Research Article

Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

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Science  13 Aug 2010:
Vol. 329, Issue 5993, pp. 811-817
DOI: 10.1126/science.1192819

Designer Anti-HIV

Developing a protective HIV vaccine remains a top global health priority. One strategy to identify potential vaccine candidates is to isolate broadly neutralizing antibodies from infected individuals and then attempt to elicit the same antibody response through vaccination (see the Perspective by Burton and Weiss). Wu et al. (p. 856, published online 8 July) now report the identification of three broadly neutralizing antibodies, isolated from an HIV-1–infected individual, that exhibited great breadth and potency of neutralization and were specific for the co-receptor CD4-binding site of the glycoprotein 120 (gp120), part of the viral Env spike. Zhou et al. (p. 811, published online 8 July) analyzed the crystal structure for one of these antibodies, VRC01, in complex with an HIV-1 gp120. VRC01 focuses its binding onto a conformationally invariant domain that is the site of initial CD4 attachment, which allows the antibody to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. The epitopes recognized by these antibodies suggest potential immunogens that can inform vaccine design.


During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

Successful vaccine development often takes advantage of clues from humoral responses elicited by natural infection. For HIV-1, neutralizing antibody responses elicited within the first year or two of infection are generally strain-specific (1) and thus provide limited insight into vaccine design (2). A few monoclonal antibodies from HIV-1–infected individuals, however, are broadly neutralizing, and an effort has been made to facilitate vaccine design by defining their structures (3, 4).

The well-studied broadly neutralizing antibodies to HIV-1—2G12, 2F5, 4E10, and b12—have unusual characteristics that have posed barriers to eliciting similar antibodies in humans (5). Thus, in addition to having broad capacity for neutralization, an appropriate antibody should be present in high enough titers in humans to suggest that it can be elicited in useful concentrations. We and others have screened cohorts of sera from infected individuals to find broadly neutralizing responses that are detectable in a substantial percentage of subjects (610). One serum response that satisfies these criteria has been mapped to the site on the HIV-1 gp120 envelope (Env) glycoprotein that binds to the CD4 receptor (8).

Although potentially accessible, the CD4-binding site is protected from humoral recognition through glycan and conformational masking (11). The identification of monoclonal antibodies against this site is described in a companion manuscript (12). In brief, we created resurfaced, conformationally stabilized probes, with antigenic specificity for the initial site of CD4 attachment to gp120 (13). This site, a conformationally invariant subset of the CD4-binding surface, is vulnerable to antibody-mediated neutralization (13), and we used probes specific for this site to identify antibodies that neutralize most viruses (12). In this work, we analyzed the crystal structure for one of these antibodies, VRC01, in complex with an HIV-1 gp120 core. We deciphered the basis of VRC01 neutralization, identified mechanisms of natural resistance, showed how VRC01 minimizes such resistance, examined potential barriers to elicitation, and defined the role of affinity maturation in gp120 recognition.

Similarities of Env recognition by CD4 and VRC01 antibody. To gain a structural understanding of VRC01 neutralization, we crystallized the antigen-binding fragment (Fab) of VRC01 in complex with an HIV-1 gp120 from the clade A/E recombinant 93TH057 (14, 15). The crystallized gp120 consisted of its inner domain–outer domain core, with truncations in the variable loops V1/V2 and V3 as well as the N- and C-termini, which are regions known to extend away from the main body of the gp120 envelope glycoprotein (16). Diffraction to 2.9 Å resolution was obtained from orthorhombic crystals, which contained four copies of the VRC01-gp120 complex per asymmetric unit, and the structure was solved by means of molecular replacement and refined to a crystallographic R value of 19.7% (Fig. 1 and table S1) (17).

Fig. 1

Structure of antibody VRC01 in complex with HIV-1 gp120. Atomic-level details for broad and potent recognition of HIV-1 by a natural human antibody are depicted with polypeptide chains in ribbon representations. The gp120 inner domain is shown in gray, the bridging sheet in blue, and the outer domain in red, except for the CD4-binding loop (purple), the D loop (brown), and the V5 loop (orange). The light chain of the antigen-binding fragment (Fab) of VRC01 is shown in light blue, with complementarity-determining regions (CDRs) highlighted in dark blue (CDR L1) and marine blue (CDR L3). The heavy chain of Fab VRC01 is shown in light green, with CDRs highlighted in cyan (CDR H1), green (CDR H2), and pale yellow (CDR H3). Both light and heavy chains of VRC01 interact with gp120: The primary interaction surface is provided by the CDR H2, with the CDR L1 and L3 and the CDR H1 and H3 providing additional contacts.

