Adjuvant effect and TLR4 mediated activation of macrophages by polysaccharide from Polyporus albicans

Dr. Antik Kiron Bose1 Senior Scientist and Project Advisor, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109.

Abstract:
A water soluble polysaccharide from mycelium of Polyporus albicans has been identified for its potency in stimulating cellular and humoral immune response. The adjuvant potential of this polysaccharide was identified and found to be higher than conventional adjuvants. The 37 KDa polysaccharide was purified using different macroporous absorption resins and Reverse phase HPLC. Structural determination was done by sugar and methylation analysis, 13C NMR, FTIR, MALDI-TOF,ESI-IT, MALDI-PSD TOF spectroscopy. The molecular size of the polysaccharide was determined by Zetasier nanosystem. It showed TLR4 dependent NO, IL-1 and TNF- production.

Abbreviation:
HPLC- High Performance Liquid Chromatography SLS- Static lights scattering DLS- Dynamic lights scattering DMSO- Dimethyl sulfoxide TFA- Trifluoro acetic acid NMR- Nuclear magnetic resonance FTIR- Fourier- transform infra red MALDI-TOF- Matrix assisted laser desorption/ionization on time of- flight ESI-IT- Electro spray ion trap HPAEC-PAD- high performance anion exchange chromatography with pulsed amperometric detection MALDI-PSD- Matrix assisted laser desorption/post- source decay TLR- toll- like receptor NO- Nitric oxide NOS- Nitric Oxide Synthatase

Introduction: Vaccination remains the most cost effective biomedical approach for the control of infectious disease. The safety of the traditional vaccines based on live attenuated or killed microbes is questionable due to risk of virulence reversion (H.-X.Sun et al;2009). New generations of vaccine based on purified recombinant proteins , synthetic peptides and plasmid DNA despite their better tolerability are unfortunately often much less reactogenic and immunogenic (H.-

X.Sun et al;2009). The majority of these vaccines require association with adjuvant capable of increasing the potency or stimulating the appropriate immune response.( Y.Lu, D.X Wang , Y.L Hu, X.Y Huang, J.M Wang;2008). The benefits flowing from adjuvant incorporation into any vaccine formulation have to be balanced with the risk of adverse reactions induced by these compounds. Unfortunately strong adjuvant activity is often correlated with increased toxicity. Fruends complete adjuvant (FCA) remains the most potent of unknown adjuvants and particularly powerful stimulant of both cellular and humoral immunities (H.-X.Sun et al ; 2009). Unfortunately FCA causes severe reaction and is too toxic for human use. The unique capacity of the extract Quil A from bark of Quillaja saponaria and its purified saponin QS-21 to stimulate both the TH1 immune response and production of cytotoxic T-lymphocyte against exogenous antigens , makes them ideal for use in subunit vaccines and vaccines directed against intracellular pathogens as well as for therapeutic cancer vaccines ( BP daSilva , G Madeiros de Silva, J.P Parente ; 2009). However , in addition to pain on injection , severe local reactions and granulomas, toxicity includes severe hemolysis (K.M Lima et al ;2004), making such adjuvants unsuitable for human uses other than for life threatening diseases, such as HIV infection and cancer. Although muramyl dipeptide (MDP) and other derivatives from Gram negative bacteria, such as LPS and monophosphoryl lipid A

have also been used as human adjuvants, their toxicities remain the single biggest barrier to the use of such adjuvants for human prophylactic vaccines (N. Byars et al ; 1990). A major challenge in adjuvant research is to increase adjuvant activity while reducing toxicity ( R.K. Gupta, E.H. Relyveld, E,B Lindblad, B. Bizzini, BenEfraim, S.Gupta; 1993). Currently aluminium compounds (Alum) are the only adjuvants licensed by the Food and Drug Administration (FDA) for use in human (D.M. Pascual, R.D Morates ;2006). While alum is safe ; it is a relatively weak adjuvant particularly when used with subunit Antigens. Moreover, the Alum is mild TH2 adjuvant that can effectively enhance IgG1 Antibody responses, but it is rarely associated with TH1 type immune response (H.HogenEsch ;2002). Furthermore Alum is poor at stimulating cell-mediated immune responses, and may actively block activation and differentiation of cytotoxic Tlymphocytes (R. Achirmbeck et al ;1994). Hence, there is a major unmet need for a safe and efficacious adjuvant capable of boosting cellular plus humoral immunity (N. Petrovsky;2006). Most polysaccharides from higher plants are relatively non-toxic and do not cause significant side effects , which is a major problem associated with immune modulatory bacterial polysaccharides and synthetic compounds. A water soluble polysaccharide from mycelium of Polyporus albicans was identified and characterized. It has a potent

stimulating effect on murine lymphocyte proliferation induced by mitogen. Immunomodulatory effect and adjuvant potential of the polysaccharide on cellular and humoral immune responses of ICR, C3H/HeN and C3H/HeJ mice were investigated. It can be a safe and efficacious adjuvant capable of boosting cellular and humoral immunity without toxicity. Adjuvants bared on the Polyporus albicans polysaccharide have enormous potential for use in vaccine against both pathogens and cancer. Materials Required:

