Synthetic peptides representing discontinuous CD4 binding epitopes of HIV-1 gp120 that induce T cell apoptosis and block cell death induced by gp120

^a Department of Pathology, Edinburgh University, Scotland, United Kingdom
^b Centre for Protein Technology, Edinburgh University, Scotland, United Kingdom

	ABSTRACT

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A vaccine against HIV-1 virus would block initial infection and must target conserved residues. Since initial infection depends on binding of the viral envelope protein gp120 to CD4 on the cell surface, the CD4 binding site of gp120 is a target for vaccine design. To identify the optimal biologically active site, we synthesized a series of 32-mer peptides, based on conserved residues in the C3 and C4 regions of gp120. These included three of five sequence discontinuous residues known to be involved in CD4 binding, one or two of which were substituted with alanine. We also synthesized a 44-mer peptide with an additional branch to incorporate an extra C4 region sequence including a fourth CD4 binding residue. All these peptides used an oxidized Cys-X-Cys bridge to link the discontinuous sequence elements in a manner suggested by the known conserved disulfide bridges in gp120. Polyclonal sera raised to these peptides indicate that they all contain both B and T lymphocyte epitopes. Binding of the peptides to CD4-transfected HeLa cells reveals a hierarchy dependent on the number of relevant CD4 binding residues present. Furthermore, antibody cross-linking of peptides bound to the surface of human T cells results in apoptosis that is similar to the known properties of gp120. The peptide incorporating three CD4 binding residues competitively inhibited gp120-induced T lymphocyte apoptosis. Thus, we have synthesized novel, branched peptides incorporating conserved discontinuous sequences from two different conserved domains of HIV-1 gp120 that contain T and B lymphocyte epitopes and mimic biological functions of the native protein. These synthetic peptides are candidates for future vaccine development.—Howie, S. E. M., Cotton, G. J., Heslop, I., Martin, N. J., Harrison, D. J., Ramage, R. Synthetic peptides representing discontinuous CD4 binding epitopes of HIV-1 gp120 that induce T cell apoptosis and block cell death induced by gp120. FASEB J. 12, 991–998 (1998)

Key Words: TFA • phycoerythrin • IgG • optical density

	INTRODUCTION

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THE HUMAN IMMUNODEFICIENCY virus (HIV)² infects CD4+ve cells (1, 2). The initial stage of insertion of the virion into the host cell is via high-affinity binding of the HIV-I coat protein gp120 to the surface receptor CD4. This ultimately leads to progressive loss of CD4+ve T lymphocytes and the immune system breakdown seen in AIDS. The production of virion particles and the destruction of CD4+ve T cells in HIV-1 infection is a dynamic process, with many CD4+ve T cells being destroyed and replaced each day (3, 4), including destruction of cells by viral cytotoxicity. It has been proposed that activation-induced cell death by apoptosis may contribute significantly to the continued depletion of CD4+ve T cells seen in HIV-1 infection (5–9). This apoptosis is mediated by signals transduced initially as a consequence of HIV-1 gp120 binding to CD4 in the T cell membrane (10–13).

The CD4 binding site on gp120 is highly conserved, conformationally restricted, and involves discontinuous regions of the molecule. Initially, Cordonnier et al. (14) showed that four conserved regions of gp120 were needed for high-affinity binding. Additional mutation and epitope mapping studies (15–19) have shown that amino acids essential for correct binding of CD4 are spread throughout the conserved regions of gp120. Based on the numbering for the HIV-1 IIIB sequence, the five amino acids thought to be most important for the high-affinity binding of gp120 to CD4 are Thr²⁵⁷ in the C2 region, Asp³⁶⁸ and Glu³⁷⁰ in the C3 region, and Trp⁴²⁷ and Asp⁴⁵⁷ in the C4 region. These residues are highly conserved in HIV-1 isolates and fall within the epitope of an antibody that blocks binding to CD4 (20).

We previously described the synthesis, immunogenicity, and CD4 binding of a synthetic 32-mer peptide that incorporated Asp³⁶⁸ and Glu³⁷⁰ in the C3 region and Asp⁴⁵⁷ in the C4 region (21). This peptide, GC1, incorporated an oxidized Cys-Val-Cys turn to bring the critical residues topographically close in a manner based on the known conserved disulfide link in the gp120 molecule between Cys³⁷⁸ and Cys⁴⁴⁵ (22). We have extended this work to examine the effects of alanine-substituting the CD4 binding residues and of adding an additional 12-mer branch to the oxidized Cys-X-Cys turn (to incorporate an extra CD4 binding residue, Trp⁴²⁷, in the C4 region) on immunogenicity and biological function. The last modification of the turn region to oxidized Cys-Lys-Cys was required so that the extra 12-mer could be appended to the N of the newly introduced Lys (peptide 3.7).

