Broad and potent neutralization of HIV-1 by a gp41-specific human antibody

Introduction

Induction of an antibody response capable of neutralizing diverse HIV-1 isolates is a critical goal for vaccines that protect against HIV-1 infection. Potentially the greatest obstacle to achieving this goal is the extraordinary diversity that develops in the target of neutralizing antibodies, the envelope glycoprotein (Env). Although vaccines have thus far failed to induce broadly neutralizing antibody responses, there are examples of chronically infected patients with sera that neutralize highly diverse HIV-1 isolates^1–8. These individuals provide evidence that it is possible for the human antibody response to neutralize highly diverse strains of HIV-1, though the mechanisms by which such responses are induced or mediated remain incompletely understood^9,10.

Recently, isolation and characterization of human monoclonal antibodies from cells of chronically infected patients have provided considerable advances in understanding the specificities and mechanisms underlying broadly neutralizing antibody responses to HIV-1. Env exists on the virion and infected-cell surface as a trimer of heterodimers made up of gp120 and gp41 subunits. For some time, only a small number of broadly neutralizing monoclonal antibodies (mAbs) had been isolated consisting of one antibody that binds the CD4-binding site on gp120 (b12), one that binds a glycan configuration on the outer domain of gp120 (2G12) and three that bind the membrane-proximal external region (MPER) on gp41 (2F5, Z13e1, and 4E10) ^11–13. More recently, considerably more broad and potent antibodies have been discovered that target the CD4-binding site of the envelope protein^14–17 (for which VRC01 is a prototype) and glycan containing regions of the V1/V2 and V3 regions of gp120^4,18–20 (for which PG9 and PGT128 are prototypes). The specificities of these new antibodies are providing important information regarding antigen targets on Env to which the humoral immune response might be directed to mediate broad and potent neutralization. However, evidence for these specificities in many chronically infected patients within our cohort is lacking, suggesting that broad and potent neutralization may be mediated by other specificities.

Here we report isolation of a broad and potent gp41 MPER-specific human mAb, 10E8, from an HIV-1-infected individual with high neutralization titers. 10E8 is among the most broad and potent antibodies thus far described, and lacks many of the characteristics previously thought to limit the usefulness of MPER-specific antibodies in vaccines or passive therapies, including lipid binding and autoreactivity. In addition, the crystal structure of 10E8, along with biochemical binding studies, demonstrate that the breadth of 10E8 is mediated by its unique mode of recognition of a structurally conserved site-of-vulnerability within the gp41 MPER.

10E8 isolation and neutralizing properties

To understand the specificities and binding characteristics that underlie a broadly neutralizing antibody response we developed techniques that permitted isolation of human monoclonal antibodies without prior knowledge of specificity²⁰. Serum from one donor, N152, exhibited neutralizing breadth and potency in the top 1% of our cohort against a 20 cross-clade pseudovirus panel (Supplementary Table 1) ¹. Peripheral blood CD19+IgM-IgD-IgA- memory B cells from this patient were sorted and expanded for 13 days with IL-2, IL-21, and CD40-ligand expressing feeder cells. The supernatants of ~16,500 B cell cultures were screened and IgG genes from wells with neutralization activity were cloned and re-expressed²¹ and two novel antibodies (10E8 and 7H6) were isolated.

Nucleotide sequence analysis of DNA encoding 10E8 and 7H6 revealed that both were IgG3 antibodies and were somatic variants of the same IgG clone. These antibodies were derived from IGHV3-15*05 and IGLV3-19*01 germline genes, and were highly somatically mutated in variable genes of both heavy chain (21%) and lambda light chain (14%) compared to germline. These antibodies also possessed a long heavy-chain complementarity-determining region (CDR H3) loop composed of 22 amino acids (Fig. 1a). The heavy chains of 10E8 and 7H6 were identical and there were only two residue differences in the light chain (Supplemental Fig. 1)²².