A. Chemicals: AB-8(purchased from Nankai chemical factory, HPD-450 and HPD600( purchased from Hebei Cangzhou Chem Co.Ltd, Japan),Macroporous adsorption resins, Vydac C-18 Reverse Phase HPLC column (Grace Vydac), ID SUPERCOSILTM LC-18-D-8 HPLC column (sigma Aldrich; catalogue no.195867), Sep-PakR plus Environmental C18 cartridge, (55105 m pore size product no.WAT023635, Bridge column), 9% Ficoll 400 and 15% sodiumdiatrizoate( density 1.13g/ml); Hypaque(sigma), Percoll (sigma, density 1.064g/ml), Modified Neuman and Tytells serumiess medium ( United States Biological, part no.S1012), Griess Reagent

system (Promega), MTT assay using cell titer 96TM non-radioactive cell proliferation assay kit (Promega), anti OVA-IgG Ab assat kit(catalogue # 3011, chondrex assay kit), anti IgG1 Ab [MOPC-21,ab10616, Abcam) anti IgG2b and antiIgG1bAb conjugated to HRP(Millipore), Mouse IgG2b ELISA(cat no. KT-405, Kamiya Biomedical company), 3,3,5,5 tetramethyl benzidine (TMB) (sigma), Mouse IgG1b ELISA (cat no. KT-406, Kamiya Biomedical( company, seattle, WA), N-G-monomethyl-L-arginine (L-NMMA) (sigma) anti IL-1 and anti TNF- Abs conjugated to HRP (Millipore), IFN (1000/ml) (sigma), anti TLR2, anti TLR4 anti CR3 Abs (sigma), Dead EndTM colorimetric TUNEL system (Promega)

c. Instruments: Rotary Evaporator RE-52 type (Shanghai Sing Pu Haxi instrument factory). HP1090A HPLC fitted with vydac C18 reverse phase column ( 2.1 X25 cm) (grace Vydac), Zetasier nanosystam MRK 577-01 (Malvern Instrument Ltd, Enigma Business park, UK), Bruker AvanceTM DRX NMR spectrometer, Fourier Transform TR spectroscopy (Shimadzu scientific instruments) Bruker ( ultraflex) MALDITOF mass spectrometer electrospray ion trap multi stage (ESI-IT) (1200 series high throughput MS system, Agilent Technologies), MALDI-PSD TOF MS (deep Dyve instruments), ELISA plate reader (chanel optical system 5sec/24 wells, single wavelength, standard 4 titers, Seeuco Electronics Technology co Ltd), chemiluminescence NO analyzer ( Seivers instrument , Boulder, CO).

B. Inbred Laboratory Mice: Belkin F5L051-ICR retractable comfort Mouse C3H/HeNCrl(strain code 025, coat colour Agouti, MHC haplotype H2K, Agouti, MHC haplocyte H2K, Charles River products and services), C3H/HeJ (coat colour Agouti, Related genotype A/A, stock no.000659,strain C3H/HeJ BirLtJ, JAXR surgical model,carrying a mutation in toll like receptor 4 gene, (Tlr4Lps-d)

Procedure: A. Preparation of mycelia cultureMycelial culture of Polyporus albicans (Imaz) Teng was used as a source for water soluble polysaccharide. Mycelial culture was prepared on SYP medium containing starch (16g/l), Fructose (4g/l), peptone (1.5g/l), formic acid (0.1g/l), KH2PO4 (1g/l), MgSO4 (0.5g/l), FeSO4 (0.01g/l) and Yeast extract (1.5g/l). Medium pH was adjusted to 4.5 before sterilization.

One 1litre flask containing 400ml of basal medium with 5% (v/v) of mycelia of P. albicans was cultured on a rotary shaking incubator at 110 rpm. Cultivation was done for 10 days at 25C (Belinkyet et al, 1994: Wi Young Lee, Young Ki Park, Tin Kwon Ahn; 2007). B. Purification of the polysaccharideThe mycelia of P. albicans were homogenized in 60% Ethanol solution at 60C for 2 hrs. The process is repeated 2 times. When the concentrated liquid viscosity is 30 pas, 30% chitosan solution was added at 60C and pH 5.5 to remove most proteins. The extract is centrifuged at 10,000 rpm for 15 mins and the pellet is discarded. The supernatant was concentrated using the Rotary Evaporator RE-52 type (Shanghai Qing Pu Haxi Instrument Factory) at oscillations of about 100 times/min for 24 hours. 1 gm of pre-dry resins AB-8 (purchased from Nankai Chemical Factory), HPD-450 and HPD-600 (purchased from Hebei Cangzhou Chem Co. Ltd. , Japan) macroporous adsorption resins were transferred to chromatographic columns separately. 20 ml of extract was added to each of the columns and eluted with 30%, 50%, 70% and 90% ethanol. Carbohydrate content of each of the eluted fractions were measured by phenol sulphuric acid method (M. Dubiol, K.A. Giller, J.K.

