ARTICLE pubs.acs.org/bc
Synthesis and Immunogenicity of a Glycopolymer Conjugate Tomasz Lipinski, Pavel I. Kitov, Adam Szpacenko, Eugenia Paszkiewicz, and David R. Bundle* Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 ABSTRACT:
A protective β-mannan trisaccharide epitope from the Candida albicans cell wall phosphomannan has been synthesized and activated for copolymerization with acrylamide. The resulting glycopolymer displayed 33 trisaccharide haptens and was derivatized for conjugation to the immunogenic carrier protein, chicken serum albumin. The resulting conjugate achieves a high degree of oligosaccharide substitution while limiting the sites of substitution on the protein. The murine immune response against this conjugate was compared with the response to a trisaccharide-tetanus toxoid conjugate vaccine. The glycopolymer was shown to induce a more robust immune response with higher trisaccharide-specific antibody titers and with a significantly larger proportion of responding mice developing antibodies that bound the target, native cell wall antigen of C. albicans.
’ INTRODUCTION We report the synthesis of a protein-polyacrylamide conjugate displaying small molecular weight hapten and the ability of this conjugate to induce a robust T-cell dependent immune response to a small carbohydrate hapten. A linear polyacrylamide bearing pendant oligosaccharides and functionalized with a reactive end group was conjugated to a protein carrier enabling presentation of multiple B-cell epitopes in a fashion similar to the multiple repeating carbohydrate epitopes present in polysaccharideprotein conjugate vaccines. The approach constitutes a convenient platform for raising antibodies to small oligosaccharides as well as other low molecular weight haptens such as peptides, antibiotics, pesticides, and drugs. Conjugate vaccines based on microbial polysaccharides have proven their efficacy in combating many infectious diseases.1 While high molecular weight polysaccharides are typically T-independent antigens and activate B-cells directly resulting in production of mainly IgM antibodies, vaccines of this type are ineffective in infants and are incapable of induction of immunological memory. Conjugation of polysaccharides to a carrier protein converts these T-independent antigens and poor immunogens into highly r 2010 American Chemical Society
immunogenic T-dependent antigens. The repeating unit nature of bacterial polysaccharides results in a conjugate vaccine that presents approximately 50 to 100 B-cell epitopes even when only one polysaccharide chain (MW ∼100 000) is attached to a carrier protein.2 When small molecular weight haptens are conjugated to protein carriers, typical incorporation levels fall in the range 10-20 haptens per protein. In rabbits, such conjugates yield robust immune responses,3-5 but in mice, antibody responses are often poor to modest.6 This manuscript describes one of our strategies to secure consistently robust murine antibody responses to the small molecular weight β-(1f2) mannan trisaccharide hapten derived from Candida albicans. The increasing incidence of hospital acquired Candida infections underscores the need for a viable anti-Candida vaccine and has triggered extensive study conducted by several groups.7 Monoclonal antibodies specific for the C. albicans β-mannan are protective for mice challenged with live Candida8 and these Received: September 3, 2010 Revised: November 29, 2010 Published: December 27, 2010 274
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performed overnight at 37 °C. Glycopolymer was dialyzed against water in a Slide-A-Lyser cassette (Thermo Scientific). Glycopolymer Fractionation. The glycopolymer was fractionated by gel filtration on a Sephacryl-400 column (1.6 100 cm) with water as eluent. Polymer Characterization. Number average molecular weight of polymers and glycopolymer fractions was estimated by determination of the content of amino groups per gram of a dry polymer using 2,4,6-trinitrobenzenesulfonic acid (Sigma).12 Gel filtration analysis was performed on tandem Ultrahydrogel 1000 and Ultrahydrogel 500 7.8 300 mm HPLC columns (Waters) in 0.1 M Tris HCl pH 9.0, 0.2 M LiNO3 running buffer and monitored