Anti-HIV type 1 (HIV-1) human monoclonal antibody 2F5 (20
) and human HIV immunoglobulin G (IgG) were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Anti-HIV p24 antibody KC57-RD1 was obtained from Beckman Coulter, Inc.
Cell and virus stocks.
Human embryonic kidney 293 cells were purchased from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum (FBS) and 100 μg of penicillin-streptomycin/ml. The human T-cell leukemia cell line MT-2 and the HeLa-derived cell line MAGI-CCR5 were obtained from the AIDS Research and Reference Reagent Program.
HIV-1 isolates (ADA, JRCSF, JRFL, Bal, SF162, and 89.6) were obtained from the AIDS Research and Reference Reagent Program. Primary isolates 6101 (previously called P15) and 1168 were obtained from David Montefiori of Duke University (3
). The viruses were expanded by two or three cycles of growth on phytohemagglutinin- and interleukin-2 (IL-2)-stimulated peripheral blood mononuclear cells (PBMC). For the production of the working stock virus, PBMC were exposed to undiluted virus for 2 h at a concentration of 107
cells/ml. IL-2 culture medium was added to bring the concentration to 106
cells/ml. The IL-2 culture medium was changed every 2 days, and supernatants were collected during the peak of p24 expression, usually 5 to 10 days after infection. Virus stocks were made cell free by centrifugation at 1,000 × g
and filtration through a 0.45-μm-pore-size filter. In some cases, viral stocks were concentrated as much as 10-fold by using a 100-kDa cutoff polyethersulfone filter (Centricon Plus Biomax filter; Millipore, Bedford, Mass.) according to the manufacturer's instructions. Virus aliquots were stored in the vapor phase of liquid nitrogen. Viruses BL01 and BR07 were provided by Dana Gabuzda of the Dana-Farber Cancer Institute. Both are chimeric infectious molecular clones of HIV strain NL4-3 that contain the full-length env
genes from primary HIV-1 isolates (18
). After the initial plasmid transfection of 293 cells, these viruses were expanded in PBMC as described above.
Buoyant density gradient analysis of lentiviral vectors.
293T cells (3 × 106) were transfected with 3 μg each of the relevant Gag and Env expression vectors in a 100-mm-diameter tissue culture dish with Dulbecco's modified Eagle's medium. Three days later, the cell supernatants were collected and mixed with 60% OptiPrep (iodixanol) medium (Invitrogen); the final concentration of OptiPre was adjusted to a 30% density gradient, formed by centrifugation at 45,000 × g for 6 h in a VTI50 rotor (used according to the manufacturer's instructions; Invitrogen); and each fraction was collected according to the indicated density. Lentiviral vector proteins were separated in a sodium dodecyl sulfate-4 to 15% polyacrylamide gel electrophoresis (SDS-4 to 15% PAGE) gel, transferred onto an Immobilon-P membrane, and blotted for the expression of Gag (human HIV IgG, used at 1:5,000) and Env (human HIV IgG, used at 1:5,000).
Construction of recombinant adenoviruses.
Adenovirus type 5 (Ad5)-based first-generation (ΔE1 and ΔE3) recombinant adenoviruses expressing different V loop deletions of gp140(ΔCFI) were constructed as described previously (1
). In brief, PacI-linearized shuttle vectors containing V loop deletions of gp140(ΔCFI) were recombined with the right side of Ad5 genomic DNA carried in a cosmid by use of Cre recombinase (Novagen, Madison, Wis.). The resulting recombinants were ethanol precipitated, dissolved in Tris-EDTA, and transfected into 293 cells. Recombinant adenoviruses were observed based on plaque formation 10 to 14 days after transfection. Viruses were amplified, purified two times through a CsCl gradient, and stored in phosphate-buffered saline (PBS) plus 15% glycerol at −20°C.
Production of pseudotyped lentivirus.
