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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.

Copyright © 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND)., Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A Neisseria meningitidis (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be O-acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the O- and N-acetyl moieties through either H-bonding or CH–π interactions. In silico docking showed that 3-O-acetylation of the upstream residue is essential for antibody binding, while O-acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies., This work was sponsored by GlaxoSmithKline Biologicals and has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement 675671.

Modification of surface antigens and differential expression of virulence factors are frequent strategies pathogens adopt to escape the host immune system. These escape mechanisms make pathogens a “moving target” for our immune system and represent a challenge for the development of vaccines, which require more than one antigen to be efficacious. Therefore, the availability of strategies, which simplify vaccine design, is highly desirable. Bacterial Outer Membrane Vesicles (OMVs) are a promising vaccine platform for their built-in adjuvanticity, ease of purification and flexibility to be engineered with foreign proteins. However, data on if and how OMVs can be engineered with multiple antigens is limited. In this work, we report a multi-antigen expression strategy based on the co-expression of two chimeras, each constituted by head-to-tail fusions of immunogenic proteins, in the same OMV-producing strain. We tested the strategy to develop a vaccine against Staphylococcus aureus, a Gram-positive human pathogen responsible for a large number of community and hospital-acquired diseases. Here we describe an OMV-based vaccine in which four S. aureus virulent factors, ClfAY338A, LukE, SpAKKAA and HlaH35L have been co-expressed in the same OMVs (CLSH-OMVsΔ60). The vaccine elicited antigen-specific antibodies with functional activity, as judged by their capacity to promote opsonophagocytosis and to inhibit Hla-mediated hemolysis, LukED-mediated leukocyte killing, and ClfA-mediated S. aureus binding to fibrinogen. Mice vaccinated with CLSH-OMVsΔ60 were robustly protected from S. aureus challenge in the skin, sepsis and kidney abscess models. This study not only describes a generalized approach to develop easy-to-produce and inexpensive multi-component vaccines, but also proposes a new tetravalent vaccine candidate ready to move to development.

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell–cell communications and in the regulation of immune responses. Through the interaction with glycan‐binding receptors, glycans are able to affect the activation status of antigen‐presenting cells, leading either to induction of pro‐inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco‐chemists and glyco‐immunologists to develop glycan‐based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan‐modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen‐presenting cells, like dendritic cells. In addition, we address how glycan‐based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan‐based therapies, including chimeric antigen receptor (CAR)‐T cells to achieve targeting of tumor‐associated glycan‐specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity., Glycans are macromolecules that perform crucial roles in development, in cell–cell communication, and in the regulation of immune responses. In this review, we highlight how glycans can be exploited and empowered as treatment or vaccine modalities, particularly in the fight against cancer and autoimmune diseases.

Glycoconjugate vaccines are among the most effective interventions for preventing several serious infectious diseases. Covalent linkage of the bacterial capsular polysaccharide to a carrier protein provides CD4(+) T cells with epitopes that facilitate a memory response to the polysaccharide. Classically, the mechanism responsible for antigen processing was thought to be similar to what was known for hapten-carrier conjugates: protease digestion of the carrier protein in the endosome and presentation of a resulting peptide to the T cell receptor on classical peptide-recognizing CD4(+) T cells. Recently, an alternative mechanism has been shown to be responsible for the memory response to some glycoconjugates. Processing of both the protein and the polysaccharide creates glycopeptides in the endosome of antigen-presenting cells. For presentation, the peptide portion of the glycopeptide is bound to MHCII, allowing the covalently linked glycan to activate carbohydrate-specific helper CD4(+) T cells (Tcarbs). Herein, we assessed whether this same mechanism applies to conjugates prepared from other capsular polysaccharides. All of the glycoconjugates tested induced Tcarb-dependent responses except that made with group C Neisseria meningitidis; in the latter case, only peptides generated from the carrier protein were critical for helper T cell recognition. Digestion of this acid-sensitive polysaccharide, a linear homopolymer of α(2 → 9)-linked sialic acid, to the size of the monomeric unit resulted in a dominant CD4(+) T cell response to peptides in the context of MHCII. Our results show that different mechanisms of presentation, based on the structure of the carbohydrate, are operative in response to different glycoconjugate vaccines.

