Prospects for the Use of Marine Sulfated Fucose-Rich Polysaccharides in Treatment and Prevention of COVID-19 and Post-COVID-19 Syndrome.

Symptoms of the new coronavirus infection that appeared in 2019 (COVID-19) range from low fever and fatigue to acute pneumonia and multiple organ failure. The clinical picture of COVID-19 is heterogeneous and involves most physiological systems; therefore, drugs with a wide spectrum of mechanism of action are required. The choice of the treatment strategy for post-COVID-19 syndrome is still a challenge to be resolved. Polysaccharides with a high fucose content derived from seaweed and marine animals can form the basis for the subsequent development of promising agents for the treatment of COVID-19 and post-COVID-19 syndrome. This class of biopolymers is characterized by a variety of biological activities, including antiviral, antithrombotic, anticoagulant, hemo-stimulating, anti-inflammatory and immune-regulatory. Low molecular weight derivatives of these polysaccharides, as well as synthetic oligosaccharides with a sufficient amount and sulfation type may be considered as the most promising compounds due to their better bioavailability, which undoubtedly increases their therapeutic potential.

Cross reacting material (CRM197) as a carrier protein for carbohydrate conjugate vaccines targeted at bacterial and fungal pathogens.

This paper gives an overview of conjugate glycovaccines which contain recombinant diphtheria toxoid CRM197 as a carrier protein. A special focus is given to synthetic methods used for preparation of neoglycoconjugates of CRM197 with oligosaccharide epitopes of cell surface carbohydrates of pathogenic bacteria and fungi. Syntheses of commercial vaccines and laboratory specimen on the basis of CRM197 are outlined briefly.

Erratum to: Prospects for the Use of Marine Sulfated Fucose-Rich Polysaccharides in Treatment and Prevention of COVID-19 and Post-COVID-19 Syndrome.

[This corrects the article DOI: 10.1134/S1068162022060152.].

Synthetic Analogs of Streptococcus pneumoniae Capsular Polysaccharides and Immunogenic Activities of Glycoconjugates.

Streptococcus pneumoniae is a Gram-positive bacterium (pneumococcus) that causes severe diseases in adults and children. It was established that some capsular polysaccharides of the clinically significant serotypes of S. pneumoniae in the composition of commercial pneumococcal polysaccharide or conjugate vaccines exhibit low immunogenicity. The review considers production methods and structural features of the synthetic oligosaccharides from the problematic pneumococcal serotypes that are characterized with low immunogenicity due to destruction or detrimental modification occurring in the process of their preparation and purification. Bacterial serotypes that cause severe pneumococcal diseases as well as serotypes not included in the composition of the pneumococcal conjugate vaccines are also discussed. It is demonstrated that the synthetic oligosaccharides corresponding to protective glycotopes of the capsular polysaccharides of various pneumococcal serotypes are capable of inducing formation of the protective opsonizing antibodies and immunological memory. Optimal constructs of oligosaccharides from the epidemiologically significant pneumococcal serotypes are presented that can be used for designing synthetic pneumococcal vaccines, as well as test systems for diagnosis of S. pneumoniae infections and monitoring of vaccination efficiency .

Glycoconjugate Vaccines for Prevention of Haemophilus influenzae Type b Diseases.

This review summarizes the experience in laboratory- and industrial-scale syntheses of glycoconjugate vaccines used for prevention of infectious diseases caused by Haemophilus influenzae type b bacteria based on the linear capsular polysaccharide poly-3-ß-D-ribosyl-(1→1)-D-ribitol-5-phosphate (PRP) or related synthetic oligosaccharide ligands. The methods for preparation of related oligosaccharide derivatives and results of the studies evaluating effect of their length on immunogenic properties of the conjugates with protein carriers are overviewed.

Synthesis of biotinylated pentasaccharide structurally related to a fragment of glucomannan from Candida utilis.

