Soluble trimeric gp140s (homotrimeric or heterotrimeric) were generated using envelope sequences from the clade A Envs Q168, Q259, and Q461 (7
) and the clade B Env SF162 (14
). Stop codons were introduced immediately upstream from the transmembrane regions of gp41, and the gp120/gp41 cleavage site was eliminated by mutagenesis, as previously described (28
). The gp140 sequences were inserted in the mammalian expression vectors pEMC* and COpTT3 (73
The SF162 gp140 was 6× His tagged (HHHHHH) at its carboxy terminus, while the clade A gp140s were 3× FLAG tagged (DYKDHDGDYKDHDIDYDDDDK) at their carboxy termini. Two versions of the SF162 gp140 were constructed. One version, SF162gp140L (L, linker), contained a 14-amino-acid (aa) linker peptide (SILEVLFQGPLGSP) between the C terminus of gp140 and the His tag. The other version, SF162gp140NL (NL, no linker), did not have a linker peptide, and the His tag was added directly to the C terminus of SF162 gp140. In the case of the clade A gp140s, the FLAG tags were added to the C terminus of gp140 without a linker.
Transfection of 293F cells.
gp140 proteins were produced by transient transfection of 293F suspension cells (Invitrogen, Carlsbad, CA) in serum-free medium, using a high-density transfection protocol (2
). In total, five homotrimeric (SF162L, SF162NL, Q168, Q259, and Q461) and four heterotrimeric (Q168/SF162L, Q461/SF162L, Q259/SF162NL, and Q461/SF162NL) gp140s were produced.
The transfection conditions for the production of the homotrimeric gp140 proteins were previously described (69
). For the generation of heterotrimeric gp140s, the plasmids expressing the clade A gp140 and the clade B gp140 were first mixed at a predetermined ratio that resulted in the equal expression of the two gp140s and then the mixtures of plasmids were added to the cells. For the Q168/SF162L heterotrimer, a 10:1 clade A/clade B ratio was used; for Q259/SF162NL, a clade A/clade B ratio of 1:5 was used; and for the Q461/SF162L and Q416/SF162NL heterotrimers, a 1:10 clade A/clade B ratio was used. A 6.25-mg amount of total DNA was added to 5 × 109
293 F cells in a volume of 250 ml (2 × 107
cells/ml) and swirled gently. A 12.5-mg amount of polyethylenimine Max (PEI Max) transfection reagent (Polysciences, Warrington, PA) was added, and the transfection reagents and the cells were placed on an orbital shaker at 125 rpm in a 37°C incubator. Following an incubation period of 3 h, the cell culture volume was expanded to 5 liters (1 × 106
cells/ml) and protein expression was carried out for 6 days, at which point the cell supernatants were collected and protease inhibitors [phenylmethylsulfonyl fluoride (PMSF) (0.5 mM), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) (0.5 mM), aprotinin (0.3 μM), E-64 (1 μM), pepstatin (1 μM), leupeptin (10 μM), and bestatin (1 μM) (Sigma, St. Louis, MO)] were added.
Purification of soluble trimeric gp140s.
The transfection supernatants were first clarified by centrifugation at 1,000 × g
for 10 min at room temperature (RT). The clarified supernatants of the homotrimeric gp140 transfections were then concentrated and purified by Galanthus nivalis
lectin affinity chromatography and gel filtration, as described previously (69
). The only variation from this protocol was with the SF162NL gp140 homotrimeric protein, which was not purified by lectin affinity chromatography but instead using Ni2+
Sepharose 6 Fast Flow (GE Healthcare, Waukesha, WI) chromatography followed by gel filtration on a Superdex 200PG 26/60 column (GE Healthcare).