Hamilton, P.A.Robers, F. Smith, 1956). Saccharide content of all four eluted fractions was also measured by combining 4 eluted fractions to calculate the total carbohydrate content of the extract by the same method. 70% ethanol extract with highest carbohydrate content was further purified by Reverse -Phase HPLC. 20 l of the extract was loaded in HP 1090A HPLC fitted with Vydac C-18 Reverse-Phase 2.1mmx25cmcm column (Grace Vydac). Separation was achieved with a linear gradient of 5-50% acetonitrile containing 0.1% Trifluoroacetic acid over a period of 60 mins at flow rate of 0.2 ml/ min. 25% acetonitrile elutent with maximum carbohydrate content (measured by phenol sulphuric acid method ) was passed through 2.1mm 1D SUPELCOSIL TM LC-18-D8 HPLC columns ( Sigma Aldrich; catalogue no. 7195867) using 1050% Triethyl amine and acetic acid. 25% Triethyl amine and acetic acid fraction gave a single peak containing the polysaccharide. C. Molecular mass determination of the polysaccharide- 25% Triethyl amine and acetic acid fraction from ID SUPELCOSILTM LC-18-D8 HPLC column was subjected to static light scattering (SLS) for molecular mass determination and dynamic light scattering (DIS) for mean diameter calculations using Zetasier Nano

System MRK 577-01 (Malvern Instruments Ltd., Enigma Business Park, UK). It combines SLS, DLS and electrophoretic light scattering technologies to measure particle size, Zeta potential and molecular mass. In SLS, a beam of monochromatic light is directed through a sample dissolved in toluene and intensity of light scattered at angle of 173 by the molecule is measured. The intensity of light scattered over a period of 10 seconds is measured for 0.10,0.11,0.12,0.13,0.14,0.15,0.16,0. 17 and 0.18 gm/ml of the sample using 633 nm wavelength of laser beam. MW is given by Rayleigh equation KC/R =( 1/M+2A2C)P() Where R = Rayleigh ratio = ratio of scattered light to incident light of the sample. M = Molecular weight A2 = 2nd Virial coefficient C = concentration P = Scattering angle K= optical constant A plot of KC/ R versus C was made. From the intercept 1/M was calculated and the slope was nd equivalent to 2 virial coefficient (A2) i.e. Debye plot. In DLS, the constructive and destructive interferences between

changing intensities of different particles lead to fluctuations of total intensity which is measured by Autocorrelation function. Mean diameter of the polysaccharide was measured at pH4.6, 7.6 and 9.6 to measure degree of aggregation as a function of pH (J.P. carver, 1991). D. Structure determination of the polysaccharide 1. Sugar Analyses 1ml of 25% Triethylamine and acetic acid fraction from ID SUPELCOSILTM LC-18-D8 HPLC column (containing 0.2gm/ml polysaccharide) was transferred to a 13 x 100 mm Screw cap tube. 50g of Rha, Fuc, Ara, Rib, Xyl, Man, Gal, Glc, GlcNAC and GalNAC (90.5gm/ml) were added as internal standards. They were hydrolysed in 0.3ml 2 M TFA at 120C for 2 hrs. The solution was evaporated to dryness by a stream of compressed air and 0.5 ml Methanol. The step was repeated. The solution was reduced with 0.3 ml Fresh solution of 50mM NaBH4 in ammonia for 30 mins at 20C. It was quenched with 0.5 ml 10% glacial acetic acid in methanol and evaporated to dryness. The step was repeated twice. Acetylation was done with 0.1ml 10% acetone and 0.1ml of 1N pyridine at 100C for 20 mins. 50l of water was added and the

solution was evaporated to dryness after adding 0.5ml toluene. The step was repeated. Partition was done between 0.5ml H2 O and 0.5ml ethylacetate by stirring fast using a triangular magnetic rod in conical vial (Reacti-Vial type) for 3 mins. The upper ethylacetate phase was transferred to the old rinsed tube and 0.5 ml ethylacetate was again added. It was concentrated to dryness and transferred into the sample tube and concentrated to 50l (PerErik Jansson, 1976). 2. Methylation Analyses 1mg of dry sample was transferred to a5ml serum flask. 1cm magnetic rod was added and the flask was flushed with N2 and then sealed with a rubber septum. The flask was kept in a fumehood due to odor and toxic fumes. 0.5ml of DMSO was added to the flask using a syringe while releasing the pressure in the flask by inserting another needle through the septum. The sample was stirred and sonicated for 30mins. 0.5ml of 2M dimsyl sodium was added and stirred at room temperature for 5hrs. The sample was frozen and 0.25ml methylacridone was added. The sample was melted for 1 hr by stirring. Excess pressure was relieved through

