Amphoteric Mannan as an Immune Response Modifier. New Model Immunobiologically Active Candida albicans Mannan-Based Formula

ABSTRACT Background Currently, the incidence and prevalence of serious fungal infections is increasing, especially in immunosuppressed individuals. The co-administration of antibiotic and immunosuppressive therapies has driven the emergence of new multidrug-resistant fungal pathogens. Their significant increase and their ability to form biofilms is associated with rising morbidity and mortality. Research into novel synthetically prepared immunomodulators as potential immune response modifiers and prospective participants in drug delivery systems is of interest. Microbial polysaccharides with zwitterionic charge motifs were shown to be promising candidates. Methods Native and ultrasonically treated mannan from the yeast Candida albicans were chemically modified to contain both positive and negative charges in a nearly equimolar ratio mimicking the zwitterionic polysaccharides. RAW 264.7 macrophages and Balb/c mice were subjected as in vitro and in vivo models. Macrophage exposure to the set of amphoteric derivatives of mannan induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The functionality of the exposed macrophages was assayed by cell proliferation and phagocytosis. Results The Th1 and Th17 dominance was over Th2. The phagocytosis and respiratory burst, together with the viability based on cell proliferation supported the bioavailability of formulas. Mouse immunization induced humoral immune responses with high titers of the IgM isotype with the IgM/IgG shift. Conclusion Our study demonstrated the immunobiological activities of amphoteric derivatives of mannan from Candida albicans. Amphoteric derivatives can be considered as bioavailable formulas with an effective immunomodulatory potency, prospectively applied as a subunit formula in the design of a mannan-based platform for drug and vaccine delivery systems. Graphical abstract


Introduction
Generally, mycobiome represents 0.1% of the human microbiome, and includes various fungal species. Numerous Candida spp. are the major reason for diverse site-specific mycoses and invasive fungal diseases with different organ involvements (Seed 2015;Tiew et al. 2020) (Table S1, Supplementary data). Currently, more than 90% of systemic infections are caused by Candida albicans, and non-albicans species, such as C. glabrata, C. parapsilosis, C. tropicalis, C. krusei etc. Mortality between 19% and 30% has been estimated especially in the susceptible immunosuppressed population. The fungal cellwall polysaccharides represent virulence factors, sensed by the germ-line encoded pattern recognition surface receptors of epithelial and myeloid cells. Mannan forming a fibrillary structures represents the outermost layer of Candida yeast cells and is the dominant (essential) antigenic and virulence factor. Its structure consists of a backbone composed of α-(1,6)-linked mannose residues branched with side manooligosaccharide substituents built up from α-(1,2)-, α-(1,3)-, α-(1,6)-, and β-(1,2)-linked mannose residues. The acid labile part of mannan contains exclusively the β-(1,2)-mannooligosaccharide sequences linked through the phosphodiester bond to side chains (Gow et al. 2017;Shibata et al. 2007). The host's immune response to Candida infection mainly depends on the mannan antigenic determinants recognition and on effector cells activation. The majority of fungi are detected and identified by the cells of the innate immune system, which contain specific membrane-bound pattern recognition receptors (Netea et al. 2008(Netea et al. , 2015Poulain and Jouault 2004;Romani 2004;Vendele et al. 2020).
Furthermore, cell-wall mannan components are highly immunogenic and stimulate cellular and humoral immune responses during infection (Erwig and Gow 2016). Cellwall mannans, along with antibodies directed against them, are effective in vitro diagnostic tools as they are detectable during the whole course of candidiasis (Meng et al. 2020;Paulovičová et al. 2015aPaulovičová et al. , 2015bPaulovičová et al. , 2019a. cellular mannan from fresh biomass. Mannan was isolated as a mannan-copper complex (Jones and Stoodley 1965) from the yeast cell wall of C. albicans, serotype A. Mannan (M; 10-1000 kDa, PDI ≈ 3.3) was ultrasonically degraded (MUS; ≈ 35 kDa, PDI = 1.6) by a method described elsewhere (Čížová et al. 2015). The detailed procedure of isolation, degradation, and the characterization of mannan is given in the Supplementary data. The prepared mannan was free of proteins or chitoligomers since no nitrogen was detected by elemental analysis (Table S2, Supplementary data). The glucose content in the isolated mannan was less than 1.7 mass % as confirmed by monosaccharide composition analysis (Table S2, Supplementary data). Contamination with lipids was further excluded by FTIR and NMR spectroscopies confirming the structure and purity of the isolated mannans ( Fig.  S1 and S2, Supplementary data).
The degree of carboxymethylation (DS CM ) was determined by the potentiometric titration according to (Rinaudo and Hudry-Clergeon 1967). The degree of quaternization (DS Q ) was estimated based on the nitrogen content, which was determined by elemental analysis using a FLASH 2000 CHNS/O organic elemental analyzer (Thermo Fisher Scientific, Waltham, MA, USA) as described elsewhere in detail (Čížová et al. 2016, 2019). The results are shown in Table 1. Silver-stained sodium dodecyl sulfate 15% polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad Mini-PROTEAN Cell system (Bio-Rad Laboratories, Hercules, CA, USA) was used to confirm the absence of LPS in native and amphoteric mannans M-Z, MUS-Z1, and MUS-Z2. The LPS content in all mannan preparations (10 µg of polysaccharide per lane) was under the detection limit of the method used (0.1 ng).

