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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:
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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 immunogenic T-dependent antigens. The repeating unit nature

We report the synthesis of a protein-polyacrylamide con-
jugate 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 polysaccharide-
protein conjugate vaccines. The approach constitutes a conve-
nient 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
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 infec-
tions 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

r 2010 American Chemical Society 274 dx.doi.org/10.1021/bc100397b | Bioconjugate Chem. 2011, 22, 274–281
Bioconjugate Chemistry
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 trisac-
charide 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)-man-
nan 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 produc-
ing significantly improved murine antibody levels when com-
pared 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). Cyste-
amine 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 Ham-
ilton syringe and polymerization was initiated by addition of
potassium persulfate solution (10.4 μL). Polymerization was
275
ARTICLE
performed overnight at 37 °C. Glycopolymer was dialyzed against
water in a Slide-A-Lyser cassette (Thermo Scientific).
Glycopolymer Fractionation. The glycopolymer was fraction-
ated 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 determi-
nation 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 Ultra-
hydrogel 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. Gly-
copolymer (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 (∼1000-
fold 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. Mono-
meric 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 albu-
min (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 over-
night. The resulting solution was dialyzed against water and
lyophilized.
Azide-Alkyne Huisgen Cycloaddition Conjugation. Con-
jugation of the glycopolymer to ChSA was performed with minor
modifications according to literature procedures.10,14 Bathophe-
nantroline/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
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Bioconjugate Chemistry ARTICLE

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 Table 1. Polyacrylamide of Different Molecular Weight Was
protein. Obtained by Polymerization at Different Ratios of Acrylamide
Vaccine Formulations. Vaccine doses (regardless of adjuvant Monomer (aa) to Chain Transfer Reagent (cys)a
used) were formulated to contain 3 μg of β-mannan trisaccharide
in 300 μL of formulation. Alum suspensions were prepared molar ratio acrylamide/cysteamine number average molecular weight
according to ref 15. Emulsions with Freund's complete and 2000:1 212 000
incomplete adjuvant (FA) were prepared by intense vortexing 1000:1 130 000
of equal volumes of glycopolymer conjugate in PBS and FA until 500:1 75 000
a stable emulsion was formed.
333:1 52 000
Immunization. CD1 female mice (5 to 7 weeks old) were
200:1 39 000
immunized three times at 3 week intervals with vaccine formulation
(300 μL) administered by 100 μL intraperitoneal and 200 μL a
133:1 29 000
subcutaneous injections. Mice immunized with Freund's adju- Mn: number average molecular weight.
vant 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
√ 0.1% Tween (PBST), wells were filled with 100 μL of
containing
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 Figure 1. GPC traces of polymers obtained by polymerization with
temperature. After washing with PBST, a reporter antibody, cysteamine (Table 1). Mn = 212 000 (1); 130 000 (2); 75 000 (3); 52 000
anti-Mouse IgG, HRP conjugate from KPL (Kirkegaard & Perry (4); 39 000 (5); 29 000 (6).
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. Antipolyacrylamide antibodies were detected by the same
Plates were washed again with PBST and color developed with protocol but using plates coated with an acrylamide-peptide
HRP substrate system (KPL) for 15 min. The reaction was stopped copolymer. Acrylamide was copolymerized with peptide epi-
with 1 M phosphoric acid, and absorbance was measured. tope fragment (p458m) derived from heat shock protein,
276 dx.doi.org/10.1021/bc100397b |Bioconjugate Chem. 2011, 22, 274–281
Bioconjugate Chemistry
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
’ 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
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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 Sephacryl-
S400 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
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Bioconjugate Chemistry
Figure 2. Glycopolymer (solid line) and two standard polymers 130
(dotted line) and 75 kDa, (dash line).
Figure 3. Ultrahydrogel HPLC traces of the unfractionated glycopoly-
mer (1), and the high (2) and lower (3) molecular weight fractions
obtained by gel permeation chromatography on Sephacryl-S400 col-
umn.
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 trisaccha-
ride 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 trisaccha-
ride 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
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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.
the glycopolymer by reductive amination using excess 6-azido-
6-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 corre-
sponding 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 glycopolymer-
ChSA 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 trisac-
charide loading was used to evaluate immunogenicity by vaccina-
tion 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 immuni-
zations 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 cross-
reacted with the native β-mannan of the C. albicans cell wall
(Figure 7, panel a).
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Bioconjugate Chemistry
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 formula-
tion: (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
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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
’ 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. Glycopol-
ymers 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 vacci-
nation 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.
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Bioconjugate Chemistry
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 trisaccharide–tetanus toxoid
adjuvant min.
FA 2nd inj. 2976
3rd inj 17 661
Alum 2nd inj. 1190
3rd inj. 6806
a
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 Trisac-
charide-Tetanus Toxoid Conjugate and Assayed against a
Trisaccharide-BSA Conjugate and against the C. albicans
Phosphomannan Complexa
titer vs trisaccharide-BSA titer vs C. albicans mannan
adjuvant min. max median min. max
FA 0 720 386 13 926 0 72 899
alum 0 273 658 2339 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 anti-
gen 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 polyacryl-
amide. All groups of vaccinated mice, those that were given the
ARTICLE
titer vs C. albicans mannan
max median min. max median
230 4708 118 665 0 174 252 0
30 152 082 882 254 0 429 248 0
7212 2159 0 0 0
33 558 13 922 0 0 0
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 polyacryl-
amide antigen,16 and titers in all mice were very high, frequently
exceeding 1 000 000 with the highest values in the FA group
median
(data not shown). Polyacrylamide was previously considered a
nonimmunogenic carrier23 for the T-independent type of im-
0 munogens; however, it appears that conjugation to a carrier
0 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 multi-
ple 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 acrylamide-
specific antibodies in human subjects has been observed in
patients with severe fibromyalgia.28 Alternate polymers that
allow similar presentation of pendant groups could be se-
lected 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
’ AUTHOR INFORMATION
Corresponding Author
*Corresponding author. Dr. David R. Bundle, Department of
Chemistry, University of Alberta, Edmonton, Alberta, Canada
T6G 2G2. E-mail: dave.bundle@ualberta.ca. Phone: 780-492-
8808. FAX: 780-492-7705.
280 dx.doi.org/10.1021/bc100397b |Bioconjugate Chem. 2011, 22, 274–281
Bioconjugate Chemistry
Funding Sources
The research was made possible by grants awarded to D. R.
Bundle; a Discovery grant from the Natural Science and En-
gineering Research Council of Canada and support from the
Alberta Ingenuity Centers Program.
’ ACKNOWLEDGMENT
We thank Dr. Tahanh Luu for a gift of 6-azido-6-deoxy-
1
D-galactose and Dr. Margaret Johnson for recording H NMR
spectra of the glycopolymer and its protein conjugate.
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