Identification of bNAbs that kill HIV-1 infected lymphocytes

We examined the ADCC activity of bNAbs against HIV-1-infected cells. We first investigated the ability of a panel of ten anti-HIV-1 bNAbs to induce signalling through FcγRIII (or CD16). The FcγRIII is the main receptor on NK cells that detects antibody-opsonized targets, and initiates the signalling that leads to ADCC. We previously showed that most of the selected bNAbs neutralize HIV-1 cell-to-cell transmission7. These antibodies are IgG1 and contain the same Fc region. They target the CD4-binding site (VRC01, NIH 45–46, 3BNC117, 12A12), the glycan-dependent V1/V2 loops (PG16), the V3 loop (PGT121, 10–1074), the gp120/gp41 interface (8ANC195) and the gp41 membrane-proximal external region (MPER)(10E8 and 4E10)2,3,4,5,28,29. As controls, we added two non-bNAbs antibodies, 5–25 (recognizing the gp41 immuno-dominant epitope) and 11–340 (a cross-neutralizing anti-V3 crown isolated from an elite neutralizer)30. To determine how the antibodies bridge HIV-infected cells to FcγRIII-expressing cells, CD4+ lymphoid cells (MT4) infected with the prototypic R5-tropic NLAD8 or X4-tropic NL4.3 HIV-1 were incubated with bNAbs before co-culture with Jurkat NFAT-luc FcγRIII cells, which express an NFAT-luciferase reporter activated by FcγRIII stimulation23. NLAD8-infected cells induced FcγRIII stimulation with 8 out of 12 antibodies, with variable efficiencies (EC 50 varying from 0.015 to 4.2 μg ml−1 for the active antibodies; Fig. 1a and Supplementary Fig. 1B). Similar results were obtained with NL4.3, with the exception of V3-specific bNAbs, which were poorly active (Fig. 1a and Supplementary Fig. 1A).

Figure 1: Analysis of the ADCC activity of bNAbs. (a) Analysis of the ability of bNAbs bound to infected cells to signal through FcγRIII. MT4C5 cells infected with HIV-1 (NLAD8 or NL4.3 strains) were incubated with the indicated antibodies and with a Jurkat indicator cell line expressing FcγRIII. Upon FcγRIII binding, activation of the NFAT transcription factor induces luciferase. Raw results are presented in Supplementary Fig. 1. The heat map represents the fold increase of the signal over background. (b) CEM-NKR cells infected with HIV-1 (NL4.3 strain) were incubated with NIH45–46 bNAb or with the mGO53 isotype antibody and with NK cells. After 4 h, the % of Gag+ CEM-NKR target cells (indicated in blue) was measured by flow cytometry. One representative experiment (out of six) is shown. FSC, forward scatter. (c) The viability of infected CEM-NK cells was assessed by flow cytometry using the live/dead cell marker. One representative experiment (out of six) is shown. (d) CEM-NKR cells infected with NL4–3 encoding IRES-GFP were incubated with NIH45–46 bNAb and plated with primary NK cells. To distinguish dead cells, DAPI dye was added and cells were imaged by time-lapse microscopy. The green cell represents infected live CEM-NKR cells, and turn blue when dying (blue arrow). NK cells are smaller in size. One representative field (corresponding to Supplementary Movie 1) is shown. The arrow indicates a contact between CEM-NKR and NK cells. Scale bar, 2 μm. Full size image