The polysaccharide mannan is the main surface antigen of the cell wall of Candida fungi, playing an important role in the pathogenesis of diseases caused by these mycopathogens. Mannan has a complex, comb-like structure and includes a variety of structural units, with their combination varying depending on the Candida species and strain. Glucomannan, a polysaccharide from Candida utilis, contains terminal α-d-glucose residues attached to oligomannoside side chains. This paper describes the first synthesis of a pentasaccharide structurally related to C. utilis glucomannan fragment, which is an α-(1→2)-linked tetramannoside terminated at the non-reducing end by an α-d-glucopyranosyl residue. The pentasaccharide was obtained as a 3-aminopropyl glycoside, which made it possible to synthesize also its biotinylated derivative, suitable for various glycobiological studies. The most complicated step in the pentasaccharide synthesis was stereoselective 1,2-cis-glycosylation to attach the α-d-glucopyranosyl residue. This was accomplished using a glucosyl donor specially developed in our laboratory, the protecting groups of which provide the necessary α-stereoselectivity. The target biotinylated pentasaccharide thus obtained will be used in the future as a model antigen for the detection of immunodeterminant epitopes of Candida mannans.

Synthetic Oligomers Mimicking Capsular Polysaccharide Diheteroglycan are Potential Vaccine Candidates against Encapsulated Enterococcal Infections.

Infections caused by Enterococcus spp. are a major concern in the clinical setting. In Enterococcus faecalis, the capsular polysaccharide diheteroglycan (DHG), composed of ß-d-galactofuranose-(1 → 3)-ß-d-glucopyranose repeats, has been described as an important virulence factor and as a potential vaccine candidate against encapsulated strains. Synthetic structures emulating immunogenic polysaccharides present many advantages over native polysaccharides for vaccine development. In this work, we described the synthesis of a library of DHG oligomers, differing in length and order of the monosaccharide constituents. Using suitably protected thioglycoside building blocks, oligosaccharides up to 8-mer in length built up from either Galf-Glcp or Glcp-Galf dimers were generated, and we evaluated their immunoreactivity with antibodies raised against DHG. After the screening, we selected two octasaccharides, having either a galactofuranose or glucopyranose terminus, which were conjugated to a carrier protein for the production of polyclonal antibodies. The resulting antibodies were specific toward the synthetic structures and mediated in vitro opsonophagocytic killing of different encapsulated E. feacalis strains. The evaluated oligosaccharides are the first synthetic structures described to elicit antibodies that target encapsulated E. faecalis strains and are, therefore, promising candidates for the development of a well-defined enterococcal glycoconjugate vaccine.

Carbohydrate Specificity and Isotypes of Monoclonal and Polyclonal Antibodies to Conjugated Tetrasaccharide, a Synthetic Analogue of Repeating Unit of Capsular Polysaccharide of Streptococcus Pneumoniae Serotype 14.

We studied carbohydrate specificity and isotypes of antibodies to BSA-conjugated tetrasaccharide, a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 14, in mouse polyclonal sera and hybridoma-synthesized products. Natural IgM antibodies to the tetrasaccharide containing epitopes similar to surface carbohydrate structures of mammalian and human cells in low titers were determined in native mouse serum by ELISA using biotinylated tetrasaccharide and synthetic capsular polysaccharide as the solid-phase antigens. Polyclonal sera to the conjugated tetrasaccharide contained IgM and all subclasses of IgG antibodies, which were detected in a higher titer when the biotinylated tetrasaccharide was used as a solid phase antigen compared to synthetic capsular polysaccharide. Monoclonal antibodies to S. pneumoniae serotype 14 tetrasaccharide were identified in an equivalent titer using either biotinylated tetrasaccharide or synthetic capsular polysaccharide. Monoclonal antibodies obtained in vitro belonged to IgM isotype and cross-reacted with secondary full-size IgG antibodies. In the serum of mice inoculated with hybridoma, IgM and IgG2a antibodies recognizing the tetrasaccharide epitope in the structure of synthetic capsular polysaccharide were simultaneously determined.

Synthesis of oligosaccharides related to galactomannans from Aspergillus fumigatus and their NMR spectral data.

The synthesis of model oligosaccharides related to antigenic galactomannans of the dangerous fungal pathogen Aspergillus fumigatus has been performed employing pyranoside-into-furanoside (PIF) rearrangement and controlled O(5) → O(6) benzoyl migration as key synthetic methods. The prepared compounds along with some previously synthesized oligosaccharides were studied by NMR spectroscopy with the full assignment of 1H and 13C signals and the determination of 13C NMR glycosylation effects. The obtained NMR database on 13C NMR chemical shifts for oligosaccharides representing galactomannan fragments forms the basis for further structural analysis of galactomannan related polysaccharides by a non-destructive approach based on the calculation of the 13C NMR spectra of polysaccharides by additive schemes.