To assess neutralization activity of the clonal variants, they were initially tested against 5 Env-pseudoviruses (Supplementary Table 1a), and mAb 10E8 was selected for further study. To determine if the neutralization activity of 10E8 was representative of the overall neutralization specificity present in patient N152 donor serum, the neutralization panel was expanded to 20 Env-pseudoviruses, and 10E8 was tested in parallel with N152 donor serum. Although there were some similarities in the pattern of neutralization of highly resistant variants, a correlation of the neutralization IC₅₀ of mAb 10E8 and ID₅₀ of N152 serum did not achieve statistical significance (p=0.11; Supplementary Fig. 2 and Supplementary Table 1b). This finding suggests that although 10E8 may play a major role, the full breadth of neutralization of by N152 serum is likely mediated by an amalgam of 10E8-like or other antibodies.

To compare the neutralization potency and breadth of 10E8 with other broadly neutralizing anti-HIV-1 antibodies, 10E8 was then tested in a 181-isolate Env-pseudovirus panel in parallel with 4E10, 2F5, VRC01, NIH45-46, 3BNC117, PG9, and PG16 (Fig. 1b and Supplementary Table 1c). At an IC₅₀ below 50 μg/ml, 10E8 neutralized 98% of the tested pseudoviruses compared to 98% for 4E10 and 89% for VRC01. However, at an IC₅₀ below 1 μg/ml, 10E8 neutralized 72% of the tested viruses compared to 37% for 4E10. The median and geometric mean IC₅₀ values for 10E8 were below 1 ug/ml. Thus, 10E8 mediates broad and potent neutralization against a large range of viruses and the potency is comparable to some of the best available monoclonal antibodies.

10E8 epitope specificity and binding

To map the epitope of the 10E8 antibody, we tested binding to different subregions of Env by enzyme-linked immunosorbent assay (ELISA). 10E8 bound strongly to gp140, gp41, and the 4E10-specific MPER peptide, but not to gp120 (Fig. 2a). To further map the 10E8 epitope within the MPER, we examined binding of 10E8 to overlapping peptides corresponding to the 2F5 (656–671), Z13e1 (666–677), and 4E10 (671–683) specificities. 10E8 bound to the full MPER and the 4E10-specific peptides, but not 2F5- or Z13e1-specific peptides. Within the 4E10 epitope, when a peptide with a truncated C-terminus was tested, 4E10.19 (671–680), 10E8 binding was weakened considerably, suggesting that the three terminal amino acids of the MPER, Tyr681, Ile682, and Arg683, were crucial for 10E8 binding (Supplementary Fig.3a). Consistent with these results, only the full MPER and 4E10-specific peptides blocked 10E8-mediated neutralization of the chimeric C1 virus, which contains the HIV-2 Env with the HIV-1 MPER (Supplementary Fig. 3b). Taken together, these data suggest that the minimal 10E8 epitope is located within residues 671–683 of the MPER although additional contacts toward the amino terminus of the MPER could not be excluded.

To more precisely map the epitope of 10E8, a panel of alanine mutant peptides scanning MPER residues 671–683 was used to block 10E8 neutralization of the chimeric C1 virus in a TZM-bl assay (Fig. 2b)²³. MPER peptides with alanine substitutions at Trp672, Phe673 or Thr676 failed to block 4E10 or 10E8 neutralization, suggesting that these residues are critical for both 4E10 and 10E8 binding. Residue Asn671 and residue Arg683, both of which are not required for 4E10 binding, were found to be critical for 10E8 binding and neutralization (Supplementary Table 2 and Fig. 2b). We also tested the ability of 10E8 to neutralize HIV-1_JR2 pseudoviruses with alanine substitutions in MPER residues 660–683 (Supplementary Table 3). Consistent with the effects of alanine substitutions on peptide binding, residues Asn671 and Arg683 were critical for 10E8, but not 4E10, neutralization. Individual alanine substitutions at residues 671–673, 680 and 683 resulted in reduced neutralization sensitivity to 10E8 most apparent at the IC₉₀ level rather than the IC₅₀ level. Although the mechanism for this phenomenon is unclear, a similar effect has been observed previously when MPER mutations cause partial resistance to 4E10²⁴. Taken together, these results suggested that 10E8 recognized a novel epitope, which overlaps the known 4E10 and Z13e1 epitopes, but differs in a critical dependence on binding to Asn671 and Arg683, the last residue of the MPER.

