Directing the Immune Response to Carbohydrate …2001/05/30 · the immune response (8-12). Peptide...
Transcript of Directing the Immune Response to Carbohydrate …2001/05/30 · the immune response (8-12). Peptide...
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Directing the Immune Response to Carbohydrate Antigens
Gina Cunto-Amesty1, Tarun K. Dam2 , Ping Luo1, Behjatolah Monzavi-Karbassi1, C. Fred Brewer2,
Thomas C. Van Cott3, Thomas Kieber-Emmons1*
1Department of Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia, PA. 19104. 2Department of Molecular Pharmacology and Microbiology and
Immunology, Albert Einstein College of Medicine, Bronx, New York 10461. 3Henry M. Jackson
Foundation, Rockville, Maryland 20850
*Address all correspondence to:
Thomas Kieber-Emmons, Ph.D.
Department of Pathology and Laboratory Medicine
Room 205, John Morgan Building
36th and Hamilton Walk
Philadelphia, PA 19104-6082
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on May 30, 2001 as Manuscript M103257200 by guest on O
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Running Title: Peptide mimetics of Concanavalin A
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Summary
Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At
present, the structural and immunological aspects of antigenic mimicry, which translate into
immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to
screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine
design approach to identify functional mimeotopes. Using Concanavalin A (Con A), as a recognition
template, peptide mimetics reactive with Con A were identified. Designed peptides were observed to
compete with synthetic carbohydrate probes for Con A binding, as demonstrated by ELISA and
Isothermal Titration Calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form
of one particular mimetic binds to Con A with similar affinity as does trimannoside. Splenocytes
from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell
dependent nature for the mimetic. As Con A binds to the Envelope protein of the Human
Immunodeficiency Virus type 1 (HIV-1), we observed that mimeotope-induced serum also binds to
HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-Cell Line Adapted HIV-
1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is more based upon
functional rather than structural determinates that regulate mimeotope-induced T-dependent antibody
responses to polysaccharide, and emphasizes that rational approaches can be employed to further
develop vaccine candidates
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Introduction
Targeting carbohydrate antigens is a major challenge in vaccine design. Carbohydrates fail to elicit
memory responses, as they are T-cell independent (TI) antigens (1-3). Conversion of a
polysaccharide (PS) antigen to a thymus-dependent (TD) antigen, by covalent coupling to an
immunogenic protein carrier, alters the response to PS in several important ways (4-6). However,
conjugation strategies that elicit carrier-specific T- and B-cell responses do not necessarily enhance
PS immunogenicity (7), nor do PS-conjugates elicit responses in immunodeficient mice.
Furthermore, in cases where a large number of carbohydrate antigens are required to afford
protection, much like that representative of the large number of pneumococcal carbohydrate
serotypes, PS-conjugates will be far more complicated to produce (6).
Immunization with peptide mimetics of carbohydrate antigens can overcome the TI nature of
the immune response (8-12). Peptide antigens have an absolute requirement for T cells that can
mediate memory responses upon carbohydrate boosting (11,13). In contrast to carbohydrate-
conjugates, peptide mimetic-conjugates can facilitate cognate interactions between B and T cells after
immunization of immunodeficient mice that lack Bruton's tyrosine kinase (10). Peptide mimetics
therefore afford a vaccine approach to break tolerance to carbohydrate self-antigens (13).
While peptide library screening has led to the identification of a variety of peptide mimetics
of carbohydrate antigens (14,15), concepts described for the design of small molecules may equally
well apply to the design of mimetics of carbohydrate antigens (16,17). To further facilitate concepts
for structure-assisted vaccine design we considered, as a model system, small molecule interactions
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with Concanavalin A (Con A). The mannose/glucose-specific lectin Con A is the most extensively
studied plant lectin, known for its application as a biochemical tool and as a model protein to gain
further knowledge about lectin-ligand interactions (18-20). Lectins are also particularly relevant to
Human Immunodeficiency Virus (HIV) pathogenesis. Lectin-induced inhibition of syncytium
formation and infection of cells by both, T-Cell Line Adapted (TCLA) and primary isolates (21-27)
focuses attention on oligomannosidic glycans, such as those characterized by interaction with Con A
(Figure 1). More recently the lectin DC-SIGN, expressed on dendritic cells, has been recognized to
participate in facilitating HIV transmission (24,28,29). Consequently, defining peptide mimetics
reactive with Con A may facilitate the development of immunogens to augment carbohydrate
responses to HIV in future vaccine applications.
Structural studies of peptidyl-Con A complexes suggest that the carbohydrate-binding site on
Con A can accommodate an extended array of carbohydrate antigens that might lend to its biological
properties (30,31). We have further defined a peptide that binds at or near the carbohydrate-binding
site of Con A that displays a free energy of association comparable to those reported for core
trimannoside-Con A and pentasaccharide-Con A interactions. The designed peptide elicits a robust
TD response, stimulating splenocytes from peptide-immunized mice. We observe that the peptide,
rendered as a Multiple Antigenic Peptide (MAP), used to emulate the clustered array of Envelope
protein of HIV (Env)-associated carbohydrates (32) induced an antibody response reactive with cell-
bound Env protein. We observe that serum induced to the peptide mimetic paralleled neutralization
results obtained by using a mannan preparation from Saccharomyces cerevisiae or from Candida
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albicans (33,34), which further suggests that carbohydrate cross-reactive responses induced by
peptide mimetics might be rendered even more effective immunogens.