the septum and methylacridone was taken away by pushing a needle through the septum and applying vaccum. The Sep-Pak column (Sep-Pak plus Environmental C18 cartridge, 55105 m pore size, product no. WAT023635, Bridge columns fitted in 10ml syringe was preconditioned by passing 10ml ethanol and 2ml H2O. The sample was applied in DMSO/H2O (1:1 ratio ) solution and eluted using 8ml H2O and 8ml 15% CH3CN solution to separate the methylated carbohydrates (Per-Erik Jansson, 1976). 2ml CH3CN and 2ml ethanol were transferred into a 13x100mM screw cap tube and concentrated to dryness. It is hydrolysed in 0.3 ml 2M TFA at 120C for 2 hrs. The solvent is evaporated and 1ml methanol was added and the solution is reduced with 0.3 ml fresh solution of NaBH4 for 1hr at 20C. The solution was quenched with glacial acetic acid and evaporated with 0.5ml 10% glacial acetic acid in methanol. The solution was acetylated with 100ml acetone and 100l 50mM pyridine at 100C. for 20 min and then 50 l water was added. After cooling, the solvent was evaporated and 1ml toluene was

added. The solution was partitioned between 0.5ml H2O and 0.5ml ethylacetate and the organic phase was transferred to sample tube and concentrated to 0.2 ml. 13 C- NMR (Bruker AVANCE TM DRX NMR spectrometer) and Fourier transform Infrared spectroscopy (FT-IR) (Shimadzu Scientific Instruments) of methylated derivatives were performed. Methylated and acetylated derivatives were also analysed by Matrix-assisted laser desorption/ionization on time of flight (MALDI-TOF) [Bruker (UltraFlex) MALDI-TOF mass spectrometer] and electrospray ion trap multi-stage (ESI-IT) (ESI IT 1200 series high throughput MS system, Agilent Technologies) Mass spectrometer. MALDI-TOF was employed to determine chain distribution. The technique was compared with high performance-anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Methylated derivatives were investigated by MALDI- TOF MS and MALDI post source decay mass spectrometer (MALDI-PSD TOF MS) (Deep Dyve). (Susanna Broberg, 2000). E. Maintenance of Inbred Mice

C3H/HeNCrl (Strain code 025, coat color Agouti, MHC haplotype H2K, Charles River Products and Services) and C 3H/HeJ (Appearance Agouti, Related genotype A/A , stock no. 000659, strain C3H/HeJ BirLtJ, JAXR surgical Models, carrying a mutation in toll-like receptor 4 gene, Tlr$ Lpsd) mice were fed an atherogenic diet (1.25% cholesterol, 0.5% cholic acid and 15% fat) for 2 weeks.A. Belkin F5LO51-ICR(Imprinting control region) Retractable comfort mouse was also fed atherogenic diet for 5 weeks. F. Study of Adjuvant effect of the water soluble polysaccharide of Polyporus albicans 5 weeks old ICR mice weighing 1822gm were immunized subcutaneously with ovalbumin (0.1mg) alone (control) or ovalbumin(0.1mg) dissolved in saline containing alum(0.2mg), or ovalbumin (0.1mg) +0.5mg /1mg/2mg of the polysaccharide separately or with ovalbumin (0.1mg) + Lipopolysaccharide (LPS) (0.5mg) on day 1 and 15. On day 18 sera were collected from ICR mice and IgG1 and IgG2 specific for ovalbumin were isolated from different mouses by Affinity chromatography using ovalbumin as ligand (W.B. Jakoby, 1974) and measured by indirect ELISA using antiisotypic IgG1 and IgG2b

monoclonal Abs (J.B. Kwapinski, 1972) G. Isolation of monocytes from C3H/HeNCrl and C3H/HeNCrl and C3H/HeJ mice Blood was obtained from both the mice and immediately mixed with 0.2volume of anticoagulant (0.8% citric acid) and then diluted 1:1 with Ca2+ and Mg2+ free phosphate buffered saline. A mononuclear cell fraction containing monocytes was obtained by layering diluted blood on a cushion consisting of a mixture of 9% Ficoll 400 and 15% sodiumdiatrizoate (density= 1.13g/ml) (Hypaque) (Sigma) and centrifuged at 400xg for 30 min at room temperature. Cells were washed and centrifuged in a gradient of Percoll (Sigma) (density= 1.064g/ml) for 45 min at 800xg. An isoosmotic percoll was prepared by mixing 1 volume of NaCl 1.5(M) with 9 volumes of percoll (Pharmacia, density =1.13g/ml). The percoll gradient was done by mixing 1:1 (V/V) osmotic percoll with PBS/citrate (1.49mM NaH2PO4, 9.15mM Na2HPO4, 139.97mM NaCl, 13mM C6H5Na3O7 2H20, pH -7.2). The monocyte enriched fraction was washed twice and plotted in 100mm plastic tissue culture dishes 7 (3.4x10 cells/dish) in modified Neuman and Tytells serumless medium supplemented with 2% new born calf serum, gentamycin

