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Candida albicans mannan–protein conjugate as vaccine candidate
Immunology Letters 85 (2003) 251 /255 www.elsevier.com/locate/
Candida albicans mannan protein conjugate as vaccine candidate /
Slavomı´r Bystricky´ a,, Ema Paulovicˇova´ b, Eva Machova´ a a
b
Institute of Chemistry, Slovak Academy of Sciences, Du´bravska´ cesta 9, 842 38 Bratislava, Slovakia Department of Clinical Immunology and Allergy, National Institute of Tuberculosis and Respiratory Diseases, Krajinska´ 99, 82556 Bratislava, Slovakia Received 23 July 2002; received in revised form 18 September 2002; accepted 6 October 2002
Abstract Mannan */branched polysaccharide with a-(10/6)-linked mannose residues in the linear backbone, is a major virulence and protective factor of yeast Candida albicans . Injected alone does not induce sufficient level of protective antibodies. In this study, we have conjugated mannan to protein carrier by simple one step reaction using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) activation reagent. Prepared mannan conjugate is immunogenic in rabbits and reinjection elicited a booster response with significant increase of serum IgG level. Anti-C. albicans effectiveness of immune serum is clearly demonstrated. Based on these results, the mannan conjugate synthesized by this scheme can be considered as actual vaccine candidate for clinical evaluation. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Conjugate vaccine; Candida albicans ; Serum IgG; Mannan
1. Introduction Candida albicans , opportunistic pathogen, remains leading cause of fungal diseases, frequently mortal in immunocompromised individuals. There is no licensed vaccine yet, despite the fact that candidiases occur at 75% of all females during the lifetime [1]. Prolonged antifugal therapy, to prevent recurrence, can not be without side effects. The serious problem is increased drug resistance [2]. Search for alternative way of fighting with the disease is very actual now. The preventive, protective and therapeutic use of antiCandida antibodies was suggested and tested recently [3]. Adhesive molecules raised antibodies, especially mucosal specific ones against surface mannan polysaccharide were tested [4]. Some of the monoclonal antibodies enhanced resistance of mice against disseminated candidiases [5]. These antibodies were immunoglobulin IgM serotype and were raised by vaccination with liposome /mannan complex [6]. Compared with antibodies induced with protein, the antibodies against mannan are more homogenous, do not contain fractions
Corresponding author. Tel.: /412-2-5941-0221; fax: /421-25941-0222. E-mail address: [email protected] (S. Bystricky´).
with potential autoimmune side effects. The effectiveness of the polysaccharide vaccine was unambiguously confirmed at bacterial pathogens [7]. Despite their success, polysaccharide vaccines have limitation because of their T-cell independence. The consequence is unresponsiveness at infants, elderly and immunocompromised individuals */the most vulnerable groups. This deficiency can be overcome by covalent coupling of saccharides to proteins [8]. The different routes of immunization were used and different isotypes of antibodies were followed for evaluation. Regulatory agencies, however, standardize vaccine only by their ability to elicit protective level of serum antibodies in order to predict their effectiveness. Serum IgG fulfills this role by ability to kill and neutralize the inoculum [9]. Critical level of serum IgG will certainly confer protection against disseminated candidiases [10]. Moreover, appearance of IgG on the mucosal surface, as was found on the cervix [11], implicates that IgG isotype probably secretoric form can eliminate also local vaginal infections. In this study we prepared mannan */protein conjugate in simple ‘one pot’ chemical reaction and tested the ability of conjugate induce the serum IgG antibodies in animal model.
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 2 4 1 - 9
252
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
2. Materials and methods
mannan/HSA conjugate was above 800 kDa [12] and this fraction was used for immunization of rabbits.