Experimental Procedures
Epitope mapping of Con A-ligand binding site
Using the crystallographically positioned pentasaccharide structure within the Con A-
combining site, we implemented the program Ligand-Design (LUDI (35) MSI/Biosym
Technologies), as previously described (36-39), to search a fragment library and identify amino acid
residue types able to interact with Con A. This program identifies small molecular fragments in a
database and then docks them into the protein-binding site, in such a way, that hydrogen bonds and
ionic interactions can be formed between the protein and the molecular fragments. The positioning
of the small fragments is based upon rules about energetically favorable non-bonded contacts, and on
geometry between functional groups of the protein and the ligand. The center of search was defined
using the crystallographic position of the central Mannose residue and searching 15 A surrounding
the centroid of the sugar for potential contact sites on Con A.
The search was performed using standard default values and a fragment library supplied with
the program. Peptides were built using INSIGHTII (MSI/Biosym Technologies), and accommodated
in relation to the docked LUDI fragments. The peptide backbone and side chain torsion angles were
rotated using a fixed docking algorithm (Affinity program) within INSIGHTII, until the side chains
of the peptide were approximated to the corresponding LUDI fragments. The peptide-Con A
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complex was subjected to energy optimization and molecular dynamic simulations, as previously
described (36-39).
Reagents and immunizations.
Multivalent carbohydrates Lewis Y (LeY), α-D-Mannose (Man-9), Disialyl-biantennary (A2),
Asialo-biantennary (NA-2) and Oligomannose 9 (Man-9), each attached to a polyacrylamide polymer
(PAA) of approximately 30 Kd, were purchased from GlycoTech Corporation, Rockville, MD.
Methylα-D-mannopyranoside (MeαMan) was purchased from the Sigma Chemical Co. (St. Louis,
MO). Peptides were synthesized as MAP (Research Genetics, Huntsville, AL), by Fmoc synthesis on
polylysine groups, resulting in the presentation of 8 peptide clusters. Linear peptides were
synthesized by standard solid-phase (Research Genetic, Huntsville, AL) and HPLC-purified. The
structures were confirmed by fast-atom bombardment mass spectrometry.
Con A was either prepared from Jack bean (Canavalia ensiformis) seeds (Sigma Chemical
Co, St Louis, MO), as previously described (20), or obtained from Sigma. The concentration of Con
A was determined spectrophotometrically, at 280 nm, using A 1%,1 cm = 13.7 (at pH 7.2) and 12.4 (at
pH 5.2) and expressed in terms of monomer (Mr = 25,600).
Balb/c mice (n=4 per group), 4-6 weeks of age, were immunized intraperitoneally three times,
at intervals of two weeks, with 100 µg of a respective peptide, or 50 µg of LeY, each combined with
20 µg of the adjuvant QS21 (Aquila Biopharmaceuticals Inc, Framingham, MA). A control group
was immunized with QS21 alone. The LeY-expressing cell line MCF7 (ATCC Rockville MD) (40),
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without adjuvant, was also used to immunize groups of mice four times. Serum was collected at days
7th and 14th after the last immunization, and stored at –80o C until use.
ELISA assays.
ELISA assays were performed as described (41). Immulon-2 plates were coated overnight, at 4o C,
with 100 mM of a selected peptide or carbohydrate probe, to assess the binding of sera to these
antigens. After blocking the plates (PBS, 0.5 % FCS and 0.2 % Tween 20), serial dilutions of sera
were added and resolved with anti-mouse isotype matched-HRP (Sigma, St. Louis, MO). To assess
the binding of Con A to carbohydrates and peptides, serial concentrations (10 to 0.6 ng/ml) of Con A
biotin-labeled (Sigma, St. Louis, MO), were added to pre-coated plates and reacted with Streptavidin-
HRP (Sigma, St. Louis, MO). All results were calculated from triplicate measurements.
Peptide-carbohydrate competition assay.
Immulon-2 plates pre-coated with 20 µg/ml of peptide were blocked. The binding of Con A (0.4
µg/ml) biotin-labeled (Sigma, St. Louis, MO) to peptides was assessed in the presence of serial
concentrations of MeαMan (0.8 to 50 mM). Control wells with Con A, but not MeαMan were also
run. Plates were reacted with Streptavidin-HRP (Sigma, St. Louis, MO) and results calculated from
triplicate measurements. Percentage of inhibition of Con A binding to peptides was calculates as 1-
(mean of test well/mean of control wells) x 100.
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Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) was performed using a MCS isothermal titration calorimeter
(Microcal, Inc., Northampton, MA). In individual titrations, injections of 5 µl of peptides (0.4 mM to
5.2 mM) and concentration of Con A ranging from 0.025 mM to 0.2 mM, were added from the
computer-controlled microsyringe, at an interval of 4 minutes, into the lectin solution (cell volume =
1.3582 ml), while stirring at 350 rpm, at 27o C. Both, lectin and peptide, were dissolved in 100 mM
HEPES buffer (pH 7.2) or 100 mM Sodium Acetate buffer (pH 5.2), containing 5 mM CaCl2 and
MnCl2. Control experiments were performed by making identical injections of peptide into a cell-
containing buffer, where protein showed insignificant heats of dilution. The experimental data were
fitted to a theoretical titration curve using software supplied by Microcal. The quantity c = Ka Mt (0),
where Mt (0) is the initial macromolecule concentration, is of importance in titration calorimetry. All
experiments were performed with c values 1 < c < 200. The instrument was calibrated using the
calibration kit containing ribonuclease A (RNase A) and cytidine 2’-monophosphate (2’-CMP),
supplied by the manufacturer. Thermodynamic parameters were calculated from the equation, ∆G = -
RT lnKa where Ka and ∆G are the association constant and changes in free energy, respectively. T is
the absolute temperature and R = 1.98 cal mol-1 K-1 .
