The HIV-1 gp120 CD4-Bound Conformation Is Preferentially Targeted by Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Sera from HIV-1-Infected Individuals

293T human embryonic kidney and HOS cell lines (obtained from the ATCC and NIH AIDS Research and Reference Reagent Program, respectively) were grown at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Sigma) and 100 μg/ml of penicillin-streptomycin (Wysent). CEM.NKr cells (obtained from David Evans, Harvard Medical School) were grown at 37°C and 5% CO₂ in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum and 100 μg/ml primocine (Invitrogen).

Ficoll density gradient isolated and cryopreserved human peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed and kept at 37°C and 5% CO₂ in RPMI 1640 containing 10% FBS and 100 μg/ml penicillin-streptomycin for at least 16 h before subsequent experiments.

Mutations were introduced into the previously described pNL43-ADA-GFP.IRES.Nef proviral vector (23). To generate env mutants, the SalI-BamHI fragment of pNL43-ADA-GFP.IRES.Nef was subcloned in a pUC19 intermediate before being subjected to site-directed mutagenesis using the QuikChange II XL protocol (Stratagene). The mutated insert was then cloned back into pNL43-ADA-GFP.IRES.Nef. Mutations in nef were introduced by a two-step PCR strategy using primers having 18-nucleotide overlaps and cloned back into the proviral construct using XhoI and NcoI restriction sites. All mutations were confirmed by Sanger DNA sequencing. The codon-optimized pcDNA3.1-HIV-1_YU2 ΔV1V2V3V5 expression construct was made by replacing the sequence encoding residues 124 to 198 from the V1/V2 loop with a sequence encoding a GG linker and the sequence encoding residues 302 to 323 from the V3 loop with a sequence encoding a GGSGSG linker (24). ΔV5 was made by replacing residues 460 to 465 with a GSG linker into pcDNA3.1-HIV-1_YU2 ΔV1V2V3. The D368R mutation was introduced into pcDNA3.1-HIV-1_YU2 V1V2V3V5 by site-directed mutagenesis as described above.

Informed consent was obtained from all study participants (the Montreal Primary HIV Infection Cohort [25, 26] and the Canadian Cohort of HIV-Infected Slow Progressors [27–29]), and research adhered to the ethical guidelines of CRCHUM. Sera were collected during Ficoll isolation of PBMCs and conserved at −80°C. Serum aliquots were heat inactivated for 30 min at 56°C and stored at 4°C until they were used in subsequent experiments. A random-number generator (GraphPad QuickCalcs) was used to randomly select a number of sera from each cohort.

FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 10⁶ cells/ml at 37°C with 8% CO₂ with regular agitation (125 rpm). Cells were transfected with a pCDNA3.1 plasmid encoding codon-optimized His₆-tagged wild-type (wt) or mutant HIV-1 YU2 gp120 using the 293Fectin reagent as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. The supernatants were filtered (0.22-μm-pore-size filter) (Corning), and the gp120 glycoproteins were purified by nickel affinity columns according to the manufacturer's instructions (Invitrogen). The gp120 preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80°C. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie blue.

Detection of trimeric Env at the surface of HOS cells was performed by cell-based ELISA, as previously described (18, 30, 31). Briefly, HOS cells were seeded in 96-well plates (2 × 10⁴ cells per well) and transfected the next day with a cytoplasmic tail-deleted HIV-1 Env_YU2 variant alone or together with a human CD4 expressor using a standard polyethylenimine transfection method. Two days later, transfected cells were washed and then incubated with 1:1,000 dilutions of sera from HIV-infected cells or 1 μg/ml of relevant MAbs. Env-specific IgGs were detected using a horseradish peroxidase (HRP)-conjugated anti-human IgG-specific secondary Ab (Pierce) with a TriStar LB 941 luminometer (Berthold Technologies).

Vesicular stomatitis virus G (VSV-G)-pseudotyped NL4.3 green fluorescent protein (GFP)-encoding ADA-Env-based viruses were produced as previously described (18). Briefly, our panel of pNL4.3-GFP-ADA-based HIV-1 proviral vectors and VSV-G-encoding plasmid were cotransfected in 293T cells by standard calcium phosphate transfection. Two days after transfection, cell supernatants were harvested, clarified by low-speed centrifugation (5 min at 1,200 rpm), and concentrated by ultracentrifugation for 1 h at 4°C at 143,260 × g over a 20% sucrose cushion. Pellets were harvested in fresh RPMI, and aliquots were stored at −80°C until use. Viral preparations were normalized before infection according to reverse transcriptase activity or using a standard 50% tissue culture infectious dose (TCID₅₀) procedure using TZM-bl cells (32). Viruses were then used to infect approximately 20% to 30% of CEM.NKr cells by spin infection at 800 × g for 1 h in 96-well plates at 25°C.

