Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1

ABSTRACT

Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes). Our previous studies defined the A32-like epitope within the cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions of gp120. Here, we elucidate additional cluster A epitope structures, including an A32-like epitope, recognized by human monoclonal antibody (MAb) N60-i3, and a hybrid A32-C11-like epitope, recognized by rhesus macaque MAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like subregion and the seven-layered β-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function in the cluster A region depends on precise epitope targeting—a combination of epitope footprint and mode of antibody attachment. All together these findings help further an understanding of how cluster A epitopes are targeted by humoral responses.

IMPORTANCE HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication, no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV)-infected nonhuman primates (NHPs), and HIV-1-infected humanized mouse models, passive transfer studies in infants born to HIV-infected mothers, and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with postinfection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the nonneutralizing CD4-dependent epitopes in the gp41-reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine.

INTRODUCTION

Antibodies (Abs) must bind conserved domains on viral envelope (Env) glycoproteins during key points in retroviral replication in order to broadly protect against human immunodeficiency virus type 1 (HIV-1) infection. Their contribution to protection may result from a variety of antiviral mechanisms, including direct neutralization of virus and Fc receptor-dependent effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-mediated phagocytosis (1–4). Antibodies that directly neutralize HIV can provide protection, as evidenced in several nonhuman primate studies with passively transferred monoclonal antibodies (MAbs) (5–8), although their role in preventing natural HIV transmission remains equivocal (reviewed in reference 9). On the other hand, a growing body of evidence indicates that direct neutralizing activity is not an absolute requirement for humoral protection against HIV infection. The RV144 vaccine trial in humans (10–13), vaccine trials in nonhuman primates (14–17), early passive immunization studies against simian immunodeficiency virus (SIV) using polyclonal sera (18, 19), and a breast milk transmission study of mother-infant pairs (20, 21) have linked Fc receptor-mediated effector functions with control or prevention of infection, often in the absence of neutralization. Finally, the Fc effector functions' contribution to the blocking of viral entry, the suppression of viremia, and the therapeutic activity of several different anti-Env broadly neutralizing Abs (bnAbs) was confirmed recently in both a mouse model of HIV-1 entry and a model of MAb-mediated therapy using HIV-1-infected humanized mice (22). Overall, these findings suggest that a vaccine capable of generating both neutralizing and nonneutralizing humoral responses will provide the broadest measure of protection at the population level.

While the neutralizing epitopes have been examined in much detail (23–34), relatively little is known about epitopes that are targets for antibodies acting through Fc receptor-dependent effector functions, their degree of overlap with neutralizing epitopes, the immunological rules underlying their selection during anti-Env antibody responses, or their precise locus of action (e.g., transmission blocking or postinfection viral control). While neutralization and Fc receptor-dependent processes of antibodies can be coincident for a given specificity, as has been reported for antibodies targeting the gp120 variable loops, the coreceptor binding site, or the V2 loop region (35–38), they can also be dissociated. Epitopes on both gp120 and gp41 are known that are targeted by antibodies lacking neutralizing activity but capable of potent Fc-mediated effector function (reviewed in references 37 38). In this group, nonneutralizing, CD4-inducible (CD4i) epitopes in the C1-C2 region of gp120 (A32-like epitopes) have recently received much attention as potent ADCC targets (39–42). RV144 analyses implicated this gp120 region as a target of ADCC responses that correlated with reduced infection. In addition, a number of MAbs specific for A32-blockable epitopes were recovered from vaccinated subjects (43) which mediated cross-clade ADCC activity and synergized with V2-specific MAbs to mediate ADCC against the tier 2 isolate AE.CM235 (44). Lastly, the protective vaccine efficacy due to ADCC responses of C1-region-specific MAbs was greatly attenuated by the presence of IgA MAbs incapable of NK cell-mediated effector function but competing for the same Env binding sites (45).

Previously, we designated C1-C2 epitopes mapping to the gp41-reactive face of gp120 cluster A, the canonical examples being MAbs A32 and C11 (41). These epitopes are exposed after envelope trimers engage target cell CD4 and persist on freshly infected cell surfaces for extended periods of time postinfection (46–48). They are also exposed on the surfaces of persistently infected cells. In general, cluster A epitopes are naturally immunogenic as HIV-1-infected individuals frequently elicit C1-C2-specific antibodies (39, 40, 42, 49). We along with others have shown that these epitopes become major targets for ADCC responses during HIV-1 infection (39, 41, 42, 50), and ADCC responses to this region are also subject to immune escape early in infection (51). Recently, it was also shown that exposure of cluster A epitopes is modulated by downregulation of CD4 on the surface of the infected target cell by host factors Nef and Vpu (42, 52). This points toward the possibility of Nef and Vpu evolving as viral defenses against the exposure of these epitope targets during virion release and as an ADCC evasion mechanism preventing antibody-mediated clearance of virus-infected cells (42, 52).

We previously reported that cluster A is comprised of at least three epitope subregions, as defined by enzyme-linked immunosorbent assay (ELISA) competition with MAbs A32 and C11 for binding to CD4 triggered gp120 (41). One subgroup competes with only A32 (A32-like epitopes), the second competes with only C11 (C11-like epitopes), and the third competes with both A32 and C11 (hybrid A32-C11-like epitopes). Recently, we defined the A32-like epitope subregion at the atomic level by describing structures of Fab fragments of two A32-like antibodies in complexes with the CD4-triggered gp120 cores (53). These studies mapped the A32-like epitope to the mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions. They also pointed toward a role of precise epitope targeting and mode of antibody binding in the Fc-mediated effector functions of antibodies against HIV-1. Here, we elucidate two more epitope structures within the cluster A region and provide a more comprehensive understanding of how these epitopes are recognized by a human MAb and a rhesus macaque MAb, both capable of potent ADCC function.

MATERIALS AND METHODS

Protein purification.

JR4 and N60-i3 monoclonal antibodies (MAbs) were purified by HiTrap protein A column (GE Healthcare) chromatography from 293T supernatants prepared by transfecting plasmids carrying the heavy- and light-chain genes of the respective Abs. The Fabs of both the MAbs were prepared from the purified IgGs (10 mg/ml) by proteolytic digestion with immobilized papain (Pierce, Rockford, IL) and purified using a protein A column to remove Fc (GE Healthcare, Piscataway, NJ), followed by gel filtration chromatography on a Superdex 200 16/60 column (GE Healthcare, Piscataway, NJ). The elution peak of each of the Fabs corresponded to a molecular mass of approximately 50 kDa and was collected and concentrated for use in the crystallization trials.