2.1. Chemicals 2.4. Immunization Human serum albumine, HSA (Fluka); Folin’s reagent (Labora, a.s., Bratislava Slovakia); Fehling’s reagent prepared from copper sulfate, sodium potassium tartrate, sodium hydroxide and distilled water; 1-cyano4-dimethylaminopyridinium tetrafluoroborate, CDAP (Sigma); triethylamine, ethanolamine, acetonitril, methanol, acetic acid (Merck); NaOH, NaCl, Na2CO3, CuSO4 ×/ 5 H2O, Na2SO4 anhydrous, NaIO4 (Chemapol a.s. Czech Republic); Agar (Serva); Immulon 4 HBX, Goat Anti-Rabbit IgG (H/L), BluePhos, Milk Diluent Block (KPL).
Rabbits (male, 8 weeks old, 2000 g of weight, variety HYLA) were from Research Institute of Animal Production (Nitra, Slovakia). Hyperimmune rabbit sera were prepared by intravenous injection of heat killed C. albicans cells (107 cells per ml). For evaluation of immunogenicity of mannan/HSA conjugate, rabbits were injected seven times with 0.312 mg of conjugate in 1ml saline per dose in week’s intervals. The blood was collected before every next injection, centrifuged 10 min at 1500 g at 4 8C and sera were stored at /20 8C.
2.2. Preparation of mannan
2.5. Serology
The yeast strain C. albicans CCY 29-3-32 from Culture Collection of Yeasts (Institute of Chemistry of Slovak Academy of Sciences, Bratislava, Slovakia) was used for preparation of mannan. Cellular mannan was isolated and purified from the fresh biomass using Fehling’s reagent. Briefly: 100 g of yeast biomass was suspended in 400 ml 2% KOH and heated for 1 h at 100 8C. Insoluble residue was separated by centrifugation and mannan was precipitated from supernatant with Fehling’s reagent. Sedimented mannan /copper complex was dissolved in minimum volume of 3 N HCl and added dropwise to methanol /acetic acid mixture (8:1, v/v). The procedure of dissolution and precipitation of mannan were repeated two times. Finally, the white sediment was separated, dissolved in distilled water, dialyzed for 24 h and the dialysate was lyophilized. Mannan contained no nitrogen as determined by elementary analysis.
Quatitative precipitation was performed using 100 ml of hyperimmune rabbit serum and mannan C. albicans containing from 2 to 1000 mg of mannan per 0.1 ml at 37 8C for 1 h and then 6 days at 6 8C. The precipitates were washed in cold saline three times, dissolved in 200 ml 1 M NaOH and the content of protein was determined [14].
2.3. Conjugation of mannan with human serum albumin Mannan/HSA conjugate was prepared as described elsewhere [12]. The solution of CDAP, hydroxyl group activator (20 mg/0.2 ml acetonitril) was added to water solution of mannan (30 mg/3 ml). After 1 min pH was raised to 9.5 by 0.3 M triethylamine and activation was performed during 3 min. Then pH was adjusted to 8.0 using 1 M acetate buffer and HSA (15 mg/ml 0.15 M NaCl) was added. The reaction was carried out overnight and quenched with 1 ml of 1 M ethanolamine. The reaction mixture was dialyzed and applied to a column of Sepharose CL 6B (110 /1.5 cm, flow rate 0.2 ml/min) in 0.1 M phosphate buffer pH 7.5. Two fractions of conjugate were obtained, desalted on Sephadex G-25 column (10 /1.5 cm) and analyzed for polysaccharide/ protein ratio. The amount of mannan was determined by phenol /sulfuric methods [13] and the content of HSA by Lowry method [14]. Mw of the larger peak of