Precipitation Study.
Measured volumes of known concentrations of lectin and peptide solutions (in 100 mM HEPES
buffer -pH 7.2- containing 150 mM NaCl, 5 mM CaCl2 and MnCl2) were mixed in a quartz cuvette, at
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room temperature, and the time-dependent development of turbidity was measured at 420 nm (42).
Absorbencies were monitored continuously, until they remained constant. A portion of the precipitate
was treated with 400 mM MeαMan, to check whether or not the precipitation was due to the binding
of the peptide at the carbohydrate binding sites of the lectin. Absorbency of the solution was recorded
at 420 nm, before and after the addition of MeαMan.
Cell proliferation assay
Spleens were aseptically removed and splenocytes, as the responder cells, isolated by lysis of
erythrocytes. Responder cells were used for detection of cell proliferation using CellTiter 96R
Aqueous One Solution (Promega, Madison, WI), based on the manufacturer’s instructions. Briefly,
cells (2.5 x 105 / well) were cultured in flat bottom 96-well plate with 106-MAP, 911-MAP or only
medium RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 5% heat-inactivated
fetal calf serum (FCS), 1 % L-Glutamine, 100 IU/ml of Penicillin and 100 µg/ml of Streptomycin.
At the third day of incubation, the provided solution was added to each well and plates incubated for
additional 1-2 hours, in a humidified 5% CO2 incubator, at 370C. As indicator of cell proliferation,
absorbency was measured at 490nm, using a 96-well plate reader (Spectra Fluor, Tecan, Triangle
Park, NC),
Cells and antibodies for FACS.
Sup-T1, a non-Hodgkin’s T-cell lymphoma cell line (43) and the same cells stably infected with HIV
type 1 (HIV-1) III-B (A1953), were kindly provided by Dr J. Hoxie. The mouse monoclonal antibody
902, specific for gp120 of HIV-1 III-B (44,45), was used to differentiate infected versus non-infected
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cells. Mouse sera were tested with dilutions ranging between 1:10 to 1:100. Secondary antibody used
was anti-mouse IgG (γ specific), FITC-conjugated (Sigma, St. Louis, MO). Cells were fixed for 30
minutes with 4 % paraformaldehyde diluted in PBS. Acquisition of data was performed by using the
FACSCAN flow cytometer, and histogram analysis by using the CELLQuest software (Becton
Dickinson Immunocytometry Systems, Mansfield, MA).
Propagation of HIV-1 isolates.
CEMx174 cells (1x105/ml), in RPMI 1640 media with 20 % FCS, 100 IU/ml of Penicillin, 100
µg/ml of Streptomycin, 1% L-Glutamine and 1% Hepes (R-20), were used to propagate the HIV-1
strains III-B (46-48) and MN (49-51). When most cells evidenced virus-induced cytopathic effect
(CPE), virus-containing supernatants were collected after centrifugation (200 x g for 10 minutes) and
filtration (0.45 nm filters), to be stored at –80o C, until use.
Determination of the TCID50.
The procedure was performed as reported (52). Briefly, 200 µl/well of a viral isolate, diluted 1:3 in
R-20, were added in sextuplicates in flat-bottomed 96-well plates. 50 µl from these wells were
admixed with 150 µl of R-20 in wells of the next row, and so, in successive rows (total of 20 serial
dilutions). 2x104 MT2 cells (53,54) per well, in 50 µl of R-20, were added and incubated at 37o C, in
a humidified atmosphere with 5% CO2. Cells were fed as necessary, all wells at the same time, and
observed daily for presence of CPE. When no further migration of CPE was evident, wells were
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scored either positive (presence of CPE) or negative (absence of CPE), and TCID50 calculated by
using the method of Reed and Muench (52).
Neutralization assays.
Test serum were obtained from immunized mice immunized and control serum from pre-immune
mice (normal mouse serum –NMS-), and from normal and HIV-infected human individuals (NHS
and IHS, respectively). Serum were inactivated at 56o C for 1 hour and sterilized by exposure to UV
light. Determined dilutions of sera and viral outputs were admixed and incubated for 1 hour at 37o C,
and then added (25 µl) in triplicated or sextuplicated wells (round-bottomed 96-well plates)
containing 104 CEMx174 cells resuspended in 175 µl of R-20. Plates were incubated for 24 to 40
hours. Control wells without virus or serum (uninfected wells), or without serum but with a selected
viral isolate (infected wells), were performed. After incubation, cells were washed, resuspended in
200 µl of R-20 and transferred to homologous flat-bottomed 96-well plates. Media was replaced at
the same time in all plates, as necessary. Cultures were maintained, until no further progression of
CPE was observed in infected control wells, time when 25 µl of supernatant per well were admixed
with 225 µl of 0.5 % triton x-100 (lysing solution), for p24 antigen detection, by ELISA. Samples
were stored at -80o C, until use. Percentage of neutralization was calculated as 1-(mean absorbency of
test wells/mean absorbency of infected control well x 100.