For crystallographic studies, the gp120 extended core (core_e) protein of clade A/E strain 93TH057 (gp120_93TH057 core_e; gp120 lacking the N and C termini and variable loops 1, 2, and 3 [V1V2V3]) and the CD4-mimetic miniprotein M48 (F²³M47) or M48U1 (54, 55) were used to prepare the ternary complexes of JR4 and N60-i3, respectively. gp120_93TH057 core_e was prepared and purified as previously described (53). Deglycosylated gp120_93TH057 core_e was first mixed with the CD4-mimetic peptide M48 or M48U1 at a molar ratio of 1:1.5 and purified through gel filtration chromatography using a Superdex 200 16/60 column (GE Healthcare, Piscataway, NJ). After concentration, the gp120_93TH057 core_e-M48 or gp120_93TH057 core_e-M48U1 complex was mixed with a 20% molar excess of JR4 Fab or N60-i3 Fab, respectively, and passed again through the gel filtration column equilibrated with 25 mM Tris-HCl buffer, pH 7.2, with 0.35 M NaCl for the JR4 Fab-gp120_93TH057 core_e-M48 complex and with 0.15 M NaCl for the N60-i3 Fab-gp120_93TH057 core_e-M48U1 complex. The purified complexes were concentrated to ∼10 mg/ml for crystallization experiments.

Crystallization.

Initial crystal screens were done in robotic vapor diffusion sitting-drop trials using commercially available sparse-matrix crystallization screens and then reproduced and optimized using the hanging-drop vapor diffusion method (drops of 0.5 μl of protein and 0.5 μl of precipitant solution equilibrated against 700 μl of reservoir solution). JR4 Fab crystals were obtained from a solution containing 0.2 M ammonium sulfate, 1.0 M sodium cacodylate trihydrate, pH 6.5, and 30% (wt/vol) polyethylene glycol (PEG) 5000. Prior to being frozen, the crystals were transferred into a crystallization solution containing 15% (vol/vol) glycerol. Crystals of JR4 Fab-gp120_93TH057 core_e-M48 were grown from 16.6% PEG 400, 13.3% PEG 3350, 0.1 M MgCl₂, and 0.1 M Tris (pH 8.5) and soaked in mother liquor supplemented with 20% 2-methyl-2,4-pentanediol (MPD) prior to being frozen for data collection. Crystals of N60-i3 Fab-gp120_93TH057-M48U1 were grown in 10 to 16% PEG 8000 or PEG 10000, 0.065 M NaCl, and 0.1 M Tris-HCl (pH 8.5) at 22°C and cryoprotected in 18% MPD, 16% PEG 8000 or 10000, 0.1 M Tris-HCl (pH 8.5), and 0.065 M sodium chloride.

Data collection and structure solution.

Diffraction data were collected at the Stanford Synchrotron Radiation Light Source (SSRL) at the beam lines BL9-2 (JR4 Fab), BL12-2 (JR4 Fab-gp120_93TH057 core_e-M48), and BL7-1 (N60-i3 Fab-gp120_93TH057 core_e-M48U1), equipped with MAR325, Pilatus 6M PAD, and ADSC Quantum 315 area detectors, respectively. All data were processed and reduced with HKL2000 (56). Structures were solved by molecular replacement with Phaser (57) from the CCP4 suite (58) based on the coordinates of gp120 (PDB accession number 3TGT) and the N5-i5 Fab (PDB 4H8W) and the coordinates of the CD4-mimetic peptide M48 (PDB 4K0A) and M48U1 (PDB 4JZW). Refinement was carried out with Refmac (59) and/or Phenix (60). Refinement was coupled with manual refitting and rebuilding with COOT (61). Data collection and refinement statistics are shown in Table 1.

TABLE 1.

Crystallographic data collection and refinement statistics

Parameter	Value for:a
	Fab N60-i3-gp12093TH057 coree-M48U1	Fab JR4	Fab JR4-gp12093TH057 coree-M48
Data collection
Wavelength (Å)	1.127	1.045	1.127
Space group	P2(1)2(1)2	P1	P2(1)
Cell parameters
a, b, c (Å)	98.3, 102.6, 108.0	79.4, 79.6, 82.0	110.3 77.8 127.6
α, β, γ (°)	90, 90, 90	78.8, 82.9, 65.2	90, 114.3, 90
No. of molecules/AUb	4	8	8
Resolution (Å)	50–3.20 (3.26–3.20)	50.0–1.91 (1.95–1.91)	50–3.17 (3.23–3.17)
No. of reflections
Total	66,561	220,077	145,898
Unique	17,516	129,457	39,432
Rmerg (%)c	12.6 (89.9)	12.5 (60.1)	25.2 (87.1)
I/σ	10.7 (1.3)	7.35 (1.5)	6.9 (1.4)
Completeness (%)	93.7 (96.0)	93.6 (95.5)	99.8 (100)
Redundancy	3.8 (3.8)	1.7 (1.6)	3.7 (3.7)
Refinement statistics
Resolution (Å)	36.01–3.2	40.2–1.91	45.0–3.21
R (%)d	22.0	19.2	27.3
Rfree (%)e	27.7	24.6	33.2
No. of atoms
Protein	6,010	12,736	12,060
Water	1	1456
Ligand	158	25	278
Overall B value (Å2)
Protein	117.1	21.1	94.6
Water	44.1	29.6
No. of ligands/No. of ions	112.0	40.9	96.7
Root mean square deviation
Bond lengths (Å)	0.007	0.018	0.007
Bond angle (°)	1.41	1.77	1.48
Ramachandran plotf
Favored (%)	72.9	90.4	90.2
Allowed (%)	24.0	9.1	9.3
Outliers (%)	3.1	0.5	0.5

^aValues in parentheses are for highest-resolution shell.

^bAU, asymmetric unit.

^cR_merge = Σ |I − <I>|/ΣI, where I is the observed intensity and <I> is the average intensity obtained from multiple observations of symmetry-related reflections after rejections.

^dR = Σ ||F_o| − |F_c||/Σ |F_o|, where F_o and F_c are the observed and calculated structure factors, respectively.

^eR_free, calculated as defined by Brünger (73).

^fCalculated with MolProbity (62).

Structure validation and analysis.

The quality of the final refined models was monitored using the program MolProbity (62). Structural alignments were performed using the Dali server and the program lsqkab from the CCP4 suite (58). PISA (63) and PIC (64) web servers were used to determine contact surfaces and residues. All illustrations were prepared with the PyMol molecular graphics suite (http://pymol.org) (DeLano Scientific, San Carlos, CA, USA).