2.6. Biotinylation of mannan Mannan was oxidized by standard procedure with sodium periodate to yield an oxidized mannan containing reactive functional carbonyl groups with degree of oxidation /10% determined by Park /Johnson assay [15]. Biotinylation reaction of oxidized mannan was performed with biotinhydrazide using sodium cyanoborohydride (NaBH3CN) as a reductant of resulting Schiff’s base. Degree of biotinylation (/4%) was determined by HABA test [16], based on the use of the dye 4?-hydroxyazobenzene-2-carboxylic acid (HABA). Standard dilutions of biotinylmannan was determined based on the antigenic activity in the ELISA (aliquotes 100 ml per well, 4 and 10 mg/ml, respectively). 2.7. ELISA Cellular mannan triggered production of rabbit antiC. albicans IgG (H/L) antibodies was detected using heterogenous enzyme */linked immunosorbent test (ELISA). Immulon 4 HBX microplates were coated with avidin (4 and 10 ug/ml in PBS pH 7.2, 0,5% v/v Tween 20 and milk diluent 2% w/v) 18 h at 4 8C. Nonfat milk (2% w/v) was used as blocking solution to minimize nonspecific binding. Washing and blocking procedure was repeated three times for 30 min at room temperature. Checkerboard titrations of hyperimmune rabbit antiserum and pooled one were performed stepwise with different dilutions 1:500 etc. up to 1:8000
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
with biotinylated mannan. The avidin /biotin binding was completed after 18 h at 4 8C. Immune complexes were detected by alkaline phosphatase conjugate goat anti-rabbit IgG (H/L) serum (dilution 1:1000 v/v in blocking buffer) for 18 h at 4 8C. Enzyme reaction was developed with BluePhos Microwell phosphatase substrate system (KPL) within time interval 30 /90 min and plates were continuesly scanned at 630 nm (microplate reader, Dynatech). The results were expressed as absorbance units. 2.8. Fungal inhibition test The dense suspension of C. albicans cells was poured out on the 1% malt agar. The Whatmann No. 1 rings (d/5 mm) soaked into rabbit serum with different concentrations (dilutions of serum with saline were 1:10; 1:100; 1:1000; 1:10 000) were placed on the surface of agar and the growth of C. albicans and their effect was observed during 3 days at 37 8C.
3. Results and discussion The water soluble mannan /HSA conjugate was prepared by simple one step reaction at mild reaction
253
conditions. The hydroxyl groups of mannan were transformed to active form by novel non hazardous reagent CDAP and then coupled with terminal amino groups of lysines on the protein carrier [12,17]. New chemical coupling method presented here has several valuable advantages. In comparison with the popular periodate oxidation reaction [18], this reaction is not destructive for polysaccharide immunological determinants. Also, it is less hazardous as widely used CNBr activation. Moreover, it is supposed that linkages of mannan to HSA are located mainly in terminal saccharide units of mannan branches. It implicates that polysaccharide and protein could be linked directly without any spacer because branches of mannan could serve as distance spacers. The content of saccharide as well as protein in mannan/HSA conjugate was determined and saccharide/protein average weight ratio was found to be 3/1. The molecular size of mannan/HSA conjugate was higher than the size of original polysaccharide as observed by HPLC and PAGE in our previous paper [12]. As reported previously, yeast’s mannans can elicit antibodies in experimental animals when administrated in certain constitutions: as liposome /mannan complex [6] or mannan /BSA conjugate with adjuvants [19]. In
Fig. 1. Serum IgG antibodies (ELISA absorption units) elicited after immunization with prepared mannan /HSA conjugate (black bars) and after immunization with whole cells of C. albicans */hyperimmune serum (grey bars) at different sera dilution.
254
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
Fig. 2. Serum IgG antibodies (ELISA absorption units) elicited after different number of injections of mannan /HSA conjugate determined at different sera dilutions: 1:1000 (diamonds), 1:2000 (squares), 1:4000 (triangles) 1:8000 (circles).