Two different protocols were developed for the purification of the heterotrimeric gp140s (protocols I and II). Protocol I was used to purify heterotrimers containing SF162L, while protocol II was used to purify heterotrimers containing SF162NL. In protocol I, the clarified transfection supernatants were concentrated ~20× using a 30-kDa-molecular-mass cutoff (MMCO) on a tangential flow filtration device (Ultrasette 30K; Pall Life Sciences, Ann Arbor, MI) and buffer exchanged 5× into 20 mM Tris (pH 7.4), 100 mM NaCl (G. nivalis agglutinin [GNA] binding buffer). The concentrated and buffer-exchanged samples (~250 ml) were loaded at 3 ml/min onto an XK-26 column packed with 20 ml of GNA resin equilibrated with GNA binding buffer (20 mM Tris, pH 7.4, 100 mM NaCl) and washed until all unbound proteins were eliminated. The GNA-bound Env glycoproteins were eluted with 4 column volumes of GNA elution buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 1 M methyl-α-d-mannopyranoside). Env-containing fractions were pooled and concentrated to ~10 ml using a Centricon Plus 70 centrifugal concentrator (30-kDa MMCO; Millipore, Billerica, MA). They were then loaded onto the Superdex 200PG size exclusion chromatography (SEC) column equilibrated in 20 mM monobasic sodium phosphate, pH 7.5, 500 mM NaCl, and 10 mM imidazole (His tag binding buffer). The Env oligomer-containing fractions were pooled and loaded directly onto a 5-ml HisTrap FF cartridge (GE Healthcare) column at 1 ml/min. The column was washed until the absorbance at 280 nm returned to baseline. At this stage, gp140 proteins that are exclusively FLAG tagged and which do not bind to the HisTrap column are removed in the wash. Only the His-tagged proteins (homotrimeric and heterotrimeric) remain on the column. The bound Env proteins were eluted at 5 ml/min with elution buffer (20 mM sodium phosphate, pH 7.5, 500 mM NaCl, and 250 mM imidazole). The eluted Env protein-containing fractions were then buffer exchanged into 1× phosphate-buffered saline (PBS) using 10-kDa-MMCO SnakeSkin dialysis tubing (Pierce, Rockford, IL). The dialyzed elution pool from the Ni2+ column was loaded at 1 ml/min onto a 2.5-ml anti-FLAG M2 resin (Sigma, St. Louis, MO) packed in a Tricorn 10/50 column (GE Healthcare). The resin was washed until the absorbance at 280 nm returned to baseline. During this step, gp140s that are exclusively His tagged do not bind to the anti-FLAG M2 resin column and thus are removed in the wash. At this step, only the hetero-oligomeric FLAG-tagged proteins remain on the column. The bound Env proteins were eluted with PBS plus 0.5 mg/ml FLAG peptide (3× DYKDDDDK) (Sigma). The eluate was concentrated as described above (30-kDa MMCO, 4,000 × g) and loaded onto an SEC column using the same column as described above but equilibrated with PBS. This “polishing” SEC step isolates the heterotrimers while simultaneously removing the FLAG peptide. Protocol II was similar to protocol I, with the following modifications: GNA, HisTrap FF, and anti-FLAG followed by SEC.
Blue native (BN) PAGE was used to determine the fractions containing trimers (which at this point were all in a heterotrimeric configuration). These fractions were then pooled. All purified samples were analyzed by SDS-PAGE, blue native PAGE, and standard and native Western blotting. The bicinchoninic acid (BCA) assay (Pierce) was used to determine the protein concentration using bovine serum albumin (BSA) as a standard.
Two micrograms of purified homotrimeric gp140 Env and 4 μg of heterotrimeric gp140 Env were loaded per lane of a 4 to 12% NuPAGE gel (Invitrogen). Electrophoresis took place at 200 V for 60 min. Proteins were visualized with Simply Blue stain (Invitrogen).
Blue native PAGE.
BN PAGE electrophoreses were performed as follows: 2.5 μg/well of purified homotrimeric gp140 proteins and 5 μg/well of purified heterotrimeric gp140 proteins in PBS were mixed 3:1 (vol/vol) with 4× native PAGE loading buffer and loaded on a 3 to 12% native gel (Invitrogen). Electrophoresis assays were run at 150 V for 2 h. The gels were fixed for 15 min (40% methanol [MeOH], 10% acetic acid) and then destained (8% acetic acid) for 2 to 3 h.
Native Env Western blotting.