We then asked whether FcγRIII signalling was associated with a killing activity of bNAbs. We first assessed the activity of NIH45–46, to determine the optimal conditions of the assay. CEM-NKR cells infected with NLAD8 or NL4.3 were pre-incubated with NIH45–46 before co-culture with NK cells for 4 h. We evaluated the disappearance of Gag+ target cells, as readout for ADCC activity (Fig. 1b). A typical experiment showed that the unrelated control antibody mGO53 was inactive, whereas NIH45–46 (at 1.5 μg ml−1) induced the disappearance of about 40% of NL4.3-infected cells (Fig. 1b). This disappearance was primarily due to killing, as demonstrated by the presence of dying Gag+ cells (Fig. 1c), and by upregulation of the degranulation marker CD107a on NK cells (Supplementary Fig. 1C). Of note, the disappearance of Gag+ cells was not due to the neutralization activity of the bNAbs, as no decrease of Gag+ cells was observed when NK cells were omitted in the co-culture (Supplementary Fig. 1D). We also visualized the killing of infected cells using time-lapse microscopy. The addition of 4,6-diamidino-2-phenylindole (DAPI), which stains the nucleus of dying cells, allowed us to monitor in real time the fate of cells infected with an IRES-GFP-encoding NL4.3 HIV-1. In the presence of NIH45–46, infected cells rapidly changed morphology and stained for DAPI following interaction with NK cells (see an example Fig. 1d and Supplementary Movie 1). Of note, the non-infected bystander cells present in the co-culture were not killed by the bNAb (Fig. 1d and Supplementary Movie 1).

We next evaluated the ADCC capacity of the full panel of antibodies (Fig. 2). To facilitate comparisons, the antibodies were first used at 1.5 μg ml−1. Most of the 12 antibodies (including 6 with NLAD8 and 4 bNAbs with NL4.3) triggered a significant disappearance of infected cells (20–50% decrease of Gag+ cells in 4 h). The most active bNAbs corresponded to those which efficiently induced FcγRIII stimulation: NIH45–46 and 3BNC117, which target the CD4bs, the clonally related anti-glycan/V3 antibodies 10–1074 and PGT121, and the MPER targeting 10E8. In contrast, other bNAbs were less active (PG16, VRC01) or inactive (12A12, 4E10 and 8ANC195). The disappearance of Gag-expressing cells required interaction with FcRs, as demonstrated using the L234A-L235A (LALA) mutation, which abrogates FcR binding9,23. The LALA mutants of five bNAbs maintained their ability to neutralize HIV-1 virions, and hence to bind Env, but lost ADCC potency (Supplementary Fig. 2A). Altogether, there results indicate that only a fraction of the bNAbs induces FcγRIII stimulation and killing of HIV-infected cells.

Figure 2: Identification of bNAbs that kill HIV-1-infected lymphocytes. The 12 indicated antibodies were tested at 1.5 μg ml−1 on CEM-NKR cells infected with NLAD8 or NL4.3 strains. ADCC was calculated as the disappearance of Gag+ cells with or without antibody (N=6–8 experiments). Each dot represents a single NK donor. Significance was determined by comparing each antibody to mGO53; ***P<0.001; **P<0.01; *P<0.05, Wilcoxon test). Full size image

Binding of bNAbs at the surface of HIV-1-infected T cells

To examine the mechanism of ADCC by bNAbs, we assayed their ability to bind HIV-1-infected cells. As previously shown with sera from infected individuals19,22,31, flow cytometry indicated that the bNAbs displaying strong ADCC activity efficiently bound (at 4 °C) HIV-1-infected lymphocytes (Fig. 3a,b and Supplementary Fig. 2B for the gating strategy). The bNAbs primarily bound to Gag+ cells and not to bystander cells. Up to 70% of Gag+ cells exposed detectable Env epitopes, when the antibodies were used at the highest concentration of 15 μg ml−1. The steady-state levels (corresponding to the Median Fluorescence Intensity) varied with each bNAb (Fig. 3c). In contrast, ADCC-inactive bNAbs 4E10 and 8ANC195 did not detectably bind infected cells (Fig. 3). PGT121, which displayed ADCC activity against NLAD8 and not NL4.3, selectively bound NLAD8-infected cells. Of note, with NL4.3, a ‘diagonal’ intermediate population, which corresponded to Gag-low cells, was detected with two antibodies (5–25 and 10E8; Fig. 3). This diagonal population was not observed in non-infected cells. It may correspond to cells infected at low levels, and/or to cells which may have recently bound incoming viral particles and expose epitopes recognized by these antibodies.

