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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The yeast Candida albicans is a common component of the human commensal flora colonizing mucocutaneous surfaces and gastrointestinal tract of the healthy humans. C. albicans is also an important opportunistic fungal pathogen in immunocompromised individuals, being responsible for superficial and systemic infections. Numerous studies have confirmed the importance of adaptive immunity for host protection against invasive fungal infections. There is widespread consensus in the field of medical mycology that cellular immunity is critical for successful host defence against fungi. However, in recent years, several studies have established the potential importance of humoral immunity in host protection against Candida infection [1]. Both C. albicans mannan-specific immune serum and short-chain β-1,2-linked oligomannosides-specific monoclonal antibodies generated from vaccinated mice were protective against experimental disseminated candidiasis [2, 3] and C. albicans vaginal infection [4]. In this studies, antibody efficacy was dependent upon epitope specificity [5], the presence of complement [6] and neutrophils [7].

The objective of the present study was to analyse the immunogenicity of two synthetic α-1,6-branched oligomannoside – BSA conjugates (pentamannoside: M5-BSA and hexamannoside: M6-BSA) and to study the ability of antibodies induced by immunization to recognize relevant antigenic structures in purified acid-stable mannan moiety and in natural cell wall mannan of yeast and hyphal C. albicans serotype A cells. The immunogenicity and induction of appropriate immune response of different saccharide – protein conjugates – depend upon structural arrangement and selection of well-defined saccharide antigen. The synthetically prepared oligomannosides provide unique possibility to study the generation of protective anti-Candida humoral immune response by exactly defined mannan-derived moieties. Mannan, a predominant polysaccharide on the surface of Candida cells, is involved in several types of interactions of fungal cells with host immune system. The mannan polysaccharide has a comb-like structure with an α-1,6-linked backbone and various oligomannosyl side chains mainly containing α-1,2-, α-1,3- and β-1,2-linked mannose residues. From the published analysis of the 1H-NMR signals of the side chains in the mannan of C. albicans serotype A [8], oligomannosyls corresponding to M5 oligomer represent 8% and oligomannosyls corresponding to M6 oligomer represent 3% of mannan side chains. Also C. albicans serotype B mannan side chains are branched by the addition of α-1,6-linked mannose units to make the epitope corresponding to antigenic factor 4 [9]. The α-linked mannosyl side chains are more prevalent in the mannan structure of both C. albicans serotypes [8, 10]. Analysis of C. guilliermondii mannan suggests significant amount of branched side chains in mannan of this strain [11]. According to the presence of antigenic factor 4–related antigenic determinants in mannan of both C. albicans serotypes and in mannan of C. guilliermondii [8, 9] antibodies induced by immunization with glycoconjugates bearing α-1,6-branched oligomannosides should have the capacity to recognize corresponding structures in acid-stable mannan moiety and also in native cell wall mannan of intact C. albicans cells. C. guilliermondii mannan has besides the antigenic factor 4 also antigenic factor 9. Antigenic factor 9 corresponds to α-1,6-branched side chain structure, which is similar to antigenic factor 4, but terminated with β-1,2-linked mannose units [11]. The α-1,6-branched side chains are over synthesized under acid conditions (pH 2.0) of C. albicans serotype A cells cultivation. Their molar ratio in mannan raised 5.7 times compared with mannan of cells cultured under conventional conditions (pH 5.9) [12].

Our previously published studies revealed that antibodies induced by synthetic oligomannoside – BSA conjugates – had the capacity to induce the candidacidal activity in vitro [13, 14]. Relative efficiency of prepared α-1,6-branched oligomannoside – BSA conjugates to induce production of potentially protective antibodies with capacity to enhance C. albicans opsonophagocytic killing by polymorphonuclear cells (PMN) – was analysed and compared with previously obtained results with conjugates containing linear mannooligosaccharides.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Oligosaccharide-conjugate preparation

Conjugation of BSA with spacered oligosaccharide derivatives (compounds a on Fig. 1) bearing synthetic pentamannoside (M5: α-D-Man-(1[RIGHTWARDS ARROW]3)-[α-D-Man-(1[RIGHTWARDS ARROW]6)]-α-D-Man-(1[RIGHTWARDS ARROW]2)-α-D-Man-(1[RIGHTWARDS ARROW]2)-α-D-Man) and hexamannoside (M6: α-D-Man-(1[RIGHTWARDS ARROW]2)-α-D-Man-(1[RIGHTWARDS ARROW]3)-[α-D-Man-(1[RIGHTWARDS ARROW]6)]-α-D-Man-(1[RIGHTWARDS ARROW]2)-α-D-Man-(1[RIGHTWARDS ARROW]2)-α-D-Man) ligands was performed by squarate method [15, 16]. Thus, the treatment with diethyl squarate at pH 7 gave corresponding monosubstituted adducts (b on Fig. 1). Their subsequent coupling with BSA at pH 9 resulted in the formation of conjugates (c on Fig. 1) designed as M5-BSA and M6-BSA (Fig. 1). According to MALDI-TOF mass spectrometry, M5-BSA conjugate contained on the average 10 pentasaccharide residues and M6-BSA conjugate contained on the average 8.5 hexasaccharide residues per one BSA molecule [16]. Selected oligomannosides mimic natural structures of Candida antigenic factor 4 [9, 11] in acid-stable mannan part of both C. albicans serotypes [8, 9] and C. guilliermondii [11].

