Alexander A. Karelin

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.

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.

The evaluation of β-(1 → 3)-nonaglucoside as an anti-Candida albicans immune response inducer.

Synthetically prepared bovine serum albumin (BSA) conjugate of linear β‐(1 → 3)‐nonaglucoside ligand (G9) has been applied as a biological response immunomodulator in vivo and ex vivo. Active immunization of Balb/c mice revealed effective induction of specific humoral responses in comparison with Candida β‐D‐glucan and Candida whole cells. Induced post‐vaccination serum exhibited a growth‐inhibition effect on the multi‐azole‐resistant clinical strain Candida albicans CCY 29‐3‐164 in experimental mucocutaneous infection ex vivo. Evaluation of immune cell proliferation and the cytotoxic potential of the G9‐ligand has revealed its bioavailability and an immunostimulative effect in vaccination‐sensitized Balb/c mice splenocytes and RAW 264.7 macrophages.

Immune cell response to Candida cell wall mannan derived branched α-oligomannoside conjugates in mice

BACKGROUND Constructs composed of cell wall mannan-derived moieties conjugated to immunogenic proteins could be promising agents for induction of protective anti-Candida immune responses. METHODS This report is focused on the cellular immune response differences induced by BSA-based conjugates bearing synthetic α-1,6-branched oligomannosides. For monitoring of the immune responses following active immunization we evaluated changes in the frequencies of T and B lymphocytes and their activation status in the blood and spleen. We compared the immunization-induced changes of co-stimulatory molecules CD80 and CD86 expression on blood neutrophils and Th1/Th2 polarization of the immune response based on IFN-γ, TNF-α (pro-Th1), IL-4, and IL-10 (pro-Th2) cytokines levels and induction of IL-17. RESULTS The results pointed out a comparable effect of the conjugates on the modulation of T and B lymphocytes frequencies in blood and spleen. Both conjugates induced upregulation of CD25 surface antigen on CD4(+) T lymphocytes, independently on the structural differences of oligosaccharides. The differences in structure of oligomannoside antigens or conjugate constructs were reflected in the increase of co-stimulatory molecules CD80 and CD86 expression on neutrophils, and in induced cytokine response. M5-BSA conjugate induced only a slight increase in CD80 expression but a significant increase in IFN-γ, TNF-α, and IL-10. M6-BSA conjugate induced a significant increase of CD80 expression and increase of TNF-α, IL-4, and IL-10. CONCLUSION Obtained data demonstrate the importance of cellular immune response analysis for investigation of immunomodulatory properties of oligomannoside-protein conjugates.

Synthesis, structure and proton conductivity of 2,4,5-trimethylbenzenesulfonic acid dihydrate

2,4,5-Trimethylbenzenesulfonic acid dihydrate was isolated in the monocrystalline state. Crystal and molecular structures of the compound were determined by X-ray diffraction analysis and vibrational spectroscopy. The compound crystal system is monoclinic: a =7.7445(3) A, b = 7.5360(3) A, c = 9.9477(4) A, α = γ = 90°, β = 92.123(4)°, V = 580.17(4) A3, P21, Z = 2, R1 = 0.0323, dcalc. = 1.353 g cm−3, T = 150 K. A 2D hydrogen bond network formed of a dioxonium ion and oxygen atom of a sulfogroup was identified in the crystal structure. IR absorption spectra, Raman spectra and ATR IR spectra of 2,4,5-trimethylbenzenesulfonic acid dihydrate and deuterodihydrate have been obtained in the region 50–4000 cm−1. It was found that the proton and deuterium in the H5O2+ and D5O2+ cations shifted from the midpoint of the strong hydrogen bond toward one of two water molecules according to the scheme O⋯H+⋯O → O⋯H+–O and O⋯D+⋯O → O⋯D+–O. Spectroscopic data concerning dioxonium ion formation show satisfactory agreement with X-ray diffraction analysis. The effect of environment humidity on the state of 2,4,5-trimethylbenzenesulfonic acid dihydrate aggregation was investigated. It was shown that the number of water molecules increases up to 2.5 per sulfogroup in the 7–84 rel.% air humidity interval through additional water molecules adsorption. A further humidity increase led to compound deliquescence. The proton conductivity as measured by impedance spectroscopy is 10−9–10−5 S cm−1 in the 7–58 rel.% humidity interval at 298 K with a [H2O]/[SO3−] = 2.2 ratio at maximum conductivity.