RATIONAL DESIGN AND SYNTHESIS OF A GLYCOCONJUGATE VACCINE AGAINST GROUP A STREPTOCOCCUS

Glycoconjugate vaccines are one of the most effective ways of preventing bacterial and fungal infections. They are generally composed of a saccharide antigen covalently linked to a carrier protein to convert the saccharide from a T-independent to a T-dependent antigen, resulting in increased immunogenicity from infancy and memory response. Currently, most of the conjugate vaccines are produced from polysaccharides extracted and purified from bacterial cultures. However, use of synthetic oligosaccharides constitutes a promising alternative approach. Synthetic saccharides have well-defined structure and can be equipped with a linker at their reducing end for selective conjugation allowing sugar epitopes preservation. Moreover, synthetic conjugates allow to avoid the handling of pathogens and presence of bacterial impurities and are characterized by minimal batch-to-batch variability and higher quality control standards compared to conjugates with native polysaccharides. New techniques such as solid phase automated synthesis and chemo-enzymatic approaches reduce reaction time and use of protective groups during synthetic steps and favor oligosaccharides production at large scale. In addition, nanoparticles have been recently proposed as potential multivalent delivery systems also for carbohydrate-based vaccines. Gold nanoparticles (AuNPs) have attracted great attention in this field due to their unique characteristics of biocompatibility and easy production. In the last years, AuNPs have been tested as carrier for short synthetic oligosaccharides in animal models. Natural nanoparticles can be obtained by the methodology called Generalized Modules for Membrane Antigens (GMMA). GMMA are Outer Membrane Vescicles (OMV) naturally released from Gram-negative bacteria genetically manipulated to increase blebbing and reduce toxicity. Similar to other nanoparticle systems, GMMA combine the multivalent display of carbohydrates, favoring B-cell activation and in a conformation that resembles on native bacteria, with optimal size for immune stimulation. GMMA also work as self-adjuvants, due to the presence of structures acting as agonist of Toll-like receptors 2 and 4, two receptors expressed on antigen presenting cells surface playing a crucial role in the innate immune response. Here the chemical synthesis to obtain oligosaccharide fragments of Group A Carbohydrate (GAC) and the preparation of the corresponding glycoconjugates with CRM197 (cross-reacting material 197, a non-toxic mutant of diphtheria toxin) carrier protein have been reported. The main aim was to explore such synthetic structures for the development of a vaccine against Group A Streptococcus (GAS) disease for which no vaccine is currently available. The use of well-defined synthetic structures will help to better understand the impact of GAC structural features on the immune response induced. Synthetic oligomers have been conjugated to GMMA and AuNPs, with the aim to i) compare GMMA and CRM197 as carrier systems, and ii) to explore multivalency on AuNPs to increase antibodies binding affinity

Glyco-gold nanoparticles: Synthesis and applications

Glyco-gold nanoparticles combine in a single entity the peculiar properties of gold nanoparticles with the biological activity of carbohydrates. The result is an exciting nanosystem, able to mimic the natural multivalent presentation of saccharide moieties and to exploit the peculiar optical properties of the metallic core. In this review, we present recent advances on glyco-gold nanoparticle applications in different biological fields, highlighting the key parameters which inspire the glyco nanoparticle design

Glyco-gold nanoparticles : synthesis and applications

Glyco-gold nanoparticles combine in a single entity the peculiar properties of gold nanoparticles with the biological activity of carbohydrates. The result is an exciting nanosystem, able to mimic the natural multivalent presentation of saccharide moieties and to exploit the peculiar optical properties of the metallic core. In this review, we present recent advances on glyco-gold nanoparticle applications in different biological fields, highlighting the key parameters which inspire the glyco nanoparticle design