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Figure 1. Structures of BSA-based glycoconjugates M5-BSA and M6-BSA. Conjugates were prepared by squarate method [16].

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Mannan preparation

Yeast strains C. albicans CCY 29-3-32 (serotype A), C. albicans CCY 29-3-102 (serotype B) and C. guilliermondii CCY 29-3-20 (Culture Collection of Yeast, Institute of Chemistry of Slovak Academy of Science, Bratislava, Slovakia) were used in all experiments. Cell wall mannans were isolated and purified using Fehling's reagent according literature [17].

Immunization

Mice (female, 6-week-old, variety BALB/c) were from Research Institute of Animal Production (Velaz, Prague, Czech Republic). The mice had free access to standard pelleted diet and tap water. The animal facilities comply with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes. The experimental protocol was approved by the Ethics Committee and by the Slovak State Veterinary Committee of Animal Experimentation. Mice (40 mice per one conjugate) were subcutaneously (sc) primary immunized (1st dose) with conjugate without adjuvant (6 μg oligosaccharide per dose) and subsequently primary sc boosted (2nd dose) without adjuvant 2 weeks after primary injection. Two weeks after primary booster injection, mice were divided into two groups and were secondary boosted by sc (3rd sc dose) or intraperitoneal (ip, 3rd ip dose) administration of the same conjugate dose without adjuvant. Sera samples were collected at day 14 following each injection. Mice (10 mice in group) three times sc injected with saline in the same time schedule were used as controls.

Micro-organisms growth conditions and viability

Yeast strain C. albicans CCY 29-3-100 (serotype A) (Culture Collection of Yeasts, Institute of Chemistry, Slovak Academy of Sciences, Slovakia) was cultured on 7% malt extract agar at 28 °C. After 48 h, static cultivation cells were harvested in saline, washed twice with PBS pH 7.4. Viability was specified by flow cytometry with propidium iodide negative staining >99%. Fixation of Candida cells was carried out by mixing with 60% ethanol (45:5 v/v) and incubating 15 min at 25 °C, washed twice with PBS and adjusted to 5 × 106 cells/ml with PBS. Ethanol-killed Candida cells were used as control sample in flow cytometric analysis for electronic gates set-up.

Determination of mannan and C. albicans whole cell–specific sera immunoglobulin levels

Levels of induced anti-mannan sera immunoglobulins (IgG, IgM and IgA) were determined by ELISA test, using C. albicans serotype A, C. albicans serotype B or C. guilliermondii mannan in coating step [18]. Antibodies levels were detected at serum dilution 1:100 (n = 10 mice from each group). For the exact expression of IgG, IgM and IgA levels, quantification (in ng/ml) using appropriate calibration curve based on reference mouse serum (Mouse Reference Serum; Bethyl Laboratories, Inc., Montgomery, TX, USA) was done. Statistic analysis was performed using one-way ANOVA test. All data were expressed as mean ± SD. P-values <0.05 were considered statistically significant. Induced C. albicans CCY 29-3-100 (serotype A) whole cell–specific sera immunoglobulin levels (IgG, IgM and IgA, n = 10 mice from each group) were determined by whole cell ELISA test, using C. albicans serotype A cells as yeast and hyphal morphological forms in plate-coating step. The concentrations of coated substances and C. albicans cells used as antigens were optimized, and for determination of mannan and Candida whole cell–specific antibodies, immune and control sera dilution 1:100 was selected as optimal according to location in the linear range of dilution curve.

IgG subclasses determination

Mouse IgG subclasses IgG1, IgG2a, IgG2b and IgG3 were examined with strip-immobilized goat anti-mouse antibodies (Serotec, Raleigh, NC, USA) according literature [19, 20]. The intensity of the resulting bands indicated specific antibody concentrations in the tested antisera (n = 5 mice from each group). Evaluation was done by calculated integral optical density (IOD) (software Gel-Pro Analyser 3.1; Media Cybernetics, Santa Barbara, CA, USA).

