Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice.

α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson's disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4-5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies.

Effects of Banafine® , a fermented green banana-derived acidic glycoconjugate, on influenza vaccine antibody titer in elderly patients receiving gastrostomy tube feeding.

Immunosenescence can negatively affect cytokine production in elderly and may impair poor antibody responses to influenza vaccination and infection. Herein, the effects of Banafine® administration on influenza vaccine antibody titer in elderly patients (average age ∼80 years) receiving gastrostomy tube feeding were examined. In the double-blind, single-center, randomized clinical studies, 30 elderly bedridden patients were administered Banafine® or placebo for 8 weeks. At week 4, all patients received influenza vaccination against H1N1, H3N2, B/Yamagata, or B/Victoria. Blood biochemical indices and serum antibody titers were assessed. Banafine® administration significantly increased hemagglutination inhibition titers in response to vaccination against H1N1, H3N2, and B/Yamagata in the elderly patients (P < 0.05). Moreover, the seroconversion rate against H1N1 (47.1%) and H3N2 (29.4%) and seroprotection rate against H1N1 (71.4%) and both B strains (31.3% and 12.5%, respectively) were increased for the Banafine® group. These results suggest that Banafine® administration can increase antibody responses to influenza vaccination in bedridden hospitalized patients, and potentially modulate immune function in the elderly. PRACTICAL APPLICATION: Literature review suggested that most of the synbiotics are based on innate immunity, strain specific (probiotics), and are not consistently observed. Herein, in clinical studies we demonstrate that administration of Banafine® , a plant-based glycoconjugate, can increase antibody levels in bedridden hospitalized elderly patients following influenza vaccination.

Potential of carbohydrate-conjugated graphene assemblies in biomedical applications.

Graphene displays various properties like optical, electrical, mechanical, etc. resulting in a large range of applications in biosensing, bio-imaging, medical and electronic devices. The graphene-based nanomaterials show disadvantages like hydrophobic surface, degradation of biomolecules (proteins and amino acids) and toxicity to the human and microbes by permeating into the cells and thus, limiting the use in the biomedical field. Conjugation of carbohydrates like chitin, cyclodextrins and cellulose with graphene results in thermal stability, oxygen repulsive ability, fire-retardant and gelling properties with better biodegradability, biocompatibility and safety leading to the formation of environment-friendly biopolymers. This article delivers an overview of the molecular interaction of different carbohydrates-derived from natural sources like marine, plants and microbes with graphene nanosheets to extend the applications in tissue engineering, surgical materials, biosensing and novel drug delivery for prolonged action in the treatment of breast and hepatic cancers.

High efficiency biosynthesis of O-polysaccharide-based vaccines against extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.

New Small-Molecule Glycoconjugates of Docetaxel and GalNAc for Targeted Delivery to Hepatocellular Carcinoma.

In this work, we have developed covalent and low molecular weight docetaxel delivery systems based on conjugation with N-acetyl-d-galactosamine and studied their properties related to hepatocellular carcinoma cells. The resulting glycoconjugates have an excellent affinity to the asialoglycoprotein receptor (ASGPR) in the nanomolar range of concentrations and a high cytotoxicity level comparable to docetaxel. Likewise, we observed the 21-75-fold increase in water solubility in comparison with parent docetaxel and prodrug lability to intracellular conditions with half-life values from 25.5 to 42 h. We also found that the trivalent conjugate possessed selective toxicity against hepatoma cells vs control cell lines (20-35 times). The absence of such selectivity in the case of monovalent conjugates indicates the effect of ligand valency. Specific ASGPR-mediated cellular uptake of conjugates was proved in vitro using fluorescent-labeled analogues. In addition, we showed an enhanced generation of reactive oxygen species in the HepG2 cells, which could be inhibited by the natural ligand of ASGPR. Overall, the obtained results highlight the potential of ASGPR-directed cytostatic taxane drugs for selective therapy of hepatocellular carcinoma.

Biodegradable pH-responsive micelles loaded with 8-hydroxyquinoline glycoconjugates for Warburg effect based tumor targeting.