our study the mannan/HSA conjugate was injected without adjuvants. The quantitative content of total induced rabbit antimannan sera immunoglobulins was conceived by weights of precipitates. We used hyperimmune serum for quantitative precipitation with mannan and the precipitates obtained with different concentrations of mannan were analyzed on the content of protein antibodies. Characteristic precipitine curve (not shown) enables to determine the weight content of precipitated antimannan immunoglobulins at maximum amount of mannan. With increased amounts of mannan, precipitated immunoglobulins raised simultaneously with maximum at 275 per 500-mg mannan. The only IgG serum level can be exactly correlated with protective immunity of organism. According this statement we tested in our ELISA test the serum anti-C. albicans IgG antibodies. The level of IgG (H/L) immunoglobulins were tested by sensitive ELISA test using biotinylated mannan as antigen for coating step. The results from ELISA test obtained for mannan / HSA conjugate antisera were compared with the value of hyperimmune serum. The Fig. 1 documents that the sera after immunization with mannan /HSA conjugate contained significantly higher levels of antimannan IgG antibodies (P B/0.05 and P B/0.01, respectively) at 1:4000 and 1:8000 dilutions compared with hyperimmune serum. During immunization the rabbits were injected seven times and sera were obtained 7 days after each injection. The time course of IgG level after the repeated immunization is demonstrated on Fig. 2. The first one
injection of mannan /HSA conjugate elicited only slight increase of IgG in comparison with preimmune level (baseline). This is obviously caused by early reaction of immune system producing mainly IgM type of antibodies. The IgG isotype level is increased after each further injection, more significantly after second and
Fig. 3. C. albicans growing inhibition test. The growth of C. albicans on the surface of 1% wort agar in presence of mannan /HSA conjugate serum applied in different dilutions: not diluted serum (A), diluted 1:10 (B) 1:100 (C) 1:1000 (D), 1:10 000 (E).
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
third one (P B/0.001 and P B/0.0001). The booster responses are typical for T-cell dependent immune reaction, but not for polysaccharides alone. The followed fourth and fifth injections did not increase IgG level significantly compared with previous ones. For immunization protocol three injections should be optimal. The antifungal quality of the sera was tested in C. albicans growth inhibition test. The strong inhibition effectivity of antisera on growth of yeast C. albicans on agar medium was evidently documented (Fig. 3). The complete inhibition of C. albicans growth was reached with non-diluted serum and the strong inhibition zones were still observed with all used dilutions.
Acknowledgements This work was supported by the Grant Agency of Slovak Academy of Sciences, grant VEGA No.2/1048/ 21.
References [1] J.D. Sobel, Pathogenesis and epidemiology of vulvovaginal candidiasis, Ann. NY Acad. Sci. 544 (1988) 547 /557. [2] J.R. Wingard, Infections due to resistant Candida species in patients with cancer who are receiving chemotherapy, Clin. Infect. Dis. 19 (Suppl. 1) (1994) S49 /S53. [3] Y. Han, J.E. Cutler, Assessment of a mouse model of neutropenia and the effect of an anti-candidiasis monoclonal antibody in these animals, J. Infect. Dis. 175 (1997) 1169 /1175. [4] E. Lett, C. Klopfenstein, J.-P. Klein, M. Scho¨ller, D. Wachsmann, Mucosal immunogenicity of polysaccharides conjugated to a peptide or multiple-antigen peptide containing T- and B-cell epitopes, Infect. Immun. 63 (7) (1995) 2645 /2651. [5] Y. Han, R.P. Morrison, J.E. Cutler, A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection, Infect. Immun. 66 (12) (1998) 5771 /5776.