FRET-FCS competition assay.

Alexa 488-Alexa 568 donor-acceptor pairs were used for competition assays using fluorescence resonance energy transfer (FRET)-fluorescence correlation spectroscopy (FCS). For FRET measurements, the Fabs (C11, A32, N60i3, and JR4) were labeled with either donor (Alexa 488) or acceptor (Alexa 568) probes (Invitrogen MAb labeling kit). Briefly, the Alexa Fluor 488 or 568 reactive dye has a succinimidyl ester moiety that reacts efficiently with primary amines of Fab to form stable dye-protein conjugates. The dye-labeled Fabs were purified using 10-kDa cutoff spin columns. Purified Alexa 488- or 568-labeled Fab was quantified by a UV-visible light (UV-Vis) spectrometer (NanoDrop 2000). Dye-to-protein ratios were determined by measuring absorbance at 280 nm (protein) versus 488 or 577 nm (dye). The dye-to-protein ratios were between 1 and 2. We specifically aimed to keep this low level of dye labeling as we were using a single-molecule fluorescence method to minimally perturb the functionality of the protein. FRET measurements were performed in a confocal microscope (MicroTime 200; PicoQuant). PicoQuant Symphotime software was used to generate the FRET histograms and for further analyses. FRET measurements were performed after an immune complex with full-length single-chain gp120_BaL-soluble CD4 (sCD4) (FLSC; gp120_BaL is gp120 of the HIV-1 BaL isolate) was formed with donor-labeled Fab and acceptor-labeled Fab. In all of our measurements each Fab concentration was 1 μg/ml, and the FLSC concentration was 1.5 μg/ml. The immune complexes were made by incubating Fabs with the FLSC at 20°C for 1 h. Fluorescence responses from the donor and the acceptor molecules were separated by a 50/50 beam splitter and detected by two avalanche photodiode detectors (APD) using the method of time-correlated single-photon counting and the time-tagged time-resolved (TTTR) mode of the PicoHarp 300 board. High-quality bandpass (Chroma) filters were used for recording donor and acceptor fluorescence in two separate detection channels. The collected single-photon data were binned by a 1-ms bin in each channel (donor or acceptor), which resulted in intensity-time traces and count-rate histograms. Threshold values in each channel were used to identify the single-molecule bursts from the corresponding background signal level. Fluorescence bursts were recorded simultaneously in donor and acceptor channels, and FRET efficiencies were calculated using E = I_A/(I_A + γI_D) where I_D and I_A are the sums of donor counts and acceptor counts for each burst, respectively, taking into account the possible difference in the detection efficiency (γ) values in two separate channels (65). The donor-to-acceptor distance (r) in terms of efficiency of energy transfer (E) and Förster distance (R₀) is given by r = R₀(1/E − 1)^1/6. We have used an R₀ value of 62 Å for the Alexa 488 (donor) and Alexa 568 (acceptor) pair for estimating the donor-to-acceptor distances. In addition to FRET measurements, we have also performed FCS measurements to assess in vitro binding of single or multiple Fab fragments to the FLSC. We determined translational diffusion coefficients of Alexa 488- or 568-labeled Fabs and the corresponding immune complexes from FCS measurements. The FCS measurements and analyses were performed as previously reported (47).

SPR competition analysis.

The binding footprints of MAb N60-i3 and JR4 in relation to MAb C11 and A32 were assessed by surface plasmon resonance (SPR) competition on a Biacore T-100 (GE Healthcare) at 25°C with buffer HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% surfactant P-20). Protein A was first immobilized onto the second of two flow cells on a CM5 chip to ∼3,000 response units (RU), and the first flow cell was blocked with a standard amine coupling protocol (GE Healthcare). IgGs to be evaluated were then captured onto the second flow cell by flowing a 5 to 10 nM solution of MAb at a 10-μl/min flow rate for 30 s. The antibody concentration was varied to give an RU in the range of 150 to 400. The single-chain gp120_BaL-sCD4 complex (FLSC) (66) was then passed over the same flow cell at a flow rate of 10 μl/min for 30 s. An FLSC concentration was chosen to give an RU in the range of 150 to 400, comparable to the RU for the antibody. Various concentrations of MAb Fab were then passed over both flow cells at a flow rate of 30 μl/min for 200 s and allowed to dissociate by passing the buffer over both cells at the same flow rate for 800 s. The cells were regenerated between concentrations with a 30-s injection of 0.1 M glycine, pH 3.0, at a flow rate of 100 μl/min. The antibody and FLSC were then reloaded onto the second flow cell for the next concentration. Blank sensorgrams were obtained by injection of HBS-EP buffer in place of Fab. Sensorgrams of the concentration series (flow cell two minus flow cell one) were corrected with a corresponding blank.

ADCC assays.

ADCC assays were carried out using the rapid fluorescence ADCC method (67) modified to reduce prozone effects. All ADCC studies used CEM-NKr-CCR5 target cells sensitized with recombinant gp120 from the HIV-1 BaL (HIV-1_BaL) isolate or spinoculated with AT-2-inactivated BaL HIV-1 virus (kindly supplied by Jeffrey Lifson, National Cancer Institute) at 3,000 rpm for 2 h at 12°C. gp120-sensitized or virus-spinoculated cells were then washed twice and added to a 96-well V-bottom plate (5,000 cells/well). The gp120-sensitized or virion-bound target cells were incubated with MAb dilutions for 15 min and washed with culture medium before the addition of peripheral blood mononuclear effector cells from healthy donors at a final ratio of 50:1. The effector and target cells were pelleted by centrifugation and incubated for 2 to 3 h at 37°C, followed by fixation and cytolysis determined by flow cytometry as described in Gomez-Roman et al. (67). The absolute cytotoxicity values were normalized using the MAb C11 as described previously (41).

Protein structure accession numbers.

Structures of Fab N60-i3-gp12093TH057-M48U1, Fab JR4, and Fab JR4-gp12093TH057-M48 were deposited in Protein Data Bank with accession codes 4RFO, 4RFE, and 4RFN, respectively.

RESULTS

MAb origin and epitope cluster A assignment.