Peripheral polymorphonuclear cells preparation and candidacidal activity assay

Peripheral blood leucocytes population was obtained from the heparinized complete peripheral blood of mice as described before [14]. Briefly, polymorphonuclear cells (PMN) were isolated by Ficoll-Urografin gradients following dextran sedimentation of erythrocytes and finally adjusted to 1 × 106 cells/ml in RPMI 1600. C. albicans CCY 29-3-100 (serotype A) cells (100 μl, 5 × 106 cells/ml) were pre-incubated with 100 μl of heat non-inactivated serum samples and heat-inactivated serum samples (n = 5 mice from each group, final serum dilution 1:50) and PBS as control for 30 min at 37 °C. Next, C. albicans cells samples were washed with PBS and incubated with isolated PMN (1 × 106 cells/ml), to obtain target cells to effector cells ratio 5:1, for 60 minutes at 37 °C. After incubation, PMN were lysed with sodium deoxycholate [13, 14, 21]. Propidium iodide (PI, 0.02 μg/ml, redistilled water, Sigma) and fluorescein diacetate (FDA, 5 mg/ml stock solution in acetone, 50 μg/ml, redistilled water, Lachema) staining was carried out by incubating 100 μl of the Candida suspension with 50 μl of PI and 50 μl of FDA for 30 min at room temperature in darkness. Incubations and staining steps were done under static conditions.

Preparation of splenocytes

Spleens aseptically removed from immunized and control mice were placed in ice-cold PBS. Spleens were washed out with PBS (5-ml syringe, 1 ml per spleen) to rinse cells. The cell suspension was centrifuged at 800 × g for 10 min at 4 °C. The cell pellet was resuspended in 5 ml of ACK lysing buffer (0.15 m NH4Cl, 1 m K2CO3, and 0.01 m EDTA, pH 7.2) and incubated at room temperature for 5 min to lyse the red blood cells. The cell suspension was washed twice with PBS and resuspended in RPMI-1640 containing 10% foetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin sulphate. The cell density was adjusted to 1 × 106 cells per ml with RPMI-1640 after determination of cell viability using trypan blue dye exclusion method.

Analysis of mannan-specific antibody-secreting cells

The ELISPOT assay was used to analyse mannan-specific antibody-secreting cells in spleen of immunized mice. C. albicans serotype A or C. albicans serotype B purified mannan was diluted in carbonate – bicarbonate coating buffer (pH 9.6) at a concentration 10 μg/ml and 100 μl of the solution was applied to each well. The plates were incubated at 4 °C overnight. The plates were washed three times with PBS and blocked by incubation with RPMI 1640 medium containing 10% foetal bovine serum for 2 h at room temperature. The plates were washed twice with PBS. Splenocytes (in RPMI 1640 medium) were added to the wells (1 × 105 cells per well), and the plates were incubated in a humidified incubator at 37 °C, 5% CO2 for 24 h. Samples (n = 10 mice from each group) were tested in triplicate. At the end of the incubation, the plates were washed five times with PBS and alkaline phosphatase-conjugated antibodies (goat anti-mouse IgG and goat anti-mouse IgM, dilution 1:2000, 100 μl per well) were added. The plates were incubated for 2 h at room temperature, after than washed with PBS. For detection of spots, 100 μl of BCIP/NBT substrate was added to each well. Following washing, the plates were left at room temperature to dry. The plates were examined for spots counts using an Axio Imager A1 microscope (Zeiss, Germany). Quantitative evaluation of spots and enumeration of Ig-producing cells was performed via KS ELISPOT 4.10 running under AxioVision software (Zeiss, Germany).

Statistical analysis

Data were evaluated for statistical significance of differences by one-way ANOVA followed by Bonferroni's multiple comparison tests and Spearman's rank correlation test. All data were expressed as mean ± SD.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Mannan-specific antibodies levels

To evaluate the ability of antibodies induced by immunization with M5-BSA and M6-BSA conjugates to react with mannan structure, the specific serum antibodies levels against acid-stable mannan moiety of both C. albicans serotypes and C. guilliermondii after each injection of conjugates were determined (Fig. 2). Detected acid-stable mannan-specific antibodies levels in immune sera were compared with the controls (sera obtained after immunization with saline).

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Figure 2. Mannan specific serum antibodies levels Mannan C. albicans serotype A, C. albicans serotype B and C. guilliermondii specific antibodies levels in sera were determined after the primary sc injection (1st, n = 10), the primary sc booster injection (2nd, secondary sc injection, n = 10), the secondary ip booster injection (3rd ip, tertiary ip injection, n = 10) and the secondary sc booster injection (3rd sc, tertiary sc injection, n = 10) of M5-BSA and M6-BSA conjugates. Control represents mice after tertiary sc injection of saline (n = 10). All data are presented as mean ± SD and statistical significance of differences between immune and control sera are expressed: ***< 0.001, **0.001 < < 0.01, *0.01 < < 0.05.

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M5-BSA conjugate immunization induced increase in mannan-specific IgM levels with maximal peak after the secondary sc booster injection (3rd sc) for mannan C. albicans serotype A. Immunization with M5-BSA conjugate induced slight statistically significant increase in mannan-specific IgG for mannan Calbicans serotype A and mannan C. guilliermondii. Nevertheless, mannan-specific IgG levels induced by M5-BSA conjugate immunization did not exceed the levels of mannan-specific IgM levels (Fig. 2). For mannan-specific IgA levels, we observed no increase using mannan C. albicans serotype A and slight statistically significant increase using mannans of C. albicans serotype B and C. guilliermondii as target antigen.