[SYNTHETIC CONJUGATED ANALOGUES OF CAPSULE POLYSACCHARIDES OF PNEUMOCOCCUS - AN INSTRUMENT FOR DETECTION OF POST-VACCINATION ANTIBODIES].

AIM: Evaluation ofthe ability of capsule polysaccharides (CP) of Streptococcus pneumoniae serotype 3 and 14 and their synthetic structure analogues, conjugated with bovine serum albumin (BSA), to detect antibodies in post-vaccination sera of mice. Materials andmethods. Oligosaccharides correspond- ing to one, one and a half and two repeating links of serotype 3 and 14 S. pneumoniae CP were synthe- sized, their conjugates with BSA were produced by squarate method as well. Ligand content-per BSA molecule was controlled by MALDI-TOF spectrometry. Immune sera were obtained after 2 intraperi- toneal administrations to mice of glucoconjugates adsorbed on aluminum hydroxide or 13-valent pneumococcal conjugated vaccine. Determination of levels of post-vaccination class G antibodies and their sub-isotypes was carried out in EIA. RESULTS: Immunization of mice with neoglucoconjugates resulted in formation of predominantly IgGl recognizing serotype 3 and 14 S. pneumoniae C. IgG1 in mice immunized with a 13-valent conjugated vaccine recognized serotype 3 S. pneumoniae CP, but detected serotype 14 S. pneumoniae CP weakly. All the conjugated synthetic oligosaccharides were characterized by a high ability to bind antibodies in blood of mice immunized with the polysaccharide conjugated vaccine. BSA-tetrasaccharide of serotype 3 S. pneumoniae and BSA-tetrasaccharide of serotype 14 S.pneumoniae were characterized by the highest ability to detect IgG1 against C. CONCLUSION: Synthetic oligosaccharides, conjugated with BSA protein-carrier, may be used to develop diagnostic test-systems for determination of antibodies in post-vaccination sera.

Influence of fucoidans and their derivatives on antitumor and phagocytic activity of human blood leucocytes.

The immunotropic activity of structurally different fucoidans and their derivatives towards isolated immune blood cells, effectors of innate immune system, was studied. The most potent effect was observed for high molecular weight fucoidan CF from the alga Chordaria flagelliformis, whose backbone is built of (1→3)-linked units of α-L-fucopyranose, and branches included residues of α-D-glucuronic acid and α-L-fucofuranose. This compound at the concentration of 0.05 mg/ml potentiated phagocytosis of Saccharomyces cerevisiae and Lactobacillus acidophilus by neutrophils, increasing relative quantity of phagocytes as well as their effectiveness. Along with this, 14% increase in the concentration of membrane-bound integrin CD11c molecules was observed. The systemic effect of CF at the dose of 0.01 mg/mouse i.p. led to potentiation of cytotoxic activity of spleen mononuclear leucocytes towards melanoma cells of line B16 by 1.9-fold and towards chronic myelogenous leukemia cells of line K-562 by 1.7-fold. These results indicate that fucoidan CF can stimulate anti-infective and antitumor activity of effectors of the innate immune system via CD11c integrins.

[EPITOPIC SPECIFICITY OF A SYNTHETIC DISACCHARIDE, RECURRING LINK OF CAPSULE POLYSACCHARIDE CHAIN OF SEROTYPE 3 STREPTOCCUS PNEUMONIAE].