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as “tumor-associated carbohydrate antigens”. Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy.

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Bibersteinia trehalosi, and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis., IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry.

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 CRM197 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 CRM197, eliciting anti-MenA protective murine antibodies, comparably to the vaccine benchmark., The Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) is a component of commercial vaccines, but is unstable. Here, the authors generate glycomimetic oligomers that demonstrate higher stability than their natural counterparts and induce protective antibodies in mice.

The neutrophil-to-lymphocyte ratio (NLR) was shown correlate with disease activity in systemic autoimmunity and MS. In a cohort of 62 children with demyelinating diseases; NLR was high both acutely (P.001) and during remission (P=.01) in AQP4-Ab NMOSD; higher only at time of clinical attack (P.001) but not at time of remission in MOGAD and not different to that of controls neither during relapse nor remission in MS. The temporal correlation of NLR with MOGAD activity could be used as a supportive biomarker for acute relapse, given that the clinical differentiation between relapses and pseudo-relapses can be challenging.

The structures of bacterial cell surface carbohydrates such as capsular polysaccharides, lipopolysaccharides, and teichoic acids can be determined using nuclear magnetic resonance (NMR) spectroscopy. The one-dimensional (1D) proton spectrum constitutes a structural fingerprint of the glycan present which can be assigned using an array of 1D and two-dimensional (2D) NMR homo- and heteronuclear techniques to establish through-bond and space connectivities and thus determine the structure of the repeating unit. The surface carbohydrates can be profiled by high-resolution magic angle spinning (HR-MAS); they play a key role in protecting bacteria from host defenses and so many of these antigens are vaccine targets. Polysaccharide vaccines are being replaced by the more immunogenic glycoconjugate vaccines in which the carbohydrate is covalently attached to an appropriate carrier protein. NMR establishes the identity of the polysaccharide vaccine and can be used to monitor the structural integrity of the attached glycan chains in the conjugate and identify key epitopes.

Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.

Physicochemical technologies are a powerful tool for the structural characterization of vaccine antigens both at bulk level as well as on the final formulation. High-field Nuclear Magnetic Resonance (NMR) spectroscopy has been found to be an extremely and robust tool for tracking the industrial process manufacturing of carbohydrate-based vaccines. I have applied NMR spectroscopy to the characterization of a penta-valent conjugate vaccine against Neisseria meninigitidis group A, C, W, Y (MenACWY) and Haemophilus influenzae type b (Hib) infections, constituted of capsule derived polysaccharide fragments independently conjugated to CRM197 protein carrier (CRM-MenA, CRM-MenC, CRM-MenW, CRM-MenY, CRM-Hib). 1H NMR has been used for the identity testing of the carbohydrate antigens and of the vaccine formulation. The application of NMR-based assays on multivalent conjugate vaccines looks to be a promising approach for identity and stability analyses useful for future vaccines development.