(50g/ml) and Andamphotericin B (0.25g/ml) (Benett & Breit 1994; Haskill et al. 1988). After 30mins at 37C, the culture fluid and nonadherent cells were removed. Adherent mono-layer of spreading monocytes (>90% pure) was rinsed twice and reincubated in fresh culture medium and stimulated with 50 g/ml and 100 g/ml P. albicans polysaccharide , 10U/ml IFN- or 5 g/ml LPS in both C3H/HeN and C3H/HeJ isolated monocyte cultures for 24 hours. A control set was prepared where C3H/HeN and C3H/HeJ derived monocyte culture was stimulated with buffer for 24 hours. H. Polyporus albicans derived polysaccharide induced peripheral macrophage proliferation was measured by measuring NO production and IL-1 and TNF- production P. albicans polysaccharide , LPS and dextran stimulated proliferation of NO production in both C3H/HeN and C3H/HeJ mouse, were measured using Griess Reagent system (Promega). It is based on chemical diazotization using 50mM sulfanilamide and 50mM N-1napthylethylenediamine dihydrochloride (NED) in presence of phosphoric acid using the Dead EndTM colorimetric TUNEL system to measure NO at 520 nm. (D.S. Bredt, S.H. Snyder, 1994). The viable cells were measured by (3-(4,5- dimethyl

thiazol-2yl)-2,5 diphenyl tetrazolium bromide) (MTT) assay using a cell titre 96TM non-radioactive cell proliferation assay kit (Promega) by reading absorbance at 490nm (Tim, Mossmann, 1983). Similarly P. albicans polysaccharide, LPS and dextran stimulated IL-1 and TNF- production in C3H/HeN and C3H/HeJ derived monocyte culture was measured by ELISA using anti IL-1 and anti TNF- antibodies (J.B. Krabinsky, 1972). 50 g/ml P. albicans polysaccharide and 200 g/ml anti TLR4 were used to stimulate C3H/HeN derived monocyte culture for 24 hrs and monocyte proliferation was measured similarly. Monocyte stimulation of C3H/HeN mice was also studied in presence of antiTLR2 and anti CR3 antibodies (Sigma) (200 g/ml). Results: Table 1 Carbohydrate content in 30%, 50%, 70% and 90% ethanol elutant of AB8, HPD-450 and HPD-600 macroporous absorption resins (as determined by phenol-sulphuric acid method) (M. Dubios et al, 1956). Percentage of ethanol elutant Carbohydrate content (g/ml) Name of resin AB-8 HPD450 48 HPD600 43

50% 70% 90%

60 350 70

63 360 73

63 358 75

Table-2 Carbohydrate content of 25% acetonitrile elutant from HP 1090A HPLC fitted with Vydac C-18 Reverse Phase column (Grace Vydac) and 25% triethylamine and acetic acid fraction of ID SUPELCOSILTM LC-18-D8 HPLC column (Sigma-Aldrich)

Carbohydrate content (g/ml) 300 1. 25% Acetonitrile elutant from HP1090 A HPLC fitted with Vydac C18 Reverse phase column 2. 25% Triethyl amine 250 and acetic acid fraction of ID SUPELCOSILTM LC18-D8 HPLC column

30%

50

Ray leigh ratio of toluene at 633 nm Scattering angle Duration of recording Scattered intensity Molecular weight 2nd Virial coefficient (A2)

1.3522x10 -5 cm-1 173 10 sec

37KDa 2.37x10-2

Fig 1. Elution profile of Polyporus albicans polysaccharide in C-18 Vydac Reverse phase HPLC (Grace Vydac) (Fig 1a) and ID SUPELCOSILTM LC-18-D8 HPLC (Fig 1b). Table-3. Molecular weight (KDa) and 2nd Virial coefficient (A2) determination of P. albicans polysaccharide from 25% triethylamine and acetic acid elution fraction of IDSUPELCOSILTM LC-18-D8 HPLC using Zetasier Nano system MRK 577-01

Fig 2. Debye plot where KC/R is plotted along Y-axis and concentration ( C ) (mg/ml) along X- axis. The intercept is equivalent to 1/M (M= 37 KDa) and slope equals to 2nd virial coefficient (A2) = 2.37 x10-2 as determined by Zetasier Nano System MRK 577-01 (Malvern Instruments) [ Where R = Rayleigh ratio, K- optical constant]

Character

Result

Differential refractive index increment (dn/dc)

0.14ml/gm

Fig 3. Mean diameter of the polysaccharide at pH 4.6, 7.6 and 9.6 were measured by DLS using Zetasier Nano System MRK 57701 (Malvern Instruments) At pH 4.6, size distribution by intensity showed presence of both monomeric fractions with diameter in range of 10 nm and aggregates with diameter in range of 100nm in almost equal percentage . A small peak of highly aggregated portion of the polysaccharide was also observed in 1000 nm diameter range . At pH 7.6, the amount of aggregation was increased with most carbohydrate in 100nm range and the percentage of monomeric fraction with diameter 10 nm was decreased. A slight increase in % of 1000 nm diameter range highly aggregated portion was also observed. At pH 9.6, almost the entire polysaccharide was present in 100 nm diameter range and a very small fraction in 10 nm diameter range. Z- mean diameter of the polysaccharide is steadily increased from 13.5 nm at pH 4.6, 13.7 nm at pH 7.6 and 14.7 nm at pH 9.6.

Fig 4. Fourier Transform- IR spectra of P. albicans polysaccharide measured by FT-IR spectrophotometer ( Shimadzu Scientific Instruments) showed presence of OH groups, weak C-H bond, -dgalactopyranosyl and --d-mannopyranosyl residues.