Preparation of stock solutions of natural cellular mannan and synthetically prepared amphoteric mannans M-Z, MUS-Z1, and MUS-Z2
Stock solutions and dilutions of natural C.albicans CCY 29-3-100 cellular mannan and the formulas M-Z, MUS-Z1, and MUS-Z2 were prepared aseptically using presterilized disposable plastic wares and sterile, apyrogenic aqua pro injectione (Fresenius Kabi Italia S.r.l., Verona, Italy). The solutions were prepared in a laminar flow cabinet and sterilized with 0.2-μm filter (Q-Max®Syringe filter, Frisenette ApS, Knebel, Denmark) before cell exposure. The laminar flow cabinet was sterilized with 70% ethanol p.a. and UV for 30 min prior to all experiments. The stock solutions were controlled with EndoLISA® ELISA-based Endotoxin Detection Assay (Hyglos, Bernried am Starnberger See, Germany) and measured using the Cytation 5 Imager Multi-Mode Reader (BioTek, Winooski, USA) to determine endotoxin-free exposure conditions.

Cell maintenance and culture, cell exposure
The murine macrophage-like RAW 264.7 cells (ATCC®TIB-71 TM , ATCC, Manassas, USA) were cultured in complete Dulbecco's Modified Eagle Medium for 24 h and 48 h, at 37 °C under 5 vol. % CO 2 atmosphere and 90-100% relative humidity until approximately 80% confluence. The viability of the RAW 264.7 cells was assayed by the Trypan Blue dye exclusion method using a TC20 TM automated cell counter (Bio-Rad Laboratories, Inc., Hercules, USA). The starting inoculum of 3.3 × 10 5 cells/mL per well (98% of viable cells) was seeded in a 24-well cell culture plate (Sigma-Aldrich, St. Louis, USA) and exposed to 10 μg and 100 μg per well of M-Z, MUS-Z1, and MUS-Z2 for 24 h and 48 h. Cellular mitogens Concanavalin A (Con A; 10 µg/mL, Sigma-Aldrich) and lipopolysaccharide (LPS; 1 µg/mL, Sigma-Aldrich) were used as positive controls. The cell culture media were separated and stored at −20 °C till further usage. The cell morphology and viability were assayed before the supernatant collection and before cytotoxicity evaluation.

RAW 264.7 cell proliferation and cytotoxicity
The effect of M-Z, MUS-Z1, and MUS-Z2 on RAW 264.7 cell proliferation and cytotoxicity was estimated following direct 24-h and 48-h cell exposure using cell proliferation assay ViaLight TM plus kit (Lonza, Rockland, ME, USA) consistent with the manufacturer's recommendations. The RAW 264.7 cell ATP was estimated by luciferase-based luminescence quantification. The intensity of the emitted light was quantified with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc.). The light emission was recorded continuously for 1 s and peak values were estimated and expressed as relative light units (RLU). The values of untreated RAW 264.7 cells were considered as the control baseline.
The proliferation index was calculated as the ratio between the stimulated cells (M-Z, MUS-Z1, and MUS-Z2 formula-treated cells) and the baseline proliferation of untreated cells. Thus, the proliferation index of the negative control, i.e. the untreated cells, was equal to one.

Immunocytometry, immunophenotyping, phagocytosis, oxidative burst. Fluorescence quenching cytometric assay. Preparation of fluorescein-labelled C. albicans
M-Z, MUS-Z1, and MUS-Z2 exposed RAW 264.7 cells were subjected to immuno-flow cytometry using a CytoFLEX (Beckman Coulter Life Sciences, Inc., Indianapolis, US). Gates were set up to exclude the debris and damaged cells using FSC vs SSC discrimination. The settings were optimized using proper isotype control (immunophenotyping assay) and C. albicans-FITC (phagocytosis). For each sample, fluorescence histograms of 10,000 cells (immunophenotyping) or 5,000 cells (phagocytosis) were generated and analyzed (green fluorescence, 525-nm band-pass filter, FL1 channel). All of the samples were analyzed in duplicates. The data are expressed as a percentage or as the mean of fluorescence intensity (MFI ± SD).

