Immunological response from an entirely carbohydrate antigen: Design of synthetic vaccines based on Tn-PS A1 conjugates

Article abstract of DOI:10.1021/ja902607a

(Figure Presented) An entirely carbohydrate-based immunogen consisting of a zwitterionic polysaccharide (ZPS) PS A1 and the well-known tumor antigen Tn has been designed, synthesized, and studied for immunological effects. The PS A1 motif was included to act as an MHCII elicitor for a T-cell-dependent immune response with increased immunogenicity against tumor-associated carbohydrate antigens, providing an alternative to carrier proteins. Through the use of C57BL/6 mice, it has been shown that chemical modification of PS A1 does not alter the recognition sequence responsible for an MHCII-mediated, T-cell-dependent immune response. The Tn-PS A1 conjugate construct confers specificity toward the Tn antigen alone, and specific carbohydrate immunoglobulins, namely, IgG3, are generated from intraperitoneal immunizations with or without adjuvant. The properties of the vaccine candidate are attributed to a site-specific linking strategy that incurs significant incorporation of Tn antigen.

Full text of DOI:10.1021/ja902607a

Published on Web 06/24/2009
Immunological Response from an Entirely Carbohydrate Antigen: Design of
Synthetic Vaccines Based on Tn-PS A1 Conjugates
Ravindra A. De Silva, Qianli Wang, Tristan Chidley, Dananjaya K. Appulage, and Peter R. Andreana*
Wayne State UniVersity, Department of Chemistry, 5101 Cass AVenue, Detroit, Michigan 48202
Received April 1, 2009; E-mail: pra@chem.wayne.edu
The introduction of vaccines into medical practice has been one of
the most significant advancements of modern medicine. Its success
direction, we elected to focus our attention on a strategy that would
profit from the inherent MHCII-mediated immune activation by 1 and
conjugate a known TACA, Tn hapten (3), in order to probe the
immunogenicity and specificity for the development of a novel cancer
immunotherapy. We sought to exploit an entirely carbohydrate
immunogen to potentially overcome nonrelated peptide binding (cross
reactivity with “self” proteins), generate carbohydrate-specific antibody
production, and capitalize on a complete donor-acceptor sequence
of carbohydrates for increased antibody-carbohydrate binding. Our
strategy also incorporates a site-specific link between 1 and 3, negating
ambiguities in structure and composition.
1
can be attributed to the fact that vaccinology has expanded beyond its
traditional mainstays (attenuated or dead microorganisms, inactivated
bacterial toxins, and protein subunit vaccines) to the likes of recom-
binant proteins and glycoproteins, synthetic peptides, and conjugate
2
vaccines. Despite the enormous success, there remains a need for
effective vaccines for the treatment of serious diseases such as malaria,
AIDS, antibiotic-resistant infections, and cancer.
3
Carbohydrates are involved in a wide variety of biological roles.
For example, oncogenic transformation of cells is closely correlated
4
with dramatic changes in their glycosylation patterns. Aberrant
Scheme 1. Synthesis of Tn-PS A1 Conjugate 4
polysaccharides expressed on these cancer-cell surfaces [so-called
tumor-associated carbohydrate antigens (TACAs)] have been used
4b
successfully in the diagnosis and prognosis of cancer for many years.
Furthermore, TACAs can induce changes in antigenicity and immu-
nogenicity in cancer cells, which may allow TACA-derived cancer
4c
vaccines and cancer immunotherapies to be realized.