Fig 5. 13C- NMR of methylated and acetylated carbohydrate derivatives of P. albicans polysaccharide showed (1 3) linked d- mannopyranosyl, (16) linked -d-galactopyranosyl residues. (Measured by Bruker AVANCE TM DRX NMR Spectrometer at 270 MHz.

Fig 6. MALDI-TOF spectra of methylated and acetylated carbohydrate derivatives of P. albicans polysaccharide (measured by Bruker Ultra Flex MALDI-TOF mass spectrometer.

Fig 8. MALDI-PSD TOF mass spectra of methylated and acetylated derivatives of polysaccharide from P. albicans (measured by Deep Dyve MALDI-PSD TOF mass spectrometer) Combined mass spectra analysis using MALDI- TOF, ESI-IT, HPAEC- PAD and MALDI-PSD TOF showed presence of a backbone of (1 3) linked - dmannopyranosyl and (16) linked -dgalactopyranosyl residues in 3:1:1 ratio and terminated with single non reducing terminal (1)-d-mannopyranosyl residues at C6 position of (13,6)- linked- - dmannopyranosyl along the main chain.

Fig 7. High performance-anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) spectra of methylated and acetylated derivatives of P. albicans polysaccharide (measured by 1200 series high throughput MS system, Agilent Technologies)

Table-4 Amount of IgG1 and IgG2b monoclonal antibodies formed by inoculation of different antigen and adjuvant combinations as measured by Indirect ELISA

Concentration of Antigen and adjuvants inoculated subcutaneously

Concentration of Ovalbumin specific Antibodies produced in ICR mice IgG1 IgG2b 2mg/ml

1. Ovalbumin 9mg/ml (0.1 mg) (control) 2. Ovalbumin 9.5mg/ml

(0.1 mg ) dissolved in saline containing alum (0.2 mg) 3. Ovalbumin 10.6 mg/ml (0.1 mg) + 0.5 mg P. albicans polysaccharide 12.6 mg/ml 4.Ovalbumin (0.1mg) + P. albicans polysaccharide (1mg) 5. Ovalbumin 15.6 mg/ml (0.1 mg) + P. albicans polysaccharide (2mg) 6. Ovalbumin 11 mg/ml (0.1 mg) + LPS (0.5 mg) 3.5 mg/ml

Positive control contained 50 l of serum with 0.1% sodium azide. Serum was collected by venipuncture. A 1:50,000 dilution of serum sample was done and 100 l of antibodies were used. Amount of IgG1b production by 0.1mg ovalbumin dissolved in saline containing 0.2 mg alum was also measured by Mouse IgG1b ELISA (Cat No. KT 406, Kamiya Biomedical Company, Seattle, WA) to be 9.5 mg/ ml.

4.5 mg/ml

6.0 mg/ml

3.5 mg/ml

Table 5 : Amount of NO produced by monocytes derived from C3N/HeN and C3H/HeT mice as measured by Griess reagent System (Promega) which were stimulated by different antigens. The number of viable monocytes were measured by MTT assay using 96 TM nonradioactive cell proliferation assay kit (Promega). Nature and Concentration of NO produced (g/ml) Concentration of Antigen Type of Inbred Mice C3H/HeN 1. Control ( no Ag) 2. Dextran (50 g/ml) 20 C3H/HeJ 22

Mouse serum anti-ovalbumin IgG were measuered by anti-Ova-IgG Antibody Assay Kit (Catalog # 3011) (chondrex Assay Kits) using Mouse anti IgG1 antibody [MOPC-21] (ab106163, Abcam), anti IgG2b and anti IgG 1b antibodies conjugated to HRP (Millipore). AntiIgG2b was separately measured by Mouse IgG2b ELISA (Cat No. KT-405, Kamiya Biomedical Company). 3,3,5,5 tetramethyl-benzidine (TMB) and H2O2 measured as the substrate for HRP and absorbance at 450nm was taken .

80

83

3. P. albicans 100 polysaccharide

22

(50 g/ml) 4. P. albicans 140 polysaccharide (100 g/ml) 5. (10U/ml) IFN- 90 22

93 Table 6 : Amount of IL-1 and TNF- production by monocytes stimulated by different antigens as measured by ELISA using anti IL-1 and anti TNF- antibodies conjugated to HRP (Millipore). Nature and Conc. of Cytokines (pg/ml) Concentration of Antigen Type of Inbred Mice C3H/HeN TNF 1. Control (No Ag) 2. Dextran (50 g/ml 20 25 C3H/HeJ IL1- 20 TNF 21

6. LPS (5 g/ml)

50

22

When the monocyte culture was incubated with 10mM N-G-monomethyl-L-Arginine (LNMMA) (a NOS inhibitor), no NO production was observed. As >95% of total NO released from activated macrophage was converted to nitrite. NO was evaluated by measuring nitrite using Griess reagent using chemiluminescence NO analyzer (Sievers Instruments, Boulder Co.). 100 l of sample was injected into a reflux chamber containing glacial acetic acid and 1% KI, where nitrite is converted to NO . NO gas was purged into chemiluminescence NO analyzer and quantitated by reference to NaNO2 standards.