Cross-linking of cell surface CD4 by the interaction between HIV-1 gp120, anti-gp120 antibodies and CD4+ve T cells induces apoptosis both in vitro and in vivo (23, 24). This effect can be mimicked using cross-linked anti-CD4 antibodies in human and murine systems (25–28). Here we present evidence that when the GC1 and 3.7 peptides are cross-linked by immunoglobulin G (IgG) antibody on the surface of T lymphocytes, they activate the cells to undergo apoptosis. We show that an apoptosis signaling pathway transduced via CD4 can be triggered by a minimal configuration of three of five CD4 binding amino acid residues in a sequence and spatial orientation mimicking the native gp120 protein. Most significantly, the GC1 peptide also blocks the apoptosis of CD4+ve T lymphocytes induced by recombinant HIV-1 gp120. Thus, these synthetic peptides not only adopt structures that mimic the epitopes of the native molecule, but can also have similar biological functions.

	MATERIALS AND METHODS

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Rational design strategy
Large protein molecules contain functional sites that may be sequence continuous or depend on tertiary structure to bring discontinuous residues into spatial proximity. It is a simple matter to synthesis peptide analogs of known continuous regions of a protein using well-established protocols. Discontinuous functional sites present quite a different problem since the participating -amino acid residues are not all connected by an amide bond, but rather represent regions close in space within the tertiary structure of a given protein. For HIV-1 gp120, four of the five discontinuous residues known to be important for CD4 binding were located in distal regions of the primary sequence near to a conserved disulfide link between Cys³⁷⁸ and Cys⁴⁴⁵ (15–20, 22). The concept was developed by `cutting' the two peptide strands and `knotting' them together by insertion of another amino acid between the two oxidized cysteine residues, Cys-X-Cys, which translated the discontinuous region into a continuous peptide for synthesis. When X = Lys, the -amino function allows another sequence to be appended. Another consideration is that this synthetic discontinuous peptide should be capable of adopting different conformations, some of which can be induced to have the required spatial relationships of the known discontinuous functional site. This is complementary to the peptoid approach to protein-ligand design.

Synthesis of peptides
Schematic sequences of the peptides are shown in Fig. 1. The derivative of GC1 with Asp⁴⁵⁷ substituted with alanine is peptide 3.5; the derivative with both Asp⁴⁵⁷ and Glu³⁷⁰ substituted with alanine is peptide 3.6; the branched peptide with the extra 12 residues incorporating Trp⁴²⁷ is peptide 3.7.

View larger version (14K): [in this window] [in a new window]   Figure 1. Schematic representation of the peptides containing three residues (GC1), two residues (3.5), one residue (3.6), and four residues (3.7) critical for high-affinity binding of gp120 to CD4. Numbering is based on the sequence for gp120 of the HIV-1 IIIB strain.

All amino acids (L isomers) were purchased from Bachem AG (Bubendorf, Switzerland), except for Dde-Lys[Fmoc]OH, which was purchased from Novabiochem U.K. Ltd. Dimethylformamide, 1,4-dioxane, and piperidine were of peptide synthesis grade and supplied by Rathburn Chemicals (Walkerburn, Scotland). Acetic anhydride, DMAP, and HOBt were obtained from Aldrich Ltd. (Gillingham, Dorset, U.K.). Peptide synthesis grade trifluoroacetic acid (TFA) was obtained from ABI. All other reagents were obtained from Sigma U.K. Ltd. (Poole, Dorset, U.K.) unless stated otherwise.

All peptides were synthesized linearly as described by Ramage et al. (29), with modifications (21), using Fmoc solid-phase peptide synthesis with real time monitoring of deprotection efficiencies. The syntheses were carried out on a 0.25 mmol scale using Fmoc-Gly functionalized 4-benzyloxybenzyl alcohol resin (30). All amino acids were single coupled with the in situ formation of the triazol-activated ester of the required amino acids using automated solid-phase peptide synthesis, except for Dde-Lys [Fmoc]OH, which, in the synthesis of peptide 3.7, was manually coupled using fourfold excess of the activated triazole ester. All cysteines were protected as the Fmoc-Cys [Acm]OH derivative. Purity of synthesis products was monitored by amino acid analysis, mass spectrometry, analytical high-performance liquid chromatography (HPLC), and Ellman's assay for free cysteine content.