Although isolated/pure carbohydrates/polysaccharides have long
been known to be T-cell-independent and exhibit poor immunogenic-
5
ity, glycoconjugate vaccines, in which carbohydrate antigens are
tethered to strong immunogenic carrier proteins, such as bovine serum
albumin (BSA), tetanus toxoid, keyhole limpet hemocyanin, and others,
can elicit an MHCII T-cell response characterized by immunoglobulin
To garner appreciable amounts (∼500 mg) of 1, a large-scale
fermentation protocol employing the anaerobe Bacterioides fragilis
NTCT 9343 was carried out. Purified 1, devoid of any protein
6
13
G (IgG) production. Extensive research has focused on the creation
7
of synthetic vaccines based on complex carbohydrate antigens/epitopes
and lipopolysaccharide, was subjected to selective oxidative cleav-
conjugated to these carriers. Such vaccines have shown varying degrees
of promise, and as an outcome, the components necessary for eliciting
protective antibody production have become more defined. Paradoxi-
age conditions using 0.5 equiv of NaIO in 0.10 M acetate buffer
4
(pH 5.0) (Scheme 1). The oxidation protocol took advantage of
8
12
the vicinal hydroxyl relationship (1° 6-OH, 2° 5-OH) of the
cally, these conjugate vaccines can be less intrinsically immunogenic,
even as they become safer and more precisely targeted. This dichotomy
typically arises from nonrelated binding and a strong immune response
against the carrier protein, resulting in suppression of carbohydrate-
D-galactofuranose motif. The oxidation led to (hydrated) aldehyde
2, providing a chemoselective handle. The addition of 5 equiv of
1
3
KCl (to remove excess periodate as insoluble KIO4) followed by
addition of O-2-NAc-D-Galp hydroxylamine (3) afforded Tn-PS
9
1
specific antibody production. Also, TACAs are non-site-specifically
A1 oxime conjugate 4 after 18 h. The 500 MHz H NMR spectrum
coupled to carrier proteins, leading to vaccine heterogeneity. Random
coupling may also result in modification of important recognition
epitomes on the carrier protein, and conjugation chemistry is often
difficult to control, leading to glycoproteins with ambiguities in struc-
ture and composition, which may affect the reproducibility of an
immune response.
of 4 in D O showed distinct oxime doublets of the Z and E isomers
at 6.3 and 7.21 ppm, respectively.
2
10
With 1 and 4 in hand, we proceeded to test our hypothesis in 22
C57BL/6 mice. Mice were allowed to acclimate for a 1 week period,
and prior to intraperitoneal (i.p.) immunizations, mouse sera were
collected (day -1). The mice were divided into four subgroups
consisting of eight, six, four, and four mice (groups A, B, C, and
D respectively). 1 was administered i.p. to group A, 1 plus TiterMax
Gold adjuvant to group B, 4 to group C, and 4 plus TiterMax Gold
adjuvant to group D. The compounds were dissolved in PBS buffer
(pH 7.4) at a concentration of 10 µg/0.1 mL. On days 0, 7, 14, 21,
and 28, mice were immunized with 100 µL of the appropriate
solution. On days 27 and 39, blood sera from groups A-D were
collected and stored at -80 °C. All of the blood sera samples were
then analyzed by enzyme-linked immunosorbent assay (ELISA).
PS A1-poly(L-lysine) and Tn-PS A1-poly(L-lysine) conjugates
A recent report has provided convincing evidence for cases in
which zwitterionic polysaccharides (ZPSs) invoke an MHCII-
1
1
mediated T-cell response in the absence of proteins. Kasper and
5
b,12
co-workers
have identified capsular polysaccharide structures
that induce a CD4+ T-cell response known to modulate bacterial
abscess formation. An unusual ZPS, PS A1 (1), consisting of a
tetrasaccharide-core repeating unit (∼120 units) carrying an elec-
trostatic charge character on adjacent monosaccharides, elicits an
immune response similar to that for exogenous proteins.
In an attempt to overcome some of the current challenges with
protein-carbohydrate vaccine development and examine a new
1
4
were synthesized using a well-known protocol and used to coat
9
622 9 J. AM. CHEM. SOC. 2009, 131, 9622–9623
10.1021/ja902607a CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
ELISA plates. Sera from groups A and B were used to determine
the total antibody titer against 1 and the effect of the immune-
stimulating adjuvant. The data in Table 1 indicate an ∼200-fold
increase in total antibody titer production specific for 1 relative to
day -1, demonstrating a strong murine immune response. In fact,
immunization with 1 + adjuvant did not show a dramatic increase
in total antibody titer production relative to 1, indicating that 1 can
elicit an excellent immune response in the absence of adjuvant.
Data from Table 1 for trials using 10 to coat the ELISA plate
illustrate a high affinity of total antibody titer toward 3. This result
implies that the oxime link in Tn-PS A1 conjugate 4 did not
undergo hydrolysis during immune processing and that anti-Tn
antibodies were generated. 2° antibodies were used to determine
the specificity of immunoglobulin binding toward 3. The data show
IgG3 specificity and imply a T-cell-dependent immune response.