Simultaneous phagocytosis and oxidative burst
RAW 264.7 macrophages phagocytosis was determined by flow cytometry (CytoFLEX, Beckman Coulter Life Sciences, Inc., Indianapolis, US). For each sample, a fluorescence histograms and dot-plots of 5,000 cells were generated and analyzed. Gates settings were adjusted to exclude the debris and damaged cells using FSC vs SSC discrimination. Measurements of phagocytosis, i.e. the ingestion of C.albicans yeast cells took place undercontrolled conditions using a fluorescein-labeled C.albicans. Aliquots of (30 μL) M-Z, MUS-Z1, and MUS-Z2 post-exposed cells were incubated with C.albicans-FITC (3 µL) for 15 min at 37 °C. Following treatment, the reaction was stopped with ice. The mean percentage of phagocytic cells represented the percentage of cells ingesting at least one Candida cell. Throughout the phagocytosis accompanied by a respiratory burst of RAW 264.7 macrophages, the double fluorescence of fluorescein isothiocyanate (FITC)-labeled ingested C.albicans cells and that of hydroxyethidine (HE) oxidized to ethidium bromide was evaluated by flow cytometry. The metabolic activity was determined as the oxidative burst causing transformation of the originally non-fluorescent hydroxyethidine (Invitrogen Molecular Probes, USA) into ethidium following Candida-FITC ingestion.

Fluorescence quenching cytometric assay
The extracellular fluorescence of fluorescein isothiocyanate was quenched by 0.4% Trypan blue dye (Sigma-Aldrich, USA). Immunocytometric analysis of Trypan blue treated RAW 264.7 cells was performed following 15-min cell incubation in the dark at 37 °C. The proportion of membrane attached to FITC-conjugated C. albicans is expressed as the difference between the whole-cell phagocytosis, i.e., cells with cell -bound and internalized C. albicans-FITC, and the Trypan blue quenched cell population. The metabolic activity is expressed as the percentage of cells simultaneously undergoing phagocytosis and oxidative burst.

Preparation of FITC-labelled C. albicans
C. albicans (yeast and/or hyphal morphoforms) grown in a liquid YPD medium 18 h, at 28 °C, without shaking was adjusted to a concentration of 0.8 × 10 8 bb/mL and treated with 70% ethanol 1 h at room temperature. Then the suspension was then washed three times with a 0.5 m carbonate-bicarbonate binding buffer (3500 × g, 4 °C, 10 min). C.albicans cultures (yeast and/or hyphal morphoforms) were fluorescently labeled with fluorescein isothiocyanate (FITC prepared in a carbonatebicarbonate buffer: 5 mg in 50 mL buffer) and then incubated for 1 h at room temperature (in the dark) and washed four times in PBS (3500 × g, 4 °C, 10 min). The quality of the incorporated fluorescence was detected on a light-fluorescence microscope (Imager A.1 microscope, Axiovision software, C. Zeiss, Germany) and on a flow cytometer (FC 500, Beckman Coulter, CXP) analysis based on the mean fluorescence intensity.

Quantification of cytokines
The

Immunization protocol with amphoteric mannan M-Z
The experimental immunization protocol comprised 18 mice per group. After an acclimatization period of 1 week, primary immunization and the next two booster immunizations of the animals were administered subcutaneously (s.c.) at 3-week intervals (10 µg saccharide/100 µL per dose). The stock solution and individual doses were prepared aseptically using apyrogenic, sterile 0.9% saline solution (Braun) under sterile laminar flow conditions (ESCO Class II Biological Safety Cabinet). The stock solution was sterile filtered using a syringe with a 0.2 µm cellulose acetate filter (Q-Max®Syringe filter). The groups of untreated animals were used as a negative control.
Individual sera samples following the primary and following booster immunizations, were collected from the retro-orbital plexus of six of the mice, 3 weeks after each immunization. The same procedure was applied to the control groups. The blood samples were incubated for 1 h at room temperature and then overnight at 4 °C. The sera were separated by centrifugation at 600 × g for 10 min at 4 °C and were then collected and stored at −20 °C until further use.