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 streptococcal infections can cause death in neonates up to 3 months of age. Intrapartum antibiotic prophylaxis in GBS-colonized mothers has limited early infections but has no impact after the first week of life. The development of a maternal vaccine to address this unmet medical need has been identified as a priority by the World Health Organization, and the GBS CPSs are considered the best antigen targets. However, to date there are no accepted standardized assays to measure immune responses to the investigational vaccines and for establishment of serocorrelates of protection. Here, we describe the performance of two microsphere-based pentaplex immunoassays for the determination of antibodies recognizing the five most frequent GBS serotypes. Our data confirm that an assay based on biotinylated polysaccharides coupled to streptavidin microspheres would be suitable for the intended purpose., Group B Streptococcus (GBS) infections constitute a major cause of invasive disease during the first three months of life and an unmet medical need that could be addressed by maternal vaccination. The GBS capsular polysaccharides (CPSs) have shown promise as vaccine targets in clinical studies. A highly specific serological assay to quantify maternal and neonatal anti-CPS antibody levels will be instrumental for GBS vaccine licensure. Here, we describe the development and comparison of two novel multiplex immunoassays (MIAs) based on the Luminex technology for the quantification of IgG antibodies recognizing the five most frequent GBS capsular variants (Ia, Ib, II, III, and V) out of the ten types identified. The first assay is based on the use of biotinylated CPSs coupled to streptavidin-derivatized magnetic microspheres (Biotin-CPS MIA), while the second is a sandwich assay with plain CPSs coupled to magnetic microspheres coated with polysaccharide-specific mouse monoclonal antibodies (Sandwich MIA). Both assays showed good specificity, linearity, and precision, although the Biotin-CPS MIA presented higher sensitivity and lower complexity than the Sandwich MIA. A panel of human sera representing a wide range of anti-CPS IgG concentrations was tested in parallel by the two assays, which resulted in comparable titers. Our data support the preservation of antigenic epitopes in the biotinylated polysaccharides and the suitability of the Biotin-CPS MIA for the precise determination of GBS anti-CPS IgG concentrations in human sera. IMPORTANCE Group B streptococcal infections can cause death in neonates up to 3 months of age. Intrapartum antibiotic prophylaxis in GBS-colonized mothers has limited early infections but has no impact after the first week of life. The development of a maternal vaccine to address this unmet medical need has been identified as a priority by the World Health Organization, and the GBS CPSs are considered the best antigen targets. However, to date there are no accepted standardized assays to measure immune responses to the investigational vaccines and for establishment of serocorrelates of protection. Here, we describe the performance of two microsphere-based pentaplex immunoassays for the determination of antibodies recognizing the five most frequent GBS serotypes. Our data confirm that an assay based on biotinylated polysaccharides coupled to streptavidin microspheres would be suitable for the intended purpose.

Mannosylation of Lipid Nanoparticles (LNP) can potentially enhance uptake by Antigen Presenting Cells, which are highly abundant in dermal tissues, to improve the potency of Self Amplifying mRNA (SAM) vaccines in comparison to the established unmodified LNP delivery system. In the current studies, we evaluated mannosylated LNP (MLNP), which were obtained by incorporation of a stable Mannose-cholesterol amine conjugate, for the delivery of an influenza (hemagglutinin) encoded SAM vaccine in mice, by both intramuscular and intradermal routes of administration. SAM MLNP exhibited in vitro enhanced uptake in comparison to unglycosylated LNP from bone marrow-derived dendritic cells, and in vivo more rapid onset of the antibody response, independent of the route. The increased binding antibody levels also translated into higher functional hemagglutinin inhibition titers, particularly following intradermal administration. T cell assay on splenocytes from immunized mice also showed an increase in antigen specific CD8+ T responses, following intradermal administration of MLNP SAM vaccines. Induction of enhanced antigen specific CD4+ T cells, correlating with higher IgG2a antibody responses, was also observed. Hence, the present work illustrates the benefit of mannosylation of LNPs to achieve a faster immune response with SAM vaccines and these observations could contribute to the development of novel skin delivery systems for SAM vaccines.

Glycoconjugate vaccines are made of carbohydrate antigens covalently bound to a carrier protein to enhance their immunogenicity. Among the different carrier proteins tested in preclinical and clinical studies, five have been used so far for licensed vaccines: Diphtheria and Tetanus toxoids, the non-toxic mutant of diphtheria toxin CRM197, the outer membrane protein complex of Neisseria meningitidis serogroup B and the Protein D derived from non-typeable Haemophilus influenzae. Availability of novel carriers might help to overcome immune interference in multi-valent vaccines containing several polysaccharide-conjugate antigens, and also to develop vaccines which target both protein as well saccharide epitopes of the same pathogen. Accordingly we have conducted a study to identify new potential carrier proteins. Twenty-eight proteins, derived from different bacteria, were conjugated to the model polysaccharide Laminarin and tested in mice for their ability in inducing antibodies against the carbohydrate antigen and eight of them were subsequently tested as carrier for serogroup meningococcal C oligosaccharides. Four out of these eight were able to elicit in mice satisfactory anti meningococcal serogroup C titers. Based on immunological evaluation, the Streptococcus pneumoniae protein spr96/2021 was successfully evaluated as carrier for serogroups A, C, W, Y and X meningococcal capsular saccharides.