with a refractive index detector. Sugar content in glycopolymer and conjugated glycopolymer fractions was determined using the phenol-sulfuric acid method.13 Methyl R-D-mannopyranoside was used as the calibration reference. Modification of the Glycopolymer with Azide Group. Glycopolymer (5 mg/mL) and 6-azido-6-deoxy-D-galactose 5 (10 mg/ mL) were dissolved in 0.2 M phosphate buffer pH 6.5 (∼1000fold molar excess of 5). NaCNBH3 (10 mg/mL) was added, and the reductive reaction was incubated at 37 °C with stirring for 7 days. Azide-terminated glycopolymer was then dialyzed against water and lyophilized. Modification of the ChSA with Propargyl Group. Monomeric chicken serum albumin (ChSA) was obtained by gel filtration of crude albumin (Pel-Freez Biologicals) on Superdex S-200 column (1.6 100 cm) in PBS buffer. Monomer fractions were collected, dialyzed against water, and lyophilized. Purified albumin (104 mg) was dissolved in 0.1 M sodium bicarbonate/0.2 M NaCl, pH 8.14 buffer (4 mL) in a glass vial. Succinimide 4 (3.54 mg) was dissolved in DMSO (50 μL) and added to the rapidly stirred albumin solution. Stirring was continued overnight. The resulting solution was dialyzed against water and lyophilized. Azide-Alkyne Huisgen Cycloaddition Conjugation. Conjugation of the glycopolymer to ChSA was performed with minor modifications according to literature procedures.10,14 Bathophenantroline/Cu1þ catalyst was prepared as follows: CuSO4 3 5H2O (10 mg) and bathophenantroline sulfonate (64.4 mg) (GFS Chemicals Inc.) were dissolved 0.2 M Tris HCl, pH 8.0 buffer (1 mL) in a 4 mL Kimball glass vial. Copper powder (∼50 mg) was added; the vial was closed with an open-top screw cap with rubber septa and purged with argon. The vial was rotated for 2 h; the reduction of copper II to copper I by metallic copper was indicated by the appearance of a dark green color. For conjugation, the glycopolymer (13 mg) and propargylated ChSA (25 mg) were dissolved in 0.2 M Tris HCl, pH 8.0 buffer (3 mL) and placed in 10 mL Kimbal glass vial equipped with open-top screw cap with rubber septa. Copper powder (∼20 mg) and isobutanol (to reduce foaming) (50 μL) were added. The vial was closed and degassed, followed by purging with argon (3). Reaction was initiated by the addition of catalyst (75 μL) from a Hamilton syringe. After 12 h incubation, the mixture was filtered and the filtrate was dialyzed against 20 mM Tris HCl, 1 mM EDTA, pH 8.5 buffer. Dialysate was then applied on an ion exchange column (Protein-Pak DEAE-8HR, Waters) connected to HPLC (Waters) and fractionated by elution with sodium chloride gradient in the same buffer. Tetanus Toxoid Conjugate. The amino-terminated glycoside 2 was conjugated to tetanus toxoid as previously described.5 The resulting conjugate (designated trisaccharide-TT) had an average
antibodies are most effectively inhibited by short oligosaccharide sequences such as disaccharide or trisaccharide β-(1f2)-mannan oligomers.9 These results implied that β-(1f2) mannan trisaccharide coupled to tetanus toxoid (TT) would be a good candidate for a Candida vaccine.3,9 Neoglycoconjugates of this type were synthesized in our group and studied for biological activity. This conjugate vaccine showed good immunogenicity in rabbits4,5 and guinea pigs (unpublished results). Furthermore, vaccinated rabbits were able to reduce Candida burden in different organs after challenge with fungi under an immunosuppression regime.4 However, as we have shown previously6 and confirmed here, the same conjugate did not elicit a significant immune response in mice. To achieve better immunogenicity of our conjugate, we have pursued two approaches. The first utilized presentation of oligosaccharide units in a clustered form6 and the second as a linear copolymer with acrylamide. Disaccharide β-(1f2)-mannan clustered on a glucose core did not show any advantage over the conjugate presenting single disaccharide units when used in mice and rabbits.6 In contrast, we report here that incorporation of β-(1f2)-mannan trisaccharide displayed as multiple side chains on polyacrylamide and then conjugated to chicken serum albumin (ChSA) gave a powerful immunogen capable of producing significantly improved murine antibody levels when compared with our previous results.6