Figure 3: Binding of bNAbs at the surface of HIV-1-infected lymphocytes. (a) CEM-NKR cells infected with HIV-1 (NLAD8 or NL4.3) were incubated with the indicated bNAbs (15 μg ml−1) at 4 °C and surface levels were analysed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. One representative experiment (out of six) is shown. The gates were first set on the staining obtained with the mGO53 isotype control. For the bNAbs displaying background staining to the fraction of Gag-negative cells (PGT121 and 8ANC195), the gates were adjusted to decrease this background. (b,c) The binding of the 12 indicated antibodies to the surface of CEM-NKR cells infected with HIV-1 NLAD8 (blue) or NL4.3 (red) was determined by flow cytometry. The antibodies are classified according to their competence to eliminate (ADCC+: >20%) or not (ADCC−:<20%) infected cells in the ADCC assay. (b) Results and expressed as the % of Env+ cells among Gag+ cells. (c) The median fluorescence intensity (MFI) of staining among Gag+ cells is shown (N=3 experiments; error bars indicate s.e.m. and significance was determined by comparing stainings to non-infected (NI) cells, *P<0.05, Mann–Whitney test). Full size image

To document the binding and killing activities, we tested the antibodies individually at concentrations varying from 0.015 to 15 μg ml−1 (Fig. 4). Binding on CEM-NKR cells infected with NLAD8 or NL4.3 was performed either at 4 °C to assess the steady-state levels of Env epitope exposure, or at 37 °C to reflect the experimental conditions of the ADCC assay. With each antibody, the % of infected cells positive for bNAb binding increased with the concentration and often reached a plateau at 1.5–15 μg ml−1 (Fig. 4). As expected, binding was generally more efficient at 37 °C than at 4 °C. This was particularly marked with 10E8, which barely bound infected cells at 4 °C, but displayed significant opsonization at 37 °C. Exceptions were also observed with 10–1074. This antibody-bound NL4.3-infected cells more efficiently at 4 °C than at 37 °C. In contrast, a strong binding occurred at both temperatures with NLAD8-infected cells. The killing activity of the antibodies increased with the concentration and mirrored binding at 37 °C (Fig. 4). A Spearman rank analysis indicated that the two variables were often correlated (Supplementary Table 1). Again, rare discrepancies were detected. 10–1074 potently killed cells infected with NLAD8, and not with NL4.3, despite binding to the later at the highest concentration tested. The same situation was observed with PG16, which poorly bound NL4.3-infected cells but did not display detectable ADCC activity (Fig. 4). Of note, the % of bNAb+-infected cells (Fig. 4) mirrored the intensity of binding (MFI), which also increased with the concentration of antibody tested (Supplementary Fig. 3).

Figure 4: Dose–response analysis of binding of bNAbs and ADCC activity against HIV-1-infected lymphocytes. CEM-NKR cells infected with HIV-1 NLAD8 (a) or NL4.3 (b) were incubated with the indicated concentrations of antibodies at 4 or 37 °C and surface levels were analysed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells (left axis) and the % of ADCC (right axis). ADCC y axes were adjusted for each antibody to facilitate comparisons with the binding profile. For measurement of ADCC, HIV-1-infected CEM-NKR cells were incubated with the indicated antibodies and with NK cells. After 4 h, the % of Gag+ CEM-NKR target cells was measured by flow cytometry. The % of ADCC was calculated as the disappearance of Gag+ cells (N=3 independent experiments for binding; killing assays were performed using at least two NK cell donors; Error bars indicate s.e.m.). Full size image