Glycoconjugate vaccines are composed of capsular polysaccharides (CPSs) of a pathogenic bacteria covalently linked to carrier proteins. Pre-exposure to the carrier is known to influence the efficacy of the glycoconjugate, by inducing enhanced or suppressed anti-CPS response. Following our previous work on the immunogenicity of diphtheria toxin mutant CRM197 and formaldehyde-treated diphtheria toxoid (DT) as carriers for meningococcal A (MenA) conjugates in mouse model, we further investigated the role of the carrier on the immunological response to glycoconjugate vaccines. We previously showed that high dosage DT priming could result in carrier-induced epitopic suppression (CIES), an event that did not occur for CRM197 priming, and we observed that anti-DT IgGs could cross-react with DT based conjugates in vitro. Here, we confirmed the cross-reactivity of anti-carrier IgGs with DT conjugates in vivo. Furthermore, we analyzed the splenocytes of animals primed with the carrier and subsequently immunized with the MenA conjugate. Pre-exposure to the carrier protein, both CRM197 and DT, resulted in increased carrier-specific plasma and memory B cell response. However, only for CRM197 priming an enhanced carbohydrate-specific plasma cell response was observed. Analysis of circulating IgGs confirmed these observations. Memory to the CPS resulted to be non-influenced by carrier priming. Analysis of T helper response showed an enhancement effect for CRM197 priming, while DT priming resulted in constrained T cell activation. Stimulation with CRM197, which does not require formaldehyde detoxification, of splenocytes from animal immunized with DT suggested that the formaldehyde treatment used to produce DT might be the cause of limited presentation of the antigen to the T cells. We concluded that the dominant carrier-specific B cell response in case of limited T cell recruitment might explain the previously observed CIES phenomenon in case of DT priming.

Diphtheria toxin mutant CRM197 is a common carrier protein for glycoconjugate vaccines, which has been proven an effective protein vector for, among others, meningococcal carbohydrates. The wide-range use of this protein in massive vaccine production requires constant increase of production yields and adaptability to an ever-growing market. Here we compare CRM197 with the alternative diphtheria non-toxic variant DT-K51E/E148K, an inactive mutant that can be produced in the periplasm of Escherichia coli. Biophysical characterization of DT-K51E/E148K suggested high similarity with CRM197, with main differences in their alpha-helical content, and a suitable purity for conjugation and vaccine preparation. Meningococcal serogroup A (MenA) glycoconjugates were synthesized using CRM197 and DT-K51E/E148K as carrier proteins, obtaining the same conjugation yields and comparable biophysical profiles. Mice were then immunized with these CRM197 and DT-K51E/E148K conjugates, and essentially identical immunogenic and protective effects were observed. Overall, our data indicate that DT-K51E/E148K is a readily produced protein that now allows the added flexibility of E. coli production in vaccine development and that can be effectively used as protein carrier for a meningococcal conjugate vaccine.

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis , Actinobacillus pleuropneumoniae , Haemophilus influenzae , Bibersteinia trehalosi , and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis. IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry.

The incidence of infectious diseases caused by several bacterial pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, has been dramatically reduced over the last 25 years through the use of glycoconjugate vaccines. The structures of the bacterial capsular polysaccharide (CPS) antigens, extracted and purified from microbial cultures and obtained with very high purity, show that many of them are decorated by O-acetyl groups. While these groups are often considered important for the structural identity of the polysaccharides, they play a major role in the functional immune response to some vaccines such as meningococcal serogroup A and Salmonella typhi Vi, but do not seem to be important for many others, such as meningococcal serogroups C, W, Y, and type III Group B Streptococcus. This review discusses the O-acetylation status of CPSs and its role in the immunological responses of these antigens.

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.

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.

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.

Carbohydrate-based vaccines constitute a potent tool for prevention of life-threatening bacterial infectious diseases like meningitis and pneumonia. Group B Streptococcus (GBS) is a major cause of neonatal sepsis and meningitis, particularly in infants born from mothers carrying the bacteria, and no vaccine is currently available. High-field Nuclear Magnetic Resonance (NMR) spectroscopy has been found to be an extremely robust tool for tracking the industrial process manufacturing of carbohydrate-based vaccines. Here we review the differences in the repeating unit structures of GBS capsular polysaccharide (CPS) type (Ia, Ib, II–VIII) yielding unique NMR proton profiles. All the profiles provided opportunities for selecting well resolved signals, in particular in the anomeric, the methylene protons at position C 3 of N-acetyl-neuraminic acid (Neu p NAc) and the N-Acetyl regions, which could be employed to develop an identity assay for monovalent vaccine bulks. Finally we reported a preliminary proof of concept of identity testing on a GBS CPS type Ia, Ib, III trivalent vaccine as blended bulks, based on the selection of one specific signal for each type in the anomeric region.