The Asp-Gly motif present in the peptides initially led to rearrangement during the N-Fmoc base deprotection protocols; 0.1 M HOBt in the Fmoc deprotection solution of 20% piperidine in DMF was found to suppress aspartimide formation to acceptable levels.

For GC1, 3.5, and 3.6, the Acm protected peptides were not separated. Removal of the Acm group was accomplished by using the silver triflate method (31). Upon separation of the reduced peptide by HPLC, cystine formation was carried out using 2.5% dimethyl-sulfoxide in TFA (32).

For synthesis of 3.7, protection of one of the two amino groups in the central lysine residue needed to be orthogonal in relation to the other, allowing selective deprotection when required. Calculations by Hopp and Woods (33) predicted a very hydrophobic area in the oxidized Cys-Lys-Cys turn region. The peptide was assembled in the usual manner until the branching Lys residue, when Dde-Lys [Fmoc]OH was added to afford the correct orthogonality; the synthesis then continued from the N amino function until residue 421. The Dde group (34) was cleaved using 2% v/v hydrazine in DMF and the synthesis continued to the end.

An additional problem with 3.7 was the presence of methionine. Cleavage and deprotection of the completed peptide were carried out as above with a small amount of thioanisole added to suppress acid oxidation of the methionine. Acm removal was then effected by using mercuric acetate in presonicated 50% acetic acid/water under nitrogen without isolation of the Acm protected peptide. Upon Acm removal, the peptide was dissolved in 50% acetonitrile in water and left for 4 days to achieve air oxidization to form the cystine bond.

Antibodies
Female BALB/C mice (five per group) were immunized as described previously (21, 35) by intraperitoneal injection of 10 µg of purified peptides in Freund's adjuvant. A minimum of three booster intraperitoneal injections of 10 µg of peptide in saline were carried out at 3 wk intervals until the maximum endpoint titer was seen. Blood was recovered, allowed to clot, centrifuged, and the serum was removed, aliquotted, and stored at -70°C until use. For some experiments the IgG fraction of serum was isolated by column fractionation on G-protein-coupled Sepharose beads (Pharmacia, U.K., Ltd.) following the manufacturer's protocol. IgG was eluted by acid washing the column, the eluate was immediately neutralized, and the protein concentration was determined using the Bio-Rad (Hemel Hempstead, Herts, U.K.) protein assay kit.

Mouse anti-human CD4 monoclonal antibody MT310 and goat phycoerythrin (PE) -coupled Fab₂ anti-mouse IgG were purchased from DAKO Ltd. (Ely, Cambridge, U.K.). Polyclonal sheep anti-gp120 mixture D7324, which recognizes the COOH terminus region of the protein, was obtained from Aalto Bioreagents (Dublin, Ireland).

HIV-1 gp120 purification
Recombinant gp120 derived from the HIV-1 IIIB strain was initially produced as the supernatant of a CHO cell line expressing gp120 from the BH10 clone, which had been obtained from Celltech via the MRC AIDS reagent project. The gp 120 was purified from the supernatant using an affinity column that consisted of Sepharose-bound D7324 sheep anti-gp120 antibody. Bound rgp120 was eluted with 4.5 M MgCl₂, pH 7.0, and dialyzed against phosphate-bufered saline (PBS), pH 7.2.

ELISA measurement of antibody binding
The enzyme-linked immunoassay (ELISA) methods used were as previously described (21). Briefly, peptide was bound to a 96-well microtiter ELISA plate (Corning-Costar Ltd., High Wycombe, Bucks, U.K.) overnight at 4°C in carbonate/bicarbonate buffer pH 9.6. The wells were washed and incubated with serial dilutions of test anti-serum and normal mouse serum or purified IgG. Bound antibody was detected using horseradish peroxidase-conjugated goat anti-mouse Ig(G,A,M), followed by o-phenylenediamine substrate. The reaction was stopped by the addition of 2N HCl (50 µl), and the optical density (OD) of the wells was read at 490 nm. IgG antibodies to GC1 had previously been shown to detect conformational and linear epitopes and to cross react with native gp120, indicating that the peptide could adopt conformational structure (or structures) that mimicked the native protein. To determine whether this was true for the other peptides, purified IgG from sera raised against 3.7 or GC1 were further characterized using concentrations of purified IgG that gave half-maximal binding OD values (approximately 1 OD unit) against the immunizing peptide coated onto ELISA wells at a standard 0.6 µM concentration: 1 µg/well for anti-GC1 and 1.5 µg/well for anti-3.7.