80

83

83

82

3. P. albicans 160 polysaccharide (50 g/ml) 4. P. albicans 200 polysaccharide (100 g/ml) 5. (10U/ml) IFN- 120

153

20

21

193

20

21

130

110

117

6. LPS (5 g/ml)

95

80

20

21

Table : 7 Number of viable monocytes was determined using assay using 96TM non radioactive cell proliferation assay kit (Promega) in control and under stimulation of different Ags. Nature and No. of viable cells Concentration of /l Antigen Type of Inbred Mice C3H/HeN 1. Control (No Ag) 1.36 X 104
4

Table-8: Number of viable monocytes was determined by MTT assay using 96 TM non radioactive cell proliferation assay kit (Promega)( and amount NO production (by Griess Reagent (Using Dead End TM colorimetric TUNEL system). When monocytes were incubated with 20g/ml anti TLR4, anti TLR2 and anti CR3 Abs (sigma) with 50g/ml P. albicans polysaccharide separately. Nature of Antigen and Abs used C3H/HeN mice No. of viable cells/ l 1.50g/ml P. albicans 6.8 polysaccharide(contro X104 l) 2.50/ml P. albicans 6.8 polysaccharide+ anti X104 TLR 4 monoclonal Ab (20g/ml) 3. 50g/ml P. albicans 6.8 polysaccharide + anti X104 TLR 2 monoclonal Ab (20g/ml) 4. 50g/ml P. albicans 6.8 X104 polysaccharide+ 20g/ml anti CR3 monoclonal Ab

inbred

C3H/HeJ 1.36 104 X

4

Amount of NO productio n (g/ml) 100

2. Dextran g/ml

(50

5.44 X10

5.46 X10

3. P. albicans 6.8 X104 polysaccharide (50 g/ml) 4. P. albicans 13.6 X104 polysaccharide (100 g/ml) 5. IFN- (10U/ml) 6. LPS (5 g/ml) 5.44 X104 2.72 X104

1.36 X104

20*

1.36 X104

100

5.43 X104 1.36 X104

100

*N.B 20g/ml NO produced by constitrutively active monocytes have been

observed in TLR4 blocked experimental set. In other sets, NO produced by constitutively active monocytes have not been substracted from 100 g/ml.

Discussion:
A water soluble polysaccharide has been purified from mycelial culture of Polyporus albicans (Imaz) Teng grown on SYP medium (Belinkyet et al;1994, Wi Young et al ;2007). The 60% ethanol extract was purified using AB-8, HPD-450, HPD-600 macroporus adsorption resins using 30-90% ethanol. Carbohydrate content of 70% ethanol elutants from all the columns was found to be highest (350, 360, 358 g/ml for AB-8, HPD-450, and HPD-600 columns respectively) as determined by Phenolsulfuric acid method (M.Dubles et al ;1956). The static absorption quantities has been found to be 80.4, 82.26, 75.96 mg/g for AB8, HPD-450 and HPD-600 respectively and desorption rate was found to be 90.47%, 80.88% and 75.84% for AB-8, HPD-450 and HPD-600 respectively as reported by others ( Xia Li, Lilu Jiao, Liping Zhang;2008). The 70% ethanol elutant was further purified using Vydac C18 Reverse phase HPLC using 25% acetonitrile and ID SUPERCOSIL TM LC18 D8 HPLC using 25% Triethylamine and acetic acid as a single peak. Carbohydrate content were found to be 300 and 250 g/ml for Vydac reverse phase HPLC and ID SUPERCOSIL TM LC-18 D8 HPLC respectively. Molecular weight and 2nd virial coefficient were determined from Debye plot. The molecular weight was determined to be 37

KDa using Zetasier nanosystem MRK-577-01 using SLS. Similar reports have been found by others ( Yong Xu Sun, Shusheng Wang, Tianboo Li, Xia Li, Lili Jiao, Liping Zhang; 2008). The 2nd virial coefficient was found to be 2.37X10-2 suggesting that the molecule will stay in solution .Z-Mean diameter of the polysaccharide was determined by DLS using Zetasier Nano System MRK-577-01 to be 13.5 nm at pH 9.6 showing aggregation of the polysaccharide with increasing pH (4.6-9.6). FT-IR spectra of the polysaccharide ( as measured by Shimadzu Scientific instruments) showed presence of OH groups , weak C-H bonds ,-dgalactopyranosyl and -d-mannopyranosyl residues. 13C-NMR of methylated and acetylated carbohydrate derivatives of P.albicans showed (1-3) linked dmannopyranosyl, (1-6) linked -dgalactopyranosyl residues at 270 MHz. combined Mass Spectra analysis using Matrix assisted laser desorption / ionization on time of-flight (MALDI-TOF), Electrospray ion trap multi-stage MS (ESI-IT MS), high performance ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI postsource decay (PSD) TOF MS showed presence of a backbone of (1-3) linked -dmannopyranosyl, (1-3,6) linked -dmannopyranosyl and (1-6)-linked -dgalactopyranosyl residue in 3:1:1 ratio and terminated with single non-reducing terminal (1-) -d-mannopyranosyl residues at C6 position (1-->3,6) linked -dmannopyranosyl along the main chain.