MAb N60-i3 was isolated from B cells of an HIV-1-infected individual and characterized for initial reactivity using recombinant proteins based on the HIV-1_BaL isolate as described previously for other cluster A MAbs (41). MAb JR4 was derived from the peripheral blood B cells of a rhesus macaque infected with a simian-human immunodeficiency virus (SHIV) KB9 mutant with deletions of glycosylation sites in gp41. The detailed description of MAbs N60-i3 and JR4, JR4 isolation, germ line gene usage, and degree of somatic hypermutation will be published elsewhere. MAbs N60-i3 and JR4, similar to other CD4-inducible (CD4i) MAbs of cluster A (41), show preferential binding to gp120-CD4 complexes compared with monomeric gp120 and no binding to Env trimers expressed on the cell surface in the presence or absence of soluble CD4 (sCD4; domains d1 to d4 of CD4) (see Fig. S1 in the supplemental material) (47, 48, 53). The initial epitope assignments were accessed based on competition of N60-i3 and JR4 binding to the single-chain gp120_BaL-sCD4 complex (FLSC) (66) by MAbs A32 and C11, two antibodies specific for distinct (nonoverlapping) epitopes in the cluster A region (41). Recently, we defined epitopes in the A32-like region by describing epitope structures of two A32-like MAbs, N5-i5 and 2.2c, and mapped them to the C1-C2 regions of gp120 (53). In contrast, the binding site for MAb C11 is still unresolved, but it has been mapped to the seven-stranded β-sandwich of gp120 and a residue in the extended C terminus of gp120 by mutagenesis studies (68). To precisely access competition due to the overlaps of epitope footprints and eliminate a possibility of avidity effects or steric clashes outside the antigen-antibody binding interface, we have developed a new fluorescence resonance energy transfer-fluorescence correlation spectroscopy (FRET-FCS)-based competition assay in which antigen-binding fragments (Fabs) of tested antibodies compete in solution for binding to the Env antigen (Fig. 1). Additionally, we also tested the capacities of MAbs A32 and C11 to block Fab N60-i3 and JR4 binding to the FLSC in an SPR competition assay (69) (Fig. 2). Shown in Fig. 1 are the FRET histograms of Fab pairs labeled with Alexa 488 (A488) or Alexa 568 (A568) of MAbs N60-i3, JR4, A32, and C11 bound to the FLSC. When binding of C11-A488 Fab and A32-A568 Fab was tested, the data could be fitted well with a Gaussian profile showing ∼20% FRET efficiency (Fig. 1A), clearly confirming the coexistence of A32 and C11 Fabs bound to a single FLSC protein molecule with a stoichiometry of C11/A32/FLSC of 1:1:1. A similar FRET profile was observed for C11-A488 Fab and N60-i3-A568 Fab binding to the FLSC with ∼18% FRET efficiency (Fig. 1B), confirming that the C11 Fab and N60-i3 Fab bind to the nonoverlapping FLSC epitopes. Additionally, mean FRET efficiencies translated to an average distance of 78 Å between C11 and A32 Fabs and a distance of 79.8 Å between C11 and N60-i3 Fabs. It is important to note that the Fabs are not fluorescently labeled at a specific position; hence, the distance derived from our FRET measurements represents an average value between the donor- and acceptor-labeled epitope probes. However, the binding of epitope probes to the FLSC is further confirmed by the diffusion coefficients derived from the FCS measurements. In contrast, when binding of C11-A488 Fab and JR4-A568 Fab was analyzed (Fig. 1C), the efficiency of FRET was below 10%, and a Gaussian distribution could not be obtained. The autocorrelation measurements in the C11 channel also showed the presence (25%) of unbound C11. This indicated that JR4 Fab, in contrast to N60-i3 Fab, is capable of partially blocking C11 Fab binding to the FLSC antigen. On the other hand, in the same assay both N60-i3 Fab and JR4 Fab blocked the binding of A32 Fab to the FLSC, as indicated by the lack of FRET signal for the mixtures of A32-A488 N60-i3-A568-FLSC and A32-A488 JR4-A568 Fab-FLSC (Fig. 1D and E). The FRET-FCS competition data are in good agreement with the results of the SPR competition assay. With A32 IgG bound to the FLSC, neither the N60-i3 nor the JR4 Fab could bind, indicative of complete competition for the same binding site on the FLSC. With C11 IgG bound to the FLSC, N60-i3 Fab could also bind with an apparent K_D (equilibrium dissociation constant) similar to that of N60-i3 binding to the FLSC alone, suggesting no overlap in the binding sites. A32 Fab showed a similar result with C11 IgG bound to the FLSC. However, with C11 IgG bound to the FLSC, JR4's apparent K_D was approximately 38-fold lower than that of JR4 binding to the FLSC alone, suggesting a partial overlap in their binding sites (Fig. 2). Altogether, these data suggest that MAb N60-i3 recognizes an A32-like epitope, whereas MAb JR4 may recognize an A32-C11 hybrid epitope.

FIG 1.

FRET histograms of donor (A488)-labeled Fabs, acceptor (A568)-labeled Fabs, and full-length single-chain gp120_BaL-CD4 complex (FLSC) in solution as determined by the FRET-FCS approach (see Materials and Methods for details). Data in panels A and B indicate ∼20% FRET efficiency for A488-labeled C11 Fab and A568-labeled A32 Fab or N60-i3 Fab bound to FLSC, respectively. Panels D and E do not show detectable FRET signals for A488-labeled A32 Fab and A568-labeled N60-i3 Fab or JR4 Fab in the presence of FLSC in solution. From the FCS measurements, the diffusion coefficient of dye-labeled Fab is ∼83 μm²/s. This diffusion has been significantly decreased upon binding two Fabs to FLSC. The diffusion coefficient for this immune complex is ∼36 μm²/s.

FIG 2.

MAb A32/C11 competition of N60-i3/JR4 Fab binding as measured by a surface plasmon resonance competition assay. MAb A32 (A and B) and MAb C11 (B, C, and D) were immobilized on a protein A chip, and FLSC antigen was loaded to form a competitor antibody-antigen complex. Various concentrations of Fab MAb were tested. N60-i3 (A and C), JR4 (B and D), and A32 (E) were then passed over the chip. (F) Sensorgrams of the concentration series are shown. Binding affinities for MAbs A32, N60-i3, and JR4 to FLSC in the absence (blue bars) and presence (red bars) of C11 IgG were calculated with the BIAevaluation software.

MAbs N60-i3 and JR4 show potent ADCC activity.

We tested the ADCC potency of MAb N60-i3 and MAb JR4 using CEM-Nkr-CCR5 target cells sensitized with gp120 (Fig. 3A) or with AT-2-inactivated BaL virions (Fig. 3B) of the HIV-1_BaL isolate, as described in Materials and Methods. MAbs N60-i3 and JR4 are potent mediators of ADCC in both assay formats using the potency criteria described previously (41).