A dose–response analysis of the antibody concentration demonstrated that EC 50 , defined as the effective concentration mediating 50% of the maximal effect, were generally similar in the binding (at 37 °C) and killing assays (Fig. 4 and Supplementary Table 2). In both assays, the EC 50 varied from 0.2 to >15 μg ml−1, depending on the antibody (Fig. 4 and Supplementary Table 2). There was a significant correlation between the binding potency of the antibodies, at either 4 or 37 °C and their killing activities against CEM-NKR cells infected with NLAD8 or NL4.3 (Fig. 5). Similarly, the ADCC potency was generally correlated with the neutralizing activity of the antibodies (Fig. 5 and Supplementary Table 1). Notable exceptions include the non-bNAb 5–25, which did not neutralize NLAD8 or NL4.3 but displayed a potent ADCC activity against these viruses.

Figure 5: Correlates of ADCC activity of bNAbs. For each antibody, the efficacy of binding and of ADCC against CEM-NKR cells infected with NLAD8 or with NL43 was calculated. The neutralizing activity of the antibodies against cell-free HIV was tested in the TZM-bl assay. EC 50 (in μg ml−1), defined as the effective concentration mediating 50% of the maximal effect, are summarized in Supplementary Table 2. Correlations were analysed by Spearman correlation coefficient (r). Full size image

To visualize the binding of bNAbs on infected cells, we performed immunofluorescent microscopy (Supplementary Fig. 4A) and found a co-localization of NIH45–46 with mature Gag (p17) proteins, whereas immuno-gold staining and scanning electron microscopy (Supplementary Fig. 4B) indicated a preferential binding of the bNAb to viral budding sites.

Stability of bNAbs at the surface of HIV-1-infected T cells

We then measured the cell surface stability of Env-bNAb complexes at 37 °C, an additional parameter that we suspect regulates the sensitivity of infected cells to ADCC. We selected 10–1074 and PG16, which both display an ADCC activity against NLAD8 but not against NL4.3, as well as NIH45–46, which is active against both viruses. Infected cells were stained with each bNAb at 4 °C, extensively washed and the level of remaining complexes was measured at different times at 37 °C (Fig. 6). 10–1074 bound both NLAD8- and NL4.3-infected cells, the latter less efficiently (73 and 47% bNAb+ cells at steady-state levels, respectively, Fig. 6a). With NLAD8, the bNAb-Env complex was relatively stable at the cell surface, with a half-life of 2.5 h (Fig. 6a,b). This longevity was strikingly reduced with NL4.3 (half-life of 30 min), consistent with the higher binding of 10–1074 to NL4.3-infected cells at 4 °C than at 37 °C (Fig. 4). Similarly, PG16-Env complexes were less stable with NL4.3 than with NLAD8 (Fig. 6b). In contrast, NIH45–46 remained stably bound with the two viruses (half-life of 2.5 h). These results, as well as those with 3BNC117 and 10E8 (Supplementary Fig. 5A), indicate that an efficient ADCC activity is associated with a sustained presence of Env-bNAb complexes at the cell surface. It is likely that the reduced surface stability of some bNAb-Env complexes results from dissociation of the bNAb due to low affinity. It will be worth exploring whether other mechanisms, including Env endocytosis or shedding of gp120, are also involved in the turnover of surface-bound bNAbs.

Figure 6: Binding and stability of bNAbs at the surface of HIV-1-infected lymphocytes. (a) CEM-NKR cells infected with HIV-1 (NLAD8 or NL4.3) were incubated with 10–1074 bNAb (at 4 °C) and surface levels were analysed by flow cytometry after the indicated incubation times at 37 °C. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. One representative experiment (out of six) is shown. (b) Decrease of surface stainings of the indicated bNAbs after incubation at 37 °C. (N=3–6 experiments; error bars indicate s.e.m. and significance was determined by comparing NLAD8- and NL4.3-infected cells ***P<0.001 extra sum-of-squares F test). Full size image