255
[6] Y. Han, J.E. Cutler, Antibody response that protects against disseminated candidiasis, Infect. Immun. 63 (7) (1995) 2714 / 2719. [7] H.J. Jennings, Capsular polysaccharides as human vaccines, Adv. Carbohydr. Chem. Biochem. 41 (1983) 155 /207. [8] J.B. Robbins, R. Schneerson, Polysaccharide /protein conjugates: a new generation of vaccines, J. Infect. Dis. 161 (1990) 821 /823. [9] J.B. Robbins, R. Schneerson, S.C. Szu, D.A. Bryla, F.-Y. Lin, E.C. Gotschlich, Standardization may suffice for licensure of conjugate vaccines, in: S. Plotkin, F. Brown, F. Horaud (Eds.), Preclinical and Clinical Development of New Vaccines, vol. 95, Krager, Basel, 1998, Dev. Biol. Stand., pp. 161 /167. [10] R. Matthews, J. Burnie, Antibodies against Candida : potential therapeutics? Trends Microbiol. 4 (1996) 354 /358 [11] J.P. Bouvet, L. Be´lec, R. Pire´s, J. Pillot, Immunoglobulin G antibodies in human vaginal secretions after parenteral vaccination, Infect. Immun. 62 (1994) 3957 /3961. [12] S. Bystricky´, E. Machova´, P. Bartek, N. Kolarova, G. Kogan, Conjugation of yeast mannans with protein employing cyanopyridinium agent (CDAP) */an effective route of antifungal vaccine preparation, Glycoconj. J. 17 (2000) 677 /680. [13] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350 /356. [14] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 /275. [15] J.T. Park, M.J. Johnson, A submicrodetermination of glucose, J. Biol. Chem. 181 (1949) 149 /151. [16] N.M. Green, in: D.B. McCormick, L.D. Wright (Eds.), Spectrophotometric determination of avidin and biotin, Academic Press, New York, 1970, Meth. Enzymol., 18, pp. 418. [17] D.E. Shafer, B. Toll, R.F. Schuman, B.L. Nelson, J.J. Mond, A. Lees, Activation of soluble polysaccharides with 1-cyano-4dimethylaminopyridinium tetrafluroborate (CDAP) for use in protein /polysaccharide conjugate vaccines and immunological reagents, Vaccine 18 (2000) 1273 /1281. [18] J. Masa´rova´, D. Mislovie`ova´, P. Gemeiner, Optimization of dextran and mannan dialdehydes preparation and examination of their biospecific interaction with concanavalin A, Chem. Pap. 55 (2) (2001) 130 /135. [19] Y. Han, M.A. Ulrich, J.E. Cutler, Candida albicans mannan extract /protein conjugates induce a protective immune response against experimental candidiasis, J. Infect. Dis. 179 (1999) 1477 / 1484.
Candida albicans mannan protein conjugate as vaccine candidate /
Slavomı´r Bystricky´ a,, Ema Paulovicˇova´ b, Eva Machova´ a a
b
Institute of Chemistry, Slovak Academy of Sciences, Du´bravska´ cesta 9, 842 38 Bratislava, Slovakia Department of Clinical Immunology and Allergy, National Institute of Tuberculosis and Respiratory Diseases, Krajinska´ 99, 82556 Bratislava, Slovakia Received 23 July 2002; received in revised form 18 September 2002; accepted 6 October 2002
Abstract Mannan */branched polysaccharide with a-(10/6)-linked mannose residues in the linear backbone, is a major virulence and protective factor of yeast Candida albicans . Injected alone does not induce sufficient level of protective antibodies. In this study, we have conjugated mannan to protein carrier by simple one step reaction using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) activation reagent. Prepared mannan conjugate is immunogenic in rabbits and reinjection elicited a booster response with significant increase of serum IgG level. Anti-C. albicans effectiveness of immune serum is clearly demonstrated. Based on these results, the mannan conjugate synthesized by this scheme can be considered as actual vaccine candidate for clinical evaluation. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Conjugate vaccine; Candida albicans ; Serum IgG; Mannan
1. Introduction Candida albicans , opportunistic pathogen, remains leading cause of fungal diseases, frequently mortal in immunocompromised individuals. There is no licensed vaccine yet, despite the fact that candidiases occur at 75% of all females during the lifetime [1]. Prolonged antifugal therapy, to prevent recurrence, can not be without side effects. The serious problem is increased drug resistance [2]. Search for alternative way of fighting with the disease is very actual now. The preventive, protective and therapeutic use of antiCandida antibodies was suggested and tested recently [3]. Adhesive molecules raised antibodies, especially mucosal specific ones against surface mannan polysaccharide were tested [4]. Some of the monoclonal antibodies enhanced resistance of mice against disseminated candidiases [5]. These antibodies were immunoglobulin IgM serotype and were raised by vaccination with liposome /mannan complex [6]. Compared with antibodies induced with protein, the antibodies against mannan are more homogenous, do not contain fractions
Corresponding author. Tel.: /412-2-5941-0221; fax: /421-25941-0222. E-mail address: [email protected] (S. Bystricky´).