Recent Advances in the Synthesis of Glycoconjugates for Vaccine Development

During the last decade there has been a growing interest in glycoimmunology, a relatively new research field dealing with the specific interactions of carbohydrates with the immune system. Pathogens\u2019 cell surfaces are covered by a thick layer of oligo- and polysaccharides that are crucial virulence factors, as they mediate receptors binding on host cells for initial adhesion and organism invasion. Since in most cases these saccharide structures are uniquely exposed on the pathogen surface, they represent attractive targets for vaccine design. Polysaccharides isolated from cell walls of microorganisms and chemically conjugated to immunogenic proteins have been used as antigens for vaccine development for a range of infectious diseases. However, several challenges are associated with carbohydrate antigens purified from natural sources, such as their difficult characterization and heterogeneous composition. Consequently, glycoconjugates with chemically well-defined structures, that are able to confer highly reproducible biological properties and a better safety profile, are at the forefront of vaccine development. Following on from our previous review on the subject, in the present account we specifically focus on the most recent advances in the synthesis and preliminary immunological evaluation of next generation glycoconjugate vaccines designed to target bacterial and fungal infections that have been reported in the literature since 2011

Design of mechanical properties of poly(butylene-adipate-terephthalate) reinforced with Zein-TiO2 complex

The aim of this work was to realize and mechanically characterize composites based on poly(butyleneadipate-terephthalate) (PBAT) filled with micrometric particles of zein-TiO2 complex (ZTC) at different concentrations. Specimens model 1BA were obtained by injection molding and subjected to a uniaxial tensile test (UTT). The addition of the ZTC proved to have a reinforcing effect on the matrix, an increase in both Young’s modulus (E) and yield stress (σy) being observed. The mechanical properties were modeled applying Kerner’s and Pukánszky’s models, obtaining a good correspondence between theoretical experimental values and good matrix-filler interfacial interaction, respectively. Microscopical analysis revealed a good dispersion of the filler within the matrix

Analytical evaluation of carotenoids, apocarotenoids, capsaicinoids, and phenolics to assess the effect of a protective treatment on chili peppers dried at different temperatures

The most common preservation process for chili peppers is drying, which inevitably causes oxidative degradation of thermolabile molecules. The aim of this research was to evaluate the potential protective effect exerted by an active ingredient based on grape seed oil, on pepper fruits. Grapeseed oil is rich in antioxidant compounds and was applied to pepper’s surface in form of a sol–gel product, before fruit thermal treatment. In this work, chili peppers samples were preventively treated with an active solution, and controls (untreated peppers), were submitted to a drying process performed at two different temperatures: 45 and 65 °C. Analysis of capsaicinoids, carotenoids, apocarotenoids, and phenolic content was performed to evaluate possible differences between the sets of samples. Oxidative stability of oil enriched with chili pepper powder aliquots was also measured to evaluate the antioxidant power of the samples. Obtained data showed that treated samples retained a higher amount of capsaicinoids and carotenoids. Oxidative stability of pepper powder was also higher for treated samples than for controls. Furthermore, the thermal treatment performed at 45 °C caused milder modifications than the 65 °C treatment. The applied pre-drying treatment can be proposed to prevent bioactive compounds loss and to enhance product stability and shelf-life

Gold nanoparticles morphology does not affect the multivalent presentation and antibody recognition of Group A Streptococcus synthetic oligorhamnans

The development of novel delivery systems capable of enhancing the antibody binding affinity and immunoactivity of short length saccharide antigens is at the forefront of modern medicine. In this regard, gold nanoparticles (AuNPs) raised great interest as promising nano-vaccine platform, as they do not interfere with the desired immune response and their surface can be easily functionalized, enabling the antigen multivalent presentation. In addition, the nanoparticles morphology can have a great impact on their biological properties. Gram-positive Group A Streptococcus (GAS) is a bacterium responsible for many infections and represents a priority healthcare concern, but a universal vaccine is still unavailable. Since all the GAS strains have a cell wall characterized by a common polyrhamnose backbone, this can be employed as alternative antigen to develop an anti-GAS vaccine. Herein, we present the synthesis of two oligorhamnoside fragments and their corresponding oligorhamnoside-AuNPs, designed with two different morphologies. By competitive ELISA we assessed that both symmetric and anisotropic oligorhamnan nanoparticles inhibit the binding of specific polyclonal serum much better than the unconjugated oligosaccharides

Elucidating the role of N-acetylglucosamine in Group A Carbohydrate for the development of an effective glycoconjugate vaccine against Group A Streptococcus

Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha) backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the polyRha one, both in mice and in rabbits. This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as preferable saccharide antigen to include in the vaccine.Bio-organic Synthesi