Lucia Paulovičová

Synthesis and bioimmunological efficiency of poly(2-oxazolines) containing a free amino group

Novel amphiphilic copolymers on the basis of 2-oxazolines containing a free amino group were prepared. The copolymers were synthesized by the living cationic polymerization of 2-ethyl-2-oxazoline (ETOX) and 2-(4-aminophenyl)-2-oxazoline (APOX). The main goal of this work was the synthesis of water soluble polymer material with the defined number of functional groups necessary for the attachment of proteins and polysaccharides. A high concentration of free amino groups allows immobilization of various biosubstances, e.g. drugs, proteins or polysaccharides. Thermal properties have been studied with respect to the composition of the copolymers. Cytotoxicity and the bioimmunological efficiency of the selected copolymer were studied.

Immunomodulatory efficiency of poly(2-oxazolines)

Poly(2-oxazolines) represent promising polymer materials for biomedical applications. The activation of mouse lymphoid macrophage line P388.D1 (clone 3124) by two selected representatives of poly(2-oxazolines), namely poly(2-ethyl-2-oxazoline) (PETOX100) and poly[2-(4-aminophenyl)-2-oxazoline-co-2-ethyl-2-oxazoline] (AEOX10), was assessed in vitro. The immunomodulatory efficacy of both polymers was evaluated via the induced release of pro-inflammatory cytokines (TNF-α, IL-1α and IL-6) and the acceleration of reactive free radicals. The present study revealed effective structure-immunomodulating associations of AEOX10 and PETOX100, which are desirable in biomedical and pharmaceutical applications of aliphatic and aromatic poly (2-oxazolines) in vivo.

Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside-protein conjugate Immunomodulatory properties of heptamannoside-BSA conjugate

Chemically defined glycoprotein conjugate composed of synthetically prepared mannan-derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7-BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7-BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat-inactivated C. albicans whole cells immunization. The administration route of M7-BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7-BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7-BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3(+) T lymphocytes and increased the CD4(+)/CD8(+) T lymphocyte ratio. We observed also an increasing proportion of CD4(+)CD25(+) T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8(+)CD25(+) cell proportion. Immunization with M7-BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.

Participation of the Candida albicans surface antigen in adhesion, the first phase of biofilm development.

The lack of work dealing with possible ways of reducing biofilm production via inhibiting Candida albicans adherence in the first stage of biofilm formation was a motivation for this study. The study was focused on two questions: (1) can a decrease in adherence affect the quantity of mature biofilm? and (2) can blocking the surface C. albicans complement receptor 3-related protein (CR3-RP) with polyclonal anti-C3-RP antibody or monoclonal antibody OKM1 significantly contribute to a reduction in adherence during biofilm formation? The presence and quantity the CR3-RP expressed in the biofilm was confirmed by immunofluorescence, immunocytometry and enzyme-linked immunosorbent assay. To determine the changes in adherence of C. albicans CCY 29-3-162 and C. albicans catheter isolate, 30-, 60-, 90- and 120-min time points were selected and viability was determined by XTT assay. The strains were preincubated with both antibodies to block CR3-RP, which proved to be effective at reducing adhesion and the formation of a mature biofilm (64.1-74.6%). The duration of adhesion, between 30 and 120 min, seems to have a significant effect on the mature biofilm. The blocking of CR3-PR by antibodies before adherence affected the fitness of biofilm, which was not able to revitalize in the later stages.

Synthesis of a heptasaccharide fragment of the mannan from Candida guilliermondii cell wall and its conjugate with BSA

The 3-aminopropyl glycoside of a heptasaccharide fragment of the cell wall mannan from Candida guilliermondii 18, which corresponds to the antigenic Factor 9, has been synthesized by a convergent approach based on glycosylation of a tetrasaccharide acceptor with a trisaccharide donor as the key step to give a protected heptasaccharide 17. Subsequent two-step deprotection of 17 afforded the heptamannoside 18, which was then conjugated with BSA using the squarate procedure.

Synthesis of 3,6-branched oligomannoside fragments of the mannan from Candida albicans cell wall corresponding to the antigenic factor 4.

3-Aminopropyl glycosides of 3,6-branched penta- and hexamannoside fragments of the cell wall mannan from Candida albicans, corresponding to the antigenic factor 4, have been synthesized. Subsequent coupling of both oligosaccharides with BSA using the squarate procedure provided corresponding neoglycoconjugates.

Model α-mannoside conjugates: immunogenicity and induction of candidacidal activity

The effect of Candida cell wall mannan-derived alpha-oligomannoside structural components on the modulation of the immune system and their role in protective immunity are studied here. Semi-synthetic alpha-mannoside-bovine serum albumin conjugates were used for immunization of rabbits. Dimeric alpha-mannoside, representing Candida antigenic factor 1, was used as a model of linear alpha-mannoside, and pentameric alpha-mannoside was used as a model of branched oligomannoside side chain structure. The induction of humoral immune response and the functionality of the serum tested by induction of peripheral blood leukocyte (PBL) candidacidal activity are documented. Anti-Candida albicans serotype B immunoglobulins (IgG and IgM) levels were higher than anti-serotype A following immunization with both conjugates. Dimer-conjugate postimmunization sera evidently enhanced C. albicans killing activity of PBLs in candidacidal assay. The study shows the importance of alpha-mannoside structures in perspective anti-Candida vaccine with a broad spectrum of effectiveness.

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.

Recent advances in the synthesis of fungal antigenic oligosaccharides

Abstract The driving force for the constant improvement and development of new synthetic methodologies in carbohydrate chemistry is a growing demand for biologically important oligosaccharide ligands and neoglycoconjugates thereof for numerous biochemical investigations such as cell-to-pathogen interactions, immune response, cell adhesion, etc. Here we report our syntheses of the spacer-armed antigenic oligosaccharides related to three groups of the polysaccharides of the fungal cell-wall including α- and β-mannan, α- and β-glucan and galactomannan chains, which include new rationally designed synthetic blocks, efficient solutions for the stereoselective construction of glycoside bonds, and novel strategy for preparation of furanoside-containing oligosaccharides based on recently discovered pyranoside-into-furanoside (PIF) rearrangement.

Ex Vivo and In Vitro Studies on the Cytotoxicity and Immunomodulative Properties of Poly(2-isopropenyl-2-oxazoline) as a New Type of Biomedical Polymer

Poly(2-alkenyl-2-oxazoline)s are promising functional polymers for a variety of biomedical applications, such as drug delivery systems, peptide conjugates, or gene delivery. In this study, poly(2-isopropenyl-2-oxazoline) (PIPOx) is prepared through free-radical polymerization initiated with azobisisobutyronitrile. Reactive 2-oxazoline units in the side chain support an addition reaction with different compounds containing a carboxylic group, which facilitates the preparation of polymers labeled with two different fluorescent dyes. The cytotoxicities of 2-oxazoline monomers, PIPOx, and fluorescently labeled PIPOx are evaluated in vitro using an 3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and ex vivo using a cell proliferation assay with adenosine triphosphate bioluminescence. The cell uptake of labeled PIPOx is used to determine the colocalization of PIPOx with cell organelles that are part of the endocytic pathway. For the first time, it is shown that poly(2-isopropenyl-2-oxazoline) is a biocompatible material and is suitable for biomedical applications; further, its immunomodulative properties are evaluated.