In comparison with M5-BSA conjugate, structurally similar M6-BSA conjugate induced different kinetics of mannan-specific antibodies levels throughout the immunization (Fig. 2). We observed a marked increase in mannan C. albicans serotype A-specific IgM levels after the primary injection (1st) and the primary sc booster injection (2nd) of M6-BSA conjugate followed by significant decrease after the secondary booster injections (both, 3rd sc and 3rd ip administration). Mannan C. albicans serotype B and mannan C. guilliermondii-specific IgM levels induced by immunization with M6-BSA conjugate were statistically significantly higher compared with the control, but lower than using mannan C. albicans serotype A as antigen (Fig. 2).

Mannan-specific IgG antibodies levels increased after the primary sc injection (1st) and primary sc booster injection (2nd) of M6-BSA conjugate. Increasing tendency of mannan-specific IgG levels after secondary booster injection of M6-BSA conjugate was maintained only for sc route of administration (Fig. 2, 3rd sc). After secondary ip booster injection (3rd ip) of M6-BSA, conjugate levels of mannan-specific IgG antibodies decreased. Trends of IgG level changes were similar for all used mannans (Fig. 2). Increase in mannan-specific IgG levels associated with parallel decrease in mannan-specific IgM revealed induction of IgM/IgG isotype switch after secondary sc booster injection of M6-BSA conjugate (Fig. 2). Throughout immunization with M6-BSA conjugate, we did not observe a significant increase in IgA levels using C. albicans mannan. C. guilliermondii mannan-specific IgA levels increased markedly especially after secondary sc booster injection (3rd sc) of M6-BSA conjugate (Fig. 2).

The immunization with both conjugates, M5-BSA and M6-BSA, induced increase in IgG1/IgG2a antibodies ratio (Fig. 3). The IgG1/IgG2a ratio increased significantly after secondary ip booster injection, and markedly higher levels of IgG1 compared with IgG2a were induced by M6-BSA conjugate.

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Figure 3. IgG1 and IgG2a serum antibody levels ratio IgG1/IgG2a ratio after the primary sc injection (1st, n = 10), the primary sc booster injection (2nd, secondary sc injection, n = 10), the secondary ip booster injection (3rd ip, tertiary ip injection, n = 10) and the secondary sc booster injection (3rd sc, tertiary sc injection, n = 10) of M5-BSA and M6-BSA conjugate. Control represents mice after the tertiary sc injection of saline (n = 10). All data were expressed as mean ± SD, statistical significance of differences between immunized and control mice are expressed: ***< 0.001, **0.001 < < 0.01, *0.01 < < 0.05.

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Production of mannan-specific antibodies by splenocytes

Candida albicans serotype A mannan and C. albicans serotype B mannan-specific IgG and IgM antibody-secreting cells counts in response to immunization was analysed by ELISPOT assay (Fig. 4). For M5-BSA conjugate immunization, we detected marked formation of mannan-specific IgM-secreting cells after primary sc injection (1st) and primary sc booster injection (2nd) with subsequent decrease after secondary booster injection (for both routes of administration, 3rd ip and 3rd sc) for both C. albicans mannans (Fig. 4). The observed decrease in count of mannan-specific IgM-producing cells after secondary booster injection of M5-BSA conjugate was more marked after ip route of administration and was accompanied with continuous slight increase in mannan-specific IgG production (3rd ip).

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Figure 4. ELISpot analysis of C. albicans serotype A (A) and mannan C. albicans serotype B (B) IgG and IgM antibody-secreting cells in the spleen of the immunized mice The number of mannan C. albicans serotype A (A) and mannan C. albicans serotype B (B) specific IgG and IgM antibodies producing cells in controls and on day 14 after each injection of M5-BSA or M6-BSA conjugate (the primary sc injection (1st, n = 10), the primary sc booster injection (2nd, secondary sc injection, n = 10), the secondary ip booster injection (3rd ip, tertiary ip injection, n = 10) and the secondary sc booster injection (3rd sc, tertiary sc injection, n = 10)). Control represents mice after tertiary sc injection of saline (n = 10). All data were expressed as mean ± SD, statistical significance of differences between immunized and control mice are expressed: ***P<0.001, **0.001<P<0.01, *0.01<P<0.05.

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Primary administration of M6-BSA conjugate (1st) induced significant increase in mannan C. albicans-specific IgM-secreting cells count followed by significant decrease after primary sc booster injection (2nd) of conjugate. Decrease in number of mannan-specific IgM-producing cells was associated with an increase in number of cells producing mannan-specific IgG with maximal peak after secondary sc booster injection (Fig. 4).