FIG 3.

Representative ADCC curves for MAbs N60-i3 and JR4. ADCC assays were performed as described in Materials and Methods using CEM-NKr-CCR5 target cells sensitized with gp120 of HIV-1_BaL isolate (A) or spinoculated with AT-2-inactivated BaL viruses (B). MAb palivizumab (Synagis; MedImmune, Inc.) was included as a control.

Structures of MAb N60-i3- and JR4-Env antigen complexes.

In an effort to elucidate the epitopes of MAbs N60-i3 and JR4 and differences in epitope footprints, if any, that could explain differences in their A32/C11 cross-competition, we determined the crystal structures of the complexes formed between their antigen-binding fragments (Fabs) and CD4-triggered gp120 antigen. Both complexes were formed using the gp120 extended core protein (residues 44 to 492 with V1V2V3 loops deleted) (27) of the clade A/E 93TH057 isolate (gp120_93TH057 core_e) and CD4 peptide-mimetic M48U1 (54) (N60-i3 complex) or M48 (JR4 complex). His³⁷⁵ of gp120_93TH057 core_e in the N60-i3 Fab-gp120_93TH057 core_e-M48U1 complex was mutated to Ser to accommodate ligands such as M48U1 that penetrate the gp120 Phe43 cavity, as described previously (70). M48U1 is a derivative of M48 and is identical, with the exception that the phenylalanine at position 23 has been replaced with a phenyl cyclohexylmethoxy moiety, increasing its affinity for gp120 by roughly 10-fold or more, based on 50% effective concentrations (EC₅₀s), depending on the HIV-1 strain used (54). The N60-i3 Fab-gp120_93TH057 core_e-M48U1 assembly crystallized in space group P2(1)2(1)2 with one complex in the asymmetric unit (Table 1). The JR4 Fab-gp120_93TH057 core_e-M48 assembly crystallized in space group P2(1) with two almost identical copies of complex present in the asymmetric unit (see Fig. S2 in the supplemental material). Structures were solved by molecular replacement at resolutions of 3.2 Å for the N60-i3 Fab complex and 3.21 Å for the JR4 Fab complex and refined to a final R/R_free of 22.0/27.7% and 27.3/33.2%, respectively. The data collection and refinement statistics for the structures are summarized in Table 1, and the overall structures of complexes are shown in Fig. 4.

FIG 4.

Crystal structures of N60-i3 Fab-gp120_93TH057 core_e-M48U1 and JR4 Fab-gp120_93TH057 core_e-M48 complex. Complexes are shown in ribbon representation (right panels), or the molecular surface is displayed over Fab molecules (left panels), with the light/heavy chain of N60-i3 Fab and JR4 Fab shown in light green/dark green and light cyan/dark cyan, respectively. The complementarity-determining regions (CDRs) are shown in slate (CDR L1), black (CDR L2), gray (CDR L3), green (CDR H1), orange (CDR H2), and pink (CDR H3). The gp120 outer domain is shown in raspberry, and the inner domain is shown in wheat (left panels) or colored in a layered color scheme (right panels), with the seven-stranded β-sandwich in magenta, layer 1 in yellow, layer 2 in cyan, and layer 3 in light wheat. The mimetic peptides M48U1 and M48 are shown in violet.

The N60-i3 Fab-gp120_93TH057 core_e-M48U1 and JR4 Fab-gp120_93TH057 core_e-M48 complex structures revealed that both MAb N60-i3 and MAb JR4 bind at largely overlapping sites in the C1-C2 gp120 region shown previously to be recognized by A32-like MAbs N5-i5 and 2.2c (53). In both cases, a conformational epitope is formed by bridging mobile layers 1 and 2 of the gp120 inner domain involving residues of the α0- and α1-helices, the β2̄-, β1̄-, and β4-strands, and the β2̄-α0-, β1̄-β0-, and β4-β5-connecting coils (Fig. 4). The interactive surface that becomes buried due to N60-i3 Fab-gp120_93TH057 core_e interaction encompasses 1,464 Ų (770 Ų contributed by Fab and 694 Ų from gp120) (see Table S1 in the supplemental material) and is roughly half (63%) the buried surface area (BSA) of the JR4 Fab-gp120_93TH057 core_e interface (BSA of 2,340 Ų with 1,145 Ų buried by Fab and 1,195 Ų buried by gp120) (see Table S1). Despite the differences in the surface areas buried at the N60-i3 and JR4 complex interfaces, these antibodies show the same affinity for gp120 in a CD4-bound conformation, as confirmed by SPR analysis (see Fig. S1 in the supplemental material) and isothermal titration calorimetry (ITC) (see Fig. S3). The paratope terrains of N60-i3 and JR4 Fab are flat and electropositive, with the only protruding areas contributed by complementarity-determining regions (CDRs) of heavy chains 1 and 3 (CDR H1 and CDR H3), respectively, making the contacts with the α1-helix of layer 2. In both complexes, the heavy chain contributes most of the Fab binding surface (approximately 85% and 83% of BSA of N60-i3 Fab and JR4 Fab, respectively), with all three CDRs (CDRs H1 to H3) involved in the interaction (see Table S1 in the supplemental material). CDR H3s of 12 and 14 residues for N60-i3 and JR4, respectively, provide most of their heavy chain BSA (approximately 57% and 44% as calculated for N60-i3 and JR4, respectively) (see Tables S1 and S2 and Fig. S4 in the supplemental material). In both cases, the contribution of the light chain to the antibody-antigen interface is minimal, with only two CDRs (CDR light chain 1 [L1] and CDR L3 for N60-i3 and CDR L1 and CDR L2 for JR4) engaged in binding (Fig. 5; see also Fig. S4). A total of 56 (7 H bonds) and 54 (15 H bonds) contacts as defined by a 5-Å cutoff are formed at the heavy chain-gp120_93TH057 core_e interfaces of N60-i3 and JR4, respectively (Fig. 5, right panel). By comparison, the light chain contributes 4 (0 H bonds) and 5 (1 H bond) contacts to the complex interface of N60-i3 and JR4, respectively (Fig. 5, right panel).

FIG 5.