Cell lines
H9 and HeLa cell lines were obtained from the European Animal Cell Culture Collection (Porton Down, U.K.). HeLa cells transfected with human CD4 were obtained through the U.K. MRC AIDS Reagent Programme. The transfected cells were selected for high CD4 expression using anti-CD4 antibodies and the MACS cell purification system (Miltenyi Biotech Lts., Bisley, Surrey, U.K.). Over 95% of the selected cells (HeLa-CD4) were CD4+ve by flow cytometry. HeLa and HeLa-CD4 cells were routinely passaged in RPMI medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM L-glutamine, and 10% fetal bovine serum (10% RPMI); all reagents were purchased from Life Technologies Ltd. (Paisley, Scotland). H9 cells were cultured in RPMI as above, but with 3% fetal bovine serum (3% RPMI).

Flow cytometric detection of peptide bound to CD4+ve cells
For binding studies, the CD4+ve H9 T cell line, CD4+ve-transfected HeLa cells (obtained through the MRC AIDS reagent program), and the untransfected parent cell line (CD4-ve) were used as described (21). All subsequent procedures were carried out on ice. Cells in suspension in flow cytometry buffer (PBS, pH 7.4, containing 0.5% bovine serum albumin and 0.01% sodium azide) were added to 96-well microtiter plates at 10⁵ cells/well and incubated with peptides at varying concentrations for 2 h. The plates were then washed with flow cytometry buffer and the wells incubated with 1.0 µg purified normal mouse IgG or anti-GC1 IgG in 50 µl flow cytometry buffer for 1 h. The plates were washed again and the wells incubated with 0.1 µg phycoerythrin-coupled goat anti-mouse IgG for 1 h. The plates were then washed, cells were suspended in flow cytometry buffer, and positive staining was determined by flow cytometry using a Coulter (Beckman-Coulter Ltd., Bedfordshire, U.K.) EPICS-XL flow cytometer with a 15 mW single argon laser operating at 488 nm. Viable cells were gated on forward and right-angle light scatter, and the percentage of positively stained viable cells was established relative to the background with normal mouse IgG.

In vitro induction of apoptosis
Sterile (0.22 µm filtered) IgG (2 µg/well, 100 µl) was coated onto tissue culture grade, 96-well microtiter plates (Corning-Costar) in 0.05 M carbonate/bicarbonate coating buffer pH9.6 (Sigma U.K. Ltd., Poole, Dorset) overnight at 4°C. Immediately before use, the wells were washed three times with cold PBS. H9 cells passaged in RPMI containing 3% fetal calf serum (3% RPMI) separated over Ficoll/hypaque (Lymphoprep, Nycomed) were washed twice with cold PBS and counted. Cells were 95–98% viable. Cells were spun down and incubated on ice for 2 h with PBS alone (50 µl) or PBS (50 µl) containing GC1, PSS 023 (an irrelevant peptide), or recombinant gp120. The cells were then washed with cold 3% RPMI, counted, and resuspended at 10⁵/ml in 3%RPMI. Quadruplicate sets of 2 x 10⁴ cells/well were plated onto an IgG-coated microtiter plate, which was then spun at 100 x ;46g for 3 min to bring the cells into contact with the bottom of the wells, and incubated for 5 h at 37°C in 5%CO₂ in a humidified incubator. After 5 h, plates were removed from the incubator and the contents of the wells were individually cytospun, air dried, fixed in acetone for 10 min, and stained with hematoxylin. The cytospins were coded and the percentage of cells showing apoptotic morphology counted `blindly' by light microscopy (9, 26). A minimum of 400 cells per slide were counted. The apoptotic nature of the cell death was confirmed by electron microscopy and DNA laddering.

