6, which is published as supporting information on the PNAS web site) was based upon methods described (15). naive scFv libraries (Fig. 6, which is published as supporting information on the PNAS web site) was based upon methods described (15). Phage supernatants were screened by bacteriophage ELISA as described (16, 17), where the biotinylated forms of 5H and IZN36 were immobilized onto 96-well ABGene, Surrey, U.K., streptavidin plates. For viral neutralization Dasotraline assays, immobilized metal ion affinity chromatography-purified soluble scFv fragments were prepared by using standard methods (18). Antiviral Assays. IMAC-purified scFvs were tested in the HIV reporter particle (HIVRP) assay essentially as described Dasotraline (19). Measurement of HIV infection of p4-2/R5 cells by using a chemiluminescent -galactosidase substrate was done as described (20). BaL and HXB2 were purchased from Advanced Biotechnologies (Columbia, MD); 89.6 was grown in peripheral blood mononuclear cells, and vesicular stomatitis virus-G-pseudotyped HIV was made by transfection as described (21). The luciferase-based pseudotyped viral neutralization assay was done as described (7). In brief, envelope genes were amplified by PCR, cloned into an expression vector, and cotransfected with a proviral plasmid to generate pseudotyped luciferase-encoding viruses. Viruses were used to infect U87/CD4/CXCR4/CCR5 cells in the presence of varying amounts of inhibitors. Luciferase production was measured 72 h after infection and IC50s calculated as described (7). AlphaScreen-Based Peptide/D5 Interaction Assays. An AlphaScreen detection kit (PerkinElmer) was used to measure binding. Biotinylated peptides (5H, IZN36, IZN17, or IQN17) were bound to streptavidin-conjugated donor beads, and D5 IgG was bound to Protein A-conjugated acceptor beads. Beads were mixed in the presence or absence of competitors, incubated overnight at room temperature, and analyzed on a Fusion -FP HT instrument (Perkin-Elmer), as suggested by the manufacturer. Six-helical bundle formation was measured by using the peptide C34-HA (22). Serial dilutions of inhibitors (D5-IgG1, Dasotraline C34, C34AAA, and 2F5) were preincubated with biotinylated 5H (final concentration, 10 nM) for 40 min at room temperature, then C34-HA was added to a final concentration of 3.3 nM along with AlphaScreen beads for detection of HA-tagged proteins (Amersham Pharmacia) and read on the Fusion instrument. Results Selection of a Human HIV-Neutralizing Antibody. We selected human-derived scFvs from phage display libraries by binding to IZN36 and 5H, antigens designed to mimic HR1 as it may exist in the Rabbit polyclonal to APEH prehairpin intermediate (Fig. 1). IZN36 is a homotrimeric peptide in which 36 amino acids of HR1 are Dasotraline fused to a stable coiled-coil peptide (IZ) to yield a soluble discrete trimeric form of the HR1 three-stranded coiled-coil in the absence of HR2 (10). In 5H, the three-stranded HR1 core is associated with two bound HR2 peptides, presenting a single binding site for HR2 (11). As a source of antibodies, we used large diverse well characterized libraries of bacteriophage bearing scFvs derived from normal human B cells (15). From a starting population of 1011 independent scFv-displaying bacteriophage, a total of 481 target-specific scFvs were obtained after two rounds of sequential selection for binding to biotinylated forms of 5H and IZN36 (schematic shown in Fig. 6). Nucleotide sequencing identified 100 unique sequences within this population of 481 scFvs. Using the HIVRP assay (19), we screened purified scFvs produced from 5H/IZN36-binding bacteriophage and identified an scFv that blocks HIV entry. The HIVRP assay relies on incorporation of -lactamase into infectious HIV particles so that fusion of the viral and cellular membranes delivers -lactamase into the target cell, where it is detected by using a cell-permeant fluorescent -lactamase substrate. This assay is particularly well suited to screening scFvs, which are inherently less durable than IgGs, because it requires only a 3- to 4-h 37C incubation of viral particles with cells to allow viral entry. One scFv, designated 5H/I1-BMV-D5 (hereafter referred to as D5), specifically inhibited the HIVRP assay in a dose-dependent manner (Fig. 2Viral envelope D5 lgG1 lC50, g/ml D5 lgG1 lC50, nM Hxb2 46.5 310 (= 6) BaL 14 93 (= 4) 89.6 262 1750 (= 2) MN-1 59 393 (= 4) NL4-3 34 226 (= 1) VSVG Not active Not active Open in a separate window Experiments were performed as described in the legend to Fig. 4. IC50s represent the average of the indicated number of determinations (= and data not Dasotraline shown). D5-IgG1 blocked six-helix bundle assembly with IC50 1 nM, but the human IgG1 2F5, which binds to an epitope on gp41 not present in either 5H or C34-HA, did not inhibit at 100-fold higher concentrations (Fig. 3and positions, which form the trimerization interface (Fig. 4Viral envelope IC50, nM Subtype C34 T20 IgG1b12 2F5 D5-IgG1 1168 B 17 54 >667 80 >2,333 21068 C 2.5 43 18.