HIV-Luc pseudotyped with HIV gp160(89.6P) and its V3 deletion mutants were prepared according to published methods (17
). Briefly, the packaging vector pMD 8.2, pHR-Luciferase, and the envelope-expressing vector were transiently cotransfected into 293T cells by use of calcium phosphate. Supernatants were harvested 48 and 72 h after transfection, filtered, and stored at −80°C. Virus concentrations were determined by an enzyme-linked immunosorbent assay (ELISA) for the p24 antigen (Coulter). The same amount of virus was added onto MT-2 (X4 tropic) and MAGI-CCR5 (R5 tropic) cells, and cells were incubated for 2 h at 37°C. The cells were harvested 48 h after infection and lysed in cell culture lysis buffer (Promega). The luciferase assay was performed according to the manufacturer's recommendations (Promega).
Plasmids pVRC1012-gp140(ΔCFI) (HXB2/BaL chimera) and pVRC1012-gp145(ΔCFI) (HXB2/BaL chimera) have been described previously (7
). To make gp140(ΔCFI)(ΔV1
) and gp145(ΔCFI)(ΔV1
), we performed PCR to amplify an XbaI/NheI fragment covering ATG and the boundary of the V1 loop using primers 5′CCTCTAGACACCATGCGCGTGAAGGAGAAG3′ and 5′CCGCTAGCGTCGGTGCACTTCAGGCTCACGCACAGGGG3′ and an NheI/ApaI fragment covering the 3′ boundary of the V2 loop and the C3 region using primers 5′CCGCTAGCACCAGCTGCAACACCAGCGTGATCACCCAG3′ and 5′GGTGCAGGGGCCCTTGCCGTTGAACTTCTT3′. The XbaI/NheI- and NheI/ApaI-digested PCR fragments were cloned into XbaI/ApaI-digested pVRC1012-gp140(ΔCFI) and pVRC1012-gp145(ΔCFI). The resulting plasmids, pVRC1012-gp140(ΔV1
) and pVRC1012-gp145(ΔCFI)(ΔV1
), have deletions of the V1 and V2 loops as follows: CTDASTSC. Two extra amino acids (AS) were introduced due to the introduction of an NheI site. A similar approach was used to make other V loop deletion mutants of gp145ΔCFI (HXB2/BaL chimera) and gp140ΔCFI (HXB2/BaL chimera). The amino acid sequences of deleted V loops are as follows: ΔV1, CTDASKNC; ΔV2, CSFASTSC; ΔV3, CTRASAHC; and ΔV4, CNSASLPC.
V3 deletion mutants were made by using the PCR-based Quickchange (Stratagene, La Jolla, Calif.) method according to the manufacturer's instructions. Each mutant was confirmed by double strain sequencing. An ApaI/SexAI fragment containing each confirmed V3 deletion was swapped with a corresponding fragment in pVRC1012-gp140(ΔCFI)(ΔV1
) and pVRC1012-gp145(ΔCFI)(ΔV1
). The cDNA encoding gp160(89.6P)(KB9) (11
) was synthesized by using human preferred codons. Plasmids expressing different V3 deletion mutants of gp160(89.6P) were made similarly and are shown in Fig. . The details for each V3 mutant are listed in Fig. .
FIG. 1. Schematic representation of Env mutations. (A) The major structural motifs in HIV Env are shown, together with the selected expression vectors used in these studies. V1, V2, V3, and V4 indicate the respective variable regions, and the sequence of the (more ...)
FIG. 2. Mutations in the stem of the V3 loop and protein expression of various gp145ΔCFI (HXB2/BaL chimera) V3 deletion mutants. (A) Sequences of progressive V3 stem deletion mutations in Env from the HXB2/BaL chimera. (B) Protein expression of gp145ΔCFI (more ...) ELISA.