	RESULTS

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Immunogenicity studies
The endpoint titers of mouse sera after four injections were determined against the peptide to which they were raised. This was defined as the lowest concentration of test serum to give an OD reading greater than the mean + 3 SEM with normal mouse serum at the same dilution. As described previously (21) GC1 was very immunogenic with an endpoint titer of 1:15,000; 3.5, 3.6, and 3.7 all induced endpoint titers in the range 1:5000–1:10,000. All antibody detected was of the IgG subclass.

Studies with purified anti-3.7 (1.5 µg/well) and anti-GC1 IgG (1.0 µg/well) showed that anti-GC1 bound equally well to both peptides whereas anti-3.7 bound more strongly to 3.7 than to GC1. At this antibody concentration, peptide 3.5 was recognized about half as well as GC1 by both IgGs, but 3.6 was not recognized by either ( Fig. 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Binding of purified IgG against GC1 (1.0 µg/well) (A) and 3.7 (1.5 µg/well) (B) to 3.7 (); to GC1 (); to 3.5 (+); and to 3.6 (). Triplicate readings; all standard deviations <7% of mean.

Binding of peptides to cell surface CD4
HeLa and HeLa cells transfected with and expressing human CD4 on the cell surface were incubated in microtiter wells with concentrations of peptides from 1 to 40 µM on ice for 2 h. To detect all peptides, anti-GC1 IgG (after washing) was used at a concentration (20 µg/ml) that detected, by ELISA, 10 µM GC1, 3.5, and 3.7 equally (data not shown), followed by PE-labeled goat anti-mouse Ig. Fluorescence was detected by flow cytometry and compared with parallel samples incubated with peptide, followed by purified normal mouse IgG. Figure 3 shows that 3.7 bound more strongly than GC1, which bound more strongly than 3.5; none of the peptides could be detected on the surface of the CD4-ve, untransfected HeLa cells.

View larger version (24K): [in this window] [in a new window]   Figure 3. Binding of peptides 3.7 (), GC1 (), and 3.5 () to A) HeLa cells and B) CD4-transfected HeLa cells detected by anti-GC1 IgG. Means and standard errors of the mean of six experiments are shown. Anti-CD4 staining gave 1.9 ± 0.5% for HeLa cells and 89.2 ± 3.7% for CD4-transfected HeLa cells. C) Detection of 3.7 by anti-GC1 IgG on the CD4+ve H9 T cell line compared to the background with normal mouse IgG.

Under the same conditions, both GC1 and 3.7 could be detected on the surface of CD4+ve H9 T lymphocytes in a concentration-dependent manner. It was determined that maximal binding to H9 cells was obtained at a peptide concentration of 30 µM ( Fig. 3C).

Induction of apoptosis by GC1
At 30 µM (5 µg/50 µl/10⁶ cells), GC1 induced significant apoptosis in 14.1 ±1.3% and 3.7 in 13.9 ±2.2 of H9 cells after 5 h when cross-linked with specific IgG ( Fig. 4a) as compared to 2.5 ±1.2 (P<0.001) and 0.8 ±0.7 (P<0.01) with normal mouse IgG, respectively. No such induction was seen when H9 cells were incubated with 30 µM of the random peptide PSS 023. The electron and light microscopic appearance of the apoptotic cells observed is illustrated ( Fig. 4b, c). Apoptosis was also confirmed by DNA laddering (data not shown). The mitotic index assessed by counting metaphase cells was between 1 and 1.5% in all cultures (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 4. A) Percentage of apoptotic cells after 5 h at 37°C. H9 cells previously incubated for 2 h on ice with PBS only, 30 µM GC1, 30 µM PSS 023, 30µM 3.7, or 0.3 µM gp120 and incubated on uncoated cells, wells coated with 2 µg purified IgG derived from normal mouse serum or from hyperimmuni anti-GC1, or anti-gp120 serum. Values are means + 1 SEM. Confirmation of apoptotic cell death by B) light microscopy (original magnification x 400, scale bar 25 µm) and C) transmission electron microscopy (original magnification x 6000, scale bar 3 µm).

GC1 inhibits gp120-induced apoptosis
To determine whether GC1 induced apoptosis by a pathway similar to gp120 and whether GC1 would block gp120-induced apoptosis, H9 cells were incubated for 2 h on ice (as described above) either with PBS, the irrelevant peptide PSS 023, or GC1. The cells were then incubated for another 2 h with or without the addition of gp120 before being plated onto uncoated or Ig-coated microtiter wells. Table 1 shows that gp120 induced significant apoptosis when cross-linked with anti-gp120 IgG. PSS 023 neither induced apoptosis nor interfered with its induction by gp120. In contrast, preincubation of H9 cells with GC1 abrogated the ability of cross-linked gp120 to induce T cell apoptosis.