Following native PAGE and destaining, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (VWR International) at 70 V for 50 min in 1× transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol). After the molecular weight markers were marked with pencil, the PVDF membranes were blocked in 5% nonfat milk (NFM)-0.2% Tween 20 for 1 h at room temperature (RT). Different primary antibodies were used to assess the composition of the heterotrimeric Envs: anti-FLAG M2-horseradish peroxidase (HRP) MAb (1:1,000) (Sigma), tetra-His MAb (1:2,500) (Qiagen, Valencia, CA), or purified IgG from several HIV-1-infected individuals (1:2,000). The PVDF membranes were incubated with primary antibody overnight (O/N) at 4°C. The membranes were then washed 5× for 5 min each in wash buffer (1× PBS, 0.6% Tween 20). In the case of anti-FLAG M2-HRP, development took place at this stage using ECL Western blotting detection reagent (GE Healthcare) mixed 1:1. For the purified anti-HIV+ IgG and anti-His blot analyses, the secondary antibodies, rabbit anti-human-HRP (1:3,000) and goat anti-mouse-HRP (1:3,000), respectively, were added and incubated for 60 min at RT with shaking. After a final 5× wash, the blots were developed as described above.
Apparent molecular mass determination.
The apparent molecular masses of the homotrimeric and heterotrimeric Env proteins were determined by analytical SEC using a Superdex 200 10/300 GL column (GE Healthcare) with the following proteins as standards: thyroglobulin (669 kDa), apoferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and BSA (67 kDa). All standard proteins were from Sigma except lactate dehydrogenase (Lee BioSolutions, Inc., St. Louis, MO). The column was run at 0.75 ml/min in 1× PBS, and 100 μg of Env protein in 100 μl (1× PBS) was injected. A standard curve was generated by measuring the elution volumes of the standard proteins and then plotting their Kav values. Kav values were plotted against the logarithm of the corresponding molecular mass. Next, the molecular masses of the unknown Env proteins were determined from the calibration curve after calculating their respective Kav value from the measured elution volume by using the following equation: Kav = Ve − V0/Vt − V0, where Ve is the elution volume, V0 is the void volume of the column, and Vt is the column bed volume.
Relative binding of known neutralizing antibodies to gp140s.
The following anti-Env reagents were used: 2F5 and 4E10 (anti-gp41 membrane-proximal external region [MPER]; Polymun Scientific, Vienna, Austria), 2G12 (anti-terminal mannose residues on gp120; Polymun Scientific), VRC01 (anti-CD4 binding site [BS]; kindly provided by J. Mascola), b12 (anti-CD4-BS; Polymun Scientific), IgG-CD4 (anti-CD4-BS; Progenics Pharmaceuticals Inc., Tarrytown, NY), and 17b (anti-coreceptor binding site; kindly provided by J. Robinson). For these experiments, the same molar concentrations of homotrimeric and heterotrimeric gp140 were adsorbed on enzyme-linked immunosorbent assay (ELISA) plates. gp140 proteins were added in duplicate wells of 96-well high-binding polystyrene plates (Immulon 2Hb; Thermo Scientific) (10 nM in the first well) and then serially diluted 3-fold in NaHCO3 (pH 9.5). Following an O/N incubation at RT, the plates were washed 3× with wash buffer (imidazole-buffered saline with Tween 20; KPL, Gaithersburg, MD) using a Skan Washer 300 (Molecular Devices, Sunnyvale, CA) and then blocked with ELISA blocking buffer (PBS, 10% nonfat milk, and 0.3% Tween 20) for 1 h at 37°C. The plates were again washed, and monoclonal antibody (MAb) was added in each well in ELISA dilution buffer (PBS, 10% nonfat milk, and 0.03% Tween 20) and incubated for 90 min at 37°C. The plates were then washed, and the secondary antibody goat-anti-human-HRP (1:3,000) was added for 90 min at 37°C. ELISA 1-Step Ultra-TMB reagent (Thermo Scientific, Waltham, MA) was added to each well for a 4-min development. The reaction was then quenched with 1 M H2SO4. The graphed data compare the absorbances at 450 nm at the highest Env concentration (10 nM).
