INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The chemokine receptors CCR5 and CXCR4, in addition to the CD4 molecule, are required for infection by human immunodeficiency virus type 1 (HIV-1). Moreover, the importance of CCR5 in HIV-1 transmission has been reported based on the findings that individuals homozygous for a 32-bp deletion in the CCR5-coding region have a greatly reduced susceptibility to HIV-1 infection, since the protein encoded by the defective CCR5 gene cannot be detected on the cell surface and is nonfunctional as an HIV-1 coreceptor (8, 12, 13, 21, 27). Autoantibodies against CCR5 have also been reported to be induced in the sera of HIV-seronegative individuals and to strongly block HIV infection despite multiple exposures to HIV-1; these antibodies are also confirmed to block HIV-1 infection in vitro (14). CCR5 is also considered a redundant molecule in adults, since CCR5-defective individuals have normal inflammatory and immune reactions (25). Therefore, CCR5 may become an important target for receptor antagonists, including specific antibodies against native receptors. The natural ligands for CCR5 (RANTES, macrophage inflammatory protein 1 [MIP-1], and MIP-1), their modified forms (Met-RANTES and aminooxypentane-RANTES), the nonpeptide CCR5 antagonist (TAK-779), and anti-CCR5 antibodies (2D7 and PA14) are known to block HIV-1 R5 infection (3, 7, 17, 19, 23, 26).

In this study, we used a different approach by developing a cyclic closed-chain dodecapeptide (cDDR5) conjugated with a multiple antigen peptide (MAP) as a new HIV-1 defense vaccine. The cDDR5-MAP, which mimics the conformation-critical domain for HIV-1 entry (the undecapeptidyl arch [UPA] of extracellular loop 2 [ECL2]), functioned as an immunogen which induced conformation-specific anti-CCR5 antibodies, and both the antibody and cDDR5-MAP inhibited infection by HIV-1 R5 in a dose-dependent manner. Therefore, we propose that cDDR5-MAP is designed for use not only as a potential ligand for defense against HIV-1 entry but also as a new vaccine in place of viral-protein-based vaccines to overcome unprecedented scientific obstacles in the development of HIV-1 vaccines.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Preparation of cDDR5-MAP, cDDR5-Multi-Pin Block, and biotinylated cDDR5. A CCR5-derived linear dodecapeptide (linear DDR5, H₂N-D₁R₂S₃Q₄K₅E₆G₇L₈H₉Y₁₀T₁₁G₁₂-COOH) in which all side chain groups are protected (protected DDR5) was synthesized using an automatic peptide synthesizer and was cyclized by bond formation between the -carboxyl group of Gly₁₂ and the -amino group of Asp₁ after removal of the resin. The o-benzyl group of the -carboxyl group of Asp₁ in protected cDDR5 was selectively removed by reduction of palladium (0.5%) on carbon. The free -carboxyl group of Asp₁ in protected cDDR5 was separately conjugated to construct the MAP resin (Applied Biosystems Japan Ltd., Tokyo, Japan) and Multi-Pin Block according to the software manual in the Multipin peptide synthesis kit (Chiron Technologies, Clayton, Australia). It was also linked to 5-[5-(N-succinimidyloxycarbonyl)pentylamido]hexyl D-biotinamide through ethylenediamine in a similar way. cDDR5 derivatives in which all protected groups were removed by trifluoroacetic acid were used for the following purposes. Female BALB/c mice were immunized with cDDR5-MAP using the protocol of Galfre and Milstein (9). cDDR5-Multi-Pin Block was used in an enzyme-linked immunosorbent assay for screening monoclonal antibodies (MAbs) produced in hybridomas according to the experimental procedures in the Multipin peptide synthesis kit (Chiron Technologies). Biotinylated cDDR5 was used to confirm the specific binding of antibodies to cDDR5 using a BIAcore biosensor. Unless otherwise specified, all peptides used were purified by high-performance liquid chromatography (Waters), and the molecular masses of the compounds were determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Burker Franzen Analytik GmbH, Bremen, Germany).