	DISCUSSION

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The CD4 binding site of HIV-1 gp120 is a major target for potential vaccine design. In the intact protein, this site consists of highly conserved, sequence discontinuous regions of the molecule that are spatially proximate as a consequence of folding of the molecule (14–20, 36–38). Five amino acids have been shown to be particularly important for the high-affinity binding of gp120 to CD4: Thr²⁵⁷ in the C2 region, Asp³⁶⁸, Glu³⁷⁰ in the C3 region, and Trp⁴²⁷ and Asp⁴⁵⁷ in the C4 region (14–20). Here we describe the synthesis of a series of peptides that were rationally designed to investigate this region of the gp120 molecule. The peptides incorporated conserved discontinuous sequences from the C3 and C4 regions. None of these peptides could have been produced by using conventional genetic engineering technology.

Peptides 3.5 and 3.6 were designed on the original GC1 template (21) with Asp⁴⁵⁷ only (3.5) or both Asp⁴⁵⁷ and Glu³⁷⁰ (3.6) substituted with alanine residues. The branched 3.7 peptide was also based on the GC1 template with the valine residue between the two cysteines substituted by lysine to allow an extra 12 amino acids to be attached to the extra N residue. The peptides thus included one (3.6), two (3.5), three (GC1), or four (3.7) of the five amino acid residues critical for binding of the virus to the CD4 molecule on human cells (17).

All the peptides contain both T and B lymphocyte epitopes. IgG antibodies purified from the serum of mice immunized with GC1 recognized equimolar concentrations of GC1 > 3.5 but did not bind to 3.6, indicating that substitution of one critical residue left some epitopes intact whereas substitution of two residues destroyed all epitopes recognized by anti-GC1 IgG. Anti-GC1 IgG recognized the branched peptide 3.7 as well as GC1, indicating that 3.7 contained most if not all of the epitopes that GC1 could form. Anti-3.7 recognized 3.7 > GC1, indicating that the extra residues conferred extra epitopes on the molecule. Like the anti-GC1 antibody, anti-3.7 recognized GC1 > 3.5 but did not bind to 3.6.

The peptides bind to CD4+ve cells in a hierarchy depending on the number of CD4 binding residues they contain, such that peptide 3.7 with four residues is greater than GC1 with three residues, which is greater than 3.5 with two residues. Both 3.7 and GC1 induced apoptosis of CD4+ve T cells when cross-linked by IgG. In addition, GC1 blocked the apoptotic signal initiated by antibody cross-linked recombinant HIV-1 gp120, indicating it binds to the same critical region.

We conclude from these results that GC1, 3.7, and gp120 bind to CD4 in configurations that trigger apoptosis when cross-linked on the surface of T lymphocytes. However, the peptides are less efficient on a molar basis than the native protein. Although 3.7 bound more strongly to CD4 than GC1, both peptides induced similar levels of apoptosis. Thus, the three residues Asp³⁶⁸, Glu³⁷⁰, and Asp⁴⁵⁷ presented within the CD4 binding conformation of GC1 are sufficient for binding to CD4 to trigger activation-induced T cell death.

This approach demonstrates that synthetic chemistry can be used to rationally design peptides that cannot be produced by genetic engineering and that can fold to mimic the biological activity of the native protein. This has implications for HIV vaccine design and, more generally, where sequence discontinuous regions of large proteins are known to form biologically active sites. Such synthetic peptides may be valuable tools to dissect molecular interactions in a number of important inter- and intracellular signaling pathways.

	ACKNOWLEDGMENTS

 
This work was funded by the U.K. MRC. N.C.M. was a recipient of Nuffield Foundation bursary. Many thanks to Liz Ramage, Helen Caldwell, Kevin Shaw, and Brian Whigham for valuable technical assistance and to Parke-Davis for support.

	FOOTNOTES

 
¹ Correspondence: Department of Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, U.K. E-mail: s.e.m.howie{at}ed.ac.uk

² Abbreviations: OD, optical density; PBS, phosphate-buffered saline; HIV, human immunodeficiency virus; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; PE, phycoerythrin; ELISA, enzyme-linked immunoassay; IgG, immunoglobulin G.

Received for publication January 26, 1998. Accepted for publication February 27, 1998.

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