The human pathogen Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis globally. A major virulence factor of Nm is the capsular polysaccharide (CPS), which in Nm serogroup A consists of N-acetyl-mannosamine-1-phosphate units linked together by phosphodiester linkages [→6)-α-d-ManNAc-(1→OPO3−→]n. Acetylation in O-3 (to a minor extent in O-4) position results in immunologically active polymer. In the capsule gene cluster (cps) of Nm, region A contains the genetic information for CPSA biosynthesis. Thereby the open reading frames csaA, -B, and -C are thought to encode the UDP-N-acetyl-d-glucosamine-2-epimerase, poly-ManNAc-1-phosphate-transferase, and O-acetyltransferase, respectively. With the aim to use a minimal number of recombinant enzymes to produce immunologically active CPSA, we cloned the genes csaA, csaB, and csaC and functionally characterized the purified recombinant proteins. If recombinant CsaA and CsaB were combined in one reaction tube, priming CPSA-oligosaccharides were efficiently elongated with UDP-GlcNAc as the donor substrate, confirming that CsaA is the functional UDP-N-acetyl-d-glucosamine-2-epimerase and CsaB the functional poly-ManNAc-1-phosphate-transferase. Subsequently, CsaB was shown to transfer ManNAc-1P onto O-6 of the non-reducing end sugar of priming oligosaccharides, to prefer non-O-acetylated over O-acetylated primers, and to efficiently elongate the dimer of ManNAc-1-phosphate. The in vitro synthesized CPSA was purified, O-acetylated with recombinant CsaC, and proven to be identical to the natural CPSA by 1H NMR, 31P NMR, and immunoblotting. If all three enzymes and their substrates were combined in a one-pot reaction, nature identical CPSA was obtained. These data provide the basis for the development of novel vaccine production protocols.

Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.

Carbohydrates, in the form of oligosaccharides, polysaccharides and lipopolysaccharides, are ubiquitous components of the cell surface of bacteria. In the last 30 years polysaccharide-based vaccines have proven to be highly safe and efficacious against bacterial infections like meningitis and pneumonia. Surface plasmon resonance (SPR) analysers have emerged as powerful tools for the characterization of label-free biomolecular interactions, enabling the dynamics of complex formation and dissociation to be monitored in real-time. A wide variety of sensor chips are currently available for many applications. Nevertheless, biosensors with nucleophilic functionalities, such as amino groups, could be useful to extend the SPR applicability. Using selected examples, this review gives an overview of significant applications of conventional flow SPR to investigate the specific interactions of bacterial polysaccharide antigens. SPR has made significant progress in the last two decades and is now becoming a relevant technology for developing immunological assays for in-depth characterization of carbohydrate antigens.

The elucidation of the molecular details underlying the immune properties of glycoconjugate vaccines has largely focused on the carbohydrate moiety, while very little is known on the effect of the corresponding conjugation sites. Herein we constructed a set of β-(1 → 3) glucan oligosaccharide conjugates with a well-defined glycan structure, connected to patterns of predetermined tyrosine or lysine residues onto the CRM197 carrier protein. To evaluate the effect of multivalent architecture in the glycan presentation, a novel linker enabling tyrosine-directed ligation of couples of oligosaccharides was prepared. The potential of these constructs as anti-Candida vaccines was evaluated in vivo, using as controls glycoconjugates prepared by a conventional random coupling strategy, and the structure–immune properties relationship was established. We found that: (i) the tyrosine-directed ligation resulted in higher anti-glycan IgG levels in comparison to the conjugation at predetermined lysine residues; (ii) the presentation of the carbohydrate antigen with a biantennary cluster of glycans onto specific tyrosine residues did not further increase the anti-glycan antibody level; (iii) the sera deriving from immunization with defined conjugates at tyrosine and, particularly at lysine residues, were proven stronger inhibitors of fungal adhesion to human epithelial cells in comparison to those from conjugates prepared by classic random chemistry; (iv) the presence of antibodies directed to the linkers did not affect the anti-glycan immune response. These findings suggest that a careful choice of the defined sites of conjugation and the loading density of antigens are important factors to raise high-quality anti-carbohydrate antibodies.

