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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

itio

n

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Figure 5.

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Figure 6.

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Figure 7

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Figure 8

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Figure 9A

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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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