Experimental infection
Four weeks after the second booster immunization, the mice were infected with C. albicans strain CCY 29-3-100 (serotype A). The C. albicans was cultured on a YPD solid medium for 48 h at 28 °C and subcultured in a YPD liquid medium for 24 h at 28 °C under stirring. The cells were harvested by centrifugation, washed, and suspended to a concentration of 2.5 × 10 6 cells/mL in sterile saline. Each mouse received 2.5 × 10 5 viable cells in 0.1 mL of sterile saline given intravenously (i.v.) via a lateral vein. The individual sera samples were collected 3 weeks after infection as described above.

Isolation, maintenance, and cultivation of spleen cells
Throughout the active M-Z immunization (primary and two secondary boosts) and C. albicans experimental infection, Balb/c mice were used for post-mortem extirpation of spleens and further isolation of splenocytes. Their spleens were aseptically removed and were transferred into an ice-cold saline (1 mL per spleen), splenocytes were isolated by homogenization of spleen tissues with the end of the syringe plunger. The splenocytes suspension was filtered (50 μm-mesh filter; CellTrics disposable filter; Partec, Görlitz, Germany) and centrifuged at 800 × g for 10 min at 4 °C. Then after, splenocytes were resuspended for 5 min in 5 mL of ACK lysis buffer (0.15 M NH 4 Cl, 1 M K 2 CO 3 , and 0.01 M EDTA, pH 7.2) to lyse erythrocytes. Consequently, the splenocytes were washed twice with saline and resuspended in a complete RPMI-1640 medium (Lonza, City, Belgium) supplemented with 10% of fetal bovine serum, penicillin (100 U/mL), and streptomycin sulphate (100 mg/mL) (Gibco, NY, USA). Following an assessment of splenocyte viability by the Trypan blue staining method, the density of cells was determined as 2.72 × 10 8 cells/ mL with 91% viability.
M-Z vaccination sensitized immunocytes following isolation and collection were seeded (4 × 10 5 cells per well) into 24 well culture plates (Nunc, Roskilde, Denmark) and incubated for 24 h in a 37 °C incubator (5% CO 2 , humidified atmosphere). Afterwards, the culture media were separated and stored at−20 °C to determination of the interleukins and growth factors.

Quantification of antigen-specific Ig izotypes
Serum anti-M-Z specific IgG and IgM were assayed with a quantitative Invitrogen™ ELISAs IgG Mouse and IgM Mouse kit (ThermoFisher Scientific, Waltham, USA) following 1 st immunization and further two boosters. The serum levels of specific antibodies were also analyzed after C. albicans experimental infection. For all the assays, the microtiter plates (4HBX, Dynex, USA) were coated with 0.01 mg/mL per well of M-Z in coating buffer pH 7.6.

Statistical analysis
All the mouse experimental results were expressed as mean values ± SD. The normality of the data distribution was established according to the Shapiro -Wilk test at the 0.05 level of significance. Statistical comparisons were performed by one-way ANOVA and post hoc Bonferroni tests. Pearson´s correlation coefficient was used to compare the strength of the relationship between immunobiological variables. The results were significant when the differences equaled or exceeded the 95% confidence level (P < 0.05). Statistics were performed using ORIGIN 7.5 PRO software (OriginLab Corporation, Northampton, USA).

Ethics statement
All mice experimental protocols were approved by the Institutional Ethics Committee (EK SZU/002/2017). The experiments were conducted according to the GLP and OECD guidelines, based on the ethical guidelines of the Research Base of Slovak Medical University, Institute of Preventive and Clinical Medicine (Bratislava, Slovakia), the approval No. Ro 3107/17-221/3 of State veterinary and the Food Administration of the Slovak Republic. This study was performed in accordance with Slovak and European Community regulations for the use of laboratory animals and follows the criteria of experimental animal welfare.