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ELISA assays to determine HIV-1 p24.
The assay was performed by using the HIV-1 p24 Antigen Capture Assay Kit from the AIDS
Vaccine Program of the NCI-Frederick Cancer Research and Development Center (Frederick, MD).
Briefly, plates pre-coated with a monoclonal anti-HIV-1 p24 antigen were washed and blocked with
PBS 0.5 % FCS and 0.2 % Tween 20. Supernatant lysates were added in duplicates and incubated at
37o C for 2 hours. A rabbit anti-HIV-1 p24 serum and a goat anti-rabbit IgG (H&L)-HRP labeled
antibody were used in successive steps. TMB peroxidase substrate (0.1 mg/ml) (Sigma, St. Louis,
MO), in 0.05 M phosphate-citrate and 0.03 % sodium perborate buffer (Sigma, St. Louis, MO), was
allowed to react for 20 minutes. Reaction was stopped with 4N H2SO4 and plates read as described
(41).
Results
Peptidyl ligands that bind to Con A.
Crystallographic analysis of Con A complexed with the trimannoside α-D-Man (1-6)α-D-Man (1-3)
D-Man (55) and the pentasaccharide β-GlcNAc- (1-2)-α-Man- (1-3)-[ β-GlcNAc- (1-2)- α-Man- (1-
6)]-Man (56) provides a template to compare peptide-Con A complexes. Prototypic peptides that
have been defined to bind Con A include 908 and 712, in Table 1 (30). Circular Dichroism (CD)
analyses of these binding analogs indicate that they share a similar CD profile (30). Secondary
structure comparison of these two peptide sequences indicates similarities in tertiary class type,
except in the all-β prediction, in which an extended structure spans the WYPY sequence tract of
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MYWYPYASGS (Table 1)(57). While the YPY motif can be viewed as adopting a β-turn
conformation that might emulate the spatial position of the trimannoside configuration (31), an
extended conformation might also be plausible for mimetics to interact with Con A. An extended
structure conformation is observed in Sesbania mosaic virus (SMV) coat protein (deposited in
Brookhaven databank) (58) for the homologue sequence WYPY. The extended structure can over-lap
with the pentasaccharide within the Con A binding site (Figure 2a).
We attempted to identify amino acid sequences that could adopt the extended secondary
profile and display an adequate interaction with the Con A site. To identify likely residue types that
can interact with Con A, we used the program LUDI. We have previously shown that LUDI could be
used to structurally map the binding of peptide mimetics to the combining site of anti-carbohydrate
antibodies (36,39). Using this approach, LUDI identified 153 interacting ligands for Con A, with
some contacting the same sites as the pentasaccharide. In the search procedure, we identified moieties
with Tyr- and Trp-like side changes and guanidinium groups that fit within the Con A site, but not
always in the same fashion as the pentasaccharide (Figure 2b). Substitution of Arg for Pro within the
prototypic peptide 908 conserves the extended structure (peptide 909 in Table 1), as does a
concomitant substitution of the first Tyr in the 909 peptide with a Trp residue, forming the 910
sequence (Table 1).
To test the ability of the peptide analogs to bind to Con A, MAP forms of the peptides 908,
909 and 910 were synthesized. The MAP forms all bound to Con A in a concentration-dependent
manner, with parallel activity (data not shown). Competition analysis with solid phase 908-MAP
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indicated that MeαMan inhibits Con A-peptide reactivity in a concentration-dependent manner,
reaching a plateau of about 60% inhibition at 1.6 mM concentration of MeαMan, a 100 fold less
concentration effect than previously reported (30) (Figure 3). As expected, Lactose, as control
inhibitor, did not affect Con A–peptide binding (data not shown). The variant MAPs 909 and 910
displayed some differences in the MeαMan inhibition profile compared to 908-MAP. At 1.6 mM
concentration, MeαMan inhibited about 20% and 45% of Con A binding to 910-MAP and 909-MAP,
respectively.
We further defined a putative peptide sequence, RYGRY, in which the Pro residue in the
WYPY motif was replaced by Gly, with the first and fourth Tyr replaced by Arg, and the addition of
a Tyr at the fifth position. This putative sequence was chosen because of the identification of these
residues and their Con A-reactive positions by LUDI analysis. The peptide motif represented in
Figure 2c, involving the putative RYGRY tract of peptide 912 (Table 1), maintains an extended
secondary structure profile spanning these residues (Table 1). Relative to the other class types, this
peptide sequence is the same as that for peptide 908 and 712, and is perhaps more like 712, as
represented in the all beta class (Table 1). The putative RYGRY tract makes contact with 3 residues
within Con A, as does the central Mannose residue. A bifurcated hydrogen bond between the
guanidinium group of Arg, at the fourth position, and Ser 21 and Tyr 12 side chains of Con A, and a
bifurcated hydrogen bond between the guanidinium group of Arg, at the first position, and Ser 223
and Ser 168, are observed (Table 2). The root mean square (RMS) deviation of the Con A-peptide
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complex, after minimization and dynamics calculations, was found to be 1.2A, compared with the
Con A-pentasaccharide complex, indicating that the extended structure is readily accommodated
within the Con A site.