To evaluate how the affinity of a bNAb towards Env regulates its binding and killing activity, we selected 10–1074 and the related 10–1369 antibody. They target the same epitope, but displaying a fivefold difference of affinity to YU-2b gp140 trimers (KD of 4 × 10−9 and 2 × 10−8 M, respectively29). As expected, both 10–1074 and 10–1369 neutralized infection with cell-free YU-2b virions (IC 50 of 1 and 3 μg ml−1, respectively, Supplementary Fig. 5B). However, 10–1369 poorly bound to YU-2b-infected cells, and did not trigger ADCC, when compared with 10–1074 (Supplementary Fig. 5B). Thus, results obtained with these two antibodies suggest that efficient killing is associated with a stronger affinity than that necessary for inhibiting cell-free infection.

Binding and ADCC activity of bNAbs against various HIV-1

Primary HIV-1 isolates, including Transmitted/Founder (T/F) viruses may be less sensitive to neutralization by bNAbs than laboratory-adapted strains32,33. To explore the sensitivity of primary HIV-1 to ADCC, we first measured the exposure of Env epitopes at the surface of CEM-NKR cells infected with five T/F strains (WITO, THRO, REJO, CH077, RHPA)32, using five bNAbs (Fig. 7a). The T/F viruses were selected based on their ability to efficiently replicate in vitro. Levels of accessible Env epitopes were lower with T/F viruses than with NLAD8. There was a strong variability in the pattern of bNAb binding. In contrast to NLAD8, which was recognized by all five bNAbs, cells infected with T/F viruses generally bound only 1–3 antibodies (Fig. 7a). There was no single bNAb recognizing all T/F viruses. We thus mixed the five bNAbs (MixA; NIH45–46, 3BNC117, 10–1074, PG16 and 10E8, each at 1.5 μg ml−1) and analysed the ability of MixA to bind infected cells and to perform ADCC. MixA efficiently bound cells infected with NLAD8 and, to a lower extent, with T/F viruses (Fig. 7b), reflecting the profile observed with individual bNAbs. Interestingly, MixA displayed an ADCC activity against cells infected with some, but not all T/F viruses (Fig. 7c). The killing activity against WITO, REJO and THRO was less potent than with NLAD8, correlating with opsonization levels. CH040 and RHPA-infected cells, which expose lower levels of Env epitopes, were barely sensitive to killing by MixA (Fig. 7c).

Figure 7: Binding of bNAbs and ADCC activity against lymphocytes infected with various HIV-1 strains. (a) CEM-NKR cells infected with the indicated HIV-1 (NLAD8 or five Transmitted/Founder HIV-1) were incubated with the five indicated bNAbs at 4 °C and surface levels were analysed by flow cytometry. The radar plots indicate the % of bNAb+ cells among infected (Gag+) cells. One representative experiment (out of four) is shown. (b) The binding of a combination of the five bNAbs (MixA: NIH45–46; 3BNC117; 10E8; 10–1074 and PG16) to the surface of CEM-NKR cells infected with various HIV-1 (NLAD8 or five T/F HIV-1) was determined by flow cytometry among Gag+ cells. One representative experiment (out of four) is shown. (c) CEM-NKR cells infected with the indicated HIV-1 strains were incubated with MixA or with mGO53 control antibody and with NK cells. After 6 h, the % of Gag+ CEM-NKR target cells was measured by flow cytometry. The % of ADCC was calculated as the disappearance of Gag+ cells (N=6 experiments; Error bars indicate s.e.m. and significance was determined by comparing MixA to mGO53; **P<0.01; *P<0.05, Wilcoxon test). (d) The binding of MixA to the surface of primary CD4+ T cells infected with various HIV-1 (NLAD8 or five T/F HIV-1) was determined as in b. (e) The ADCC activity of MixA against primary CD4+ T cells infected with the indicated HIV-1 strains was determined as in c. Full size image

MixA-bound primary CD4+ T cells infected with T/F with variable intensities (Fig. 7d). Primary lymphocytes infected with NLAD8, and to a lower extent with T/F viruses, were killed by MixA (Fig. 7e). Thus, T/F HIV-1 isolates poorly expose Env epitopes at the surface of infected lymphocytes. When combined, bNAbs act in a complementary manner to bind infected cells, providing increased ADCC breadth against primary HIV-1 isolates. These results are in line with experiments demonstrating that bNAb combinations control HIV-1 replication in humanized mice27, and block cell–cell transmission of T/F in culture34.