with potential autoimmune side effects. The effectiveness of the polysaccharide vaccine was unambiguously confirmed at bacterial pathogens [7]. Despite their success, polysaccharide vaccines have limitation because of their T-cell independence. The consequence is unresponsiveness at infants, elderly and immunocompromised individuals */the most vulnerable groups. This deficiency can be overcome by covalent coupling of saccharides to proteins [8]. The different routes of immunization were used and different isotypes of antibodies were followed for evaluation. Regulatory agencies, however, standardize vaccine only by their ability to elicit protective level of serum antibodies in order to predict their effectiveness. Serum IgG fulfills this role by ability to kill and neutralize the inoculum [9]. Critical level of serum IgG will certainly confer protection against disseminated candidiases [10]. Moreover, appearance of IgG on the mucosal surface, as was found on the cervix [11], implicates that IgG isotype probably secretoric form can eliminate also local vaginal infections. In this study we prepared mannan */protein conjugate in simple ‘one pot’ chemical reaction and tested the ability of conjugate induce the serum IgG antibodies in animal model.
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 2 4 1 - 9
252
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
2. Materials and methods
mannan/HSA conjugate was above 800 kDa [12] and this fraction was used for immunization of rabbits.
2.1. Chemicals 2.4. Immunization Human serum albumine, HSA (Fluka); Folin’s reagent (Labora, a.s., Bratislava Slovakia); Fehling’s reagent prepared from copper sulfate, sodium potassium tartrate, sodium hydroxide and distilled water; 1-cyano4-dimethylaminopyridinium tetrafluoroborate, CDAP (Sigma); triethylamine, ethanolamine, acetonitril, methanol, acetic acid (Merck); NaOH, NaCl, Na2CO3, CuSO4 ×/ 5 H2O, Na2SO4 anhydrous, NaIO4 (Chemapol a.s. Czech Republic); Agar (Serva); Immulon 4 HBX, Goat Anti-Rabbit IgG (H/L), BluePhos, Milk Diluent Block (KPL).
Rabbits (male, 8 weeks old, 2000 g of weight, variety HYLA) were from Research Institute of Animal Production (Nitra, Slovakia). Hyperimmune rabbit sera were prepared by intravenous injection of heat killed C. albicans cells (107 cells per ml). For evaluation of immunogenicity of mannan/HSA conjugate, rabbits were injected seven times with 0.312 mg of conjugate in 1ml saline per dose in week’s intervals. The blood was collected before every next injection, centrifuged 10 min at 1500 g at 4 8C and sera were stored at /20 8C.
2.2. Preparation of mannan
2.5. Serology
The yeast strain C. albicans CCY 29-3-32 from Culture Collection of Yeasts (Institute of Chemistry of Slovak Academy of Sciences, Bratislava, Slovakia) was used for preparation of mannan. Cellular mannan was isolated and purified from the fresh biomass using Fehling’s reagent. Briefly: 100 g of yeast biomass was suspended in 400 ml 2% KOH and heated for 1 h at 100 8C. Insoluble residue was separated by centrifugation and mannan was precipitated from supernatant with Fehling’s reagent. Sedimented mannan /copper complex was dissolved in minimum volume of 3 N HCl and added dropwise to methanol /acetic acid mixture (8:1, v/v). The procedure of dissolution and precipitation of mannan were repeated two times. Finally, the white sediment was separated, dissolved in distilled water, dialyzed for 24 h and the dialysate was lyophilized. Mannan contained no nitrogen as determined by elementary analysis.