The interaction surface between VRC01 and gp120 encompasses almost 2500 Å2, with 1244 Å2 contributed by VRC01 and 1249 Å2 by gp120 (18). On VRC01, both heavy chain (894 Å2) and light chain (351 Å2) contribute to the contact surface (table S2), with the central focus of binding on the heavy chain–second complementarity–determining region (CDR H2). Over half of the interaction surface of VRC01 (644 Å2) involves CDR H2, a mode of binding that is reminiscent of the interaction between gp120 and the CD4 receptor; CD4 is a member of the V-domain class of the immunoglobulin superfamily (19), and the CDR2-like region of CD4 is a central focus of gp120 binding (Fig. 2A and table S3) (20). For CD4, the CDR2-like region forms antiparallel, intermolecular hydrogen bonds with residues 365gp120 to 368gp120 of the CD4-binding loop of gp120 (20) (Fig. 2B); with VRC01, one hydrogen-bond is observed between the carbonyl oxygen of Gly54VRC01 and the backbone nitrogen of Asp368gp120. This hydrogen bond occurs at the loop tip, an extra residue relative to CD4 is inserted in the strand, and the rest of the potential hydrogen bonds are of poor geometry or distance (Fig. 2C and table S4). Other similarities and differences with CD4 are found: Of the two dominant CD4 residues (Phe43CD4 and Arg59CD4) involved in interaction with gp120, VRC01 mimics the arginine interaction but not the phenylalanine one (Fig. 2, B and C). Lastly, substantial correlation was observed between gp120 residues involved in binding VRC01 and CD4 (fig. S1).

Fig. 2

Structural mimicry of CD4 interaction by antibody VRC01. VRC01 shows how a double-headed antibody can mimic the interactions with HIV-1 gp120 of a single-headed member of the immunoglobulin superfamily, such as CD4. (A) Comparison of HIV-1 gp120 binding to CD4 (N-terminal domain) and VRC01 (heavy chain–variable domain). Polypeptide chains are depicted in ribbon representation for (right) the VRC01 complex and (left) the CD4 complex with the lowest gp120 root mean square deviation (table S5). The CD4 complex (3JWD) (16) is indicated in yellow for CD4 and red for gp120, except for the CDR-binding loop (purple). The VRC01 complex is colored as in Fig. 1. Immunoglobulin domains are composed of two β sheets, and the top sheet of both ligands is labeled with the standard immunoglobulin-strand topology (strands G, F, C, C′, C″). (B and C) Interface details for (B) CD4 and (C) VRC01. Close-ups are shown of critical interactions between the CD4-binding loop (purple) and the C″ strand as well as between Asp368gp120 and either Arg59CD4 or Arg71VRC01. Hydrogen bonds with good geometry are depicted by blue dotted lines, and those with poor geometry are depicted in gray. Atoms from which hydrogen bonds extend are depicted in stick representation and colored blue for nitrogen and red for oxygen. In the left panel of (C), the β15-strand of gp120 is depicted to aid comparison with (B), although because of the poor hydrogen-bond geometry, it is only a loop. (D) Comparison of VRC01- and CD4-binding orientations. Polypeptides are shown in ribbon representation, with gp120 colored the same as in (A) and VRC01 depicted with heavy chain in dark yellow and light chain in dark gray. When the heavy chain of VRC01 is superimposed onto CD4 in the CD4-gp120 complex, the position assumed by the light chain evinces numerous clashes with gp120 (left). The VRC01-binding orientation (right) avoids clashes by adopting an orientation rotated by 43° and translated by 6 Å.

Superposition of the gp120 core in its VRC01-bound form with gp120s in other crystalline lattices and bound by other ligands indicates a CD4-bound conformation [Protein Data Bank (PDB) ID number 3JWD] (16) to be most closely related in structure, with a Cα-root-mean-square deviation of 1.03 Å (table S5). Such superposition of gp120s from CD4- and VRC01-bound conformations brings the N-terminal domain of CD4 and the heavy chain–variable domain of VRC01 into close alignment (Fig. 2), with 73% of the CD4 N-terminal domain volume overlapping with VRC01 (21). This domain overlap is much higher than observed with the heavy chains of other CD4-binding-site antibodies, such as b12, b13, or F105 (table S6). However, when the VRC01 heavy chain is superimposed—on the basis of conserved framework and cysteine residues—on CD4 in the CD4-gp120 complex, clashes are found between gp120 and the entire top third of the VRC01 variable light chain (Fig. 2D) (22). In its complex with gp120, VRC01 rotates 43° relative to the CD4-defined orientation and translates 6 Å away from the bridging sheet, to a clash-free orientation that mimics many of the interactions of CD4 with gp120, although with considerable variation. Analysis of electrostatics shows that the interaction surfaces of VRC01 and CD4 are both quite basic, although the residue types of contacting amino acids are distinct (fig. S2). Thus, although VRC01 mimics CD4 binding to some extent, considerable differences are observed.

