décollement and c. 17 km of passive translation caused by the later shortening of footwall units. Deformation balancing, constrained through upper Aquitanian – upper Burdigalian (c. 21–16 Ma) biostratigraphy, provides an average shortening rate of c. 8.6 mm a–1. Internal shortening of the TTW shows an average shortening rate of c. 4 mm a–1 for this period. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 CRM197 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. [ABSTRACT FROM AUTHOR]

Group B Streptococcus type III (GBSIII) is the most relevant serotype among GBS strains causing infections and the potential of its capsular polysaccharide conjugated to a protein carrier as vaccine is well documented. Polysaccharide from GBSIII (PSIII) can form helical structures in solution where negatively charged sialic acid residues would be disposed externally providing stabilization to the helix. A peculiar high affinity to specific monoclonal antibodies (mAbs) has been reported for PSIII, and fragments of diverse size bind to mAbs in a length dependent manner. These data have been rationalized in terms of conformational epitopes that would be formed by fragments with > 4 saccharidic repeating units. Saturation Transfer Difference NMR experiments have demonstrated that the sialic acid residue is not involved in antibody recognition. However the molecular basis of the interaction between PSIII and mAbs has not been fully elucidated. An important prerequisite to achieve this would be the availability of the three possible sugar sequences representing the pentasaccharide PSIII repeating unit. Herein we established a [2 + 3] convergent approach leading to these three pentasaccharides (1-3) with the end terminal sugar bearing a linker for possible conjugation. The PSIII fragments were coupled to the genetically detoxified diphtheria toxin CRM197 to prove by ELISA that the three pentasaccharides are recognized by polyclonal anti-PSIII serum. The presence of the branching formed by a Glc residue β-(1→6) linked to GlcNAc was proven an important motif for antibody recognition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [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]

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 CRM197. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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.)

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.)

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.)

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.