We next investigated whether the greater neutralization potency of 10E8 compared to other MPER antibodies was a result of higher binding affinity to the MPER. Capture of a biotinylated peptide encompassing the full MPER (656–683) to a surface-plasmon resonance chip allowed the binding kinetics of Fabs 10E8, 2F5 and 4E10 to be examined. In contrast to its higher neutralization potency, the K_D of 10E8 to this MPER peptide was weaker than that of 2F5 and 4E10; 17 nM for 10E8 versus 3.8 nM for 2F5 and 0.74 nM for 4E10 (Supplementary Fig. 4). Therefore, the affinity of 10E8 for the MPER in a soluble peptide format did not explain its greater neutralization potency compared to other MPER-specific antibodies.

Prevalence of 10E8-like antibodies

We next investigated the prevalence of MPER-specific and 10E8-like neutralizing antibodies in our cohort of HIV-1-infected donors. We selected 78 sera from our cohort with a neutralization ID₅₀>100 against at least 1 pseudovirus in a 5-virus mini-panel¹. The median time since diagnosis of these donors was 13.5 years, median CD4 count was 557 cells/μl, median plasma HIV RNA 5573 copies/ml, and they were not receiving antiretrovirals. We tested neutralization against the HIV-2/HIV-1 chimera C1 (Supplementary Table 4)²⁵. Of 78 sera, 21 exhibited neutralization activity against the HIV-2/HIV-1 C1 virus (Supplementary Table 5). To map the region that was targeted by these sera, neutralization was measured using 7 HIV-2/HIV-1 chimeras containing subdomains of the MPER (Supplementary Table 4)²⁵. Of the 21 sera with neutralization activity against the entire MPER, 8 exhibited a neutralization pattern similar to that observed for 10E8, which entailed neutralization of only those HIV-2/HIV-1chimeric viruses that contained the terminal residue of the MPER Arg683 (C4, C4GW and C8; Supplementary Table 5). To further confirm these results, we used peptides corresponding to different portions of the MPER to block sera neutralization of the HIV-2/HIV-1 chimera C1 (Supplementary Table 6). Of the 8 sera found to have a 10E8-like pattern based upon neutralization of the chimeras, 3 were blocked by peptides consistent with 10E8-like activity. An additional 3 of the 8 10E8-like sera were blocked by peptides in a pattern consistent with a combination of 10E8 and Z13e1-like antibodies. The 6 patients whose sera had 10E8-like activity did not differ from the remaining 72 patients with regard to clinical course or HIV neutralization (Supplementary Fig. 5, legend). Overall, 27% of the tested patient sera exhibited anti-MPER neutralizing activity. This prevalence is considerably higher than observed in prior work, possibly related to selection of donors with known neutralizing activity^8,26–28. Further, 8% of the tested sera had 10E8-like antibodies (Supplementary Fig. 5), suggesting that 10E8-like antibodies are not rare.

Analysis of 10E8 autoreactivity

A property common to the previously characterized MPER mAbs 2F5 and 4E10 is that they cross-react with self-antigens²⁹. In addition, binding to both the cell membrane and the Env trimer is thought to be important for optimal neutralization by these antibodies and this autoreactivity may be an obstacle to the elicitation of similar antibodies by a vaccine^29,30. Surface plasmon resonance analysis showed that 10E8 did not bind to anionic phospholipids, such as phosphatidyl choline-cardiolipin (PC-CLP) and phosphatidyl choline-phosphatidyl serine (PC-PS) liposomes (Fig. 3a). 10E8 also did not bind HEp-2 epithelial cells, in contrast to 2F5 and 4E10 that bound in a cytoplasmic and nuclear pattern (Fig. 3b). Additionally, 10E8 did not bind autoantigens, such as Sjogren's syndrome antigens A and B, Smith antigen, ribonucleoprotein, scleroderma 70 antigen, Jo1 antigen, centromere B and histone (Supplementary Table 7). Taken together, these results suggest that 10E8, in contrast to other MPER antibodies, is not autoreactive.