AIM: Study epitopic specificity of synthetic disaccharide, recurring link of serotype 3 S. pneumoniae, conjugated with bovine serum albumin (BSA). MATERIALS AND METHODS: Conjugate of the synthetic disaccharide with BSA was obtained by squarate method. Antigenic activity of the conjugate was studied in competitive EIA. Titers of IgG against capsule polysaccharide of serotype 3 S. pneumoniae were determined in EIA by using sera of mice immunized twice with disaccharide conjugate sorbed onto aluminum hydroxide. RESULTS: Disaccharide conjugate used as a well-covering antigen (4 µg/well) in EIA was characterized by a high degree of specificity and interacted only with IgG against serotype 3 S. pneumoniae in antimicrobial sera of animals without reacting with antibodies (ABs) against other pneumococcus serotypes (6B, 10A, 19A, 19F, 23F). Disaccharide conjugated with BSA was determined in competitive EIA to inhibit bonding of ABs to disaccharide by 78.8%, bacterial capsule polysaccharide by 56.9%, BSA did not inhibit the sera activity. The study of sera of mice immunized by serotype 3 S. pneumoniae disaccharide conjugate in EIA, where capsule polysaccharide was used as a plate-sorbed antigen, has established the presence of IgG against capsule polysaccharide at a titer of 1:1600. CONCLUSION: The disaccharide that is a single recurring link of serotype 3 S. pneumoniae contains a key epitope of capsule polysaccharide. The synthetic disaccharide could be used as a component of multivalent conjugated pneumococcal vaccines and for development of diagnostic test-systems.

Convergent synthesis of isomeric heterosaccharides related to the fragments of galactomannan from Aspergillus fumigatus.

Aspergillus fumigatus is a very common fungus with high pathogenic potential for immunosuppressed hospital patients. A. fumigatus galactomannan, being the part of its cell wall, is considered as a promising candidate for vaccine and diagnostic test-systems. In this article we report the convergent synthesis of pentasaccharide fragments of the galactomannan containing the ß-(1→5)-linked galactofuranoside chain attached to O-3 or O-6 of a spacer-armed mannopyranoside residue. The synthesis of selectively protected galactofuranoside precursors has been performed using recently developed pyranoside-into-furanoside (PIF) rearrangement. For assembling the target galactomannan structures the [1 + 2 + 2]-scheme was applied. This strategy was shown to be highly efficient and can easily be extended to the synthesis of longer fragments of thegalactomannan.

Development of approaches to creation of experimental test system for evaluation of antigenic activity of synthetic oligosaccharide ligands related to fragments of the Streptococcus pneumoniae type 14 capsular polysaccharide.

A conjugate of a synthetic hexasaccharide fragment of the Streptococcus pneumoniae type 14 capsular polysaccharide with bovine serum albumin (BSA) has been prepared. The antigenic activity and specificity of this conjugate are comparable with those of natural antigens of S. pneumoniae type 14. The data suggest that the resulting synthetic conjugate can be used as a coating antigen in an experimental test system (based on enzyme immunoassay) for evaluating the antigenic activity and specificity of synthetic oligosaccharide ligands and for testing specimens of natural capsular polysaccharides and immune sera.

Effect of branched α-oligomannoside structures on induction of anti-Candida humoral immune response.

Several studies have established the potential efficacy of humoral immunity, primarily mannan-specific antibodies, in host protection against major fungal pathogen Candida albicans. In this study, we analysed humoral immune response induced by immunization with BSA-based conjugates bearing synthetic α-1,6-branched oligomannosides (pentamannosides (M5) or hexamannosides (M6)) mimicking antigenic sequences of Candida cell wall mannan. We analysed the ability of antibodies prepared by immunization to recognize relevant antigenic determinants in mannan polysaccharide structure and in C. albicans yeast and hyphal morphoforms. M6-BSA conjugate induced markedly higher levels of mannan-specific IgG compared with M5-BSA conjugate. In contrast to M5-BSA conjugate, M6-BSA conjugate induced immunoglobulin isotype class switch from IgM to IgG, as revealed also from ELISPOT analysis. Immunization-induced antibodies showed higher reactivity with hyphal form of C. albicans cells. The reduced immunogenicity of M5-BSA conjugate seems to be related to branching point location at terminal non-reducing end in comparison with M6-BSA oligomannoside with branching point at non-terminal location. Candidacidal activity assay revealed different capacity of sera prepared by immunization with M5-BSA and M6-BSA conjugates to improve candidacidal activity of polymorphonuclear leucocytes. Limited capacity of α-1,6-branched oligomannoside--BSA conjugates to induce antibodies significantly enhancing candidacidal activity of polymorphonuclear leucocytes--was presumably related to absence of antibodies with strong reactivity to corresponding antigenic determinants in natural cell wall mannan and with reduced ability to activate complement. The study documented markedly structure-dependent immunogenicity and limited capacity of branched α-mannooligosides conjugates to induce production of potentially protective antibodies.