Guinea pig anti-HIV gp140(ΔCFI) ELISA titers were measured by using a modified lectin capture method. Briefly, Immunon 2HB ELISA plates (Thermo Labsystems, Franklin, Mass.) were coated with 100 μl of Galanthus nivalis lectin (Sigma, St. Louis, Mo.) (10 μg/ml in PBS)/well overnight at 4°C. The plates were blocked with 200 μl of PBS containing 10% FBS for 2 h at room temperature and washed twice with PBS containing 0.2% Tween 20 (PBS-T). One hundred microliters of tissue culture supernatant from pVRC 1012-gp140(ΔCFI)-transfected 293 cells was added to each well and incubated at room temperature for 1 h. The plates were washed five times with PBS-T. One-hundred-microliter serial dilutions of guinea pig immune serum in PBS containing 1% FBS were then added in triplicate and incubated for 1 h at room temperature. After five washes with PBS-T, 100 μl of horseradish peroxidase-conjugated F(ab)′2 donkey anti-guinea pig IgG (1:5,000) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) in PBS plus 1% FBS was added to each well and incubated for 1 h at room temperature. The plates were washed five times with PBS-T, developed by the addition of 100 μl of o-phenylenediamine dihydrochloride (Sigma) (one gold and one silver tablet in 20 ml of water), and incubated at room temperature for 30 min. The reaction was stopped by the addition of 100 μl of 1 N H2SO4 to each well. The readout was measured at 450 nm by a SPECTRAmax plate reader (Molecular Devices, Sunnyvale, Calif.). The end-point dilution was calculated by picking the dilution for which the readout was above that of the 1:100 dilution of preimmune serum.
The single-round intracellular p24 antigen flow cytometric HIV-1 neutralization assay has been described previously (16
). Briefly, 40 μl of virus stock was incubated with 10 μl of heat-inactivated guinea pig immune serum (multiplicity of infection, approximately 0.1). After incubation for 30 min at 37°C, 20 μl of PBMC (1.5 × 105
cells) was added to each well. PBMC were maintained in IL-2 culture medium containing 1 μM indinavir, and the cells were fed on day 1 with 150 μl of IL-2 culture medium containing indinavir. One day after infection, cells were stained for intracellular p24 antigen with the KC57 anti-p24 antibody, followed by the quantitation of HIV-1-infected cells by flow cytometry. The percent neutralization was defined as the reduction in the number of p24-positive cells compared with the number for wells incubated with the corresponding preimmune serum.
To obtain 50% inhibitory concentration (IC50) and IC80 data, we incubated serial dilutions of antiserum with the virus, as described above. Antiserum dose-response curves were fit with a nonlinear function, and the IC50 and IC80 of the virus were calculated by a least-squares regression analysis. Statistical analysis of IC50 titers was performed with the nonparametric Mann-Whitney rank-order test (GraphPad Prism software package 3.0; GraphPad Software Inc., San Diego, Calif.).
Guinea pigs were immunized intramuscularly with 500 μg (in 400 μl of PBS) of the gp145 version of plasmid DNA at weeks 0, 2, and 6. At week 14, the guinea pigs were boosted with 1011 (in 400 μl of PBS) particles of recombinant Ad expressing the corresponding gp140 version of the protein. Sera were collected at weeks −2 and 16, divided into aliquots, and frozen at −20°C.
293 cells were transfected with plasmid DNA expressing each immunogen by the calcium phosphate method performed according to the manufacturer's instructions (Invitrogen). Forty-eight hours after transfection, the cells were harvested, washed once with PBS, resuspended in lysis buffer (50 mM HEPES [pH 7.0], 150 mM NaCl, 1% NP-40, 1× proteinase inhibitor cocktail), and incubated on ice for 45 min. The cell lysate was centrifuged at 16,000 × g for 10 min at 4°C. The supernatant was collected, and the protein concentration was measured. Twenty micrograms of protein was mixed with 2× sample loading buffer (100 mM Tris, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol, 0.2% bromophenol blue) and boiled for 5 min. The sample was then resolved by 4 to 15% gradient SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked twice with Tris-buffered saline containing 0.3% Tween 20, 5% skim milk, and 1% bovine serum albumin at room temperature for 10 min, followed by incubation with the 2F5 antibody (1:2,500) in blocking buffer for 1 h at room temperature. The membrane was washed twice with 100 ml of Tris-buffered saline containing 0.3% Tween 20, followed by incubation with horseradish peroxidase-conjugated goat anti-human IgG (Chemicon, Temecula, Calif.) (1:5,000) for 30 min at room temperature. After two washes with 100 ml of washing buffer, the membrane was developed by use of ECL Western blotting detection reagents (Amersham, Piscataway, N.J.) and was exposed on Hyperfilm ECL (Amersham).