Preparation and characterization of amphoteric mannans
Amphoteric mannans were prepared by the chemical modification of native and ultrasonically treated mannan from the yeast C. albicans in a sequential two-step manner (Fig. S1, Supplementary data) under previously optimized aq. alkaline conditions (Čížová et al. 2016;Machová et al. 2014).
Our former study revealed that the specific antibody-binding capacity of these derivatives depends strongly on the level of chemical modification (Čížová et al. 2016). Therefore, particular attention was paid to setting an optimal level of substitution to preserve the mannan epitopes essential for its recognition by immunoglobulins. This was achieved by a precise adjustment of the molar ratio of reactants, so as to obtain a total degree of substitution of about 0.20 and the particular desired balance between positive and negative charges. Thus, two amphoteric derivatives (M-Z and MUS-Z1) with a comparable degree of substitution but differing in molar mass were prepared and characterized (Table 1).
Compared to natural or synthetic zwitterionic polysaccharides containing at least one positive and one negative charge in each repeating unit, and composed of 3-6 monosaccharide units (Gallorini et al. 2007;Nishat and Andreana 2016), the charge density in M-Z and MUS-Z1 derivatives is lower (approximately one positive and one negative charge per 10 monosaccharide units on average). Derivative MUS-Z2 with a charge density similar to that of natural zwitterionic polysaccharides (approximately one positive and one negative charge per 4 monosaccharide units on average) was used for study and comparison.
The evaluation of the immunobiological activity of M-Z, MUS-Z1, and MUS-Z2 derivatives was performed in vitro using RAW 264.7 macrophage cells. Afterwards, in vivo active immunization of Balb/c mice was performed with M-Z formula. The assessment of the immunobiological activity of M-Z, MUS-Z1, and MUS-Z2 in RAW 264.7 macrophage cells included cell proliferation, cell phagocytosis, and the production of inflammatory and anti-inflammatory interleukins as well as growth factors.

Effect of M-Z, MUS-Z1, and MUS-Z2 derivatives on RAW 264.7 proliferation
Macrophages can adjust their activation state in response to microbes and microbial products and release various signalling molecules, cytokines, growth factors, transcription factors, and metabolites. Nowadays, the functionally distinct macrophages subpopulations are referenced (Chávez-Galán et al. 2015;Martinez et al. 2013).

Effect of M-Z, MUS-Z1, and MUS-Z2 derivatives on RAW 264.7 phagocytic activity, respiratory burst and metabolic activity
RAW 264.7 macrophage cells exposed to M-Z, MUS-Z1, and MUS-Z2 derivatives, were subjected to candida phagocytosis and respiratory burst analyses (Table 2, Fig. S4). The phagocytosis and the respiratory burst of cells exposed to LPS as cell mitogen were used as a positive control. The impact of applied concentrations of formulas on phagocytic activity mainly favored the lower (10 μg/mL) concentration. Following 48-h exposure, the highest cell bound and internalized Candida was observed for M-Z and MUS-Z1 (1.65-fold and 1.58-fold increases compared to the untreated control, P < .001). The higher internalization of Candida was also induced by M-Z and MUS-Z1 (1.67-fold and 1.63-fold increases compared to the untreated control, P < .001). Exposure to M-Z, MUS-Z1, and MUS-Z2 derivatives enhanced the respiratory burst of RAW 264.7 macrophages, more clearly