Experimentally, Con A displays higher affinity for α-D-Man (1-6)α-D-Man (1-3) D-Man
constituents over β-D-GlcNAc (1-2)α-D-Man (1-6) D-Man, paralleling the intermolecular interaction
(I.E.; interaction energy) calculation trends shown in Table 2. The calculated location for the peptide
mimetic is, however, not an optimal binding mode in terms of mimicking the conformational
properties of the pentasaccharide and in contacting the same Con A residues, as does the
pentasaccharide (Table 2). Nevertheless, the interaction energy for this Con A/peptide-binding mode
was found to be –75.9 Kcal/mol, falling within the range of interaction energies calculated for the
trimannoside constituent (Table 2).
Peptide mimetic competes for carbohydrate binding to Con A
Clustering or repeating the 912 sequence, forming the peptide 911, manifests an extended secondary
structure (Table 1). ELISA assays carried out using various concentrations of Con A showed a
concentration-dependent binding to the clustered form of the RYGRY containing peptide 911, which
was inhibited by MeαMan (Figure 4a). We observed binding of Con A to 911-MAP, at Con A
concentrations lower than those required for binding to ligands tested, which included extended
structure peptides, and better than that for binding to 908-MAP. This result suggests a high avidity
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interaction of Con A with the multivalent 911 peptide and therefore requiring a higher concentration
of MeαMan for inhibition of 911-MAP binding to Con A than that for the 908-MAP (Figure 4b).
The putative monovalent peptide 912 and 911-MAP binding to Con A was studied further by
ITC, to determine the binding parameters such as Ka and ∆G. Experimental conditions were
previously standardized for studies of multivalent interaction by ITC (9). The association constant
(Ka) (1.1 x 104 M-1) and ∆G (5.6 kcal mol-1) values of Con A for the monovalent 912 peptide was
observed to be comparable to that observed for MeαMan (Figure 5). Earlier, microcalorimetric
studies showed that the Ka and ∆G values of Con A for carbohydrate ligands, such as MeαMan,
trimannoside and pentasaccharide, were 0.82 x 104 M -1, 49 x 104 M -1 and 92 x 104 M -1 and 5.3 kcal
mol-1, 7.8 kcal mol-1, and 8.1 kcal mol-1, respectively (20). In contrast, Ka of Con A for 911-MAP was
found to be 26 x 104 M-1, with a value for ∆G of 7.4 kcal mol-1. These results indicate that the
multivalent 911 peptide displays a comparable association constant and free energy of binding, as do
oligosaccharide ligands.
To further verify the valence of the peptides for Con A, a precipitation study was carried out
(Figure 6). The number of binding site per monomer (n), as determined by ITC, suggests that peptide
912 is monovalent while 911-MAP is multivalent for Con A. Multivalent lectin-ligand interactions
often lead to the formation of insoluble cross-linked complexes, which can easily be monitored
spectrophotometrically by measuring the absorbency at 420 nm. The efficient precipitation by the
911-MAP form confirms that it possesses multiple binding sites for Con A. About 70% of the cross-
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linked complexes are re-dissolved when treated with MeαMan (400 mM). This observation clearly
indicated that 911-MAP was bound to Con A predominantly through the carbohydrate-binding sites
of the lectin, while the remaining ~30% of precipitation is probably due to protein-protein interaction.
Peptide 912 was unable to form any detectable precipitate, which confirms its monovalent nature
(Figure 6).
Binding of serum from immunized mice to HIV-1 III-B -infected cells.
The 911 peptide is predicted to have a MHC Class II motif spanning the RYRYGRYRS sequence.
Immunization with peptide 911 indicated a robust cellular response specific for peptide 911 (Figure
7). To determine if serum antibodies react with membrane-expressed gp120/gp41, we examined
serum IgG binding to constitutively infected cells compared with the binding to the same non-
infected cells. Results in Figure 8 show IgG antibody binding to chronically infected cells (A1953
cells). We observe that the MAb 902 differentiates infected from non-infected cells (panel A).
Immunization with control MCF7 cells induce serum reactive with the neolactoseries antigen LeY
(13) and also antibodies that are potentially reactive with MHC Class I, which shares some homology
with gp120, as anti-class I antibodies bind to Env protein (panel B) (59,60). Serum from 911-
immunized mice reacted stronger with infected cells than non-infected cells (panel C). IgG from mice
immunized with other formulations (LeY or QS21) did not show any significant increased binding to
A1953 cells compared to their binding to Sup-T1 cells (data not shown).
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Immune serum affects in vitro neutralization.
The neutralizing activity of the serum was assessed by p24 ELISA. In figure 9A, we observe that
serum from MCF7- and 911-MAP-immunized mice were able to neutralize a viral input of 100
TCID50 of HIV-1 III-B, up to 1:128 dilutions, with a calculated percentage of neutralization of about
80% for each, reflecting a reduction of p24 antigen in supernatants. In contrast, serum to 910 peptide
did not neutralize this viral load at 1:64 dilutions. Positive control (IHS) displayed a neutralizing
capability beyond 1:512 dilution.