The development of vaccines against polysaccharide-encapsulated pathogens (e.g. Haemophilus influenzae type b, pneumococci, meningococci) is challenging because polysaccharides do not elicit a strong and long-lasting immune response (i.e. T-cell independent). This can be overcome by conjugating the polysaccharide to a protein carrier (e.g. tetanus toxoid, cross-reacting material 197 [CRM]), which vastly improves the immune response and induces memory to the polysaccharide (T-cell dependent). Although it is well documented that protein carriers additionally induce an immune response against themselves, this potential "additional valency" has so far not been recognized. The only exception is for the protein D carrier (derived from non-typeable Haemophilus influenzae [NTHi]) used in a pneumococcal conjugate vaccine, which may have a beneficial impact on NTHi acute otitis media. In this review, we describe the immunogenicity of various protein carriers and discuss their potential dual function: as providers of T-cell helper epitopes and as protective antigens. If this "additional valency" could be proven to be protective, it may be possible to consider its potential effect on the number of required immunizations. We also describe the potential for positive or negative interference between conjugate vaccines using the same protein carriers, the resulting desire for novel carriers, and information on potential new carriers. The range of conjugate vaccines is ever expanding, with different carriers and methods of conjugation. We propose that new conjugate vaccine trials should assess immunogenicity to both the polysaccharide and carrier. Ultimately, this so-far "neglected valency" could be an exploitable characteristic of polysaccharide conjugate vaccines.

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

Vaccination is a key strategy for the control of various infectious diseases. Many pathogens, such as Streptococcus pneumoniae , Haemophilus influenzae type b (Hib), and Neisseria meningitidis produce on their surfaces dense and complex glycan structures, which represent an optimal target for eliciting carbohydrate specific antibodies able to confer protection against those bacteria. Glycoconjugates represent nowadays an important class of efficacious and safe commercial vaccines. It has been known for a long time that covalent linkage of poorly immunogenic carbohydrates to protein is fundamental to provide T cell epitopes for eliciting a memory response of the immune system against the saccharide. However, while the traditional mechanism of action of glycoconjugates has considered peptides generated from the carrier protein to be responsible of T cell help recruitment, only recently evidence of the active involvement of the carbohydrate part in determining the T cell help has been shown. In addition, zwitterionic polysaccharides have been proven to activate the adaptive immune system without further conjugation to protein. Progress in this interface area between chemistry and biology, in combination with novel synthetic and biosynthetic methods for the preparation of glycoconjugates, is opening new perspectives to clarify their mechanism of action and give new insights for the design of improved carbohydrate-based vaccines.

Invasive meningococcal disease (IMD) is a global health problem and vaccination has proven the most effective way of disease control. Neisseria meningitidis serogroup X (NmX) is an emerging threat in the African sub-Saharan meningitis belt, but no vaccine is available today. Leading vaccines against Nm are glycoconjugates, in which capsular polysaccharides isolated from large-scale pathogen cultures are conjugated to adjuvant proteins. Though safe and efficacious even in infants, high costs and biohazard associated with the production limit abundant application of glycoconjugate vaccines particularly in the most afflicted nations. An existing NmX vaccine candidate (CPSXn-CRM197) produced by established protocols from NmX capsule polysaccharide (CPSX) has been shown to elicit high bactericidal immunoglobulin G titres in mice. Here we describe the scalable in vitro synthesis of CPSXiv from chemically pure precursors by the use of recombinant NmX capsule polymerase. Application of the described coupling chemistry gives CPSXiv-CRM197, which in mouse vaccination experiments behaves identical to the benchmark CPSXn-CRM197. Excluding any biohazards, this novel process represents a paradigm shift in vaccine production and a premise towards vaccine manufacturing in emerging economies.