Scheme 2. Synthesis of ELISA Plate Coating 10 for Determining
Antibody Specificity against the Tn Hapten
Table 1. Immunization Results with 1 and 4
goat anti-mouse
, ,
a b c
kappa
ELISA
coating
a
a
d
d
c
,
d
c
,
d
c
,
d
,
c d
immunization
day -1 day 39 IgM IgG1 IgG2a IgG3
PS A1 (1)
1-poly(L-K) 0.5
101
131 101
-
-
110
-
-
-
-
-
10
4
0
-
5
e
e
1
1
1
+ adjuvant 1-poly(L-K) 0.5
+ adjuvant 4-poly(L-K)
+ adjuvant 10
0
0
e
-
-
60
2
-
70
0
0
-
0
0
e
Tn-PS A1 (4) 4-poly(L-K) 0.4
-
e
4
4
4
+ adjuvant 1-poly(L-K)
+ adjuvant 4-poly(L-K) 0.5
-
0
0
0
0
In summary, we have designed, chemically synthesized, and
immunologically evaluated an entirely carbohydrate vaccine candidate.
We have demonstrated that chemical modification of zwitterionic
polysaccharide PS A1 1 does not alter or negate an immune response.
Tn-PS A1 conjugate 4 can elicit exceptionally high titer antibodies
even in the absence of an immune stimulant. Our design for a Tn-PS
A1 conjugate takes advantage of site-specific conjugation to a modified
D-galactofuranoside. ELISA results confirm immunoglobulin specificity
toward Tn hapten 3, which makes this a viable route for further
exploration. We are currently pursuing the conjugation of other known
TACAs and determining antibody specificity produced in sera as a
measure for cross-reactivity.
e
e
200 140
99 77
0
50
6
e
e
+ adjuvant 10
0.5
0
a
b
See the Supporting Information for details. Specific for mouse κ light
c
chains. Titers were determined by linear regression analysis, plotting
dilution versus absorbance. The highest dilution yielding an optical density
OD) of g0.2 relative to normal control mouse sera (day -1) was used to
define the titers. Value shown × 10 . Negligible amount.
In order to determine the specific antibody isotypes and provide
further insight into the immune-processing pathway for PS A1 1, 2°
antibodies to determine IgM and IgG isotypes IgG1, IgG2a, and IgG3
were used in an ELISA. The data for immunization 1 + adjuvant in
Table 1 clearly illustrate that an abundance of IgG3 antibodies was
produced, indicating a murine class switch carbohydrate-based immune
(
d e
3
Acknowledgment. Prof. Zhongwu Guo (WSU) is acknowledged
for assistance with mouse studies. P.R.A. thanks WSU for start-up
funds and a WSU Research Grant (145767).
4c,d
response corresponding to a T-cell-dependent pathway.
In an attempt to determine whether selective antibodies would arise
against the Tn-PS A1 conjugate 4, we examined blood sera from
groups C and D. The data in Table 1 indicate a 200-fold increase
Supporting Information Available: Experimental protocols, NMR
spectra for all new compounds, and complete ref 7a. This material is
available free of charge via the Internet at http://pubs.acs.org.
(
relative to day -1 sera) in total antibody titers produced against 4 in
the absence of adjuvant, very similar to the result for the corresponding
immunization with 1. This implies that modification of 1 through the
process of oxidation, yielding 2, followed by oxime formation did not
alter the ability to elicit an immune response. In order to confirm a
T-cell-dependent immune response, we examined the specific antibody
isotypes. The Table 1 data show an IgG3 response to 4 + adjuvant
when 4-poly(L-K) was used to coat the plate. This result implies that
chemical modification of 1 did not alter the ability of 4 to target the
T-cell-dependent pathway, most likely because the alternating charge
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The synthesis of conjugate with BSA tethered to Tn (10) (Scheme
2
) commenced using 2-ethylacrolein (5) and thioacetic acid (6) to
(
(
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construction buffer, and the mixture was stirred at 4 °C for 18 h.
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was used to coat the ELISA plate.
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(
JA902607A
J. AM. CHEM. SOC. 9 VOL. 131, NO. 28, 2009 9623