Quatitative precipitation was performed using 100 ml of hyperimmune rabbit serum and mannan C. albicans containing from 2 to 1000 mg of mannan per 0.1 ml at 37 8C for 1 h and then 6 days at 6 8C. The precipitates were washed in cold saline three times, dissolved in 200 ml 1 M NaOH and the content of protein was determined [14].
2.3. Conjugation of mannan with human serum albumin Mannan/HSA conjugate was prepared as described elsewhere [12]. The solution of CDAP, hydroxyl group activator (20 mg/0.2 ml acetonitril) was added to water solution of mannan (30 mg/3 ml). After 1 min pH was raised to 9.5 by 0.3 M triethylamine and activation was performed during 3 min. Then pH was adjusted to 8.0 using 1 M acetate buffer and HSA (15 mg/ml 0.15 M NaCl) was added. The reaction was carried out overnight and quenched with 1 ml of 1 M ethanolamine. The reaction mixture was dialyzed and applied to a column of Sepharose CL 6B (110 /1.5 cm, flow rate 0.2 ml/min) in 0.1 M phosphate buffer pH 7.5. Two fractions of conjugate were obtained, desalted on Sephadex G-25 column (10 /1.5 cm) and analyzed for polysaccharide/ protein ratio. The amount of mannan was determined by phenol /sulfuric methods [13] and the content of HSA by Lowry method [14]. Mw of the larger peak of
2.6. Biotinylation of mannan Mannan was oxidized by standard procedure with sodium periodate to yield an oxidized mannan containing reactive functional carbonyl groups with degree of oxidation /10% determined by Park /Johnson assay [15]. Biotinylation reaction of oxidized mannan was performed with biotinhydrazide using sodium cyanoborohydride (NaBH3CN) as a reductant of resulting Schiff’s base. Degree of biotinylation (/4%) was determined by HABA test [16], based on the use of the dye 4?-hydroxyazobenzene-2-carboxylic acid (HABA). Standard dilutions of biotinylmannan was determined based on the antigenic activity in the ELISA (aliquotes 100 ml per well, 4 and 10 mg/ml, respectively). 2.7. ELISA Cellular mannan triggered production of rabbit antiC. albicans IgG (H/L) antibodies was detected using heterogenous enzyme */linked immunosorbent test (ELISA). Immulon 4 HBX microplates were coated with avidin (4 and 10 ug/ml in PBS pH 7.2, 0,5% v/v Tween 20 and milk diluent 2% w/v) 18 h at 4 8C. Nonfat milk (2% w/v) was used as blocking solution to minimize nonspecific binding. Washing and blocking procedure was repeated three times for 30 min at room temperature. Checkerboard titrations of hyperimmune rabbit antiserum and pooled one were performed stepwise with different dilutions 1:500 etc. up to 1:8000
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
with biotinylated mannan. The avidin /biotin binding was completed after 18 h at 4 8C. Immune complexes were detected by alkaline phosphatase conjugate goat anti-rabbit IgG (H/L) serum (dilution 1:1000 v/v in blocking buffer) for 18 h at 4 8C. Enzyme reaction was developed with BluePhos Microwell phosphatase substrate system (KPL) within time interval 30 /90 min and plates were continuesly scanned at 630 nm (microplate reader, Dynatech). The results were expressed as absorbance units. 2.8. Fungal inhibition test The dense suspension of C. albicans cells was poured out on the 1% malt agar. The Whatmann No. 1 rings (d/5 mm) soaked into rabbit serum with different concentrations (dilutions of serum with saline were 1:10; 1:100; 1:1000; 1:10 000) were placed on the surface of agar and the growth of C. albicans and their effect was observed during 3 days at 37 8C.