By increasing the viral load to 200 TCID50, (Figure 9B), IHS still displayed neutralizing
activity, while MCF7 serum retained some neutralizing capacity, better than that for serum raised to
911-MAP. However, in a representative neutralization assay of 50 TCID50 of HIV-1 III-B, the 911-
MAP serum displayed neutralization capability with dilutions up to 1:256 (data not shown). As with
III-B, 911-MAP serum was able to neutralize 100 TCID50 of MN, at 1:128 dilutions, with partial
neutralization activity at 1:256 dilution (data not shown). Negative controls, NHS and NMS, did not
show neutralizing capabilities to any viral isolate, even at low dilutions.
Discussion
Carbohydrate antigens are important targets in vaccine development. Vaccine design strategies have
little utilized structural concepts to develop novel carbohydrate forms (61). The clustering and
multivalent presentation of carbohydrate antigens appears relevant to induce antibody responses to
natively expressed carbohydrate antigens on cell surfaces (13,62,63). We have shown that peptide
mimeotopes can elicit carbohydrate cross-reactive immune responses to natively expressed bacterial
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and tumoral antigens related to those expressed on HIV-1 Env glycoprotein (9,13,41). To further
explore the utility of targeting the Env glycoprotein and generalizing peptide design strategies
(39,64), we are optimizing peptides reactive with Con A seen as a template. The mimetics described
here represent rationally designed candidate immunogens despite significant gaps in our knowledge
regarding the molecular and functional characteristics of PS mimeotopes (12).
We identified a peptide which, rendered as a clustered, multivalent form, was reactive with
Con A at lower concentrations than those required for reaction of some native oligosaccharide
ligands of Con A. The 911-MAP displayed competitive inhibition with carbohydrate ligands of Con
A, indicating that it binds at an overlapping carbohydrate-binding site on Con A. ITC and
precipitation experiments suggest that the putative peptide 912 is monovalent for Con A, and its
affinity is comparatively weak (similar to that of MeαMan). The MAP format of peptide 911
resulted in a higher association constant and free energy of association with Con A compared to that
found upon binding of the putative 912 peptide. Detailed thermodynamic analysis of binding of
trimannoside to Con A has been performed by ITC (18,20), and the Ka and ∆G values of 911-MAP
are comparable to those of Con A-reactive trimannoside and pentasaccharide.
While the enhancement in Ka of 911-MAP (relative to 912) is due to multivalent presentation,
the increased affinity of the two carbohydrate ligands (compared to the monosaccharide) is an
outcome of an extended site interaction. Initial analysis of binding raw data obtained from Con A-
911-MAP titration, and its comparison with the data of multivalent sugars, point to certain
fundamental differences in the overall binding mechanism. Differences between two multivalent
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binding systems are primarily attributed to the structural dissimilarities of the peptide and
carbohydrate ligands, a conclusion indicated from the molecular modeling study. It has previously
been observed that even a minor structural alteration in lectin structure profoundly affects the binding
thermodynamics. Con A, and the homologous lectin from Dioclea grandiflora (DGL), posses
conserved binding sites for the pentasaccharide, yet, minor differences in the lectin structure result in
a totally different binding energetic for the oligosaccharide. DGL binds the pentasaccharide with
much lower Ka than does Con A (20). Therefore, it is possible that structural differences between the
peptide and carbohydrate ligands contribute to the lower Ka value of the 912 peptide (compared to the
pentasaccharide). However, the affinity is significantly boosted in the MAP format, where the peptide
is functionally multivalent.
Many naturally occurring carbohydrates, including glycoproteins, are multivalent, which
results in increased avidity for lectins and antibodies. This characteristic must be emulated to affect
functional immune responses (13). Although the benefits of multivalency is well established for both
antibody and lectin binding to carbohydrates, the molecular mechanisms underlying this phenomena
is poorly understood. Presumably, the effect is not attributable to the recognition of a combined
epitope encompassing three or more sugar chains, as such a structure would exceed the size of an
antibody combining site (39). It is probable that multivalency contributes both, to structural
properties and entropy involved in binding (19). It is likely that the density of antigen expressed on
cell or pathogen surfaces can play a significant role in mediating avidity interactions.
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We have shown that the 911 peptide mimetic, formulated as a MAP form, can induce
functional carbohydrate cross-reactive antibodies, in concordance with our other studies (13,41). 911-
MAP induces serum that neutralizes HIV-1 III-B at levels comparable to serum induced by MCF7
cells, using a viral input of 100 TCID50. For lower viral inputs (50TCID50), anti-911 serum
neutralizes same isolate up to 90% at 1:256 dilution (data not shown). The presentation of the MAP
form of the mimeotopes, while emulating the multivalent carbohydrate surface may still not
effectively cope with micro-heterogeneity in carbohydrate structures. However, this same problem
exists when immunizing with carbohydrate immunogens, since, many times, synthetic carbohydrate
forms do not induce responses cross-reactive with native carbohydrate forms, requiring modifications
in synthetic strategies. Likewise, cyclization of peptide mimetic immunogens may further restrict
carbohydrate cross-reactive responses much as it does in inducing responses to protein antigens, by
limiting the polyclonal response.
In summary, these results indicate that designed peptide mimetics of carbohydrate antigens
can induce functional responses that may find utility in priming strategies to further augment
carbohydrate immune responses against pathogens or tumor cells (11). While modeling does not
account for multivalent interactions, as represented by MAP forms, modeling can define potential
binding-site constituents. Consequently, strategies that evaluate potential mimetic-binding modes
and thermodynamics of binding should further facilitate structure-assisted design of surrogates for
vaccine applications. Likewise, this study encourages further investigation to ascertain the
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mechanism(s) by which certain peptide mimics of PS antigens play their role as mimeotopes, in that
they can stimulate immunity that targets PS (12).