Binding of MAbs N60-i3 and JR4 to gp120 antigen. On the left, the N60-i3 Fab-gp120_93TH057 core_e and the JR4 Fab-gp120_93TH057 core_e interfaces are shown with the molecular surfaces displayed at the gp120 molecules and with CDRs of Fabs shown as ribbons. Only CDRs contributing to the binding are shown. The 45° rotations show close-up views of the binding interfaces of Fabs and the α1-helix of layer 2. H bonds are shown as dashes in blue. Colors are as defined in the legend of Fig. 4. On the right, networks of interactions formed between Fabs and gp120 antigen as defined by a 5-Å distance criterion cutoff are shown as solid lines. H bonds are shown as dashes in blue.

MAb N60-i3 and JR4 epitope footprints.

The epitope footprint of MAb N60-i3 maps exclusively to layers 1 and 2 of the C1-C2 region of the gp120 inner domain. MAb JR4 largely overlaps MAb N60-i3 in targeting layers 1 and 2 but also involves contacts within the seven-stranded β-sandwich and the C terminus of gp120 (Fig. 6).

FIG 6.

Structural basis for interaction of cluster A MAbs with gp120 antigen. (A) Mapping of the N60-i3 and JR4 contact residues on the primary sequence of the gp120 inner domain of the 93TH057 isolate. The topology diagram depicting a distribution of secondary structure elements is shown above the gp120 sequences. Buried residues are highlighted in green and blue. Main chain (o), side chain (+), and both side and main chain (*) interactions are shown immediately above the residues as defined by a 5-Å distance criterion cutoff and colored based on contact type: hydrophobic, blue; hydrophilic, green; or both, black. Residues forming the N5-i5 and 2.2c epitopes as described in reference 53 are indicated by blue and gray lines below the gp120 sequence, respectively. (B) Epitope footprints of MAb N60-i3, JR4, N5-i5, and 2.2c. The Cα atoms of the gp120 residues involved in Fab binding are shown as spheres and displayed over the gp120 ribbon diagram. The selected residues of layer 2 and of the seven-stranded β-sandwich contribution to Fab binding and all the residues in the α1-helix involved in N60-i3, JR4, and N5-i5 binding are labeled. (C) Comparison of binding of MAb N60-i3, JR4, N5-i5, and 2.2c to CD4-triggered gp120 antigen. The N60-i3 Fab-gp120_93TH057 core_e-M48, JR4 Fab-gp120_93TH057 core_e-M48U1, and 2.2c Fab-gp120_YU2 core_e-M48U1 complexes were superimposed based on the gp120 outer domain onto the N5-i5 Fab-gp120_93TH057 core_e-d1d2CD4 complex and oriented relative to the target cell membrane. In the 180° view only the gp120_93TH057 core_e and d1d2CD4 from N5-i5 Fab-gp120_93TH057 core_e-d1d2CD4 complex (53) are shown, with variable heavy and light (V_H and V_L) domains of Fabs displayed as balls. Insets show rotation angles calculated using gp120's center of mass as an origin and the average α-carbon position for the heavy chain framework region 2 (residues 36 to 49) as a reference point for each antibody (top) and a 90°rotation of the 180°view (bottom).

Layer 1 (C1 gp120 region) contacts.

Layer 1 of the C1 gp120 region makes up the majority of gp120 contact surface engaged in MAb N60-i3 and JR4 binding (79% and 73% of BSA for N60-i3 and JR4, respectively) (see Table S1 in the supplemental material). Layer 1 contacts for both MAbs are similar and include residues 51 to 54, 60, 68 to 69, and 71 to 79 buried at the N60-i3 Fab-gp120_93TH057 core_e interface and residues 50 to 55, 59 to 61, 68 to 69, 71 to 80, and 82 buried at the JR4 Fab-gp120_93TH057 core_e interface (Fig. 6A). There are two anchoring points that provide most of the hydrophobic surface utilized by both antibodies to attach to layer 1. These include a Thr⁵¹LeuPheCys motif of the β2̄-strand of gp120 and a Thr⁷¹HisAlaCysValPro motif at the C termini of the α0-helix and β1̄-strand. JR4 utilizes CDRs H1 to H3 to make hydrophobic contacts with the Thr⁵¹LeuPheCys motif, whereas N60-i3 contacts in this region include contributions of CDR H1 and H3 only (Fig. 5; see also Table S2, and Fig. S4 in the supplemental material). In contrast, five CDRs of N60-i3 Fab (CDRs H1 to H3 and CDRs L1 and L3) contribute to its attachment to the Thr⁷¹HisAlaCysValPro motif, whereas JR4 utilizes almost exclusively CDR H3 (with a few contacts from residues of the framework region H1 [FWRH1] and CDR H1) to contact this region (Fig. 5, right panel; see also Table S2 and Fig. S4). The Leu⁵³PheCys and Thr⁷¹HisAlaCysValPro motifs are coupled by a disulfide bond, Cys⁵⁴ to Cys⁷⁴, connecting these two anchor points. The Cys⁵⁴-Cys⁷⁴ disulfide bond plays a functional role in stabilizing the native conformation of gp120 and is highly conserved among HIV-1 isolates across clades. With the exception of His⁷² (97.8% of conservation across clades), most residues of these two motifs are invariant in greater than 99% of HIV-1 sequences, with some like Pro⁷⁶ and Pro⁷⁹ invariant in greater than 99.9% of sequences, as determined by the HIV Sequence Database Compendia (http://www.hiv.lanl.gov). JR4's reach into layer 1 is slightly longer than that of N60-i3 and continues to the edge of layer 1 residues, Asn⁸⁰, Pro⁸¹, and Gln⁸² (Fig. 5, right panel, and 6A).

Layer 2 (C1 and C2 gp120 region) contacts.