’ EXPERIMENTAL PROCEDURES Trisaccharide Monomer. The allyl glycoside 1 prepared according to a published procedure5 was reacted with cysteamine as previously described to provide the amino-terminated glycoside 2. Acylation of the amino group of the trisaccharide tether by acrylic acid -NHS ester installed the acrylamide functionality to give the activated trisaccharide 3. Alkyne 410 and azide 511 were synthesized by published methods. Polymerization of Polyacrylamide with Cysteamine as Chain Transfer Reagent. Acrylamide (1.25 g, 17.5 mmol) of was dissolved in 0.1 M acetate buffer (12.5 mL, pH 4) in a 50 mL Falcon tube, closed with rubber septum, and degassed on a vacuum line and then purged with argon. Stock solutions of cysteamine hydrochloride (50 mg/mL, 0.44 mM) and potassium persulfate (50 mg/mL, 0.18 mM) in water were prepared in the same manner in septa-closed 6 mL glass vials (Kimball). Cysteamine stock solution was added (20, 40, 80, 120, 200, and 300 μL; corresponding to 1, 2, 4, 6, 10, and 15 mg) to acrylamide solution prepared above using a Hamilton syringe, and polymerization was initiated by addition of potassium persulfate solution (300 μL). Reaction was allowed to proceed at 37 °C overnight. After polymerization samples were extensively dialyzed against water and then lyophilized. Synthesis of the Glycopolymer. Acrylamide (43.4 mg, 0.61 mmol) and acrylamide activated trisaccharide 3 (12.38 mg, 0.018 mmol) were dissolved in 0.1 M acetate buffer pH 4.0 (500 μL), the solution was transferred to a glass vial sealed with a silicone septum, and the solution was degassed and purged with argon. Stock solutions of cysteamine hydrochloride (10 mg/mL 0.088 mM) and potassium persulfate (50 mg/ mL, 0.18 mM) in water were prepared in the same manner. Cysteamine stock solution (5.8 μL) was added with a Hamilton syringe and polymerization was initiated by addition of potassium persulfate solution (10.4 μL). Polymerization was 275
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Scheme 1. Synthesis of Activated Trisaccharide Monomer 3a
a
Compounds 4 and 5 are the reagents used to functionalize the glycopolymer and the protein, ChSA for subsequent conjugation via Huisgen cycloaddition.
degree of substitution of 12 trimannosides per molecule of protein. Vaccine Formulations. Vaccine doses (regardless of adjuvant used) were formulated to contain 3 μg of β-mannan trisaccharide in 300 μL of formulation. Alum suspensions were prepared according to ref 15. Emulsions with Freund's complete and incomplete adjuvant (FA) were prepared by intense vortexing of equal volumes of glycopolymer conjugate in PBS and FA until a stable emulsion was formed. Immunization. CD1 female mice (5 to 7 weeks old) were immunized three times at 3 week intervals with vaccine formulation (300 μL) administered by 100 μL intraperitoneal and 200 μL subcutaneous injections. Mice immunized with Freund's adjuvant were given vaccine in complete Freund's adjuvant for the initial immunization, and subsequent immunizations were given with incomplete Freund's adjuvant. ELISA Assays. To avoid possible cross-reactivity between sera raised against the glycopolymer-ChSA conjugate and BSA, we used a previously described trisaccharide-TT conjugate5 to coat plates. Polystyrene 96 well plates were coated overnight with trisaccharide-TT conjugate at a concentration of 5 μg/mL in PBS or with phosphomannan complex at the same concentration in 0.05 M carbonate buffer pH 9.8. After washing with PBS containing √ 0.1% Tween (PBST), wells were filled with 100 μL of serial 10 dilutions of sera (starting from 10-3). 0.1% skim milk (Difco) in PBST was used for dilutions to prevent nonspecific binding. Plates were sealed and incubated for 2 h at room temperature. After washing with PBST, a reporter antibody, anti-Mouse IgG, HRP conjugate from KPL (Kirkegaard & Perry Laboratories, Inc.) in 0.1% skim milk PBST, at a dilution of 1/2000 was applied and plates were incubated for 1 h at room temperature. Plates were washed again with PBST and color developed with HRP substrate system (KPL) for 15 min. The reaction was stopped with 1 M phosphoric acid, and absorbance was measured.