Purification of the antibody against cDDR5-MAP, KB8C12 Ten BALB/c mice were immunized intraperitoneally with cDDR5-MAP in Freund's adjuvant at 1-week intervals and administered an intravenous boost of cDDR5-MAP 3 days prior to splenectomy. We observed no adverse effects of autoantibody induction on BALB/c mice that were monitored for 3 months from the initial immunization. Thirty-three hybridomas were generated by a standard method, by which splenocytes were fused with P3U1 cells and selected in hypoxanthine-, aminopterin-, and thymidine-supplemented media. In the screening, supernatants were tested for reactivity to cDDR5-Multi-Pin Block. Hybridomas that produced the most-potent supernatants were then cloned by limiting dilution.

Antiviral activities. The antiviral activity of cDDR5-MAP or purified KB8C12 was determined using MAGIC-5 cells, which were engineered to express CCR5 in HeLa-CD4-LTR/-Gal cells by transfection with an expression vector for CCR5 (11). MAGIC-5 cells were plated at 10⁴ cells per well (48-well plate) and incubated overnight in a culture medium (200 µl); the medium was then replaced by a medium containing either cDDR5-MAP (50, 100, 200, or 400 µM) or a KB8C12 antibody solution (0.1, 0.2, 0.4, 0.8, or 1.5 µg/ml). Cells were then separately incubated with various HIV-1 strains (200 µl each of the R5 and X4 virus suspensions) in the presence of DEAE dextran (20 µg/ml) for 2 h, washed twice with the culture medium, and cocultured in the medium (400 µl) containing cDDR5-MAP or the antibody solution for 48 h. Cells were fixed, and HIV-1-infected cells that were stained blue were counted by conventional methods. Control experiments were carried out under identical conditions, except for the omission of cDDR5-MAP or KB8C12.

Real-time biomolecular interaction analysis using surface plasmon resonance. The principle of the operation of the BIAcore biosensor and its use in analyzing antigen-antibody interactions have been described previously (4, 16, 18). All interactions were analyzed in a binding buffer (0.02% KH₂PO₄, 0.29% Na₂HPO₄ · 12H₂O, 0.8% NaCl, and 0.02% KCl) at a constant flow rate of 20 µl/min and a constant temperature of 25°C. Biotinylated cDDR5 was injected over a streptavidin-coated sensor chip (BIAcore) until a suitable level was achieved. Binding experiments were performed by injection of purified KB8C12 (0.7, 1.3, 2.6, and 5.2 pmol/ml) (see Fig. 2A). Competitive experiments were performed by injection of purified KB8C12 (0.52 pmol) treated with cDDR5 (10, 50, and 100 nmol) (see Fig. 2B) or linear DDR5 (10, 50, and 100 nmol) (see Fig. 2C). Bound antibody was eluted from biotinylated cDDR5 by a short pulse (20 µl) of 10 mM Gly-HCl (pH 2.0). This regeneration procedure did not, to any measurable extent, alter the ability of biotinylated cDDR5 to bind to the antibody in subsequent cycles.

Flow cytometry. The following antibodies were used: an anti-CXCR4 MAb (clone 12G5; PharMingen, San Diego, Calif.), an anti-CCR5 MAb (clone 2D7; PharMingen), purified KB8C12, an isotype-matched control antibody (Sigma Chemical Co., St. Louis, Mo.), and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG or anti-mouse IgM.

Chemotaxis assay. A chemotaxis assay was performed by use of the protocol of Gosling et al. (10) with THP-1 cells (10⁶ cells) treated with or without KB8C12 (0.5 or 1.0 pmol). The assay was conducted in the presence of 20 nM MIP-1 placed in the lower chamber. Transwells (pore size, 5 µm; Corning Inc., Corning, N.Y.) were incubated for 3 h at 37°C. The cells that migrated from the upper chamber to the lower chamber were quantified by trypan blue dye exclusion.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