Virion accessibility of 10E8

The 2F5 and 4E10 antibodies have been shown to bind relatively poorly to the HIV-1 envelope spike on the surface of infected cells or to cell-free virions, and react more efficiently after Env engagement of the CD4 receptor³¹. We measured binding to cleaved, full-length envelope spikes on HIV_JRFL transfected cells (Supplementary Fig. 6a). Although 10E8 bound less efficiently than other antibodies such as VRC01 or F105, where accessibility is not an issue, it bound more efficiently than either 2F5 or 4E10. In contrast to results of alanine substitution, a mutation in the 4E10 (F673S) region in full-length HIV_JRFL envelope spikes enhanced 10E8 binding although the mechanism remains unclear. A mutation in the 2F5 (K665E) region had no influence on 10E8 binding. These data suggest that 10E8 has modestly greater access to the MPER epitope on the cell surface than either 2F5 or 4E10.

To assess binding to cell-free virus, we incubated virions with antibody, washed out unbound antibody, and testing neutralization^31–33. During washing, antibodies that cannot access their Env target on free virions will be largely removed and therefore neutralization will be diminished. As a control, neutralization of the HXBc2 isolate was not diminished by washing, because the MPER region is accessible on this laboratory adapted isolate³¹. Washing also had little impact on neutralization of JRFL by VRC01. Consistent with prior work, 2F5 and 4E10 neutralization of most virus isolates tested was substantially diminished after washing (Supplementary Fig. 6b)^31,34. In contrast to 2F5 and 4E10, washing had a smaller effect on 10E8 neutralization of most viruses tested, as measured by the area under the curve or analysis of the fold-change in neutralization at a fixed inhibitory concentration (Supplementary Fig. 6c). Although 10E8 is not fully able to access its epitope on the native viral spike similarly to VRC01, under most experimental conditions tested it was better able to access its epitope than either 2F5 or 4E10.

Structure of 10E8-gp41 complex

To provide an atomic-level understanding of the interaction of 10E8 with HIV-1, we crystallized the antigen-binding fragment (Fab) of 10E8 in complex with a peptide encompassing the entire 28-residue gp41 MPER (residues 656–683). Monoclinic crystals diffracted to 2.1 Å resolution, and structure solution and refinement to R_cryst = 18.01% (R_free = 21.76%) revealed two complexes in the asymmetric unit (heretofore referred to as complexes 1 and 2) (Supplementary Table 8). Overall, 10E8 bound to one face of the MPER peptide, which formed two helices, each 15–20 Å in length and oriented 100° relative to each other (Fig. 4a). Electron density was observed for the entire MPER, ranging from Asn656-Arg683 (Leu660-Arg683 for complex 2), with the highest degree of ordered density observed from residue Trp666 within the N-terminal helix through to Arg683 of the C-terminal helix (Supplementary Fig. 7). Analysis of main-chain dihedral angles (Supplementary Table 9) indicated that the N-terminal α-helix extends from residue Asn657 to Ala667, tightens into a 3₁₀-helix between residues Ser668 and Leu669, before turning at residues Trp670 and Asn671. The C-terminal α-helix, capped by Asn671, starts at residue Trp672 and extends to residue Arg683, the final residue of the MPER (Fig. 4a,b).

The 10E8 antibody contacts the gp41 MPER primarily through its heavy chain, although crucial contacts are also mediated by the light chain CDR L3 (Fig. 4c and Supplementary Tables 10–12). Three predominant loci of interaction are observed between the antibody and gp41 (Supplementary Tables 13–14): One between residues of the tip of the CDR H3 loop and the tip of the C-terminal helix of the peptide, a second between residues of the CDR H2 loop and residues of the hinge region of the peptide, and a third at the juncture of the three heavy chain CDR loops and the light chain CDR L3, which form a hydrophobic cleft that holds residues of the beginning of the MPER C-terminal helix (Fig. 4b).