Activity of bNAbs against reactivated HIV-1-infected cells

We then asked whether bNAbs could target HIV-1 produced after stimulation of lymphocytes isolated directly from patients, as bNAbs are potential therapeutic molecules that may reach the reactivated viral reservoir in HIV-1-infected individuals. We selected infected individuals under suppressive antiretroviral treatment (viral loads <40 copies per ml, see Supplementary Table 3 for details). We used a viral outgrowth assay, in which phytohaemagglutinin (PHA) treatment activates resting CD4+ T cells and induces HIV-1 spread from latently infected cells35. HIV-1 Gag+ cells started to be detected by flow cytometry at days 7–12 post reactivation in 6 individuals (out of 20 tested, Supplementary Fig. 6A) and increased over time, indicating that reactivated viruses were infectious. Cell surface Env expression was assessed with MixA or, for some of the patients, with MixB, a second cocktail of antibodies including VRC01, PGT121, 5–25 and 11–340 (see Supplementary Fig. 6B for the gating strategy). Binding with either MixA or MixB was observed with variable intensities on reactivated cells from five out of the six individuals (Fig. 8a,b and Supplementary Fig. 6C,D). In one individual (KB12), despite ongoing viral replication, no Env signal was detected with either Mix. We then tested the sensitivity of reactivated cells to ADCC, using the two bNAb combinations (Fig. 8c and Supplementary Fig. 6E). Interestingly, in cells from four out of the five patients that bound the antibodies, an ADDC activity was detected with either MixA or MixB, leading to the disappearance of 10–50% of Gag+ cells. Thus, there is a strong heterogeneity in the levels of Env epitopes expressed at the surface of reactivated cells, which is associated with variable susceptibility to ADCC.

Figure 8: Binding of bNAbs and ADCC activity against reactivated HIV-1-infected cells from the viral reservoir in patients on HAART. (a) Purified CD4+ T cells from the six indicated patients on HAART were activated and viral replication was followed by flow cytometry. When the % of Gag+ cells was >5%, cells were stained with the MixA bNAb combination at 4 °C. The figures indicate the % of bNAb+ cells among Gag+ cells. One representative experiment (out of two or three for each patient) is shown. (b) The binding of two combinations of antibodies (MixA, red columns) and MixB (blue columns) to cells from the same patients is shown. The figures indicate the % of bNAb+ cells among Gag+ cells. One representative experiment (out of two or three for each patient) is shown. (c) The ADCC activity of MixA and MixB against cells from the indicated patients is shown. Target cells were used for ADCC experiments when the fraction of Gag+ cells was above 5%. (N=2–4 replicates with different NK cells; Error bars indicate s.e.m.). (d) The binding of individual antibodies (at 4 °C) to cells from the indicated patients is shown. The figures indicate the % of bNAb+ cells among Gag+ cells. The radar plots indicate the % of bNAb+ cells among infected (Gag+) cells. One representative experiment (out of two or three for each patient) is shown. Full size image

Env epitope exposure was further assessed by sampling reactivated cells from four donors with individual bNAbs present in the two cocktails (Fig. 8d). As expected, no single antibody bound to reactivated KB12 cells. With KB5, KB18 and KB19 samples, three to six bNAbs out of the nine tested displayed significant attachment to reactivated cells, each patient displaying a different binding profile.