For both conjugates, mannan C. albicans serotype A-specific IgG sera levels and detected specific IgG spot counts showed strong correlation (M5-BSA: r = 0.94, P = 0.017; M6-BSA: r = 0.814, P = 0.09). For M5-BSA conjugate mannan C. albicans serotype A-specific IgM, sera levels did not correlate with specific IgM-producing cells counts, but for M6-BSA conjugate immunization, we observed moderate correlation (r = 0.7, P = 0.19) between mannan C. albicans serotype A-specific IgM sera levels and specific IgM-producing cells counts. Mannan C. albicans serotype B-specific sera antibodies levels induced by immunization with M5-BSA conjugate did no correlate with specific antibody-secreting cells counts. Alteration of mannan C. albicans serotype A-specific IgM and IgG antibody production induced by immunization with M6-BSA conjugate distinctively revealed IgM/IgG isotype switch (Fig. 4).

Antibodies against branched oligomannoside structure in C. albicans yeast and hyphal cells

Purified cell wall mannan does not always maintain their native conformation. To maintain mannan native conformation intact for the analysis of antibodies generated during the immunization with conjugates, we used intact yeast and hyphal cells of C. albicans serotype A in whole cells ELISA assays to determine natural cell wall mannan-specific antibodies levels (Fig. 5).

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Figure 5. Anti – C. albicans serotype A whole cell antibodies levels C. albicans serotype A yeast (Yeast form) and hyphae (Hyphal form) specific antibodies levels detected in sera on day 14 after each injection of M5-BSA or M6-BSA conjugate (after the primary sc injection (1st, n = 10), the primary sc booster injection (2nd, secondary sc injection, n = 10), the secondary ip booster injection (3rd ip, tertiary ip injection, n = 10) and the secondary sc booster injection (3rd sc, tertiary sc injection, n = 10) of conjugate). Control represents yeast and hyphae specific antibodies in sera of mice after third sc injection of saline (n = 10). All data were expressed as mean ± SD, statistical significance of differences between immunized and control mice are expressed: ***< 0.001, **0.001 < < 0.01, *0.01 < < 0.05.

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We observed higher yeast and hyphae-specific IgM sera levels following M5-BSA immunization in comparison with control although without significant alteration throughout immunization. M5-BSA conjugate immunization induced significantly higher levels of yeast and hyphae-specific IgG antibody levels in comparison with IgG levels in sera of controls only after the primary sc injection of conjugate (Fig. 5). IgG levels induced by subsequent M5-BSA conjugate injections were comparable or lower than IgG levels in sera of controls for both morphological forms of C. albicans serotype A. Yeast and hyphae-specific IgA levels significantly increased after primary M5-BSA conjugate injection and decreased after the primary sc booster administration to the levels comparable with IgA levels in sera of controls (Fig. 5).

For M6-BSA conjugate and whole cell–specific IgM levels, we obtained similar results as for M5-BSA conjugate immunization. Hyphae-specific IgM levels in immune sera were slightly higher than or comparable with control (yeast form, secondary ip booster injection, 3rd ip) but without significant alteration throughout immunization (Fig. 5). Immunization with M6-BSA conjugate induced C. albicans serotype A yeast form specific IgG levels comparable with yeast that form specific IgG levels in sera of controls. For hyphal form of C. albicans serotype A, primary sc booster injection and secondary booster injections (both routes of administration, 3rd ip and 3rd sc) induced significantly higher IgG levels in comparison with sera of controls with maximal peak after secondary ip booster injection (Fig. 5). Only for hyphal form of C. albicans, whole cell–specific IgA levels significantly increased after primary M6-BSA conjugate injection.

The alterations in C. albicans serotype A whole cell–specific IgG levels after individual administrations of conjugates reveal differences between conjugates. Different changes of the whole cell–specific IgG levels in the course of immunization with conjugates indicated different specificity of induced IgG antibodies and indicate M6-BSA conjugate as preferable for induction of potentially opsonizing antibodies. According to high yeast and hyphae-specific IgM levels in control sera, higher level of non-specific interaction of serum IgM antibodies with C. albicans serotype A whole cells could be assumed (Fig. 5).

Serum-mediated candidacidal activity of PMN

We tested the efficacy of sera prepared by immunization with conjugates to improve the candidacidal activity of PMN by candidacidal activity assay (Fig. 6). For C. albicans serotype A cells opsonization, we used sera obtained after each M5-BSA or M6-BSA dose and as a control opsonization with sera of control group (mice immunized in the same time schedule with saline) was used. The analysis of viable and killed C. albicans cells after co-incubation with PMN was performed using two-colour staining, fluorescein diacetate (FDA, green fluorescence) and propidium iodide (PI, red fluorescence) to detect viable (FDA+PI) and death (FDAPI+) C. albicans cells with subsequent analysis using flow cytometry.

