A random linear dodecapeptide phage display library (Ph.D-12), wherein the displayed peptide (12-mer) is expressed fused to the N terminus of gIII protein, was purchased from New England Biolabs (Beverly, MA). Monoclonal antibody (MAb) 2D7 was purchased from BD Pharmingen (San Diego, CA). Immunoglobulin (Ig) and HRP (horseradish peroxidase)-conjugated secondary antibodies used for enzyme-linked immunosorbent assay (ELISA) were obtained from Jackson Immuno Research Laboratories (West Grove, PA). Buffers and substrates for ELISA were purchased from KPL Biotech (Gaithersburg, MD). New Zealand rabbits were procured from Charles River (Wilmington, MA).
Epitope mapping using phage display library.
A random, linear, dodecapeptide-phage display library (Ph.D-12; New England Biolabs) was used for MAb 2D7 epitope mapping. Affinity selection of the phage clones from the random peptide library was carried out per the manufacturer's instructions with minor modifications. Microtiter wells were coated with 200 ng MAb 2D7 in 100 μl phosphate-buffered saline (PBS), pH 7.4, at 4°C for 12 h followed by 1 h at room temperature (RT). The wells were washed with PBS containing 0.05% Tween 20 (PBST) and blocked with EMEM containing 5% fetal bovine serum (FBS) at RT for 2 h. The phage library (1 × 1010 phages/100 μl) was added to the blocked wells and incubated at RT for 1 h. Unbound phages were washed off with 10 washes of PBST followed by three washes with PBS. The bound phages were eluted at low pH by incubation with elution buffer (0.1 N Gly·HCl, pH 2.2, containing 1 mg/ml bovine serum albumin) at 37°C for 10 min. Eluent phages were neutralized with 6 μl of 2 M Tris (pH unadjusted) per 100 μl phage eluate. After four rounds of biopanning, the phage clones were analyzed by DNA sequencing and affinity-capture phage ELISA.
Phages used for ELISA were derived from clones selected after the fourth round of panning. Microtiter plates coated with 200 ng of MAb 2D7 were blocked with EMEM containing 5% FBS (blocking solution). The phages (1010/100 μl/well) diluted in blocking solution were then added and incubated for 1 h at room temperature (RT). The plates were washed three times with PBST prior to addition of HRP-conjugated anti-phage antibody (GE Healthcare, Piscataway, NJ), and the reaction was quantified using ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] substrate. Absorbance was measured at 405 nm.
Peptides were biotinylated using maleimide PEO2-Biotin reagent per the manufacturer's instructions (Pierce Biotech, Rockford, IL). The biotinylated 2D7-2SK peptide or a scrambled version of this peptide (2D7-2SK-SCR) (1 μg/well) was captured onto wells coated with 200 ng of streptavidin. After blocking with EMEM containing 5% FBS, serial dilutions of MAb 2D7 in blocking solution were added to each well and incubated for 1 h at RT, followed by addition of peroxidase-conjugated goat anti-mouse IgG. The reaction was quantified using ABTS substrate. Absorbance was measured at 405 nm. The synthetic peptides that were used in the study are outlined in Table .
Sequences of peptides used in the study
Competition phage ELISA using synthetic peptides.
Competition of peptides with selected phage clones for binding to MAb 2D7 was performed by phage ELISA. Synthetic 2D7-2SK peptide (or its biotinylated derivative), or a control peptide (CGRAARIGFPGAYTTKNG), was added to MAb 2D7-coated wells for 30 min at RT. The phages (109/100 μl/well) were added to MAb 2D7-coated wells, which were preincubated in the absence or presence of various concentrations of competing peptides. The reaction was developed as described above for phage ELISA.
The Cf2Th canine cell line and the hCCR5-transfected Cf2Th/synCCR5 derivative were obtained through the AIDS Research and Reference Reagent Program (catalog no. 4662; contributed by Tajib Mirzabekov and Joseph Sodroski [12
], McKesson HBOC BioServices, Rockville, MD). At 48 h before the ELISA, 200 μl of 105
cells/ml was plated in 96-well plates and incubated at 5% CO2
and 37°C. The plates were gently washed with Dulbecco's PBS supplemented with Ca2+
after each incubation step during ELISA. Serial dilutions of control or anti-CCR5 antibodies in EMEM containing 5% FBS were added to each well followed by addition of peroxidase-conjugated goat anti-mouse or anti-rabbit IgG. The reaction was quantified using ABTS substrate. Absorbance was measured at 405 nm.
CEM.NKR.CCR5 cells (a generous gift from John Moore, Rockefeller University) were stained with 10 μg/ml MAb 2D7 (or isotype-matched control mouse IgG) or with 25 μg/ml rabbit anti-2D7-2SK IgG (or with preimmune rabbit IgG) for 60 min on ice, followed by extensive washing and staining with fluorescein isothiocyanate (FITC) goat anti-mouse or with FITC goat anti-rabbit antibodies (Sigma). Flow cytometry was performed and analyzed with Cell Quest Software on FACSCalibur (BD Biosciences).