Some new phosphonoester-linked oligomers, stabilized analogues of the corresponding phosphate-bridged oligomers of Neisseria meningitidis A (MenA) capsular polysaccharide (CPS), were conjugated to human serum albumin (HSA), as a protein carrier model, and studied for immunological activities. We determined (i) in vitro, their biocompatibility (CAM test) and activity in inducing both T cell proliferation (CFSE method) and IL-2 release (ELISA), and (ii) in vivo, their ability to stimulate specific IgG antibody production (ELISA). All HSA-conjugated compounds induce T cell proliferation (40% of proliferation at 10(2) μM), whereas only the phosphonodisaccharide was effective (28% of proliferation at 10(2) μM) among the unconjugated forms. IL-2 release confirmed these results. In addition, the HSA-conjugated showed in vivo the capacity of eliciting the production of specific IgG antibodies. In conclusion, we obtained novel biocompatible, water-stable, and immunoactive MenA CPS analogues. A short disaccharide fragment showed the unusual behavior of triggering T cell proliferation in vitro.

Laminarin-CRM197 glycoconjugates were previously demonstrated to be immunogenic and confer protection against Candida albicans in mice. Laminarin consists of β-(1,3) glucan repeating units, with sporadic β-(1,6) branches. A set of short glucans was used to study the effect of the β-(1,6) branch on the antigenicity of linear β-(1,3) glucans. A linear β-(1,3) glucan hexasaccharide was selected as the best fragment able to inhibit the binding between laminarin and antilaminarin antibodies. The hexamer was then conjugated to CRM197 and induced, in mice, significant titers of specific antilaminarin antibodies comparable with those raised by the Lam-CRM197 conjugate.

A laminarin-diphtheria toxoid (CRM197) conjugate vaccine conferred protection against fungal infections in mice. We have now generated novel beta-glucan-CRM197 vaccines, with either natural (Curd-CRM197) or synthetic linear (15mer-CRM197), or beta-(1,6)-branched (17mer-CRM197) beta-(1,3)-oligosaccharides, formulated with the human-acceptable adjuvant MF59. Curd-CRM197 and 15mer-CRM197 conjugates, which induced high titers of anti-beta-(1,3)-glucan IgG, but no antibodies against beta-(1,6)-glucan, conferred protection to mice lethally challenged with C. albicans. In contrast, the 17mer-CRM197 conjugate, which induced anti-beta-(1,6)-glucan antibodies in addition to the anti-beta-(1,3)-glucan IgG, was non-protective. These data provide some insights on beta-glucan epitope(s) mediating antifungal protection and open the way to develop a synthetic oligosaccharide vaccine against fungal diseases.

The design of safe and potent adjuvants able to enhance and modulate antigen-specific immunity is of great interest for vaccine research and development. In the present study, negatively charged poly(lactide-co-glycolide) (PLG) nanoparticles have been combined with a synthetic immunepotentiator molecule targeting the Toll-like receptor 7. The selection of appropriate preparation and freeze-drying conditions resulted in a PLG-based adjuvant with well-defined and stable physico-chemical properties. The adjuvanticity of such nanosystem has later been evaluated in the mouse model with a diphtheria-tetanus-pertussis (DTaP) vaccine, on the basis of the current need to improve the efficacy of acellular pertussis (aP) vaccines. DTaP antigens were adsorbed onto PLG nanoparticles surface, allowing the co-delivery of TLR7a and multiple antigens through a single formulation. The entrapment of TLR7a into PLG nanoparticles resulted in enhanced IgG and IgG2a antibody titers. Notably, the immune potentiator effect of TLR7a was less evident when it was used in not-entrapped form, indicating that co-localization of TLR7a and antigens is required to adequately stimulate immune responses. In conclusion, the rational selection of adjuvants and formulation here described resulted as a highly valuable approach to potentiate and better tailor DTaP vaccine immunogenicity.