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Figure 6. Serum induced candidacidal activity of PMN Percentage of propidium iodide positive C. albicans serotype A cells after PMN's candidacidal activity without opsonisation (gray line) and induced by non-inactivated sera and heat inactivated sera opsonisation. For opsonisation non-inactivated immune sera (dark gray bars, Immune sera) or heat inactivated immune sera (diagonally striped dark gray bars, Inactivated immune sera); sera collected on day 14 after each injection of M5-BSA conjugate or M6-BSA conjugate (after the first injection (1st, n = 10), the primary booster injection (2nd, secondary sc injection, n = 10), the secondary ip booster injection (3rd ip, tertiary ip injection, n = 10) and the secondary sc booster injection (3rd sc, tertiary sc injection, n = 10) of conjugate) were used. Control of PMN's candidacidal activity represent opsonisation with non-inactivated control sera (black line, Control sera) or heat inactivated control sera (dashed black line, Inactivated control sera); sera were collected on day 14 after third sc injection of saline (n = 10). All data were expressed as mean ± SD, statistical significance of differences between complement non-inactivated immune sera and complement non-inactivated control sera are expressed: ***< 0.001, **0.001 < < 0.01, *0.01 < < 0.05, statistical significance of differences between complement inactivated immune sera and complement inactivated control sera are expressed: ###< 0.001, ##0.001 < P<0.01, #0.01 < < 0.05.

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When we compared efficacy of PMN's candidacidal activity using unopsonized (sera unpretreated, PMN, Fig. 6) and opsonized (sera pretreated, control sera, immune sera, Fig. 6) C. albicans serotype A cells, serum opsonization increased the relative numbers of PI+ C. albicans cells in comparison with unopsonized PI+ C. albicans cells. The candidacidal activity of PMN against unopsonized C. albicans cells was set as background for candidacidal assay.

Mean proportions of PI+ C. albicans cells after PMN's candidacidal activity induced by opsonization with immune sera after the 1st, the 2nd and the 3rd ip dose of M5-BSA conjugate were not statistically different from control sera–induced PMN's candidacidal activity (Fig. 6). PMN's candidacidal activity induced by sera after the 3rd sc dose of M5-BSA conjugate was statistically significantly lower than control sera–induced PMN's candidacidal activity (Fig. 6). When we analysed the ability of sera after each M6-BSA conjugate administration to increase the PMN's candidacidal activity, we obtained slightly different results as for M5-BSA conjugate immune sera. Mean values of PI+ C. albicans cells proportion opsonized by sera after the 2nd and the 3rd ip dose of M6-BSA conjugate (Fig. 6) were comparable with control sera–induced PMN's candidacidal activity and for sera after the 1st and the 3rd sc dose of M6-BSA conjugate (Fig. 6) slightly statistically significantly higher than mean percentage of PI+ C. albicans cells after control sera induced–candidacidal activity of PMN.

To assess the contribution of complement to increase in PMN's candidacidal activity, non-inactivated sera opsonization was compared with opsonization of C. albicans cells with heat-inactivated sera. After inactivation of complement, the capacity of control sera to improve the candidacidal activity of PMN markedly decreased.

Heat complement inactivation of M5-BSA conjugate immune sera showed mainly statistically significant decrease in induction of candidacidal activity of PMN except sera after primary sc booster injection (2nd) of conjugate (Fig. 6). Due to complement inactivation, mean percentage of PI+ C. albicans cells after PMN's candidacidal activity induced by sera after primary sc booster injection of M5-BSA conjugate remains the same as for sera with non-inactivated complement, although statistically not significantly higher in comparison with percentage of PI+ C. albicans cells after PMN's candidacidal activity induced by complement-inactivated control sera. PMN's candidacidal activity induced by complement-inactivated M6-BSA conjugate immune sera decreased in comparison with complement non-inactivated sera. Candidacidal activity of PMN induced by complement-inactivated M6-BSA conjugate immune sera stays statistically significantly higher than inactivated control sera for sera after secondary sc booster injection of M6-BSA conjugate (Fig. 6).

PMN's candidacidal activity assay demonstrated difference between M5-BSA and M6-BSA conjugates ability to induce production of antibodies improving killing action of PMN and reveal significant impact of active complement on C. albicans cells opsonization for PMN's candidacidal activity.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In the last few decades, the incidence of invasive candidiasis significantly increased [22-24]. This increase in Candida infection is associated with the increasing numbers of patients susceptible for the development of fungal infections, including patients undergoing major surgery (especially gastrointestinal surgery), blood and marrow transplantation and solid organ transplantation; patients with AIDS, neoplastic disease and advanced age; and patients receiving immunosuppressive therapy [22-25].

Our previously published results revealed the ability of linear α-1,2-linked mannooligomers conjugates to induce antibodies elevating candidacidal activity of leucocytes [13, 14]. The results presented here are a continuation of the immunomodulatory properties assessment of α-mannoside BSA-based glycoconjugates. For this study, two synthetically prepared oligomannosides (pentamannoside: M5 and hexamannoside: M6) with α-1,6-linked branching unit in addition to α-1,2-, α-1,3-linked mannose residues (Fig. 1) were used for preparation of BSA-based conjugates and for subsequent immunization.