MAb N60-i3 and JR4 largely overlap in binding to layer 2 (Fig. 5, right panel, and 6A). These contacts contribute 21% and 16% of BSA for the N60-i3 and JR4 complexes, respectively (see Table S1 in the supplemental material). Layer 2 residues buried at the N60-i3 Fab-gp120_93TH057 core_e interface include residues 103, 106 to 107, 114, 217, and 219 to 221, whereas the JR4 Fab-gp120_93TH057 core_e complex buries residues 103, 106 to 107, 217, and 220 to 222. Residues Gln¹⁰³, Glu¹⁰⁶, and Asp¹⁰⁷ of the α1-helix serve as the major anchor points for MAbs N60-i3 and JR4 in layer 2 of the C1 gp120 region. N60-i3 coordinates these three residues exclusively through Arg⁹⁹ of CDR H3, forming a salt bridge with Asp¹⁰⁷ and multiple H bonds with Gln¹⁰³, and Glu¹⁰⁶ JR4 uses Arg³¹ (CDR H1) to coordinate Asp¹⁰⁷ through a salt bridge and Arg³⁰ (FWRH1) to establish an H bond with Glu¹⁰⁶ (Fig. 5 and 6A; see also Fig. S4 in the supplemental material). The N60-i3 Fab-gp120_93TH057 core_e interaction also buries Gln¹¹⁴ of the α1-helix, but the contribution of this residue to binding is minimal (see Table S2). Interestingly, CDR H3 of N60-i3 and CDR H1 of JR4, by providing contacts to both the α0-helix of layer 1 and the α1-helix of layer 2, span these two layers and form a single binding surface. This mode of cross-layer attachment closely resembles binding of the potent ADCC mediator MAb N5-i5, which uses its CDR H2 to contact both the α0- and α1-helices (53). While electrostatic interactions play a major role in MAb N60-i3 and JR4 attachment to the α1-helix, the rest of the binding contacts with layer 2 are predominantly hydrophobic. These contacts center on the Tyr²¹⁷ and around the Thr²¹⁹ProAla motif of the β4-strand and β4-β5-connecting coil of gp120 (Fig. 5; see also Tables S2 in the supplemental material). The main layer 2 contact residues used by N60-i3 and JR4 are highly conserved; Gln¹⁰³, Asp¹⁰⁷, and Pro²²⁰ are invariant in greater than 99.9% of HIV-1 sequences, and Tyr²¹⁷, Thr²¹⁹, and Ala²²¹ are present in 99.8% of sequences. Thus, MAbs N60-i3 and JR4 are similar to the ADCC potent cluster A MAb N5-i5 (53) and target highly conserved elements of the HIV-1 envelope within both layers of the C1-C2 gp120 region.

Seven-stranded β-sandwich (C2 region).

Analysis of the JR4 epitope footprint (Fig. 6A) indicates that JR4 also reaches residues in the seven-stranded β-sandwich (residues 84, 223 to 224, and 246 to 247) and residue 492 of the C terminus of gp120. These contacts contribute to approximately 11% to the BSA of the JR4 complex and are not present in the N60-i3 Fab-gp120_93TH057 core_e interface (see Tables S1 and S2 in the supplemental material). CDR H3 of JR4 is anchored deeper in this area than in N60-i3 and makes multiple contacts with Gln²⁴⁶ and Cys²⁴⁷ of the seven-stranded β-sandwich. In addition Tyr²²³, Val²²⁴, and Glu⁴⁹² are buried in the JR4 Fab-gp120_93TH057 core_e interface (Fig. 5). When both N60-i3 and JR4 epitopes are mapped on the gp120 antigen and displayed over the gp120 core_e ribbon diagram (Fig. 6B), the shift of the JR4 epitope toward the seven-stranded β-sandwich and the N and C termini of gp120 is evident. In FCS-FRET and SPR cross-competition assays, MAb JR4, but not N60-i3, cross-competes with MAb C11 binding to the gp120 antigen (Fig. 1 and 2). Since the N60-i3 and JR4 epitopes in layers 1 and 2 largely overlap, we can speculate that the JR4 contacts to the seven-stranded β-sandwich account for its hybrid phenotype and its ability to cross-compete with MAb C11 binding to gp120. In this regard, the JR4 epitope represents a hybrid A32-C11 epitope within the cluster A region.

Structural basis for ADCC potency to cluster A region.

We recently reported the atomic-level definition of the A32-like region by providing epitope footprints of two human A32-like antibodies, N5-i5 and 2.2c, both specific for largely overlapping epitope surfaces in the C1-C2 region but varying in their abilities to mediate ADCC, with N5-i5 75-fold more potent than 2.2c (53). These studies pointed toward a dominant role of precise epitope targeting and mode of antibody attachment in ADCC responses when largely overlapping epitopes in the A32-like region are involved. MAb N5-i5, which engages both the α0- and α1-helices of the inner domain layers 1 and 2, respectively, was shown to cross-link antigen better on target cells and to be more effective at ADCC. On the other hand, the impaired ability of 2.2c to mediate effective Fc effector function resulted from suboptimal positioning of its CH2 domain for Fc receptor interaction within the immune complex and from poor accessibility of its epitope for antibody avidity interactions, as judged by cell surface binding and saturation studies, with the positioning having a greater impact. As shown in Fig. 3, MAbs N60-i3 and JR4 represent very potent ADCC mediators in the cluster A region capable of Fc-dependent effector function against target cells sensitized with gp120 of the HIV-1_BaL isolate, with an effectiveness comparable to that of MAbs A32 and C11 (41). To better understand the structural basis for the ADCC potency in the cluster A region, we compared the epitope footprints (Fig. 6A and B) and modes of attachment (Fig. 6C) of MAbs N60-i3 and JR4 to the previously described potent and weaker ADCC mediators, MAbs N5-i5 and 2.2c. As expected, the comparison revealed close similarities between epitope footprints of MAbs N60-i3 and JR4 to the MAb N5-i5 footprint as their epitopes largely overlap in layers 1 and 2 (Fig. 6A). Most importantly, MAbs N60-i3, JR4, and N5-i5 all engage the same residues of the α1-helix for binding that include the highly conserved Gln¹⁰³, Glu¹⁰⁶, and Asp¹⁰⁷ residues of gp120. MAb 2.2c does not contact the α1-helix and focuses its binding almost entirely onto the α0-helix and layer 1. The N60-i3, JR4, and N5-i5 contacts to the α1-helix are mediated exclusively by arginines of their heavy chains (Arg⁹⁹ in CDR H3, Arg³⁰ and Arg³¹ in CDR H1, and Arg⁵⁵ in CDR H2 of N60-i3, JR4, and N5-i5, respectively) coordinating the Asn¹⁰³-Glu¹⁰⁶-Asp¹⁰⁷ triad through an invariant salt bridge and network of H bonds (Fig. 5) (53). Thus, to reach the α1-helix through the heavy chain and target their cognate epitopes, N60-i3, JR4, and N5-i5 must approach the gp120 antigen at similar angles and contact gp120 antigen via similar variable domain contact surfaces. Although there are differences in the modes of attachment among N60-i3, JR4, and N5-i5, defined by the exact orientation of the contact surfaces of the heavy and light chain variable regions (V_H and V_L, respectively) on gp120, the heavy chain contributions are all in very close proximity (Fig. 6C). Using the conserved framework of the V_H domain with the gp120 center of mass as the origin, MAbs N60-i3 and JR4 rotate in their complex by 8.7°and 9.3°, respectively, relative to the V_H domain of N5-i5. In contrast, the V_H domain of 2.2c rotates 25.5° relative to the N5-i5-defined orientation. Furthermore, although the exact position of the target cell membrane is unknown, based on the model for cell fusion as shown in Fig. 6C, N60-i3, JR4, and N5-i5 approach the gp120 antigen at an angle 16.2° or more farther from the target cell membrane to bind their epitopes than 2.2c. We showed previously that the N5-i5 epitope is more accessible on the target cell surface than the 2.2c epitope, resulting in effective gp120-CD4 complexes cross-linking and potent Fc-mediated effector function. Indeed, although SPR studies show that there are essentially no differences in K_D values between the MAbs N60-i3, JR4, and N5-i5 and MAb 2.2c for binding to monomeric gp120-CD4 complexes (FLSCs) (see Fig. S1 in the supplemental material) (53), when tested by ELISA, MAb 2.2c can be easily cross-competed by N60-i3, JR4, or N5-i5 (data not shown). This indicates that the N60-i3/JR4/N5-i5 epitope is more accessible for antibody cross-linking in the ELISA format and that its engagement results in a more stable epitope-paratope complex. Furthermore, experiments with hybrid variants with interconverted CH2 domain orientations indicated that the mode of attachment, as defined by the relative orientations of its light and heavy chains bound to gp120 antigen, contribute to the relative impotency of 2.2c in ADCC assays. It did not affect the ADCC potency of N5-i5 (53). MAbs N60-i3 and JR4, similar to N5-i5, attach their heavy chains to the α1-helix, but the positions and gp120 binding contacts of their light chains differ noticeably (Fig. 6C). This suggests that the epitope footprint and the precise epitope targeting determine the ADCC potency for N60-i3 and JR4. By directing their heavy chains to the α1-helix, N60-i3, JR4, and N5-i5 are not only more accessible for antibody cross-linking on the target cell but also position their CH2 domains optimally for effective Fc receptor interaction within the immune complex. Analysis of the residues subject to somatic mutation from the germ line sequences revealed that N60-i3, JR4, and N5-i5 were selected to have arginines at positions Arg⁹⁹, Arg³⁰/Arg³¹, and Arg⁵⁵, respectively, enabling them to interact with the Asn¹⁰³-Glu¹⁰⁶-Asp¹⁰⁷ triad of the α1-helix. Thus, N60-i3, JR4, and N5-i5 seem to have been selected to specifically target the α1-helix and recognize an epitope that encompasses structures in two inner-domain mobile layers and utilize cooperative binding in the α0- and α1-helices to link the two layers into one binding unit.