cDDR5 synthesis and peptide analysis. It is generally thought that the conformational B-cell epitopes involved in induction of a conformation-specific antibody would be difficult to mimic using a simple synthetic linear peptide. Therefore, a linear side chain group-blocked oligopeptide with a free-amino-terminal head and a carboxyl-terminal tail was first synthesized and then cyclized by peptidyl bond formation between the -amino group of Asp₁ and the -carboxyl group of Gly₁₂ on the basis of the deduced conformation of the critical domain for HIV-1 entry (UPA of ECL2) in the CCR5 structure. After removal of the resin, both the linear DDR5 (H₂N-D₁R₂S₃Q₄K₅E₆G₇L₈H₉Y₁₀T₁₁G₁₂-COOH) and cDDR5 (cyclized at the head and tail of linear DDR5) were purified by high-performance liquid chromatography, and their molecular masses were determined by MALDI TOF-MS using -cyano-4-hydroxy-cinnamic acid as a matrix. The spectra of purified linear DDR5 and cDDR5 exhibited major peaks at m/z 1372.34 (Fig. 1, upper spectrum) and 1354.42 (Fig. 1, lower spectrum), respectively. The difference in molecular mass between linear DDR5 and cDDR5 indicates the formation of a peptide bond. On the basis of these results, it was established that the structure of cDDR5 is cyclo(DR₁₆₈S₁₆₉Q₁₇₀K₁₇₁E₁₇₂G₁₇₃L₁₇₄H₁₇₅Y₁₇₆T₁₇₇G).

View larger version (10K): [in this window] [in a new window]   FIG. 1.   MALDI-TOF MS spectrum of linear DDR5 and cDDR5. The spectra exhibited two peaks at m/z 1372.34 and 1354.42: the upper peak is that of the ion derived from linear DDR5, and the lower peak is that of the ion derived from cDDR5. The matrix was a saturated solution of -cyano-4-hydroxycinnamic acid in a solution of acetonitrile-water (1:2) containing 0.1% trifluoroacetic acid. The fraction with a molecular mass of 17.92 corresponding to H2O was deleted after cyclizing the head and tail of the peptide.

Immunochemical specificity of the anti-cDDR5-MAP MAb, KB8C12. Among many antibody-producing clones, one clone producing the antibody to cDDR5-MAP was effectively selected using cDDR5-Multi-Pin Block. The novel MAb [KB8C12, IgM()] was purified; the immunochemical specificity of KB8C12 treated with or without cDDR5 was determined using a BIAcore biosensor bound with biotinylated cDDR5. As shown in Fig. 2A, the response of the biotinylated-cDDR5-bound biosensor increased with increasing concentration of the flowing antibody. Antibody binding was competed by cDDR5 (Fig. 2B) but not by linear DDR5 (Fig. 2C). The response curve shows a typical antibody-antigen interaction in the presence or absence of cDDR5 or linear DDR5.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Immunochemical specificity of the anti-cDDR5-MAP MAb KB8C12. The binding specificity of KB8C12 was determined by real-time biomolecular interaction analysis using surface plasmon resonance with a BIAcore biosensor. Aliquots of the deprotected cDDR5 with the free -carboxyl group of Asp1 were used for the construction of biotinylated cDDR5 as described in Materials and Methods. Antigen-antibody binding and competitive experiments were carried out with purified KB8C12 alone (0.7, 1.3, 2.6, or 5.2 pmol/ml) (A) or with the antibody treated without (KB8C alone; 5.2 pmol/ml; black line) (B) or with cDDR5 (10, 50, or 100 nmol) (B) or linear DDR5 (10, 50, and 100 nmol) (C) using the biotinylated-cDDR5-bound BIAcore biosensor.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Flow cytometry of NP-2 transfectants with KB8C12. NP2/CD4 (A and D), NP2/CD4/CCR5 (B and E), and NP2/CD4/CXCR4 (C and F) were first detached by incubation in PBS containing 0.25% trypsin at 37°C for 1 min and then suspended in a cold washing buffer (PBS containing 2% fetal calf serum and 0.02% NaN3) at 106 cells/ml. These were subjected to flow cytometry as described in Materials and Methods, in which cells were separately incubated with 12G5 (1 µg; black line in Fig. 4A and C), 2D7 (1 µg; dark-gray line in Fig. 4A and black line in Fig. 4B), KB8C12 (1 µg; light-gray line in Fig. 4D, E, and F), an isotype-matched IgG antibody (control; 1 µg; light-gray line in Fig. 4A, B, and C), or an isotype-matched IgM antibody (control; 1 µ; black line in Fig. 4D, E, and F) at 4°C. Then the cells were washed with the washing buffer and resuspended in the washing buffer containing FITC-conjugated anti-mouse IgG or anti-mouse IgM.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   KB8C12 binds specifically to CCR5 on the cell surface. THP-1 cells were exposed to KB8C12 (0.05 pmol) in the absence (B) or presence (C) of cDDR5-MAP (0.5 pmol), or in the presence of an isotype-matched control antibody (A) at 4°C. Positive cells were observed within the region in each histogram.