Rabbit immunizations with gp140s.
Immunizations took place at the Pocono Rabbit Farm (Canadensis, PA). Animals were immunized with one of the following four heterotrimeric gp140s: Q168/SF162L, Q461/SF162L, Q259/SF162NL, and Q461/SF162NL. In each case, a second group of animals was immunized with the corresponding mixture of homotrimeric gp140s. Rabbits (n = 3) were immunized with 0.2 ml of a mixture of 100 μg of protein and 1 mg of 25-kDa branched polyethylenimine (Polysciences) in PBS. An 0.1-ml amount of this formulation was administered intramuscularly in each hind leg. Immunizations took place every 4 weeks (0, 4, 8, 12, and 16 weeks) with the exception of the Q461/SF162L and Q461+SF162L constructs, which were administered eight times (0, 6, 9, 21, 28, 32, 52, and 57 weeks). Blood was collected at −1, 0, 6, 10, 14, and 18 weeks for the three groups with 5 immunizations. Blood was collected at −1, 0, 8, 11, 23, 30, 34, 54, and 59 weeks for the study with 8 immunizations.
Relative endpoint antibody titers.
The relative endpoint antibody titers against the corresponding Env gp140 were determined by ELISA. Briefly, 50 ng/well of Env antigen was added O/N at RT in 100 mM NaHCO3, pH 9.5, in 96-well plates. Following washing, 5-fold-serially diluted rabbit sera were added for 90 min at 37°C. After washing, secondary antibody (1:3,000; goat anti-rabbit) was added for 60 min at RT. Endpoint titers were reported as the dilution at which the titrated curve intersected the 3× prebleed values.
Luminex multiplex assay.
Peptides derived from the V1, V2, and V3 regions of each Env gp140 tested as immunogens were individually amine coupled through a lysine at their carboxyl termini to Bioplex beads (Bio-Rad, Hercules, CA), according to the manufacturer's instructions (12 μg of peptide per 100 μl of beads). Peptide sequences are found in . Serially diluted sera (pre- and postimmunization) were incubated with the cocktail of beads for 1 h at RT. Following 4 washes, the beads were incubated with anti-rabbit-phycoerythrin (PE)-conjugated secondary MAb (1:500) for 1 h at room temperature. A final wash step was performed prior to analysis on a Luminex 200 system (Invitrogen).
V1, V2, and V3 loop peptides used for epitope mapping of immune sera
Streptavidin (SA; 50 ng/well) was applied as a coating onto high-binding polystyrene ELISA plates O/N at RT in 100 mM NaHCO3, pH 9.5. Plates were then washed prior to blocking with 3% BSA in PBS for 1 h at 37°C. Biotinylated peptides derived from the MPER of gp41 (2F5, EQELLELDKWASLWN, and 4E10, NWFDITNWLWYIRKKK) were added at 0.2 μg/well (100 μl of 2 μg/ml) for 1 h at 37°C in PBS plus 0.2% BSA. After washing, 5-fold-serially diluted rabbit serum was added for 1 h at 37°C in PBS plus 0.2% BSA. Goat anti-rabbit secondary antibody was added at 1:5,000 in PBS plus 0.2% BSA and incubated for 1 h at 37°C before development as described above.
AviTag peptides were synthesized by Genscript (Piscataway, NJ) so that targeted, residue-specific biotinylation could be accomplished in addition to presenting the affinity tag optimally in solution using an ELISA format. The peptides were designed with the AviTag at the N terminus adjacent to a 6-aa soluble linker and an alpha-helix (EAAAK)4 to control tag orientation, followed by a 6-aa soluble linker with the affinity tag comprising the C terminus. The AviTag peptides were enzymatically biotinylated (Genecopia, Rockville, MD), and the biotinylated peptides were purified using the appropriate affinity resin (FLAG-M2 for the biotinylated FLAG AviTag peptide or Sepharose 6 Fast Flow for the biotinylated His AviTag peptide). Purified peptides were then concentrated and buffer exchanged into PBS using a 3,000-molecular-weight-cutoff (MWCO) centrifugal concentrator. Peptide concentrations were determined by BCA assay (Pierce, Rockford, IL). ELISA plates were coated with biotinylated peptide as described above. Threefold-serially diluted rabbit sera were added and incubated at 37°C in PBS plus 0.2% BSA for 1 h. Secondary antibody treatment and development of ELISA plates were done as described above for the biotinylated ELISA.