Table 1. Polyacrylamide of Different Molecular Weight Was Obtained by Polymerization at Different Ratios of Acrylamide Monomer (aa) to Chain Transfer Reagent (cys)a
a
molar ratio acrylamide/cysteamine
number average molecular weight
2000:1
212 000
1000:1
130 000
500:1
75 000
333:1
52 000
200:1
39 000
133:1
29 000
Mn: number average molecular weight.
Figure 1. GPC traces of polymers obtained by polymerization with cysteamine (Table 1). Mn = 212 000 (1); 130 000 (2); 75 000 (3); 52 000 (4); 39 000 (5); 29 000 (6).
Antipolyacrylamide antibodies were detected by the same protocol but using plates coated with an acrylamide-peptide copolymer. Acrylamide was copolymerized with peptide epitope fragment (p458m) derived from heat shock protein, 276
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Scheme 2. Synthesis of Glycopolymer and Its Conjugation to Chicken Serum Albumin
functionalized with triethylene glycol acrylate spacer (kind gift from Dr. Sebastian Dziadek).16
Ultrahydrogel columns (Figure 1) by correlating molecular weight and the respective retention times. For the synthesis of glycopolymer, an acrylamide/trisaccharide acrylamide ratio of ∼34:1 was used in order to target a polymer containing 2-3% trisaccharide monomer (Scheme 2). A ratio of combined monomers/cysteamine of 530:1 was chosen, in order to target a polymer with MW of ∼100 kDa. Ultrahydrogel HPLC analysis of the resulting glycopolymer (Figure 2) revealed that its polydispersity was greater than that of previously characterized polyacrylamide polymers (Table 1, cf Figures 1 and 2). The glycopolymer was then subjected to fractionation on a SephacrylS400 column, and the eluted material was divided into two fractions, which were then analyzed by Ultrahydrogel HPLC (Figure 3). The higher molecular weight fraction had Mn of 122 000, and this fraction was used for conjugation with albumin. The approximate
’ RESULTS The trisaccharide allyl glycoside 1 was synthesized, and photochemical addition of cysteamine gave the amino-terminated glycoside 2.5 Acylation with acrylic acid afforded the monomeric trisaccharide 3 for use in polymerization reactions (Scheme 1). In order to establish the relationship between the amount of chain transfer reagent and the molecular weight of the resulting polymer, a series of experiments were performed in which acrylamide was polymerized in the presence of varying amounts of cysteamine. The number average molecular weight (Mn) was determined by quantitation of terminal amino groups (Table 1) and HPLC on 277
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Figure 2. Glycopolymer (solid line) and two standard polymers 130 (dotted line) and 75 kDa, (dash line).
Figure 5. 1H NMR (D2O, 700 MHz, 27 °C) of β-mannan trisaccharide glycopolymer conjugated to ChSA. Signals arising from aromatic amino acids of ChSA are shown as an insert (8.8-6.6 ppm). Aliphatic amino acid resonances are also visible (1.5-0.5 ppm). Signals from the amino acid R-hydrogens are visible in the region between 4 and 3.5 ppm overlapped by the protons of the pyranose rings. The formation of the higher molecular weight acrylamide-protein conjugate is accompanied by a general line broadening.
Figure 3. Ultrahydrogel HPLC traces of the unfractionated glycopolymer (1), and the high (2) and lower (3) molecular weight fractions obtained by gel permeation chromatography on Sephacryl-S400 column.
the glycopolymer by reductive amination using excess 6-azido6-deoxy-D-galactose 5 (Scheme 2). Conjugation of the glycopolymer to propargylated albumin was performed at a ∼1:1 ratio (protein to glycopolymer). The resulting conjugate was fractionated on an ion-exchange column to remove unreacted polyacrylamide and protein components. Unconjugated glycopolymer was eluted in the nonabsorbed fraction (about 22% of starting glycopolymer), and two major fractions containing both protein and sugar were collected. The fraction eluting at 0-0.165 M NaCl (fraction 1) contained mannose as determined by phenol-sulfuric acid colorimetric assay corresponding to ∼33 units of trisaccharide per molecule of albumin. The fraction eluting at higher salt concentration (0.165-0.2 M NaCl) contained about 18 trisaccharide units. A 1H NMR spectrum provided further evidence for the formation of the glycopolymerChSA conjugate (Figure 5) and SDS PAGE showed no residual unconjugated protein and diffuse, high molecular weight protein bands. The first conjugate fraction containing the higher trisaccharide loading was used to evaluate immunogenicity by vaccination of mice. The immune response to the vaccine was investigated using groups of 5 to 10 mice immunized with this conjugate at intervals of three weeks employing two different adjuvants, Freund's adjuvant and alum, and the other group was vaccinated without any adjuvant. Sera collected after the second and third immunizations were titered against synthetic β-mannan trisaccharide conjugated to tetanus toxoid (Figure 6) and native antigen consisting of a Candida albicans cell wall extract17 (Figure 7). Following the second and third immunizations, significant antibody titers frequently exceeding 1 million measured against the trisaccharide-TT conjugate were observed in the sera of all mice vaccinated with glycopolymer-ChSA conjugate. Although all mice had a robust response to the immunizing hapten, only half of the mice produced antibodies that crossreacted with the native β-mannan of the C. albicans cell wall (Figure 7, panel a).