We analysed the ability of immunization-induced antibodies to react with purified acid -stable mannan moiety and with natural form of mannan as a cell wall component of intact yeast and hyphal cells. Comparison of mannan-specific antibodies levels induced by M5-BSA conjugate and M6-BSA conjugate revealed higher immunogenicity of M6-BSA conjugate (Fig. 2). M6-BSA conjugate mannooligomers, in contrast to M5-BSA conjugate mannooligomers, possess additional α-linked mannosyl unit at non-reducing end of oligomers. Markedly more beneficial immunomodulatory effect of M6-BSA conjugate resulted also from induction of immunoglobulin isotype class switch from IgM to IgG after secondary sc booster injection, clearly detected for mannan C. albicans serotype A (Fig. 2). Isotype switching in appropriately activated cells is regulated by T helper cells through direct contact with B cells mediated by costimulatory molecules in conjunction with other signals provided by cytokines produced by these cells [26].

ELISPOT overcomes certain limitations of ELISA and combination of both techniques in one experiment supplies additional information. M6-BSA conjugate-induced IgM to IgG isotype switch was confirmed also by ELISPOT analysis of mannan-specific antibody- secreting cells (Fig. 4). The main advantage of ELISPOT is sensitivity of the method. ELISPOT allowed detection of single cell currently secreting an antigen-specific antibody and reflects varying physiological status for the cells at different time-points, as was observed for hybridoma cells [27]. Plasma cells are terminally differentiated B lymphocytes producing large amounts of antibodies. The immune response gave a rise of short-lived and long-lived plasma cells [28, 29]. After antigen stimulation, short-lived plasma cells are rapidly formed in secondary lymphoid organs, where they undergo apoptosis after a few days of intensive antibody secretion. Long-lived plasma cells are located in survival niches, especially in bone marrow and to a lesser extent in the spleen. These antibody-secreting cells could be pivotal for the maintenance of humoral immunity [28, 29]. Correlation between detected mannan-specific antibody levels in serum and number of mannan-specific antibody-secreting cells (SFCs) in spleen was not observed. The difference is most significantly evident for mannan C. albicans serotype A-specific IgM after secondary booster injection of M5-BSA conjugate. Levels of mannan-specific IgM in serum (3rd sc, Fig. 2) markedly increased in comparison with decreased mannan-specific SFCs (3rd sc, Fig. 4). Certain proportion of antibodies detected in serum may possibly produced by short-lived plasma cells, which could not be detected as mannan-specific SFCs, because they undergo apoptosis prior to ELISPOT analysis.

These results clearly indicate higher potential of M6-BSA conjugate to induce beneficial immune response, in comparison with M5-BSA conjugate and reveal more effective recognition of M6 oligomannoside-derived antigenic moieties in mannan structure despite presumed lower presence of corresponding oligomers in mannan structure. Moreover, the administration route of secondary booster injection of M6-BSA conjugate significantly affected the intensity of mannan-specific humoral immune response giving priority to sc route of administration. This observation is inconsistent with our previously published results with linear heptamannoside-BSA conjugate [14] favouring ip administration route conferring higher antibody response. Due to obtained results, we can assume oligomannoside structure-dependent difference in induced humoral immune response.

Whole cells of C. albicans represent complex mixture of antigens with the presence of specific antigens associated with yeast or hyphal cells. We observed the ability of antibodies in prepared sera to recognize relevant structures in natural mannan structure, especially for IgG antibodies induced by M6-BSA conjugate in hyphal form of C. albicans (Fig. 5). The structural and bioimmunological analysis of Candida mannans has mostly been conducted using yeast cells form grown at 28 °C. Nevertheless, Candida cells become pathogenic and invade tissue in the hyphal form at 37 °C [30, 31]. Recently, it has been shown that presence of the α-1,6-linked branching mannose residues in mannan structure is reduced in Candida hyphal form mannan [8]. IgM and IgG antibodies levels induced by both conjugates immunization were slightly higher for hyphal morphological form of C. albicans (Fig. 5). Difference in α-1,6-linked branching presence in mannan of C. albicans yeast and hyphal form and detected antibody levels indicate that recognized antigenic determinants are α-1,6-linked branching independent. We can suppose that observed difference in induction of humoral immune response by M5-BSA and M6-BSA conjugates is less related to difference in oligomannoside length and is more related to structure diversity, concretely branching difference at non-reducing end of oligomers. Generally, oligosaccharides of intermediate length are required for the carbohydrate components of conjugate vaccines to obtain conformation similar to its native state on the cell surface. In the case of β-1,2-linked mannooligomers, the size of the epitopes that are able to induce protective antibodies is 2 or 3 residues [1]. We can suppose that dominant antigenic determinants of α-1,6-branched oligomannosides are not related to branching site. In addition, whole cell ELISA assay reveal marked non-specific interaction of serum antibodies with Candida whole cells of both morphological forms. Determination of the source of non-specific interactions requires further investigation.