10E8-gp41 interface

To complement the results observed for the mutagenesis of the highly conserved 10E8 epitope (Fig. 4d and Supplementary Table 2), each residue of the 10E8 paratope, as determined from the crystal structure, was individually mutated to alanine and the resulting 25 10E8 variants assessed for affinity to a soluble MPER peptide. Overall, the most pronounced effects of the alanine mutations on the binding affinity of 10E8 to a soluble MPER peptide occurred within residues of the CDR H3 loop, though mutations within the hydrophobic cleft also showed substantial effect (Fig. 4e, Supplementary Table 15, and Supplementary Fig. 8). 10E8 residues identified by alanine scan as critical for the interaction with gp41 stretched from the cleft all the way to the tip of the CDR H3 (Fig. 4e) and were mirrored by a corresponding stretch of gp41 residues which substantially affected 10E8 binding when mutated to alanine (Fig. 4f).

The same panel of 10E8 alanine mutations was tested for neutralization potency against a panel of five Env-pseudoviruses that included both Tier 1 and Tier 2 viruses (Supplementary Table 16). Similar to the binding data, residues of the 10E8 CDR H3 had dramatic effects on neutralization, as did residues of the hydropohobic cleft (Fig. 4g). Generally, K_Ds of paratope mutants correlated with neutralization (Supplementary Fig. 9). Backbone interactions (on both 10E8 and gp41) also contribute to the interface, especially between the CDR H2 of 10E8 and the hinge region of the MPER, though these are silent in alanine scan analyses. Overall, 10E8 utilizes a narrow band of residues (~20 x 5 Å) that stretches from the CDR H1 and H2 and extends along most of the CDR H3 to recognize a string of highly conserved hydrophobic gp41 residues, and a critical charged residue, Arg/Lys683, that occurs just prior to the transmembrane region (Fig. 4f,h).

A conserved gp41-neutralization determinant

Several structures of neutralizing antibodies in complex with the MPER of gp41 have been reported previously, including those for antibodies 2F5, Z13e1 and 4E10^35–39(Supplementary Fig. 10a). The MPER adopts divergent loop conformations when bound by 2F5 and Z13e1 and an α-helix when bound by 4E10. Comparison of 2F5, Z13e1, and 4E10 epitopes with 10E8-bound gp41 revealed that only the 4E10 epitope has similar secondary structure, with superposition yielding an RMSD of 2.49 Å for all atoms of residues 671–683 and 0.98 Å for main-chain atoms (Supplementary Fig. 10b, Supplementary Table 17).

To compare further the recognition of 10E8 and 4E10, we examined their angles of epitope approach. As shown in Supplementary Fig. 10c-f, alignment of the recognized MPER helix places 10E8 and 4E10 into similar overall spatial positions. The relative orientations of the recognized helix and the heavy and light chains of the two antibodies, however, differ dramatically. With 10E8, the C-terminal helix is perpendicular to the plane bisecting heavy and light chains (Supplementary Fig. 10c,e); with 4E10, the recognized helix is at the interface between heavy and light chains (Supplementary Fig. 10d,f). Perhaps relevant to this, 10E8 utilizes CDR loops almost exclusively in its recognition of gp41, while 4E10 incorporates substantial β-strand interactions with gp41 at the interface between the heavy and light chains.

The differing modes of 10E8 and 4E10 recognition of the conserved C-terminal MPER helix result in a substantial difference in the proportion of the recognized helical face: 10E8 contacts roughly a third of the helical face, while 4E10 contacts over half (Supplementary Fig. 10g,h and Supplementary Tables 18–19). The smaller contact surface of 10E8 may provide an explanation for the reduced recognition of lipid surfaces by 10E8 versus 4E10 – providing a potential structure-based explanation for reduced autoreactivity of 10E8.