Figure 4. 1H NMR (D2O, 500 MHz, 27 °C) of β-mannan trisaccharide glycopolymer. Integration of a single mannose H-2 proton versus the acrylamide methylene protons indicates 2% incorporation of trisaccharide in the polyacrylamide backbone.
trisaccharide payload was 2.2% based on the integration of 1H NMR resonances at δ 4.36 ppm (d, J2,3 = 3.0 Hz, H-2 of one mannose residue) and δ 2.38-2.15 ppm (m, CH of the acrylamide residue) (Figure 4). These numbers translate to 31 trisaccharide haptens per molecule of glycopolymer. Comparable values were obtained using the phenol-sulfuric acid colorimetric assay. Monomeric ChSA obtained by gel filtration on Superdex-200 was reacted with the activated ester 4 to introduce propargyl groups for conjugation with the glycopolymer. MALDI analysis of the modified protein showed the presence of 8 propargyl groups per molecule of albumin. Azide groups were installed on 278
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5 mice immunized according to the protocol described in the Experimental section of this paper, which is the same as that reported.9
Figure 6. Trisaccharide specific antibody titers following vaccination of mice with glycopolymer conjugate. Sera were collected after the 2nd (circles) and 3rd (triangles) injections and assayed by ELISA on β-(1f2)-mannan trisaccharide-TT conjugate-coated microtiter plates. Panels represent experiments with different vaccine formulations: (a) FA; (b) alum; (c) no adjuvant. Data points are titers for individual mice. Horizontal lines in panels a and b represent median values.
Figure 7. Candida albicans cell wall specific antibody titers following vaccination of mice with glycopolymer vaccine. Sera were collected after the 2nd (circles) and 3rd (triangles) injections and assayed by ELISA on C. albicans cell wall phosphomannan-coated microtiter plates.17 Panels represent experiments with different vaccine formulation: (a) FA; (b) alum; (c) no adjuvant. Data points are titers for individual mice.
The glycopolymer-ChSA conjugate elicited high titers of serum IgG when administered with Freund's complete adjuvant but with alum as adjuvant; much lower titers against the synthetic β-mannan were observed, and no antibodies were detected against the native cell wall mannan. Glycoconjugate vaccine injected without any adjuvant failed to elicit a detectable antibody response against either anti β-mannan trisaccharide or native cell wall mannan (Figures 6 and 7, panels c). The antibody response of mice immunized with the β-mannan trisaccharide-tetanus toxoid conjugate (Table 3) is considerably lower than that seen for the glycopolymer-ChSA conjugate (cf Tables 2 and 3). The data presented in Table 3 are a combination of mice reported previously9 together with an addition group of
’ DISCUSSION Due to the high cost and toxicity issues of currently available antifungal drugs, there is growing interest in the potential of vaccines to combat the increasing incidence of life threatening hospital-acquired C. albicans infections in immuno-compromised individuals.18,19 Our group has focused on a glycoconjugate vaccine that targets the unique cell wall β-(1f2)-mannan of C. albicans. On the basis of the original findings of Cutler and subsequent inhibition data showing protective monoclonal antibodies specific for a di- or trisaccharide β-mannan epitope,9 we hypothesized that a synthetic β-(1f2)-mannan trisaccharide conjugated to tetanus toxoid could induce protective