For cell surface staining, infected or mock-infected CEM.NKr cells were incubated for 20 min at room temperature 48 h postinfection with 1 μg/ml OKT4 (anti-CD4 Ab; 14-0048-82; eBioscience), 2 μg/ml BST-2 (sc-99191; Santa Cruz), 1 μg/ml 2G12 (AB002; Polymun), 1 μg/ml A32 Ab (kindly given by J. Robinson), or a 1:1,000 final dilution of serum from participants in PBS. Cells were then washed once with PBS and stained with 1 μg/ml goat anti-mouse and anti-rabbit (Alexa Fluor 594; Invitrogen) or anti-human (Alexa Fluor 647; Invitrogen) secondary Abs for 15 min in PBS. After one more PBS washing, cells were fixed in a 2% PBS-formaldehyde solution.

Measurement of serum- and A32-mediated ADCC was performed with a previously described assay (18, 33). Briefly, CEM.NKr-infected cells were stained with viability (AquaVivid; Invitrogen) and cellular (cell proliferation dye eFluor670; eBioscience) markers and used as target cells. PBMC effectors cells, stained with another cellular marker (cell proliferation dye eFluor450; eBioscience), were then mixed at an effector/target (E/T) ratio of 10:1 in 96-well V-bottom plates (Corning). A 1:1,000 final dilution of serum or 5 μg/ml of the A32 MAb was added to appropriate wells. Cocultures were centrifuged for 1 min at 300 × g and incubated at 37°C for 5 to 6 h before being fixed in a 2% PBS-formaldehyde solution containing 5 × 10⁴ flow cytometry particles/ml (AccuCount blank particles; 5.3 μm; Spherotech). Samples were analyzed on an LSRII cytometer (BD Biosciences) and acquisition was set to acquire 1,000 particles, which allows the calculation of relative cell counts. Data analysis was performed using FlowJo vX.0.7 (Tree Star). The percentage of cytotoxicity was calculated with the following formula: (relative count of GFP⁺ cells in targets plus effectors) − (relative count of GFP⁺ cells in targets plus effectors plus A32 or serum)/(relative count of GFP⁺ cells in targets) according to our previously described gating strategy (18, 33).

For serum adsorption and gp120 competition assays, serum dilutions from HIV-1-infected individuals were preincubated for 30 min at room temperature with purified soluble D368R gp120 ΔV1V2V3V5 at a concentration of 83.3 pmol/μl of serum. This concentration was determined by assaying the dose-dependent reduction in gp120-coated cell staining (see Fig. S4 in the supplemental material), as previously reported (33).

HIV-1 accessory proteins Nef and Vpu are known to modulate cell surface levels of CD4 (34, 35). In addition to its role in CD4 degradation, Vpu also antagonizes a restriction factor, Tetherin/BST-2, which normally inhibits retroviral release (36, 37). Viruses lacking Vpu remain trapped at the cell surface, resulting in an accumulation of exposed Env (18, 19). Therefore, Nef and Vpu can indirectly modulate Env-CD4 interaction at the surface of infected cells through CD4 and BST-2 downregulation (18). Accordingly, we recently reported that cells infected with viruses defective for both Nef and Vpu present enhanced levels of CD4 and Env at the cell surface, resulting in the exposure of Env CD4i epitopes (18). To address whether these epitopes were recognized at the surface of infected cells by sera from HIV-1-infected individuals, we infected CEM.NKr cells with a panel of NL4.3-GFP ADA-Env-based viruses encoding either wild-type (wt) or Nef- and Vpu-defective accessory proteins as previously described (18). Two days postinfection, infected cells were stained with sera from HIV-infected individuals and then fluorescently labeled (Fig. 1). Interestingly, as we previously reported for the ADCC-mediating A32 Ab (18), we observed that a threshold of both CD4 and Env must be reached at the cell surface, and Env must be able to engage with CD4 in order for Env to be detected by A32 or sera from HIV-1-infected individuals (Fig. 1 and 2; also see Fig. S1 and S2 in the supplemental material). The majority of the sera recognized more efficiently cells infected with a virus lacking Nef and Vpu than its wild-type counterpart. Cells infected with viruses lacking Nef and Vpu (i.e., presenting high levels of CD4 and Env at the cell surface [Fig. 2A; also see Fig. S2]) but where the ability of Env to interact with CD4 was dramatically decreased by a CD4-binding site mutation (D368R) (38, 39) were poorly recognized by sera from HIV-1-infected individuals. Of note, the decreased recognition of the D368R Env variant by sera from HIV-1-infected individuals was not due to decreased levels of CD4 and/or Env at the surface of cells infected with HIV-1 lacking nef and vpu (see Fig. S2).