Sequence variation and 10E8 neutralization

To place the specificity and structural data into the context of known variation of the MPER, we analyzed viral sequences with resistance to neutralization by 10E8 (Fig. 5a). Of the 183 viruses tested, only 3 were highly resistant to 10E8 with IC₅₀ >50 μg/ml. Each of these viruses had substitutions at positions found to affect neutralization by alanine scanning (Asn671, Trp672, Phe673, and Trp680). Plasma virus of the patient N152, from whom 10E8 was cloned, is also likely resistant to 10E8 mediated neutralization⁴⁰. Sequence analysis of plasma viral RNA revealed rare substitutions at positions Trp680 and Lys/Arg683 (Fig. 5a). These residues are typically highly conserved with variation only occurring in 1.17% of 3,730 HIV Env sequences in the Los Alamos Database (www.hiv.lanl.gov). When the substitutions for the 3 resistant viruses and the patient viruses were placed on the background of the sensitive JR2 virus, substitutions at Asn671Thr, Trp672Leu, and Phe673Leu had a modest effect on the IC₅₀ but raised the IC₈₀ above 20 μg/ml. In the structural analysis, direct contacts with 10E8 were not observed at position 671 suggesting the effects on neutralization of Thr or Ala substitutions at this position are mediated by conformational or other effects within gp41. The combination of Trp672Leu and Phe673Leu conferred high-level resistance at the IC₅₀ and IC₈₀ level. Changes corresponding to the patient’s dominant circulating virus had a similar effect. Although Lys/Arg683Gln alone conferred resistance at the IC₈₀ level, together Trp680Arg and Lys/Arg683Gln resulted in greater resistance to 10E8 (Fig. 5a). When taken together with the analysis of the 10E8 paratope, these data suggest that in addition to Trp672, Phe673, and Trp680 found in the 4E10 epitope, the additional 10E8-bound residue Lys/Arg683 is critical to neutralization. In addition to other differences in binding based upon structural analyses noted above, it is possible that the additional potency of 10E8 compared to 4E10 against naturally occurring viral variants may be mediated through binding of highly conserved residues Trp680 and Lys/Arg683 that directly interact with the 10E8 CDRH3.

Discussion

10E8 is a broad and potent neutralizing antibody with important implications for efforts to stimulate such antibodies with vaccines. Previous MPER antibodies were somewhat limited in potency, and had a more limited ability to access MPER on Env of primary isolates. In addition, lipid binding and autoreactivity were thought to be characteristics of MPER antibodies and important obstacles to their elicitation by vaccines^9,29,30. However, 10E8 lacks each of these characteristics. In addition, antibodies with a similar specificity were not rare in our chronically infected cohort. This suggests that 10E8-like antibodies were not deleted from the repertoire because of autoreactivity. These results further suggest that 10E8-like antibodies might be raised in a larger fraction of HIV-uninfected persons receiving a vaccine designed to elicit these antibodies without the B cell defects of chronic HIV infection. Design of such a vaccine will likely require not only presentation of an intact 10E8 epitope but also use of a platform sufficiently immunogenic to drive the evolution of 10E8-like antibodies.

The extraordinary breadth and potency of 10E8 appears to be mediated by its ability to bind highly conserved residues within MPER. Although the epitope of 10E8 overlaps those of known mAbs such as 4E10, it differs in recognition surface, angle of approach, lipid binding, and self-reactivity. Alanine scanning, structural analysis, and paratope analysis each indicate that 10E8 makes crucial contacts with highly conserved residues Trp672, Phe673, Trp676 and Lys/Arg683. The extraordinary breadth of some potent mAbs, for example that bind the CD4 binding site, is thought to be conferred by blocking a functionally important site that is critical for viral entry. Whether 10E8 impairs Env function or simply acts by binding highly conserved residues remains to be determined. Nonetheless, the breadth and potency of 10E8 demonstrates a conserved site of gp41 vulnerability (Fig. 5b) that is an important target antigen for HIV neutralization and that will likely reinvigorate interest in MPER-based HIV vaccine design.

Methods

Supplementary Material