Effect of M-Z, MUS-Z1, and MUS-Z2 derivatives on cytokine and growth factors release by RAW 264.7 macrophages
Cytokines play an important role in the innate and adaptive immune responses as they are engaged in immune regulation, cell proliferation, cell death, inflammation, tissue repair, and cellular homeostasis. M-Z, MUS-Z1, and MUS-Z2 derivatives were evaluated according Table 3. The impact of M-Z, MUS-Z1, and MUS-Z2 on the RAW 264.7 macrophages expressing selected surface markers CD11b, F4/80, and CD14 signature antigens. Mean percent and mean fluorescence intensity of positive cells following staining with MAbs recognizing CD11b, F4/80, and CD14 after M-Z, MUS-Z1, and MUS-Z2 after 48-h stimulation. Control constituted untreated cells. All data are presented as mean ± SD values from two biological and three technical replicates. The statistical significance of differences between untreated and stimulated cells using one-way ANOVA and post hoc Bonferroni tests is expressed as: ***P < .001, **.001 < P < .01, *.01 < P < .05. Formula (μg) CD11b ( to their impact on the induced release of pro-inflammatory cytokines (TNFα, IL-6, IL-1β, IL-2, IL-17, and IL-12), anti-inflammatory cytokine IL-10, and haemopoietic growth factor GM-CSF in RAW 264.7 macrophages after exposure for 24 h or 48 h (Figure 2).
In our experiments, the relevant pro-, and, anti-inflammatory interleukins and growth factors, and signature cytokines of Th1, Th2, and Th17 polarization were assayed (Figure 2).
Following RAW 264.7 cell exposure by M-Z, MUS-Z1, and MUS-Z2 derivatives, the induced release of Th1, Th2, and Th17 signature cytokines revealed Th1 polarization of the immune response over Th2 and Th17 according to typical induced concentration levels of the signature cytokines IFN-γ (Th1), IL-4 (Th2), and IL-17 (Th17). This trend has been assessed following 24-h and 48-h exposure. The Th17 polarization over Th2 was demonstrated for M-Z and MUS-Z1. These derivatives exerted comparable DS CM+Q , unlike MUS-Z2; evidently, the structure underlines the differences between cell functionality. The same pattern of biological and functional comparability was detected for M-Z and MUS-Z1 in the processes of RAW 264.7 cell proliferation and cell phagocytosis (Figure 1 and Table 2 The next significantly increased TNF-α was mostly produced following 48-h cell exposure with 100 μg/dose of all the formulas tested ( Figure 2): M-Z vs control: 24-h treatment 1.70-fold P = .00703 and 48-h treatment 2.5-fold increase P = .00271; MUS-Z1 vs untreated control: 24-h treatment 2.43-fold P = .00649 and 48-h treatment 3.76-fold increase P = .00398; MUS-Z2 vs control: 24-h treatment 1.8-fold P = .0023 and 48-h treatment 4.474fold increase P = .00255). The degree of TNFα production reflected the structure-biological activity relations, the order: M-Z < MUS-Z1 < MUS-Z2 was determined. Evidently, the degree of substitution DS CM+Q (Table 1) is discriminative. The highest TNFα production was revealed with the MUS-Z2 with the highest degree of substitution DS CM+Q , followed by MUS-Z1 and M-Z, with a lower degree of substitution. The rather different impact of MUS-Z2 vs MUS-Z1 and M-Z on the induced production of Th1 cytokines IL-1β, IFN-γ, IL-2, IL- Figure 2. Evaluation of Th1, Th2, and Th17 signature cytokines and pro-and anti-inflammatory cytokines production by RAW 264.7 cells following exposure with 10 and 100 μg/dose of M-Z, MUS-Z1, and MUS-Z2 derivatives. Control constituted untreated cells. All data are presented as mean ± SD values from two biological and three technical replicates. Statistical significance of differences between untreated and stimulated cells using one-way ANOVA and post hoc Bonferroni tests is expressed as: ***P < .001, **.001 < P < .01, *.01< P < .05 -for 24 h and as ### P < .001, ## .001 <P < .01, # .01< P < .05 -for 48 h. 12, and on the hemopoietic growth factor GM-CSF, and Th2 cytokine IL-4 was determined. A Th1 response is characterized by cytokines, such as IFN-γ and IL-12, whereas a Th2 response usually involves the production of IL-4 and IL-6. According to the proinflammatory cytokine results, the order of pro-inflammatory activity of formulas tested was evaluated as MUS-Z2 > MUS-Z1 > M-Z. Obviously, the structure modification and the degree of substitution DS CM+Q reflected the susceptibility to stimulate the inflammatory effect in vitro. The pattern of IL-2 cell release was further evidence of the structural impact on diverse functional cell effectivity. Pro-inflammatory Th1 interleukin IL-2, is strongly engaged in cell proliferation, and it was established that IL-2 has two opposing functionsto stimulate effector cell responses and to maintain Tregs (Abbas 2020). The highest concentration levels of IL-2 after 48-h exposure to MUS-Z1 (100 μg/dose) and MUS-Z2 (10 μg/dose) are in agreement with the results of the cell proliferation, as the highest proliferation index was exerted with these derivatives and the exposition dose (Figure 1). The formulas tested did not significantly influenced the IL-10 production. The IL-17 production revealed a concentration and structure dependency, with a higher efficacy of the lower concentration (10 µg/dose) of the formulas tested. The interleukin 17 and Th17 responses were identified as crucial components of immunity to C. albicans. Interleukin IL-17 is essential in the host defense against mucocutaneous candidiasis, inducing chemotaxis, neutrophil, and antimicrobial peptide activities (Davidson et al. 2022;Whibley and Gaffen 2014).
The capability of M-Z, MUS-Z1, and MUS-Z2 derivatives to induce the production of selected cytokines was significantly reduced compared to the C.albicans CCY 29-3-100 cell mannan (

Effect of administration of M-Z formula as active immunization of Balb/c mice
The results of RAW 264.7 exposure to M-Z, MUS-Z1, and MUS-Z2 derivatives, suggested that the M-Z formula was the most effective for active in vivo mice prime-boost immunization. Balb/c mice were immunized with amphoteric mannan M-Z to follow up the Th polarization of the induced immune response (Figure 3) and induction of specific IgG and IgM class antibodies (Figure 4).