Rabbit immunization and antibody purification.
New Zealand rabbits were immunized subcutaneously with 500 μg of KLH-conjugated peptide emulsified in complete Freund's adjuvant. Two booster doses at 21-day intervals, with 500 μg antigen emulsified in incomplete Freund's adjuvant, were injected subcutaneously. The rabbits were bled 8 days after the second boost, and the isolated serum was titrated for peptide binding by ELISA. The polyclonal rabbit IgG was affinity purified on a peptide-coupled gel using a sulfolink kit (Pierce Biotech, Rockford, IL).
Affinity measurements by Biacore.
Steady-state equilibrium binding of MAb 2D7 and rabbit antibodies raised against the 2D7-2SK peptide was monitored at 25°C using a Biacore 3000 surface plasmon resonance biosensor (Biacore AB, Uppsala, Sweden). The 2D7-2SK peptide was coupled to an F1 sensor chip using sulfhydryl coupling with 60 resonance units (RU), 120 RU, and 400 RU in the three test flow cells. Samples of 100 μl of freshly prepared antibody at various concentrations were injected at a flow rate of 25 μl/min (240-s contact time). Flow was directed over a mock surface to which no protein was bound, followed by the 2D7-2SK peptide-coupled surface. Responses from the peptide surface were corrected for the response from the mock surface and for responses from a separate, buffer only, injection. Binding surfaces in the flow cells were regenerated by the injection of two 30-s injections of 10 mM glycine at pH 2.5. All injections were done three times. Rabbit anti-anthrax protective antigen or anti-ovalbumin antibodies were used as control nonspecific antibodies in various binding experiments.
For the competition experiments, various concentrations of rabbit anti-2D7-2SK peptide (diluted with nonimmune rabbit IgG so that the rabbit immunoglobulin concentration was constant) were mixed 1:1 with MAb 2D7 at a concentration of 12.5 μg/ml, and 25 μl samples were injected at a flow rate of 10 μl/min through 2D7-2SK peptide-coated chips. In these experiments, MAb 2D7-specific binding in the presence of rabbit antibodies was quantified by injection of 25 μl of goat anti-mouse IgG (25 μg/ml). The experimental conditions were identical in the reverse competition analysis of the rabbit anti-2D7-2SK binding to 2D7-2SK peptide in the presence of MAb 2D7. In that case, MAb 2D7 was diluted with nonimmune mouse IgG so that the mouse immunoglobulin concentration was kept constant. Bound rabbit antibodies were quantified by a goat anti-rabbit IgG antibody (25 μg/ml) to determine the association of rabbit anti-2D7-2SK to the 2D7-2SK peptide-coated chip in the presence of increasing concentrations of MAb 2D7.
Fusion inhibition assays.
T-cell line 12E1 was infected with HIV-1 envelope (env)-expressing recombinant vaccinia viruses at 10 PFU/cell overnight. Envelope-expressing 12E1 cells or TF228 (stably transfected cells expressing IIIB/BH10 envelope) were mixed (1:1) with the human PM1 cell line (a CD4+
derivative of the Hut 78 cell line that is susceptible to infection by both X4 and R5 strains) (10
) or with phytohemagglutinin-activated-interleukin-2-activated rhesus peripheral blood mononuclear cells (PBMC). Various antibodies were added at different concentrations to these target cells and incubated for 1 h at 37°C. The envelope-expressing 12E1 effector cells were then added (at a 1:1 effector/target ratio) as previously described (7
). The numbers of multinucleated syncytia were scored at various times after initiation of cocultures. Peak syncytium formation was usually observed between 3 and 5 h. All groups were plated at two or three replicates, and all experiments were done at least three times.
HIV-1 strains and virus neutralization assays.
HIV-1 strains IIIB (X4 virus) and BaL, JR-CSF, or primary isolate 92US657 (R5 viruses) were obtained from the AIDS Research and Reference Reagent Program (McKesson HBOC BioServices, Rockville, MD). Viral stocks were propagated, and their titers were determined in phytohemagglutinin-activated peripheral blood mononuclear cells (PBMC). The CCR5-expressing PM1 cells or human PBMCs (2.5 × 104 cells/well in 96-well plates) were preincubated with the different antibodies (twofold dilutions, starting at 50 or 17.0 μg/ml final concentration) for 120 min at 37°C, followed by addition of viruses at 50 to 100 50% tissue culture infective dose (TCID50)/well (five replicates per group). The plates were washed extensively after 24 h to remove residual virions and antibodies. Every second day thereafter, the supernatants were removed and the cultures were supplemented with fresh medium (without inhibitors). Virus production was determined by measuring p24 in the supernatants with an ELISA kit (NEN Life Sciences Products Inc., Boston, MA). Virus neutralization by the different antibodies is expressed as percent inhibition of p24 production at a given concentration.