Structural basis of VRC01 breadth and potency. When CD4 is placed into an immunoglobulin context by fusing its two N-terminal domains to a dimeric immunoglobulin constant region, it achieves reasonable neutralization. VRC01, however, neutralizes more effectively (Fig. 3A) (12). To understand the structural basis for the exceptional breadth and potency of VRC01, we analyzed its interactive surface with gp120. VRC01 focuses its binding onto the conformationally invariant outer domain, which accounts for 87% of the contact surface area of VRC01 (table S7). The 13% of the contacts made with flexible inner domain and bridging sheet are noncontiguous, and we judged these not to be critical for binding. In contrast, CD4 makes 33% of its contacts with the bridging sheet, and many of these interactions are essential (20). The reduction in inner domain and bridging sheet interactions by VRC01 is accomplished primarily by a 6 Å translation relative to CD4, away from these regions; critical contacts such as made by Phe43CD4 to the nexus of the bridging sheet–outer domain are not found in VRC01, whereas those to the outer domain (such as Arg59CD4) are mimicked by VRC01.

Fig. 3

Structural basis of antibody VRC01 neutralization breadth and potency. VRC01 displays remarkable neutralization breadth and potency, a consequence in part of its ability to bind well to different conformations of HIV-1 gp120. (A) Neutralization dendrograms. The genetic diversity of current circulating HIV-1 strains is displayed as a dendrogram, with locations of prominent clades (such as A, B, and C) and recombinants (such as CRF02_AG) labeled. The strains are colored by their neutralization sensitivity to (left) VRC01 or (right) CD4. VRC01 neutralizes 72% of the tested HIV-1 isolates with an IC80 (the concentration of a substance required to inhibit the activity of another substance by 80%) of less than 1 μg/ml; in contrast, CD4 neutralizes 30% of the tested HIV-1 isolates with an IC80 of less than 1 μg/ml (table S14). (B) Comparison of binding affinities (Kd). Binding affinities for VRC01 and various other gp120-reactive ligands as determined by means of surface-plasmon resonance are shown on a bar graph. White bars represent affinities for gp120 restrained from assuming the CD4-bound state (23), and black bars represent affinities for gp120 fixed in the CD4-bound state (13). Binding too weak to be measured accurately is shown as with an asterisk and bar at 10−5 M Kd. (C) Neutralization of viruses with altered sampling of the CD4-bound state. Mutant S375Wgp120 favors the CD4-bound state, whereas mutants H66Agp120 and W69Lgp120 disfavor this state. Neutralization by VRC01 (top) is similar for wild type (WT) and all three mutant viruses, whereas neutralization by CD4 (bottom) correlates with the degree to which gp120 in the mutant viruses favors the CD4-bound state.

To determine the affinity of VRC01 for gp120 in CD4-bound and non–CD4-bound conformations, we used surface-plasmon resonance spectroscopy to measure the affinity of VRC01 and other gp120-reactive antibodies and ligands to two gp120s: a β4-deletion developed by Harrison and colleagues that is restrained from assuming the CD4-bound conformation (23) or a disulfide-stabilized gp120 core that is largely fixed in the CD4-bound conformation in the absence of CD4 itself (13) (Fig. 3B and fig. S3). VRC01 showed high affinity to both CD4-bound and non–CD4-bound conformations, which is a property shared by the broadly neutralizing b12 antibody (13). In contrast, antibodies F105 and 17b as well as soluble CD4 showed strong preference for either one, but not both, of the conformations.

To assess the binding of VRC01 in the context of the functional viral spike, we examined its ability to neutralize variants of HIV-1 with gp120 changes that affect the ability to assume the CD4-bound state. Two of these mutations, His66Ala gp120 and Trp69Leugp120, are less sensitive (24), whereas a third, Ser375Trpgp120, is more sensitive to neutralization by CD4 (24, 25). VRC01 neutralized all three of these variant HIV-1 viruses with similar potency (Fig. 3C), suggesting that VRC01 recognizes both CD4-bound and non–CD4-bound conformations of the viral spike. This diversity in recognition allows VRC01 to avoid the conformational masking that hinders most CD4-binding-site ligands (26) and to potently neutralize HIV-1 (27).