Similar reports have been found by others in P.albicans (Yong Xu Sun et al ,2008 and in P. umbellatus ( Xingqun Li, Wen Xu;2011). 5 week old Belkin F5L051-ICR mice was immunized with ovalbumin alone (0.1mg) (control) or ovalbumin (0.1 mg) + 0.2 mg alum or ovalbumin (0.1mg) + P. albicans polysaccharide (0.5/1/2 mg) or ovalbumin (0.1 mg) + 0.5 mg LPS on day 1 and 15. On day 28, ovalbumin specific IgG1 and IgG2b Abs in sera were measured by indirect ELISA. Alum as an adjuvant with ovalbumin showed 5.2% increase in OVA-specific IgG1b Ab production in comparison with control (0.1 mg ovalbumin) but no IgG2b Ab production, showing that it is a weak adjuvant. Bacterial LPS (0.5 mg) as an adjuvant with 0.1mg ovalbumin showed 15.7% increase in OVA specific IgG Ab production and 75% increase in OVA specific IgG2b Ab production. 0.5 or 1 or 2 mg P. albicans used as an adjuvant with 0.1mg ovalbumin showed 16.8%, 36.84%, and 64.21% increase in OVA specific IgG1 Ab production respectively while it showed 75%,166.67% and 266.67% increase in OVA-specific IgG2b Ab production with 0.5/1/2 mg P. albicans polysaccharide respectively. It does not cause any toxicity even at 2 mg. so it could be a safe and efficacious adjuvant capable of boosting humoral immunity without toxicity. P. albicans polysaccharide induced monocyte proliferation was measured in C3H/HeN and C3H/HeJ mice by measuring NO production. (Griess Reagent system using Dead EndTM colorimetric TUNEL

System), number of viable monocytes (measured by MTT assay using 96TM non radioactive cell proliferation assay kit) and IL-1 and TNF- production (using ELISA). 50g/ml dextran induced 80g/ml and 83g/ml NO production (300% and 315% increase) in C3H/HeN and C3H/HeJ mice respectively. In control ( no Ag) 20 g/ml and 22 g/ml NO production was observed by constitutively activated monocytes in C3H/HeN and C3H/HeJ mice respectively. 300% and 301.47% increase in number of monocytes was observed using 50g/ml dextran in 24 hours. 10U/ml IFN- (Sigma) induced 350% and 331.8% increase in NO production and 300% and 299.26% increase in monocyte production in C3H/HeN and C3H/HeJ mice, respectively in 24 hours. 5g/ml bacterial LPS induced 150% increase in NO production and 100% increase in monocyte number in C3H/HeN and C3H/HeJ mice in 24 hours but no increase in NO production and no increase in monocyte number in C3H/HeN and C3H/HeJ mice suggesting that TLR4 ( toll like receptor) is required for LPS induced NO production and monocyte proliferation. 50g/ml P. albicans polysaccharide induced 400% increase in NO production and 544% increase in monocyte number in C3H/HeN and C3H/HeJ mice in 24 hours but no increase in NO production and no increase in monocyte number in C3H/HeN and C3H/HeJ mice. Similarly, 100 g/ml P. albicans polysaccharide induced 600% increase in NO production and 900% increase in number of monocyte in C3H/HeN mice but no increase NO

production and increase in monocyte number in C3H/HeJ mice suggesting that TLR4 is required for PAP induced NO production and monocyte proliferation. Similar results have been observed by others ( Xingqun Li, Wen Xu;2011) in P. umbellatus polysaccharide. 50g/ml PAP induced 700% and 512% increase in IL-1 and TNF- production in C3H/HeN mice respectively in 24 hours but no increase in IL-1 and TNF- production in C3H/HeJ mice. 100g/ml PAP showed 900% and 672% increase in production of IL-1 and TNF- in C3H/HeN mice respectively but no increase in IL-1 and TNF- production in C3H/HeJ mice suggesting that TLR4 is required for PAP induced IL-1 and TNF- production by monocytes. No increase in monocyte number and NO production was observed when monocyte cultures were incubated with 50g/ml PAP and 20g/ml anti TLR4 Ab for 24 hours suggesting that TLR4 is required for PAP induced NO production and monocyte proliferation but when anti TLR2 and antiCR3 Ab were used normal NO production was observed. It suggests that even in PAP induced monocyte cultures, anti TLR4 Ab can inhibit induced monocyte NO production by blocking signaling through TLR4 receptor. Similar reports have been observed by others (Xingqun Li et al;2011 and Yongxu Sun et al;2008). Contitutively activated monocyte produce NO indepecdent of TLR4 receptor but PAP induced monocytes require TLR4 signaling. N-G-monomethyl Larginine (L-NMMA), an inhibitor of Nitric oxide synthetase can completely inhibit NO

production by monocyte suggesting that NOS is the main enzyme in induced monocytes for NO production.

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