Biodegradable triblock copolymer poly(ethylene glycol)-b-polycarbonate-b-oligo([R]-3-hydroxybutyrate) was prepared via metal-free ring-opening polymerization of ketal protected six-membered cyclic carbonate followed by esterification with bacterial oligo([R]-3-hydroxybutyrate) (oPHB). Amphiphilic triblock copolymer self-organizes into micelles with a diameter of ~25 nm. Acid-triggered hydrolysis of ketal groups to two hydroxyl groups causes an increase in hydrophilicity of the hydrophobic micelle core, resulting in the micelles swell and drug release. oPHB was added as core-forming block to increase the stability of prepared micelles in all pH (7.4, 6.4, 5.5) studied. Doxorubicin and 8-hydroxyquinoline glucose- and galactose conjugates were loaded in the micelles. In vitro drug release profiles in PBS buffers with different pH showed that a small amount of loaded drug was released in PBS at pH 7.4, while the drug was released much faster at pH 5.5. MTT assay showed that the blank micelles were non-toxic to different cell lines, while glycoconjugates-loaded micelles, showed significantly increased ability to inhibit the proliferation of MCF-7 and HCT-116 cells compared to free glycoconjugates. The glycoconjugation of anti-cancer drugs and pH-responsive nanocarriers have separately shown great potential to increase the tumor-targeted drug delivery efficiency. The combination of drug glycoconjugation and the use of pH-responsive nanocarrier opens up new possibilities to develop novel strategies for efficient tumor therapy.

Improving protein glycan coupling technology (PGCT) for glycoconjugate vaccine production.

INTRODUCTION: Vaccines are one of the great success stories of modern medicine and an increasingly important strategy in the fight against antimicrobial resistance. Glycoconjugate vaccines, consisting of a protein component covalently linked to a glycan antigen, are extremely efficacious in preventing infectious disease. However, glycoconjugates have yet to reach their full potential, with currently licensed glycoconjugate vaccines available against only four pathogens. Protein glycan coupling technology, where glycoconjugates are biologically produced in purpose engineered bacterial cells, has the potential to revolutionize the field by lowering manufacturing cost and increasing flexibility for tailor-made vaccines. AREAS COVERED: This review gives an overview of the past 20 years of PGCT research, discusses the key developments and current status of the technology, and speculates on the future of PGCT-based vaccinology. EXPERT OPINION: PCGT has the potential to overcome some of the limitations of chemical conjugation production methods. The technology has undergone significant development since its inception, and new discoveries are continually driving the field forward. Vaccines currently in clinical trials have demonstrated the potential of the PGCT to deliver effective glycoconjugate vaccines for unmet medical needs.

Broadly protective semi-synthetic glycoconjugate vaccine against pathogens capable of producing poly-ß-(1→6)-N-acetyl-d-glucosamine exopolysaccharide.

Poly-ß-(1→6)-N-acetylglucosamine (PNAG) was first discovered as a major component of biofilms formed by Staphylococcus aureus and some other staphylococci but later this exopolysaccharide was also found to be produced by pathogens of various nature. This common antigen is considered as a promising target for construction of a broadly protective vaccine. Extensive studies of PNAG, its de-N-acetylated derivative (dPNAG, containing around 15% of residual N-acetates) and their conjugates with Tetanus Toxoid (TT) revealed the crucial role of de-N-acetylated glucosamine units for the induction of protective immunity. Conjugates of synthetic penta- (5GlcNH2) and nona-ß-(1→6)-d-glucosamines (9GlcNH2) were tested in vitro and in different animal models and proved to be effective in passive and active protection against different microbial pathogens. Presently conjugate 5GlcNH2-TT is being produced under GMP conditions and undergoes safety and effectiveness evaluation in humans and economically important animals. Current review summarizes all stages of this long-termed study.

Structure-based glycoconjugate vaccine design: The example of Group B Streptococcus type III capsular polysaccharide.

Microbial surface polysaccharides are important virulence factors and targets for vaccine development. Glycoconjugate vaccines, obtained by covalently linking carbohydrates and proteins, are well established tools for prevention of bacterial infections. Elucidation of the minimal portion involved in the interactions with functional antibodies is of utmost importance for the understanding of their mechanism of induction of protective immune responses and the design of synthetic glycan based vaccines. Typically, this is achieved by combination of different techniques, which include ELISA, glycoarray, Surface Plasmon Resonance in conjunction with approaches for mapping at atomic level the position involved in binding, such as Saturation Transfer NMR and X-ray crystallography. This review provides an overview of the structural studies performed to map glycan epitopes (glycotopes), with focus on the highly complex structure of Group B Streptococcus type III (GBSIII) capsular polysaccharide. Furthermore, it describes the rational process followed to translate the obtained information into the design of a protective glycoconjugate vaccine based on a well-defined synthetic glycan epitope.

Classical and novel strategies to develop a Shigella glycoconjugate vaccine: from concept to efficacy in human.