Acknowledgements
This study was supported by NIH grant AI44412. We thank Kaity Lin for technical assistance. We
thank Dr. James Hoxie of the University of Pennsylvania for the A1953 (Sup-T1 cells stably infected
with HIV-1 III-B) cells. We thank Charlotte Read Kensil of Aquila Biopharmaceuticals Inc. for the
QS21.
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Table 1. Peptides used in this study and their secondary structure properties.
Number Sequence None All alpha alpha/beta All beta
105107911908909910712912711
GGIYYPYDIYYPYDIYYPYDGGIYYRYDIYYRYDIYYRYDYRYRYGRYRSGSYRYRYGRYRSGSDVFYPYPYASGSDVFYRYRYASGSDVFWRYRYASGSMYWYPYASGSRYRYGRYRSGSGGPGQPGQPGQPGQ
---EEE-----------------EEEEEEEEEEEEEE----------------EEEE----------------------EEEE--------EEEE---------------------------------------
-------------------------H--H--H----------------------H------------------------HHHHH---------HHH--------------------------------------
-----------------------E-EE-EE--H-EE-----------------H-------------------------EE-H---------EHHH--------------------------------------
--EEEEEEEEEEEEEEE-----EEEEEEEEEEEEEEEE-----EE-EEE---EEEEE-EEE------E--E-------EEEEEE-------EEEEE------EEEE-------EE-EEE-----------------
Secondary structure profiles calculated from neural network calculations.
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Table 2. Hydrogen Bonding Scheme of Putative Carbohydrate and Peptide Constituents with Con A
Individual Residue Contact
Model | | I.E.
MannoseCore
Man(1-6) D-Man (1-3) Man -72.8
Tyr 12 (SC) Asn 14 (SC) Thr 15 (BB)
Asp 208 (SC) Thr 15 (SC)
Leu 99 (BB) Pro 13 (BB)
Arg 228 (BB)
Tyr 100 (BB)
Tri-Sac Man-(1-6) D-Man (1-2) GlcNAc -57.3
Tyr 12 OH (SC) Asn 14 (SC)
Asp 208 (SC)
Leu 99 (BB)
Arg 228 (BB)
Tyr 100 (BB)
Peptide Arg 1 Arg 4 Tyr 5 -75.9
Leu 99 (BB) Asp 16 (BB) Arg 228 (SC)
Ser 168 (SC) Ser 21 (SC)
Ser 223 (SC) Tyr 12 (SC)
Hydrogen-bonding schemes are from crystal structure contacts in the pentasaccharide complexstructure. The mannose core designation is for the trimannoside α-D-Man (1-6)α-D-Man (1-3)D-
Man constituents of the pentasaccharide. The Tri-sac designation is for β-D-GlcNAc (1-2)α-D-Man
(1-6) D-Man constituents of the full pentasaccharide. The central mannopyroside makes the most
contacts with Con A. Amino acid designation in the peptide model is positional location in theRYGRY sequence. In addition to hydrogen bonds, Tyr 2 forms a stacking interaction with Tyr 100 ofCon A. The Interaction Energies (IE) were calculated for the respective structures within the Con A
site. BB-Backbone, SC-Side Chain.
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LEGENDS FOR FIGURES
Figure 1. Representative carbohydrate antigen structures expressed on gp120, as defined by specific
molecular reagents (antibodies and lectins). Monoclonal antibodies reactive with sialyl-Tn, LeY, and
A1 neutralize HIV infection in vitro (65). Lectins reactive with mannose-containing carbohydrates
also display ability to block infection.
Figure 2. A. Overlap of the extended structure of prototypic WYPY with the pentasaccharide {β-
GlcNAc- (1-2)-α-Man- (1-3)-[ β-GlcNAc- (1-2)- α-Man- (1-6)]-Man}. The Trp overlaps with the
first GlcNAc- (1-2) on the Man (1-3) side, with the Proline residue overlapping with central α-Man-
(1-6)-moiety. The Tyr at the 4th position in the sequence tract approximates the location of the second
GlcNAc residue. Holding the Pro residue, fixed relative to the centralized mannose ring, least
squares fitting of the backbone atoms, comprising the first three residues in the WYPY motif to the
α1-6 linkage in the α-D-Man (1-6)-D-Man (1-3) α-D-Man binding mode, resulted in a root mean
square (RMS) deviation of 0.18 A. B. Representative placement of LUDI identified guanidinium-
like moieties. C. Putative 912 peptide (yellow) sitting in Con A-carbohydrate binding site,
emphasizing the extended nature of the putative interacting motif.
Figure 3. Inhibition of Con A binding to solid phase MAP by soluble MeαMan in competitive
lectin-binding assay. Biotinylated-Con A (0.4 µg/ml) was incubated with an increasing amount of
MeαMan and binding of free biotinylated lectin to MAPs was measured using peroxidase-labeled
Streptavidin. Lactose did not display any inhibition of Con A binding to the peptides.
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Figure 4. A. Serial dilutions of Con A biotin-labeled were added to ELISA plates pre-coated with
selected carbohydrate probes or peptide mimotopes (100 nM/well), and reacted with Streptavidin-
HRP. OD readings at 450 nm demonstrate that Con A binds to 911 peptide more efficiently than to
other peptides or to carbohydrate probes known to be reactive with Con A. B. Inhibition of Con A
binding to solid phase 911-MAP by soluble MeαMan in competitive lectin-binding assay.