Th polarization induced by M-Z vaccination
The splenocytes sensitized by M-Z prime vaccination and the following two boosters were evaluated for Th1, Th2, Th17, and Treg signature cytokines (Figure 3). The most effective was the increased induction of IL-10 throughout the whole vaccination route, with peak values after the second booster dose. The prime vaccination (M-Z1) induced 1.36-fold, P = 2.26 E −04 , the increase following the first booster (M-Z2) induced 1.49-fold increase, P = 2.5 E −05 . The rising tendency continued after the second booster (M-Z3), induced 2.013-fold, P = 5.43 E −06 , in comparison with baseline values (control mouse splenocytes). The next statistically significant 3.42-fold (P = 5.41 E −08 ) increase vs 2 nd boost value of IL-10 in spleen homogenates was revealed following experimental Candida infection 4 weeks after the second booster dose (Figure 3). Generally, IL-10 participated in the suppression of the differentiation between Th1-and IL-17-producing T-helper cells, and promotes the differentiation of Treg cells (Sarazin et al. 2010). Evidently, IL-10 was engaged as a pleiotropic anti-inflammatory cytokine mainly suppressing the pro-inflammatory milieu by inhibiting Candida infection induced pro-inflammatory cytokines such as IL-1, IL-6, IL-12, and TNFα in order to re-sustain homeostasis after infection (Chin et al. 2014;Saraiva and O'Garra 2010). The production of Th2 cytokine IL-4 by splenocytes exerted statistically not significant moderate increase throughout the immunization route ( Figure 3). Evidently, the Th2 polarization was not supported by the M-Z administration. IL-4 was found to have anti-inflammatory properties, with an ability to suppress the production of proinflammatory cytokines (Woodward et al. 2010). Reactively elevated IL-4 (5.39-fold, P = 1.83 E −04 ) over the 2 nd booster value was detected following the experimental Candida infection and infection inflammation.
The Control constituted splenocytes from control/untreated groups. All data are presented as mean ± SD values from two biological and three technical replicates. Statistical significance of differences between untreated control group and immunized groups using one-way ANOVA and post hoc Bonferroni tests is expressed as: ***P < .001, **.001 < P < .01, *.01< P < .05 -for prime-boost immunization and as ### P < .001, for C. albicans post-immunization infection.
experimental Candida infection. This cytokine is assumed to have an essential role in both innate and adaptive arms of the immune response to candidiasis and favors the development of a Th1 protective response (Darwich et al. 2009;Gozalbo 2014;Li and Kuemmerle 2021;Munder et al. 1998  According to our in vitro analyses, based on the determination of Th1, Th2, Treg, and Th17 signature cytokines, mice vaccination with M-Z formula favored Th1, Th17, and Treg polarization over Th2.