Production of antifungal cellobiose lipids by Trichosporon porosum.

The yeast Trichosporon porosum suppresses growth of ascomycetes and basidiomycetes belonging to 52 genera. It is due to secretion of a thermostable fungicidal agent. The suppression was maximal at pH 3.5-4.0. Fungicidal preparation obtained from the culture broth was shown to be a mixture of cellobiosides of dihydrodecane acid with different degree of acetylation of cellobiose residue. The preparation caused the death of Candida albicans and Filobasidiella neoformans cells in the concentrations of 0.2 and 0.03 mM, respectively.

Photothrombic activity of m-THPC-loaded liposomal formulations: pre-clinical assessment on chick chorioallantoic membrane model.

The objective of this study was to evaluate the ability of meso-tetra(hydroxyphenyl)chlorin (m-THPC) encapsulated into liposomal formulations to occlude neovascularization. Two m-THPC formulations including conventional or plain liposomes (Foslip) based on dipalmitoylphosphatidylcholine (DPPC) and the corresponding long-circulating poly(ethylene glycol) (PEG)-modified liposomes (PEGylated liposomes: Fospeg) were evaluated as delivery systems. Using the chick chorioallantoic membrane (CAM) as in vivo model, the fluorescence pharmacokinetic behaviour of encapsulated m-THPC reflecting the rate of the extravasation of the dye from the CAM vasculature and its photothrombic effectiveness were determined. This study was focused on the influence of the drug and/or light doses on the mean retention time of m-THPC within the CAM blood vessels after intravenous injection, and its photothrombic efficacy. Irrespective of the formulations tested and the drug doses injected, similar fluorescence pharmacokinetic profiles were obtained. The fluorescence contrast reached a steady state 30 s after injection. Constant positive values of the fluorescence contrast suggest that m-THPC is confined into the intravascular compartment during the experimental time (500 s). However, the photodynamic therapy assays showed that Foslip appears to be less potent than Fospeg in terms of photothrombic activities on the CAM model. For instance, the light dose necessary to induce the desired vascular damage with Foslip was twice (100 J/cm2) higher than with Fospeg (50 J/cm2). It can be inferred that this pre-clinical study showed that the formulation based on PEGylated liposomes technology offers a suitable delivery system for the treatment of choroidal neovascularization associated with age-related macular degeneration.

Preclinical evaluation of a novel water-soluble chlorin E6 derivative (BLC 1010) as photosensitizer for the closure of the neovessels.

In the present study, photodynamic activity of a novel photosensitizer (PS), Chlorin e(6)-2.5 N-methyl-d-glucamine (BLC 1010), was evaluated using the chorioallantoic membrane (CAM) as an in vivo model. After intravenous (i.v.) injection of BLC 1010 into the CAM vasculature, the applicability of this drug for photodynamic therapy (PDT) was assessed in terms of fluorescence pharmacokinetics, i.e. leakage from the CAM vessels, and photothrombic activity. The influence of different PDT parameters including drug and light doses on the photodynamic activity of BLC 1010 has been investigated. It was found that, irrespective of drug dose, an identical continuous decrease in fluorescence contrast between the drug inside and outside the blood vessels was observed. The optimal treatment conditions leading to desired vascular damage were obtained by varying drug and light doses. Indeed, observable damage was achieved when irradiation was performed at light doses up to 5 J/cm(2) 1 min after i.v. injection of drug doses up to 0.5 mg/kg body weight(b.w.). However, when irradiation with light doses of more than 10 J/cm(2) was performed 1 min after injection of drug doses up to 2 mg/kg body weight, this led to occlusion of large blood vessels. It has been demonstrated that it is possible to obtain the desired vascular occlusion and stasis with BLC 1010 for different combinations of drug and/or light doses.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.

Effect of enzyme preparation from the marine mollusk Littorina kurila on fucoidan from the brown alga Fucus distichus.

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units -->3)-alpha-L-Fucp-(2,4-di-SO3(-))-(1-->4)-alpha-L-Fucp-(2SO3(-))-(1-->, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.