Search

GlaxoSmithKline (GSK) is currently developing a fully liquid presentation to ease the administration of the licensed quadrivalent conjugate vaccine (Menveo) against meningococcal serogroup A, C, W, and Y (MenACWY) infections. Herein, we report a new method for determining the free saccharide (FS) content of CRM

The capsular polysaccharide of the human pathogen Group B Streptococcus is a key virulence factor and vaccine candidate that induces protective antibodies when conjugated to carrier proteins. It consists of long polymeric chains of oligosaccharide repeating units, and each of the ten capsular serotypes described so far presents a unique chemical structure with distinct antigenic properties; therefore, broad protection against this pathogen could be achieved by a combination of ten glycoconjugates. Capsular polysaccharide biosynthesis and assembly follow a polymerase-dependent pathway that is widespread in encapsulated bacteria and is encoded by a polycistronic operon. Here we exploited the sequence similarity between the capsule operons of types V and IX to generate hybrid polysaccharides incorporating epitopes of both serotypes in a single molecule, by co-expressing their specific CpsM, O, I glycosyltransferases in a single isolate. Physicochemical and immunochemical methods confirmed that an engineered strain produced a high molecular weight chimeric polysaccharide, combining antigenic specificities of both type V and IX. By optimizing the copy number of key glycosyltransferase genes, we were able to modulate the ratio between type-specific epitopes. Finally, vaccination with chimeric glycoconjugates significantly decreased the incidence of disease in pups born from immunized mice challenged with either serotype. This study provides proof of concept for a new generation of glycoconjugate vaccines that combine the antigenic specificity of different polysaccharide variants in a single molecule, eliciting a protective immune response against multiple serotype variants.

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.

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.

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.

Copper binding to α-synuclein (aS) and to amyloid-β (Ab) has been connected to Parkinson's and Alzheimer's disease (AD), respectively, because Cu ions can modulate the peptide aggregation, and these Cu ⋅ peptide complexes can catalyse the production of reactive oxygen species (ROS). In a significant proportion of AD brains, aggregation of aS and Ab has been detected, and it was proposed that Ab and aS interact with each other. Thus, we investigated the potential interactions of Ab and aS through their binding of copper(I) and copper(II). Additionally, β-synuclein (bS) was investigated, due to its additional methionine residue, a potential Cu(I) ligand. We found that: 1) the peptides containing the Cu-binding domains Ab1-16, aS1-15 and bS1-15 have similar affinities towards Cu(II) and towards Cu(I), with Ab1-16 being slightly stronger, 2) in the case of Cu(I), the additional Met residue in bS1-15 increased the affinity slightly, 3) the exchange of Cu(I/II) between the two peptides is rapid (≤ ms), 4) a/bS1-15 and Ab1-16 form a heterodimeric complex with Cu(II), 5) Cu(I) probably promotes a transient ternary complex, 6) the different Cu(I/II) coordination of Ab1-16, aS1-15 and bS1-15 impacts the capacity to produce ROS and to oxidise catechol, and 7) when Ab1-16, aS1-15 and Cu are present, the ROS production more closely resembles that by Ab1-16. The work gives insights into the coordination chemistry of these related peptides, and the relevance of coordination differences, the ternary complex and ROS production are discussed.

This article describes the characterization of the antigenic determinant of the capsular polysaccharide from the clinically relevant serotype III of group B Streptococcus (GBS). NMR and X-ray crystallography have been applied to elucidate the interaction of type III GBS oligosaccharides obtained by synthetic and depolymerization procedures of the bacterial polysaccharide with a functional monoclonal antibody. A Fab–GBS oligosaccharide complex structure has been solved at high resolution (2.7 Å). The results demonstrate the existence of a sialic acid-dependent functional epitope of GBS that is fully contained within four consecutive sugars deriving from the type III GBS polysaccharide backbone and one branched disaccharide present in this sequence. This finding has implications for the development of vaccines against GBS infection.

α-Synuclein (αS) is the main protein component of Lewy bodies, characterizing the pathogenesis of Parkinson's disease. αS is unstructured in solution but adopts a helical structure in its extended N-terminal segment upon association with membranes. In vitro the protein binds avidly Cu(II), but in vivo the protein is N-acetylated, and Cu(II) binding is lost. We have now clarified the binding characteristics of the Cu(I) complex with the truncated αS peptide 1-15, both in N-acetylated and free amine forms, in a membrane mimetic environment and found that complexation occurs with a 1:2 Cu(I)-αS stoichiometry, where Cu(I) is bound to Met1 and Met5 residues of two helical peptide chains. The resulting tetrahedral Cu(I) center is redox-stable, does not form reactive oxygen species, and is unreactive against dopamine in the presence of O2. This suggests that, unlike cytosolic Cu(I)-αS, which retains the capacity to activate O2 and promote oxidative reactions, membrane-bound Cu(I)-αS may serve as a sink for unreactive copper.