DISCUSSION

In conclusion, our findings indicate that the potent ADCC to the cluster A region focuses on a highly conserved epitope surface involving the α0- and α1-helices of the inner domains of the C1 and C2 regions of gp120, respectively. The cluster A region is buried and not accessible for antibodies in native and soluble CD4-triggered HIV-1 Env trimers and becomes exposed within a viral spike only upon binding to the cell surface form of CD4, where it is readily accessible for antibody avidity interactions and effective antigen cross-linking (47, 48, 53). Furthermore, these studies confirm our previous observation that precise epitope targeting—a combination of both the epitope footprint and the mode of antibody attachment—plays a major role in determining the potency of ADCC. Cluster A MAbs capable of potent Fc-mediated effector function cross-link the epitopes on the target cell surface by attaching their heavy chains to the α1-helix of gp120. This mode of binding allows positioning of CH2 domains for more effective Fc receptor interaction.

Epitopes in the cluster A region may be restricted to the surface-engaging residues of layers 1 and 2 only (N60-i3 epitope) and include also involvement of residues of the seven-stranded β-sandwich (JR4 epitope). MAb N60-i3, similar to MAb N5-i5, competes in the binding to the Env antigen only with A32 Fab; thus, its epitope represents the A32-like epitope in the cluster A region. In contrast, the Fab of rhesus macaque MAb JR4 competes entirely for A32 Fab binding and partially for C11 Fab binding in ELISA, FCS-FRET, and SPR competition assays. This indicates that the JR4 epitope footprint on gp120 antigen involves elements of both the A32- and C11-binding surfaces and represents a mixed A32-C11 epitope of the cluster A region. MAb JR4 was isolated from SHIV-infected rhesus macaques, but we have shown previously that antibodies of similar A32-C11 mixed specificity are also induced in HIV-1-infected individuals (41). This indicates that the A32-C11 mixed specificity could be induced in both nonhuman primates and humans following HIV-1 and SHIV infection, respectively. Since the exact epitope footprint of MAb C11 is not known, our studies allow us for the first time to define a putative contact region of MAb C11 with gp120. As shown in Fig. 7, the gp120 residues previously shown by mutagenesis to be involved in MAb C11 binding (68, 71) mapped to the seven-stranded β-sandwich in the gp41-interactive region (PDB accession number 3JWD) (72), with N60-i3 and JR4 Fabs bound as in their CD4-triggered gp120 complexes. As previously indicated, both N60-i3 and JR4 bind to gp120 in largely overlapping regions with only the protruding region of the CDR H3 of JR4 attaching to the seven-stranded β-sandwich (Fig. 7). We propose that the CDR H3 of JR4 bound to the seven-stranded β-sandwich interferes with MAb C11 binding in this area. The residues shown previously by mutagenesis to decrease C11 binding to gp120 map to this region. Thus, the putative C11 epitope involves residues of the seven-stranded β-sandwich and maps immediately adjacent to the A32-like epitope surface (Fig. 7, inset). This is also in agreement with our FCS-FRET measurements showing a distance of 79.8 Å between C11 and N60-i3 Fabs bound to FLSC.

FIG 7.

Putative binding site of MAb C11. The crystal structures of N60-i3 Fab-gp120_93TH057 core_e-M48 and JR4 Fab-gp120_93TH057 core_e-M48U1 are superimposed onto the HIV-1 gp120 with the gp41-interactive region (PDB code 3JWD) (72). Only the Fabs of complexes are shown, and a molecular surface is displayed over the N60-i3 Fab. The CDR H3 of JR4 predicted to block MAb C11 binding is shown in blue. The residue contacts of CDR H3 on the seven-stranded β-sandwich are shown as red spheres. Residues shown by mutagenesis studies to affect binding of MAb C11 to gp120 Env are shown as gray spheres. The inset shows a 45° rotation of the assembly where only gp120 and the CDR H3 of JR4 are shown, and the putative C11 epitope is encircled in red.