Neutralization assays were performed at the Stamatatos, Montefiori, and Seaman laboratories using single-round entry-competent viruses and TZM-bl cells as targets. The protocol used at the Stamatatos laboratory was previously described (20
). Briefly, a predetermined amount of virus (2 × 105
relative luminescence units [RLU]) was mixed (1:1) with a single serum dilution (1:20, final) at 37°C for 1.5 h; total volumes in duplicate wells were 60 μl. Fifty microliters of the virus/serum mixture was transferred to wells of flat-bottomed 96-well tissue culture plates containing 3 × 103
Polybrene-treated TZM-bl cells. Following a 3-day incubation at 37°C, the cell supernatants were removed and 100 μl of SteadyGlo luciferase (Promega) was added to each well for 15 min at room temperature. The number of relative luminescence units associated with 75 μl of cell lysate was determined on a Fluoroskan Ascent FL (Thermo Labsystems). Percent neutralization at 1:20 serum dilution was determined for each immune serum against the corresponding serum collected prior to immunization, based on the following equation: (RLU preimmunization − RLU postimmunization/RLU preimmunization) × 100.
The Montefiori and Seaman laboratory neutralization protocols were described previously (49
). Briefly, 200 tissue culture infective doses (TCID) of virus was incubated with serial 3-fold dilutions of test sample in duplicate in a total volume of 150 μl for 1 h at 37°C in 96-well flat-bottomed culture plates. Freshly trypsinized cells (10,000 cells in 100 μl of growth medium containing 75 μg/ml DEAE dextran) were added to each well. One set of control wells received cells plus virus (virus control), and another set received cells only (background control). After a 48-h incubation, 100 μl of cells was transferred to a 96-well black solid plate (Costar) for measurements of luminescence using the Britelite luminescence reporter gene assay system (PerkinElmer Life Sciences). Neutralization titers are the dilution at which relative luminescence units (RLU) were reduced by 50% compared to those of virus control wells after subtraction of background RLU. Assay stocks of molecularly cloned Env-pseudotyped viruses were prepared by transfection in 293T cells and were titrated in TZM-bl cells as described previously (49
The following viruses (clade) were tested: tier 1, MN.3 (B), SF162.LS (B), HxB2 (B), W61D (TCLA) 0.71 (B), Bal.26 (B), BZ167.12 (B), Bx08.16 (B), SS1196.1 (B), MW965.26 (A), Q461d1 (A), Q168b23 (A), DJ263.8 (A), Q259w6 (A), Q769.b9 (A), TV1.21 (C), and 92BR025.9 (C); tier 2, 3988.25 (B), REJO4541.67 (B), TRO.11 (B), ADA (B), JRFL (B), YU2 (B), 89.6 (B), 6535.3 (B), 7165 (B), THRO4156.18 (B), AC10.0.29 (B), QH0692.42 (B), AC10.0.29 (B), RHPA4259.7 (B), WITO4160.33 (B), Q23.17 (A), Q842.D12 (A), 3365.v2.c20 (A), 3415.v1.c1 (A), 0260.v5.c1 (A), 191955_A11 (A), 191084 B7-19 (A), 9004SS_A3A4 (A), Q168a2 (A), Q461e2 (A), Q769h5 (A), Du422.1 (C), ZM197M.PB7 (C), ZM214M.PL15 (C), ZM233M.PB6 (C), ZM249M.PL1 (C), Q259d2.17 (A), CAP210.2.00.E8 (C), Du156.12 (C), Du172.17 (C), CAP45.2.00.G3 (C), and ZM53M.PB12 (C); and tier 3, PVO.4 (B).
Statistical analyses to differentiate the neutralizing antibody responses elicited by the heterotrimers and the homotrimers were performed with Welch's t test.