Production of specific IgG and IgM class antibodies throughout the M-Z vaccination
As immune response parameters, two main IgG and IgM class antibodies towards M-Z formula were assessed (Figure 4). Control constituted sera from control/untreated groups. All data are presented as mean ± SD values from two biological and three technical replicates. Statistical significance of differences between untreated control group and immunized groups using one-way ANOVA and post hoc Bonferroni tests is expressed as: ***P < .001, **.001 < P < .01, *.01 < P < .05 -for prime-boost immunization and for Candida albicans post-immunization infection.
The assessment of specific anti-M-Z IgG and IgM class antibodies throughout the threedose immunization schedule revealed the dominant IgM isotype immune response following the first and second vaccination (Figure 4). The first s.c. administration of M-Z (M-Z1) induced a 1.42-fold increase vs untreated control (P = .08151), the dominance of IgM reflected ratio IgM/IgG = 38.87. Next, the first booster of M-Z (M-Z2) accelerated the significant rise of anti-M-Z specific IgM 1.74-fold vs untreated control (P = .01031) and 1.22-fold (P = .1319, ns) vs primo vaccination levels. The ratio between the two specific isotypes IgM/IgG = 51.36 is suggestive of the persisting dominance of the IgM class antibody. The specific IgM response slightly decreased following the second booster (M-Z3) i.e., 1.49-fold vs baseline values (P = .17575) and 0.857-fold (P = .19176) compared to the values after the first booster dose. The ratio between the two specific isotypes IgM/IgG = 25.84 decreased in comparison with previous values following primo booster. The experimental infection (section 2.10) was manifested by a 1.6-fold rise (P = .31819) in specific anti-M-Z IgM compared to its values following the second booster. The M-Z specific IgG exerted the most significant rise following the second booster (1.92-fold, P = .0188) vs baseline values and a significant 1.7-fold (P = 8.26585 E −4 ) compared to the primo booster. The experimental infection resulted in a 3.85-fold rise (P = .32502) in specific IgG in comparison with the second booster. The engagement of IgG and IgM class antibodies and the induced changes of the IgM/IgG ratio, are suggestive of isotype switching of M-Z specific antibodies from IgM to IgG after active immunization. Generally, polysaccharides stimulate B-lymphocytes by cross-linking antigen cell receptors. This arrangement resulted in initial IgM mediated response, which generally switches to an IgG response upon repeated antigen boosters. The IgG-specific response is characterized by long-term immunological memory to antigen (Harmer and Chahwan 2016;Jones et al. 2020;Kurosaki et al. 2015).
Numerous experimental vaccines against Candida based on cell-wall mannan were designed to generate protective immune responses (Shukla et al. 2021). The induction of humoral and cellular responses with natural mannan and various mannan conjugates was studied either in vivo or in vitro using various experimental models. Liposome-encapsulated mannan was demonstrated to exert neutrophil candidacidal activity and performed agglutination of Candida cells (Han et al. 1999;Xin and Cutler 2011). Mannan extracts were reported to have anti-adhesion or anti-germ tube formation effects (Cassone et al. 1995), class antibodies switching was demonstrated with mannan-BSA conjugate (Han and Rhew 2012;Han et al. 1999), mannan-HSA conjugate caused an inhibition of Candida growth, B-cell immune-enhancement, and antifungal activity (Bystrický et al. 2003;Machova et al. 2015;Paulovičová et al. 2007).
Furthermore, various synthetically prepared vaccine candidates were evaluated e.g. glycopeptide vaccine combining beta-mannan and peptide epitopes (Xin et al. 2008(Xin et al. , 2012, α-, β-mannooligosaccharides-protein conjugated (Paulovičová et al. 2012(Paulovičová et al. , 2019b(Paulovičová et al. , 2019a. Xin et al. demonstrated immunological activities of β-1,2-mannotriose-Eno1 peptide (Xin and Cutler 2011;Xin et al. 2008) and of Fba peptide conjugated with β-1,2-mannotriose conjugate vaccine (Xin and Cutler 2011;Xin et al. 2008Xin et al. , 2012. The effectiveness of mannosylation in vaccination or for target drug-delivery can be ascribed to specific recognition of and processing of mannose and mannans by pattern recognition receptors on the relevant cells of immune systems such as macrophages, dendritic cell, etc. (Gupta et al. 2009;Irache et al. 2008;Netea et al. 2008Netea et al. , 2015Romani 2004;Vendele et al. 2020). Mannosylated constructs were suggested as drug-carriers and a platform for cell-specific delivery of bioactive agents (Tiwari 2018). The complex of mannan-coated nanoliposomes has the potential to be used as a self-adjuvanted nanocarrier for the construction of recombinant vaccines (Bartheldyová et al. 2019). Mannosylated conjugates were suggested as vaccines against pathogens, for the treatment of autoimmune diseases or for the development of cancer vaccines especially in combination with nanoparticles, liposomes, or niosomes (Irache et al. 2008).

Conclusion
The present study investigated the effects of amphoteric mannan and derivatives on immune modulation in murine macrophage RAW 264.7 cells and in vivo in Balb/c mice models. Our results demonstrated effective bioimmunological activity either in vitro or in vivo. Active mouse immunization resulted in IgM/IgG shift. Moreover, the Th1, Th17, and Treg polarization over Th2 during vaccination was demonstrated in splenocytes. Enhancement of phagocytosis by amphoteric mannan was assessed by RAW 264.7 cells engulfment and the internalization of Candida cells. The biocompatibility of amphoteric mannan and derivatives was confirmed by cell viability, proliferation, and cytotoxicity. Acquired results supported further immunobiological investigation of the immunological properties of Candida cell wall carbohydrate moieties and their modifications toward the selection of the most effective structures appropriate for application as immunomodulative agents prospectively for participation in drug delivery systems, Candida diagnostics, and monitoring of Candida immune responses.

Disclosure statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding
This work was supported by the Grant Agency of the Slovak Academy of Sciences VEGA [grant number 2/0076/21], the Slovak Research and Development Agency [grant number APVV-15-0161], Operational Program Integrated Infrastructure for the project Study of structural changes of complex glycoconjugates in the process of inherited metabolic and civilization diseases, ITMS: 313021Y920, co-financed by the European Regional Development Fund and is based upon work from COST Action CA16231 ENOVA (European Network of Vaccine Adjuvants). This publication is the result of the project implementation CEMBAM -Centre for Medical Bio-Additive Manufacturing and Research, ITMS2014+: 313011V358 supported by the Operational Programme Integrated Infrastructure funded by the European Regional Development Fund.