antibody. Although this idea is in contrast to currently deployed conjugate vaccines, which are based on polysaccharides, recent research examples exist where protective conjugate vaccines might be composed of relatively small oligosaccharide epitopes.20,21 Oligosaccharides of this limited size are attractive candidates for a vaccine, since they can be produced economically by synthetic approaches. Chemical synthesis of the β-(1f2)-mannan trisaccharide and its conjugation to proteins BSA and tetanus toxoid5,9 gave conjugate vaccines that induce a robust immune response in rabbits with protective efficacy in an immuno-compromised animal model of disease.4 However, the same trisaccharide-TT conjugate was poorly immunogenic in mice (Table 3). In search of a better immunogen, we decided to employ a glycopolymer as a carrier for oligosaccharide haptens. Glycopolymers can be easily synthesized and manipulated to present a controlled number of saccharide haptens thus allowing for precise tuning of the carrier protein to carbohydrate ratio. With tetanus toxoid as carrier, payloads higher than the 8-12 trisaccharides per protein are not attainable, since tetanus toxoid contains only 12 amino groups per molecule of protein (this precise number varies according to the source of the toxoid). We reasoned that improving the sugar/protein ratio might increase immunogenicity of the vaccine while preserving potential T-cell peptides by limiting the sites of attachment to protein. Further, application of glycopolymer technology leads to a fully synthetic, well-defined conjugate which can also be conjugated to synthetic peptides as a source of T-helper epitopes.22 Our results show that a polyacrylamide glycopolymer conjugated to chicken serum albumin is highly immunogenic in mice and induces a more robust immune response than haptenated tetanus toxoid (cf Tables 2 and 3). Mice given the glycopolymer vaccine with FA had high IgG titers against the synthetic trisaccharide haptens after 2 injections (median ∼118 000), while the third injection resulted in a further increase of titer by more than a factor of 7 (median ∼880 000). By comparison, the IgG response after vaccination with tetanus toxoid-β-(1f2)-mannan trisaccharide conjugate (after two immunizations as reported in ref 9 and confirmed by immunization of an additional 5 mice for this work) was observed in only 50% of mice with a median IgG titer of 14 000 (Table 3). The vaccine construct prepared for this study contained ∼33 trisaccharide molecules per molecule of protein compared to only 8-12 per tetanus toxoid conjugate. Determination of whether the improved immunogenicity of the glycopolymer vaccine arises from the higher payload of sugar in the construct or better presentation of haptens linked to a long, linear, flexible polymeric backbone requires more detailed study. 279
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Table 2. Antibody Titers of Mice Immunized with the Glycopolymer Vaccine Assayed against a Trisaccharide-Tetanus Toxoid Conjugate and against the C. albicans Phosphomannan Complexa titer vs C. albicans mannan
titer vs trisaccharide–tetanus toxoid adjuvant FA
min. 2nd inj. 3rd inj
Alum a
max
median
min.
max
median
2976
230 4708
118 665
0
174 252
0
17 661
30 152 082
882 254
0
429 248
0
1190 6806
7212 33 558
2159 13 922
0 0
0 0
0 0
2nd inj. 3rd inj.
Mice vaccinated without any adjuvant failed to produce detectable antibody levels and are not included in the table.