Shigella are gram-negative bacteria that cause severe diarrhea and dysentery, with a high level of antimicrobial resistance. Disease-induced protection against reinfection in Shigella-endemic areas provides convincing evidence on the feasibility of a vaccine and on the importance of Shigella lipopolysaccharides as targets of the host humoral protective immune response against disease. This article provides an overview of the original and current strategies toward the development of a Shigella glycan-protein conjugate vaccine that would cover the most commonly detected strains. Going beyond pioneering "lattice"-type polysaccharide-protein conjugates, progress, and challenges are addressed with focus on promising alternatives, which have reached phases I and II clinical trial. Glycoengineered bioconjugates and "sun"-type conjugates featuring well-defined synthetic carbohydrate antigens are discussed with insights on the molecular parameters governing the rational design of a cost-effective glycoconjugate vaccine efficacious in preventing diseases caused by Shigella in the most at risk populations, young children living in endemic areas.

Semisynthetic Glycoconjugate Vaccines To Elicit T Cell-Mediated Immune Responses and Protection against Streptococcus pneumoniae Serotype 3.

Streptococcus pneumoniae serotype 3 (ST3) is one of the main pneumococcal strains that can cause severe invasive diseases, but its current vaccines are relatively inefficient. To develop more effective ST3 vaccines, tetanus toxoid (TT) conjugates of the synthetic penta-, hexa-, hepta-, and octasaccharide analogs of ST3 capsular polysaccharide (CPS) were systematically studied. These conjugates, especially those of penta- and hexasaccharides, were demonstrated to induce extremely robust T cell-dependent immune responses in mouse. Various studies also revealed that the induced antibodies could recognize ST3 CPS and mediate in vitro opsonophagocytic killing of ST3 cells. It was proved ultimately that immunization with the hexasaccharide-TT conjugate could completely protect mice from ST3-caused infection and lung damage and significantly elongate mouse survival. It was proposed that this conjugate functions through the help of CD4+ T cells and via promoting Th cell differentiation into carbohydrate antigen-specific Th2 cells to establish humoral immunity. In conclusion, ST3 CPS hexasaccharide-TT was identified as a particularly promising ST3 vaccine candidate worthy of further investigation and development.

Immunogenicity and protective efficacy against Salmonella C2-C3 infection in mice immunized with a glycoconjugate of S. Newport Core-O polysaccharide linked to the homologous serovar FliC protein.

Nontyphoidal Salmonella (NTS) are important human enteric pathogens globally. Among the different serovars associated with human NTS disease, S. Newport (a serogroup C2-C3Salmonella) accounts for a measurable proportion of cases. However, to date there are no licensed human NTS vaccines. NTS lipopolysaccharide-associated O polysaccharides are virulence factors and protective antigens in animal models. As isolated molecules, bacterial polysaccharides are generally poorly immunogenic, a limitation overcome by conjugation to a protein carrier. We report herein the development of a candidate serogroup C2-C3 glycoconjugate vaccine based on S. Newport Core-O polysaccharide (COPS) and phase 1 flagellin (FliC). S. Newport COPS and FliC were purified from genetically engineered reagent strains, and conjugated at the polysaccharide reducing end to FliC protein lysines with thioether chemistry. S. Newport COPS:FliC immunization in mice improved anti-polysaccharide immune responses, generated high anti-FliC IgG titers, and mediated robust protection against challenge with both the homologous serovar as well another serogroup C2-C3 serovar (S. Muenchen). Analyses of S. Newport COPS:FliC induced sera found that the anti-COPS IgG antibodies were specific for serogroup C2-C3 lipopolysaccharide, and could promote bactericidal killing by complement and uptake into phagocytes. These preclinical studies establish the protective capacity of serogroup C2-C3 OPS glycoconjugates, and provide a path forward for the development of a multivalent Salmonella vaccine for humans that includes serogroup C2-C3.

Synthesis of cholesterol-based neoglycoconjugates and their use in the preparation of liposomes for active liver targeting.

Carbohydrate receptors on liver represent attractive targets for receptor-mediated delivery of nanostructured therapeutics. In this study, two new cholesterol-based glycoconjugates derived from d-galactose and N-acetylglucosamine were synthesized and incorporated into liposomes. 99mTc-Cholesterol-DTPA complex was used for radiolabeling experiments in vivo with high radiochemical yields and stability. Biodistribution studies confirmed the targeting of galactosylated liposomes (GalL) to liver cells. These results indicated that GalL could be considered a promising drug delivery system for liver diseases therapy.

Immunogenicity and efficacy following sequential parenterally-administered doses of Salmonella Enteritidis COPS:FliC glycoconjugates in infant and adult mice.

In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[5],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa.

Biomedical Applications of Glycoconjugates.

Glycans expressed on the forefront surface of cells participate in many key biological processes via forming various glycoconjugates. The recognition of complex glycoconjugates as mediators of important biological processes has stimulated investigation into their therapeutic potential. As the increasing accessibility of glycoconjugates, it has been widely applied in the field of drug delivery. This review particularly refers to the constitutive glycoconjugates of receptor-mediated binding of glycoprotein, glycolipids and glycopeptides for cell-selective drug delivery, in order to broaden the future therapeutic scope of drug delivery system and effective cancer therapy.