Biotinylated-Con A (0.4 µg/ml) was incubated with an increasing amount of MeαMan and binding of
free biotinylated lectin to 911-MAP was measured using peroxidase-labeled Streptavidin. Lactose did
not display any inhibition of Con A binding to the 911-MAP.
Figure 5. ITC profile of Con A (0.2 mM) with 912 peptide (5.2 mM), at 27o C. Top, data obtained
from 20 automatic injections, 6 micro liters each, of 912 peptide, and bottom, the integrated curve
showing points (the squares) and best fit (the line). The buffer was 0.1 M HEPES with 5 mM each of
CaCl2 and MnCl2.
Figure 6. Profile for the kinetics of precipitation of Con A (60 µM) in presence of 911-MAP (15
µM) and peptide 912 (Squares 911-MAP, circles 912.)
Figure 7. 911 peptide-stimulated proliferation of splenocytes from 911 peptide-immunized mice.
Mice were immunized with MAP form of 911 peptide two times at a three-week intervals. 7 days
after the boost, splenocytes were collected and used for detection of cell proliferative response to
MAPs 106 and 911, using CellTiter 96R Aqueous One Solution (Promega, Madison, WI). MAPs
were used at 5 and 1 µg/ml final concentrations. Results are given as mean ± SD based on three
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replications. Experiment was repeated three times with comparable results using pooled splenocytes
from four mice. Peptide 106 (GGIYWRYDIYWRYDIYWRYD) is also in the MAP format and
displays a MHC binding score of 2000.
Figure 8. Binding of serum IgG from mice immunized with 911 and MCF7 to Sup-T1 cells
(column 1) or to A1953 (Sup-T1 cells infected with HIV-1 III-B) (column 2). Dotted lines represent
the binding of IgG from pre-immune mouse serum. Part A shows the binding of the mAb 902
(mouse IgG1-k, specific for gp120 of HIV-1 III-B) to the respective cells; part B, the binding of
MCF7-IgG and Part C, the binding of 911-IgG. Serum dilution used in these assays was 1:100.
Figure 9. The activity of sera from mice immunized with different formulations of immunogens to
neutralize HIV-1 III-B was assessed by p24 ELISA. Percentage of neutralization was calculated. A.
The data using a viral input of 100 TCID50. B. The data with a viral input of 200 TCID50.
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Figure 1.
Antigen Structure
Fucα1Fucα1
Fucα1
||
|
Fucα1-2Galβ1-4GlcNAc β1-R3
3
GalNAcα1-3Galβ1-4GlcNAc β1-R2
Lacto-N-difuconeohexose I (LeY)
GalNAcα1-3Galβ1-3GlcNAc β1-R2|
Fucα1
A1
ALeY
Antibody/lectin
Siayl-Tn NeuAcα2-6GalNAc α1- O-Ser/Thr ΤΚΗ2 (IgG1)
BM1 (IgM)
AH21 (IgM)
AH16 (IgG3)
AH16BR55-2 (IgG3)
B72.3 (IgG1)
Manα1-2Manα1-3Manβ1-4(GlcNAc)-R
Manα1-3Manα1-4(GlcNAc)-R
Galβ1-4GlcNAc β1-2Manα1|6Manβ1-4(GlcNAc)-R3|
Galβ1-4GlcNAc β1-2Manα1
Man3GlcAc
Man2GlcAc
Asialo-biantennary (NA-2)
ConA
ConA
ConA
Neu5Aca2-3/6Gal β1-4GlcNAc β1-2Manα1
Manβ1-4GlcNAc β1-4GlcNAC
Neu5Aca2-3/6Gal β1-4GlcNAc β1-2Manα1
|
|
6
3ConADisialylyl-biantennary (A2)
Oligomannose 9 (Man 9)
Manα1-2Manα1-2Manα1
Manα1-2Manα1-6Manα1-6Manβ1-4GlcNAc β1-4GlcNAC
Manα1-2Manα1
|
|
3
3ConA
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Figure 2A. Figure 2B.
Figure 2C.
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Figure 3
0
20
40
60
80
0 5 10 15 20 25 30
Me Man (mM)
% I
nhib
itio
n
908
909
910
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Figure 4A
Figure 4B.
0
0.1
0.2
0.3
0.4
0.5
10 5 2.5 1.25 0.6
Con A (ng/well)
911
908
909
107
LeY
711
mannose-9
D-mannose
NA-2
blocking
0
20
40
60
80
0 5 10 15 20 25 30
Me Man (mM)
% I
nhib
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41
Figure 7
0.000
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106 pep 911 pep
Antigen
OD a
t 490
5 ug
1 ug
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1 2
Figure 8
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Figure 9A
Figure 9B
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Serum dilution
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910911MCF7HISNHS
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Serum dilution
Neutr
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910911MCF7I HS
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Brewer, Thomas C. Van Cott and Thomas Kieber-EmmonsGina Cunto-Amesty, Tarun K. Dam, Ping Luo, Behjatolah Monzavi-Karbassi, C Fred
Directing the immune response to carbohydrate antigens
published online May 30, 2001J. Biol. Chem.
10.1074/jbc.M103257200Access the most updated version of this article at doi:
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