Table 3. Antibody Titers of Mice Vaccinated with a Trisaccharide-Tetanus Toxoid Conjugate and Assayed against a Trisaccharide-BSA Conjugate and against the C. albicans Phosphomannan Complexa titer vs trisaccharide-BSA
vaccine with Freund's and alum adjuvants, as well as mice that were vaccinated without any adjuvant, exhibited strong titers against acrylamide; however, precise end point titers were not measured. ELISA plates were coated with peptide polyacrylamide antigen,16 and titers in all mice were very high, frequently exceeding 1 000 000 with the highest values in the FA group (data not shown). Polyacrylamide was previously considered a nonimmunogenic carrier23 for the T-independent type of immunogens; however, it appears that conjugation to a carrier protein transforms polyacrylamide into a potent antigen. To the best of our knowledge, this is the first report of fully functional glycopolymer conjugate vaccine construct deriving B-cell epitopes from the glycopolymer and T-cell peptides from the carrier protein. However, it has been shown that polyacrylamide modified with small haptens;DNP and FITC or DNP attached to polymers produced by ring-opening metathesis polymerization; are able to stimulate B-cells for production of IgM antibodies in a T-independent manner.23-26 Human blood group Lea antigen and peptides attached to polyacrylamide induced a good immune response to the corresponding haptens.27 A polyacrylamide polymer with pendant hapten and phosphatidylethanolamine adsorbed on killed Salmonella Minnesota cells has been used to generate Ley specific monoclonal antibodies.28 Dendrimers such as multiple antigenic peptides (MAPs) have also been used as scaffolds to enhance the immune response to antigens.29 Conjugation of a small trisaccharide hapten to polyacrylamide, which in turn is covalently linked to a carrier protein, creates a potent T-dependent conjugate vaccine. These results suggest that glycoconjugates of the type described here should be useful tools for the generation of high titer sera or monoclonal antibodies specific for oligosaccharide epitopes, and most likely also other low molecular weight haptens. In the context of conjugate vaccines, it is desirable to replace polyacrylamide by other polymeric carriers, since the incidence of high levels of acrylamidespecific antibodies in human subjects has been observed in patients with severe fibromyalgia.28 Alternate polymers that allow similar presentation of pendant groups could be selected from suitable polysaccharides or other polymers.24 In the case of C. albicans, one candidate could be β-glucans. Since glucans of that type also constitute components of the fungal cell wall, antibodies to them would also provide additional protection.29
titer vs C. albicans mannan
adjuvant
min.
max
median
min.
max
median
FA alum
0 0
720 386 273 658
13 926 2339
0 0
72 899 0
0 0
a
The table contains data presented in ref 6 combination with an additional group of 5 CD1 mice vaccinated with same antigen (TT conjugate) and Freund's adjuvant and the same immunization protocol (only two vaccinations) as cited.9
In order to be an effective vaccine, it is crucial that the conjugate induces antibodies capable of recognizing the cell wall β-mannan. Antibodies that bound native Candida albicans cell wall antigen were observed in only 50% of vaccinated mice. Since we used CD-1 outbreed mice in our experiment, this effect may reflect variability in the genetic background. Another potential source of variability in immunogenicity is the effect of the tether. Our previous study in rabbits showed that all vaccinated animals were able to raise antibodies recognizing the native cell wall antigen, but this antibody type constituted only 10-20% of the antibodies that bound the synthetic trisaccharide.4 We conclude that antibody raised by vaccination with synthetic trisaccharide tends to recognize the sugar portion together with a part of the tether. Consistent with this interpretation, the protective monoclonal antibody C3.1 raised against native antigen bound equally well to ELISA plates coated with a cell wall β-mannan preparation or synthetic glycoconjugate (BSA–β-(1f2)trisaccharide conjugate). In previous work, we did not observe significant differences between FA or alum adjuvants when mice were immunized with a trisaccharide-tetanus toxoid conjugate.6 In this study, alum was a much less effective adjuvant with the glycopolymer vaccine. With FA as adjuvant, median IgG titers reached ∼118 000 after two injections and ∼880 000 after a third injection (Table 2), while mice receiving the vaccine with alum had a median titer of ∼2000 after the second injection and ∼14 000 after the third injection. This difference in the adjuvant potency of alum for these two types of vaccines is explained by the lack of a depot effect for a glycopolymer vaccine. When we tested binding of both vaccines to alum, tetanus toxoid conjugate showed good adherence to alum particles, whereas the glycopolymer vaccine remained mainly as an unbound fraction. For FA, the oil emulsion ensures a depot effect regardless of the vaccine type. Our construct induced high antibody titers against polyacrylamide. All groups of vaccinated mice, those that were given the
’ AUTHOR INFORMATION Corresponding Author
*Corresponding author. Dr. David R. Bundle, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2. E-mail:
[email protected]. Phone: 780-4928808. FAX: 780-492-7705. 280
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Funding Sources
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The research was made possible by grants awarded to D. R. Bundle; a Discovery grant from the Natural Science and Engineering Research Council of Canada and support from the Alberta Ingenuity Centers Program.
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dx.doi.org/10.1021/bc100397b |Bioconjugate Chem. 2011, 22, 274–281