The impact of Env-CD4 interaction on the binding of serum IgGs from 163 HIV-infected individuals was also evaluated by cell-based ELISA, as previously described (18, 30, 31). Briefly, HOS cells were transfected with a cytoplasmic tail-deleted HIV-1 Env_YU2 variant alone or with a human CD4 expressor as reported previously (18, 30). Two days later, transfected cells were washed and then incubated with 1:1,000 dilutions of sera from HIV-infected individuals. Env-specific IgGs were detected using an anti-human IgG-specific secondary Ab. Interestingly, the recognition of the Env trimer by sera from all of the clinical categories tested was significantly increased by the coexpression of CD4, while the outer domain, recognizing Ab 2G12, was not affected (see Fig. S3 in the supplemental material), suggesting that sera from HIV-1-infected individuals contain a significant portion of CD4i Abs, as previously proposed (40).

We previously described that Env-CD4 interaction modulates the susceptibility of HIV-1-infected cells to ADCC by CD4i Abs (18). However, whether this was also the case for sera from a large number of HIV-1-infected individuals remains unknown. Therefore, we sought to determine if the overall ADCC activity potential in sera from HIV-infected individuals would be associated with efficient Env-CD4 interaction. Using infected CEM.NKr cells as described above, we measured serum-mediated ADCC with our previously described fluorescence-activated cell sorting (FACS)-based ADCC assay (18, 33). Thus, we determined the ability of 48 randomly chosen sera (including those tested in Fig. 1B) from HIV-infected individuals to mediate ADCC against infected cells (Fig. 3). Strikingly, while cells infected with wt virus were slightly more susceptible to ADCC killing than mock-infected cells, those infected with a virus lacking Nef and Vpu were dramatically more susceptible to ADCC (Fig. 3A). Interestingly, the slight increase in ADCC killing of cells lacking Nef was similar to that obtained with cells infected with a Nef variant (L166A-L168A) unable to downregulate CD4 from the cell surface (34), further stressing the importance of CD4 downregulation to protect infected cells from ADCC. However, the increase in ADCC observed with cells lacking Nef or Vpu alone was significantly lower than ADCC levels reached with cells infected with viruses lacking both accessory proteins. Moreover, the ADCC potential of different sera correlated with their ability to recognize Env at the surface of cells infected with viruses lacking both Nef and Vpu (Fig. 3B), suggesting that the efficient recognition of HIV-1-infected cells by sera is required for their ability to mediate ADCC. Finally, introducing the CD4-binding site D368R mutation in Env dramatically decreased the sensitivity of infected cells to killing by ADCC (Fig. 3A and C), further stressing the importance of Env-CD4 interaction for exposure of epitopes recognized by ADCC-mediating Abs normally elicited in the course of HIV-1 infection.

Recent reports indicate that various vaccine-elicited Abs targeting conserved (CD4i) or variable epitopes (V1V2) as well as rare broadly neutralizing Abs can mediate ADCC against HIV-1-infected cells (17, 18, 21, 22, 41). To investigate whether, aside from gp120 CD4-induced antibodies, epitopes recognized by anti-gp41, variable regions, or quaternary-dependent antibodies contributed to serum-mediated ADCC responses, we designed an antibody competition assay using purified, soluble gp120 lacking variable regions V1, V2, V3, and V5 while presenting a D368R mutation (ΔV1V2V3V5 D368R) making it unable to bind cell surface CD4 (33). Of note, variable region 4 (V4) could not be removed without affecting the structural integrity of the protein (42 and data not shown). The preincubation of sera with either full-length or ΔV1V2V3V5 D368R gp120 recombinant proteins captured anti-Env antibodies and prevented the recognition of gp120-coated cells by serum Abs (see Fig. S4 in the supplemental material). Interestingly, this was also observed at the surface of HIV-1-infected cells where competition with the ΔV1V2V3V5 recombinant gp120 almost completely abrogated cell surface staining of cells infected with virus unable to express Nef and Vpu (Fig. 4A) and correlated with decreased ADCC activity (Fig. 4B), indicating that a recombinant protein lacking the V1V2V3V5 variable regions is sufficient to absorb the majority of ADCC activity present in sera from HIV-1-infected individuals.