IgGl and IgG2a subclass antibodies play a significant role in the opsonization either in the presence or absence of complement [32]. A comparison of the levels of IgGl and IgG2a indicates poor correlation between the putative Th responses initiated and mice strain susceptibility to infection [33]. Experimental infection of BALB/c mice with low susceptibility to Candida infection produced increased levels of IgGl instead of IgG2a [33]. By immunization with semi-synthetic oligomannoside-BSA conjugates M5-BSA and M6-BSA, we observed in agreement with mentioned report increase in IgG1 levels instead of IgG2a.

The ability of immune sera to enhance the candidacidal activity of PMN was studied according to previously published candidacidal assay [14]. The published observations of efficient yeast cells opsonophagocytosis revealed ability of mannan-specific antibodies alone to serve as sufficient opsonins [34]. These results are supported by an earlier report of C. albicans yeast cells opsonophagocytic killing by human neutrophils induced by natural anti-mannan antibodies [35]. Our previously published studies revealed that antibodies induced by synthetic oligomannoside – BSA conjugates – have the capacity to induce the candidacidal activity in vitro [13, 14]. Presented results showed that C. albicans cells opsonization with sera significantly improved the killing efficiency of PMN. The ability of immune sera prepared by immunization with M5-BSA conjugate to induced PMN's killing activity was comparable to or statistically significantly lower (3rd sc dose) than capacity of placebo control sera. The lower efficiency of immune sera to induce candidacidal activity is probably related with lower capacity of specific antibodies to recognize corresponding antigenic structures in cell wall of C. albicans cells and to activate complement, which leads to limited opsonization and reduced induction of PMN's candidacidal activity. Sera obtained by immunization with M6-BSA conjugate slightly improved the candidacidal activity of PMN with statistically significantly higher effect than control sera for sera after the 1st and the 3rd sc dose of conjugate. Comparison of obtained results revealed different functionality of antibodies induced by these two conjugates containing structurally similar α-1,6-branched oligomannosides. Mannan is also able to contribute to the resistance of C. albicans to complement activation through the alternative pathway in the absence of mannan-specific antibodies [36]. Han and Cutler described protection by a murine IgM and IgG3 antibodies requiring an intact complement system in a mouse model of disseminated candidiasis [6]. We observed mainly decrease in PMN's candidacidal activity using complement-inactivated sera in comparison with non-inactivated sera; thus, inactivation of complement in sera obtained by immunization with conjugates mainly reduced effectiveness of sera to induce candidacidal activity (Fig. 6). Upon obtained results, we assume different specificity and different potential protective efficacy of antibodies induced by immunization with M5-BSA and M6-BSA conjugates. The importance of antibodies specificity seems to be critical for induction of candidacidal activity and obtained result confirmed low correlation between protection and mannan or whole cell–specific antibodies levels alone [13, 14]. In addition, results obtained with M5-BSA and M6-BSA conjugates revealed lower ability of α-1,6-branched oligomannoside – BSA conjugates in comparison with linear oligomannoside – BSA conjugates to induce production of antibodies with strong reactivity to corresponding antigenic determinants in natural cell wall mannan and lower capacity to induce antibodies significantly enhancing candidacidal activity of PMN in comparison with previously published results obtained with linear oligomannoside – BSA conjugates [13, 14]. Moreover, immunogenicity and efficiency of PMN's candidacidal activity induction were more negatively affected by oligomannoside branching point location at terminal non-reducing end (M5-BSA) in comparison with M6-BSA oligomannoside with branching point at non-terminal location. Several authors [2, 37, 38] described protective effect of antibodies against experimental disseminated candidiasis in vivo. Prepared monoclonal antibodies showed enhanced ingestion and killing of yeast cells by PMN (MAb B6.1) or macrophages (MAb C7) in the presence of serum complement [37, 38]. They proposed that complement activation might contribute to the protection by antibodies in vivo and that during initiation of candidiasis protective antibodies induce prompt complement opsonization, which results into an association of Candida cells with host phagocytes. Non-protective antibodies may lead to reduced complement activation kinetics. According these results, we could assume enhanced candidacidal activity induced by serum opsonization in vitro as a possible precondition for protection in vivo.

Differences concerning the antibody quantity, specificity and isotype composition of polyclonal sera could explain why antibody protection against Candida infection has been observed in some studies but not in the others. Presented study indicates limited effectiveness of branched α-mannooligosides to induce production of highly protective antibodies. Additional and more detailed immunomodulatory properties investigation of α-mannooligosides of different structure should bring significant information to successful protective anti-Candida subcellular vaccine development.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This project was supported by grants from Grant Agency of Slovak Academy of Sciences VEGA No. 2/0026/13, by the Slovak Research and Development Agency under the contract No. APVV- 0032-06. This contribution is the result of the project implementation: Centre of excellence for Glycomics, ITMS 26240120031, supported by the Research & Development Operational Programme funded by the ERDF.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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