Biomedical applications of glyconanoparticles based on quantum dots.

BACKGROUND: Quantum dots (QDs) are outstanding nanomaterials of great interest to life sciences. Their conjugation versatility added to unique optical properties, highlight these nanocrystals as very promising fluorescent probes. Among uncountable new nanosystems, in the last years, QDs conjugated to glycans or lectins have aroused a growing attention and their application as a tool to study biological and functional properties has increased. SCOPE OF REVIEW: This review describes the strategies, reported in the literature, to conjugate QDs to lectins or carbohydrates, providing valuable information for the elaboration, improvement, and application of these nanoconjugates. It also presents the main applications of these nanosystems in glycobiology, such as their potential to study microorganisms, the development of diseases such as cancer, as well as to develop biosensors. MAJOR CONCLUSIONS: The development of glyconanoparticles based on QDs emerged in the last decade. Many works reporting the conjugation of QDs with carbohydrates and lectins have been published, using different strategies and reagents. These bioconjugates enabled studies that are very sensitive and specific, with potential to detect and elucidate the glycocode expressed in various normal or pathologic conditions. GENERAL SIGNIFICANCE: Produce a quick reference source over the main advances reached in the glyconanotechnology using QDs as fluorescent probes.

Alternative vaccine administration by powder injection: Needle-free dermal delivery of the glycoconjugate meningococcal group Y vaccine.

Powder-injectors use gas propulsion to deposit lyophilised drug or vaccine particles in the epidermal and sub epidermal layers of the skin. We prepared dry-powder (Tg = 45.2 ± 0.5°C) microparticles (58.1 µm) of a MenY-CRM197 glyconjugate vaccine (0.5% wt.) for intradermal needle-free powder injection (NFPI). SFD used ultrasound atomisation of the liquid vaccine-containing excipient feed, followed by lyophilisation above the glass transition temperature (Tg' = - 29.9 ± 0.3°C). This resulted in robust particles (density~ 0.53 ±0.09 g/cm3) with a narrow volume size distribution (mean diameter 58.1 µm, and span = 1.2), and an impact parameter (ρvr ~ 11.5 kg/m·s) sufficient to breach the Stratum corneum (sc). The trehalose, manitol, dextran (10 kDa), dextran (150 kDa) formulation, or TMDD (3:3:3:1), protected the MenY-CRM197 glyconjugate during SFD with minimal loss, no detectable chemical degradation or physical aggregation. In a capsular group Y Neisseria meningitidis serum bactericidal assay (SBA) with human serum complement, the needle free vaccine, which contained no alum adjuvant, induced functional protective antibody responses in vivo of similar magnitude to the conventional vaccine injected by hypodermic needle and syringe and containing alum adjuvant. These results demonstrate that needle-free vaccination is both technically and immunologically valid, and could be considered for vaccines in development.

T Cell-Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of Streptococcus pneumoniae.

Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine targets because they are easily accessible and recognizable by the immune system. However, CPS-specific adaptive humoral immune responses can only be achieved by the covalent conjugation of CPSs with carrier proteins to produce glycoconjugate vaccines. We previously described a mechanism by which a model glycoconjugate vaccine can activate the adaptive immune system and demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells. In this study, we use glycoconjugates of type 3 Streptococcus pneumoniae CPS (Pn3P) to assess whether the carbohydrate-specific adaptive immune response exemplified in our previous study can be applied to the conjugates of this lethal pathogen. In this article, we provide evidence for the functional roles of Pn3P-specific CD4+ T cells utilizing mouse immunization schemes that induce Pn3P-specific IgG responses in a carbohydrate-specific T cell-dependent manner.

A smart gene delivery platform: Cationic oligomer.

Low transfection efficiency and high cytotoxicity of polymeric gene carriers have hampered the application of numerous polycations for gene therapy. To overcome this barrier, a cationic glycoconjugate of kanamycin and di(ethylene glycol) diacrylate was prepared via a facile approach. Nuclear magnetic resonance, Fourier transform infrared spectroscopy, and size exclusion chromatography were employed to investigate the resulting materials. Agarose gel electrophoresis, atomic force microscopy, and circular dichroism spectroscopy were used to record the interaction of the cationic oligomer and plasmid DNA. Finally, the cytotoxicity and transfection efficiency were evaluated by using COS-7 cells. The results indicated that cationic oligomers had been obtained and plasmid DNA was condensed into nanocomplexes, with a high transfection efficiency of the oligomer and a low toxicity in COS-7 cell line. It provided a novel perspective to develop gene carrier, with better safety and greater transfection efficiency, compared to traditional high molecular weight polymers.