3. Results and discussion The water soluble mannan /HSA conjugate was prepared by simple one step reaction at mild reaction
253
conditions. The hydroxyl groups of mannan were transformed to active form by novel non hazardous reagent CDAP and then coupled with terminal amino groups of lysines on the protein carrier [12,17]. New chemical coupling method presented here has several valuable advantages. In comparison with the popular periodate oxidation reaction [18], this reaction is not destructive for polysaccharide immunological determinants. Also, it is less hazardous as widely used CNBr activation. Moreover, it is supposed that linkages of mannan to HSA are located mainly in terminal saccharide units of mannan branches. It implicates that polysaccharide and protein could be linked directly without any spacer because branches of mannan could serve as distance spacers. The content of saccharide as well as protein in mannan/HSA conjugate was determined and saccharide/protein average weight ratio was found to be 3/1. The molecular size of mannan/HSA conjugate was higher than the size of original polysaccharide as observed by HPLC and PAGE in our previous paper [12]. As reported previously, yeast’s mannans can elicit antibodies in experimental animals when administrated in certain constitutions: as liposome /mannan complex [6] or mannan /BSA conjugate with adjuvants [19]. In
Fig. 1. Serum IgG antibodies (ELISA absorption units) elicited after immunization with prepared mannan /HSA conjugate (black bars) and after immunization with whole cells of C. albicans */hyperimmune serum (grey bars) at different sera dilution.
254
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
Fig. 2. Serum IgG antibodies (ELISA absorption units) elicited after different number of injections of mannan /HSA conjugate determined at different sera dilutions: 1:1000 (diamonds), 1:2000 (squares), 1:4000 (triangles) 1:8000 (circles).
our study the mannan/HSA conjugate was injected without adjuvants. The quantitative content of total induced rabbit antimannan sera immunoglobulins was conceived by weights of precipitates. We used hyperimmune serum for quantitative precipitation with mannan and the precipitates obtained with different concentrations of mannan were analyzed on the content of protein antibodies. Characteristic precipitine curve (not shown) enables to determine the weight content of precipitated antimannan immunoglobulins at maximum amount of mannan. With increased amounts of mannan, precipitated immunoglobulins raised simultaneously with maximum at 275 per 500-mg mannan. The only IgG serum level can be exactly correlated with protective immunity of organism. According this statement we tested in our ELISA test the serum anti-C. albicans IgG antibodies. The level of IgG (H/L) immunoglobulins were tested by sensitive ELISA test using biotinylated mannan as antigen for coating step. The results from ELISA test obtained for mannan / HSA conjugate antisera were compared with the value of hyperimmune serum. The Fig. 1 documents that the sera after immunization with mannan /HSA conjugate contained significantly higher levels of antimannan IgG antibodies (P B/0.05 and P B/0.01, respectively) at 1:4000 and 1:8000 dilutions compared with hyperimmune serum. During immunization the rabbits were injected seven times and sera were obtained 7 days after each injection. The time course of IgG level after the repeated immunization is demonstrated on Fig. 2. The first one
injection of mannan /HSA conjugate elicited only slight increase of IgG in comparison with preimmune level (baseline). This is obviously caused by early reaction of immune system producing mainly IgM type of antibodies. The IgG isotype level is increased after each further injection, more significantly after second and
Fig. 3. C. albicans growing inhibition test. The growth of C. albicans on the surface of 1% wort agar in presence of mannan /HSA conjugate serum applied in different dilutions: not diluted serum (A), diluted 1:10 (B) 1:100 (C) 1:1000 (D), 1:10 000 (E).
S. Bystricky´ et al. / Immunology Letters 85 (2003) 251 /255
third one (P B/0.001 and P B/0.0001). The booster responses are typical for T-cell dependent immune reaction, but not for polysaccharides alone. The followed fourth and fifth injections did not increase IgG level significantly compared with previous ones. For immunization protocol three injections should be optimal. The antifungal quality of the sera was tested in C. albicans growth inhibition test. The strong inhibition effectivity of antisera on growth of yeast C. albicans on agar medium was evidently documented (Fig. 3). The complete inhibition of C. albicans growth was reached with non-diluted serum and the strong inhibition zones were still observed with all used dilutions.
Acknowledgements This work was supported by the Grant Agency of Slovak Academy of Sciences, grant VEGA No.2/1048/ 21.
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