KB8C12 inhibits the chemotactic response to MIP-1. To determine whether KB8C12 affects cell functions following the blockade of the CCR5 receptor, the migration of THP-1 cells induced by a chemokine, MIP-1, was investigated. It is known that ECL-2 of CCR5 is critical for high-affinity binding of MIP-1 (22). As expected, KB8C12 significantly interfered with chemotaxis induced by MIP-1 in a dose-dependent manner (Fig. 5). Incubation of cells with 1.0 pmol of KB8C12 was sufficient to achieve complete inhibition of THP-1 cell migration induced by MIP-1.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   KB8C12 interferes with MIP-1-induced THP-1 chemotaxis. KB8C12 inhibits the THP-1 chemotactic response in a dose-dependent manner. Results are expressed in terms of the chemotaxis index, which represents the fold increase in the number of cells migrating in response to MIP-1 over the number migrating spontaneously in the control medium.

Antiviral activity. Because CCR5 is the main coreceptor for HIV-1 R5, we investigated whether KB8C12 could also inhibit HIV-1 entry via CCR5. Thus, the anti-HIV-1 activities of KB8C12 were also determined using MAGIC-5 cells that express CCR5. These cells were separately inoculated with various strains of HIV-1 (the R5 HIV-1 strain JRFL and the X4 HIV-1 strain LAV-1) in the presence of the antibody (twofold serial dilution). As expected, KB8C12 markedly suppressed infection by JRFL, but not by LAV-1, in a dose-dependent manner (Fig. 6).

View larger version (59K): [in this window] [in a new window]   FIG. 6.   Antiviral activities of KB8C12. MAGIC-5 cells were separately incubated with HIV-1 strains JRFL (amount of HIV-1 p24 antigen, 5.6 ng) (A) and LAV-1 (amount of HIV-1 p4 antigen, 56 ng) (B) and cocultured in the KB8C12-containing medium (400 µl) for 48 h as described in Materials and Methods. No significant cytotoxicity of the antibody-containing medium was observed. Values are means of triplicate determinations.

Inhibition of HIV-1 R5 infection by cDDR5-MAP. cDDR5 was conjugated to MAP to develop a new vaccine in place of viral-protein-based vaccines. Interestingly, cDDR5-MAP also functions as a potential ligand for defense against HIV-1 entry. We evaluated the effect of cDDR5-MAP on HIV-1 replication in MAGIC-5 cells that express CCR5. Cells were inoculated with JRFL in the presence or absence of cDDR5-MAP (0, 50, 100, 200, or 400 µM). The number of cells infected (blue-stained) in the presence of cDDR5-MAP was determined microscopically and expressed as a percentage of the number of infected cells in the control. cDDR5-MAP immediately suppressed infection by JRFL in a dose-dependent manner (Fig. 7) but did not suppress infection by HIV-1 X4 (data not shown).

View larger version (77K): [in this window] [in a new window]   FIG. 7.   Antiviral activities of cDDR5-MAP. MAGIC-5 cells were separately incubated with various HIV-1 strains (JRFL; amount of HIV-1 p24 antigen, 5.6 ng) and cocultured with cDDR5-MAP (50, 100, 200, and 400 µM) for 48 h as described in Materials and Methods. No significant cytotoxicity of the cDDR5-MAP-containing medium was observed. Values are means of triplicate determinations.

Potential usefulness of antibodies raised against cDDR5-MAP. The chemokine receptor CCR5 binds MIP-1, MIP-1, and RANTES and constitutes the major coreceptor that allows infection of CD4⁺ T lymphocytes, macrophages, and microglial cells by macrophage(M)-tropic strains of HIV (22). In particular, the second extracellular loop of CCR5 is critical for high-affinity binding of MIP-1, MIP-1, and RANTES and for the functional response to these chemokines. However, some studies have suggested that the multiple domains of CCR5 are used for gp120 binding (1, 2, 5, 15, 20). For example, Rucker et al. have reported that the NH₂ terminus of CCR5, as well as the first extracellular loop, is important for M-tropic HIV-1 binding (20). Bieniasz et al. have indicated that the second extracellular loop is important for binding (5). Therefore, it is difficult to develop a small agonist or antagonist which by itself can inhibit HIV-1 entry via CCR5.