The tetrasaccharide (2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-&alpha, d-glucopyranosyl-(1&rarr, 3)-&alpha, l-rhamnopyranosyl-(1&rarr, 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 &alpha, and &beta, glycoforms have been synthesized. Herein, we prepared neoglycoconjugates from a series of fragments of the tetrasaccharide, including the complete &alpha, tetrasaccharide glycoforms, a 2-demethoxylated version of the &alpha, tetrasaccharide, and the &alpha, trirhamnosides and CRM197. By immunization of mice, we showed that the anti &alpha, tetrasaccharide serum equally recognized both glycoforms. In contrast the sera produced following immunization with the &alpha, trirhamnoside fragments exhibited higher recognition for their own antigens than for their anomeric counterparts. The anti &alpha, tetrasaccharide sera recognized Sterne spores in a comparable fashion. &Delta, BclA 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 &alpha, tetrasaccharide are equally potent immunogens.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of abnormal α-synuclein (αS) deposits in the brain. Alterations in homeostasis and metal-induced oxidative stress may play a crucial role in the progression of αS amyloid assembly and pathogenesis of PD. Contrary to αS, β-synuclein (βS) is not involved in the PD etiology. However, it has been suggested that the βS/αS ratio is altered in PD, indicating that a correct balance of these two proteins is implicated in the inhibition of αS aggregation. αS and βS share similar abilities to coordinate Cu(II). In this study, we investigated and compared the interaction of Cu(I) with the N-terminal portion of βS and αS by means of NMR, circular dichroism, and X-ray absorption spectroscopies. Our data show the importance of M10K mutation, which induces different Cu(I) chemical environments. Coordination modes 3S1O and 2S2O were identified for βS and αS, respectively. These new insights into the bioinorganic chemistry of copper and synuclein proteins are a basis to understand the molecular mechanism by which βS might inhibit αS aggregation.

Furin-dependent maturation of the BRI2 protein generates the Bri2-23 fragment that is able to arrest the aggregation of amyloidβ, the peptide implicated in Alzheimer's disease (AD). Bri2-23 contains cysteines at positions 5 and 22, which are likely to bind to metal ions such as Cu(i). Metal ions may play a role in the etiology of neurodegenerative disorders such as AD, and in this work we explore the metal ion induced folding and aggregation of Bri2-23 using Hg(ii) and Ag(i) as spectroscopic probes with structural and ligand preferences similar to those of Cu(i), while not displaying redox activity under the experimental conditions. In general, interaction of Bri2-23 with soft metal ions changes the structural properties and solution behavior of the peptide that tune to increasing metal to peptide stoichiometry. Potentiometric, (199m)Hg PAC and ESI-MS data indicate that addition of up to 0.5 equivalents of Hg(ii) to Bri2-23 yields a two-coordinated HgS2 structure at the metal site. While the free peptide is inherently unstructured, the presence of Ag(i) and Hg(ii) gives rise to β-sheet formation. NMR spectroscopy supports the formation of β-sheet structure in the presence of 0.5 equivalents of Hg(ii), and displays an interesting and marked change in the TOCSY spectra when increasing the Hg(ii) to peptide stoichiometry from 0.5 to 0.7 equivalents, indicating the equilibrium between two structural analogues of the complex. Addition of more than 0.7 equivalents of Hg(ii) gives rise to line broadening, presumably reflecting aggregation. This is further supported by ThT fluorescence studies showing that the Bri2-23 peptide does not aggregate over 24 hours, while addition of over 0.7 equivalents of Ag(i) or Hg(ii) leads to increase of fluorescence, indicating that these metal ions induce aggregation. Thus, a model integrating all data into a coherent picture is that the metal ion binding to the two thiolates gives rise to folding of the peptide into a structure that is prone to aggregation, forming aggregates with a considerable amount of β-sheets. Molecular dynamics simulations initiated with structures that agree with NMR data additionally support this model.

Essential main-group elements [e.g., Na(I) and Ca(II)] and transition metal ions [e.g., Cu(II)/Cu(I), Fe(III)/Fe(II), Zn(II), and Ni(II)] play key roles in the structural organization and biological function of many macromolecules such as proteins, DNA, and RNA. Healthy conditions require tight regulation of metal concentrations inside and outside cells, and both metal deficiency and overload can lead to cellular dysfunction. Altered metabolism of transition metal ions is implicated in severe and chronic diseases, including cancer, neurodegenerative disorders, and microbial infection. Nature has developed a sophisticated machinery to balance the content of transition metal ions. Many enzymes, transporters, and chaperones are involved in these complex processes and control metal uptake and delivery to specific cellular domains. Many efforts have been devoted to clarifying metal interactions with amyloidogenic proteins, metal transporters, and metal storage proteins via different spectroscopic techniques. In this review we describe the application of NMR to determine the metal coordination spheres and structural features of flexible and disordered regions of proteins that are either involved in neurodegenerative processes or are derived from metal chaperones. The systems investigated include (i) copper, iron, and zinc binding to unstructured regions of prion protein, α-synuclein, and amyloid β; (ii) zinc binding to extracellular domains of ZIP proteins; and (iii) zinc and nickel binding to the loop region of HypA, HspA, and SlyD proteins. The NMR behavior of these systems is compared and discussed. The benefits and drawbacks of the methodology are addressed by stressing the tricks and pitfalls encountered.

Des-acyl-ghrelin is a 28 amino acid peptide secreted by both human and rat stomach. Together with ghrelin and obestatin, it is obtained by post-translational modification of a 117 aminoacid prepropeptide mainly expressed in distinct endocrine cell type in the stomach. Although its receptor has not been unambiguously identified so far, des-acyl-ghrelin is considered one of the strongest antagonists of ghrelin in activating the growth hormone secretagogue receptor (GHS-R). Here the secondary structure of des-acyl-ghrelin in different experimental conditions has been investigated and compared with that of obestatin, a bioactive peptide having similar biological functions. CD and NMR techniques have been combined for gaining the desired conformational features. The obtained structures support a steady alpha-helix structure spanning residues from 7 to 14, very similar to that observed for obestatin at the same experimental conditions, leading to suggest that a similar secondary structure can be associated with the similar biological role.