Multicomponent constructs, obtained by coupling different glycans to the carrier protein, have been proposed as a way to co-deliver multiple surface carbohydrates targeting different strains of one pathogen and reduce the number of biomolecules in the formulation of multivalent vaccines. To assess the feasibility of this approach for anti-microbial vaccines and investigate the potential immunodominance of one carbohydrate antigen over the others in these constructs, we designed a bivalent unimolecular vaccine against serogroup A (MenA) and C (MenC) meningococci, with the two different oligomers conjugated to same molecule of carrier protein (CRM197). The immune response elicited in mice by the bivalent MenAC construct was compared with the ones induced by the monovalent MenA and MenC vaccines and their combinations. After the second dose, the bivalent construct induced good levels of anti-MenA and anti-MenC antibodies with respect to the controls. However, the murine sera from the MenAC construct exhibited good anti-MenC bactericidal activity, and very low anti-MenA functionality when compared to the monovalent controls. This result was explained with the diverse relative avidities against MenA and MenC polysaccharides, which were measured in the generated sera. The immunodominant effect of the MenC antigen was fully overcome following the third immunization, when sera endowed with higher avidity and excellent bactericidal activity against both MenA and MenC expressing strains were elicited. Construction of multicomponent glycoconjugate vaccines against microbial pathogens is a feasible approach, but particular attention should be devoted to study and overcome possible occurrence of immune interference among the carbohydrates.

The Group B Streptococcus capsular polysaccharide type IX was isolated and purified, and the structure of its repeating unit was determined. Type IX capsule → 4)[NeupNAc-α-(2 → 3)-Galp-β-(1 → 4)-GlcpNAc-β-(1 → 6)]-β-GlcpNAc-(1 → 4)-β-Galp-(1 → 4)-β-Glcp-(1 → appears most similar to types VII and V, although it contains two GlcpNAc residues. Genetic analysis identified differences in cpsM, cpsO, and cpsI gene sequences as responsible for the differentiation between the three capsular polysaccharide types, leading us to hypothesize that type V emerged from a recombination event in a type IX background.

BACKGROUND: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy to distinguish serotypes 11A and 11E. METHODS: A collection of 48 (34 invasive and 14 carriage) S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA) based on monoclonal antibodies. RESULTS: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates. CONCLUSIONS: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new serotyping approaches.

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

Primary examples in vaccine design have shown good levels of carbohydrate-specific antibody generation when raised using extracted or fully synthetic capsular polysaccharide glycans covalently coupled to a protein carrier. Herein, we cover recent clinical developments of carbohydrate-based vaccines and describe how novel cutting-edge methodology for the total synthesis of oligosaccharides and for the precise placement of carbohydrates at pre-determined sites within a protein may be used to further improve the safety and efficacy of glycovaccines.

Primary examples in vaccine design have shown good levels of carbohydrate-specific antibody generation when raised using extracted or fully synthetic capsular polysaccharide glycans covalently coupled to a protein carrier. Herein, we cover recent clinical developments of carbohydrate-based vaccines and describe how novel cutting-edge methodology for the total synthesis of oligosaccharides and for the precise placement of carbohydrates at pre-determined sites within a protein may be used to further improve the safety and efficacy of glycovaccines., Chemical Science, 4 (8), ISSN:2041-6520, ISSN:2041-6539

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.

Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. difficile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM(197) elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates.

Prior to the introduction of the MenAfriVac™ serogroup A glycoconjugate vaccine in September 2010, serogroup A was the major epidemic disease-causing meningococcal serogroup in the African meningitis belt. However, recently serogroup X meningococcal (MenX) disease has received increased attention because of outbreaks recorded in this region, with increased endemic levels of MenX disease over the past 2 years. Whereas polysaccharide-protein conjugate vaccines against meningococcal serogroups A, C, W and Y (MenA, MenC, MenW, MenY) are on the market, a vaccine able to protect against MenX has never been achieved. The structure of serogroup A, C, W and Y meningococcal polysaccharides has been already fully elucidated by NMR. MenX capsular polysaccharide (MenX CPS) structure is also documented but fewer characterization data have been published. We have applied here (1)H NMR, (31)P NMR and HPLC to evaluate the stability of MenX CPS in aqueous solution as compared to MenA capsular polysaccharide (MenA CPS). The stability study demonstrated that MenA CPS is more susceptible to hydrolytic degradation than MenX CPS. The different stereochemistry of the N-acetyl group at position C(2) of mannosamine (MenA CPS) and glucosamine (MenX CPS) respectively might play a fundamental role in this susceptibility to polysaccharide chain degradation. The satisfactory stability of MenX CPS predicts the possibility that a stable fully-liquid MenX polysaccharide or glycoconjugate vaccine could be developed.

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae , pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.