Precise targeting by VRC01. Prior analysis of effective and ineffective CD4-binding-site antibodies suggested that precise targeting to the vulnerable site of initial CD4 attachment is required to block viral entry (11, 28). This site represents the outer domain contact for CD4 (13). Analysis of the VRC01 interaction with gp120 shows that it covers 98% of this site (Fig. 4, A and B, and fig. S4), comprising 1089 Å2 on the gp120 outer domain, which is about 50% larger than the 730 Å2 surface covered by CD4. The VRC01 contact surface outside the target site is largely limited to the conformationally invariant outer domain and avoids regions of conformational flexibility. This concordance of binding is much greater than for ineffective CD4-binding-site antibodies as well as for those that are partially effective, such as antibody b12 (11, 13) (fig. S4).

Fig. 4

Natural resistance to antibody VRC01. VRC01 precisely targets the CD4-defined site of vulnerability on HIV-1 gp120. Its binding surface, however, extends outside of the target site, and this allows for natural resistance to VRC01 neutralization. (A) Target site of vulnerability. The CD4-defined site of vulnerability is the initial contact surface of the outer domain of gp120 for CD4 and comprises only two thirds of the contact surface of gp120 for CD4 (13). The molecular surface of HIV-1 gp120 has been colored according to its underlying domain substructure: red for the conformationally invariant outer domain, gray for the inner domain, and blue for the highly mobile bridging sheet. Regions of the gp120 surface that interact with VRC01 have been colored green, with the CD4-defined site of vulnerability outlined in yellow. The view shown here is rotated 90° about the horizontal from the view in Figs. 1 and 2. (B) VRC01 recognition. The molecular surface of gp120 in the VRC01 bound conformation is colored as in (A). The variable domains of VRC01 are shown in ribbon representation, with the heavy and light chains colored as in Fig. 1 and extensions to constant regions indicated. (C) Antigenic variation. The polypeptide backbone of gp120 is colored according to sequence conservation: blue if conservation is high and red if conservation is low. (D) Molecular surface of VRC01 and select interactive loops of gp120. Variation at the tip of the V5 loop is accommodated by a gap between heavy and light chains of VRC01.

The outer domain–contact site for CD4 is shielded by glycan (11, 20). Contacts by the VRC01 light chain (Tyr28VRC01 and Ser30VRC01) are made with the protein-proximal N-acetyl-glucosamine from the N-linked glycan at residue 276gp120 (29). Thus instead of being occluded by glycan, VRC01 makes use of a glycan for binding. Other potential glycan interactions may occur with different strains of HIV-1 because the VRC01 recognition surface on the gp120–outer domain extends further than that of the functionally constrained CD4 interaction surface, especially into the loop D and the often-glycosylated V5 region (fig. S5).

Natural resistance to antibody VRC01. In addition to conformational masking and glycan shielding, HIV-1 resists neutralization by antigenic variation. In a companion manuscript, we show that of the 190 circulating HIV-1 isolates tested for sensitivity to VRC01, 173 were neutralized and 17 were resistant (12). To understand the basis of this natural resistance to VRC01, we analyzed all 17 resistant isolates by threading their sequences onto the gp120 structure (fig. S5). Variation was observed in the V5 region in resistant isolates, and this variation—along with alterations in gp120 loop D—appeared to be the source of most natural resistance to VRC01 (Fig. 4C and figs. S5 and S6).

Because substantial variation exists in V5, structural differences in this region might be expected to result in greater than 10% resistance. The lower observed frequency of resistance suggests that VRC01 employs a recognition mechanism that allows for binding despite V5 variation. Examination of VRC01 interaction with V5 shows that VRC01 recognition of V5 is considerably different from that of CD4 (fig. S7), with Arg61VRC01 in the CDR H2 penetrating into the cavity formed by the V5 and β24 strands of gp120 (fig. S8). The V5 loop fits into the gap between heavy and light chains; thus, by contacting only the more conserved residues at the loop base, VRC01 can tolerate variation in the tip of the V5 loop (Fig. 4D).

Unusual VRC01 features and contribution to recognition. We examined the structure of VRC01 for special features that might be required for its function. A number of unusual features were apparent, including a high degree of affinity maturation, an extra disulfide bond, a site for N-linked glycosylation, a two-amino-acid deletion in the light chain, and an extensively matured binding interface between VRC01 and gp120 (Fig. 5 and fig. S9). We assessed the frequency with which these features were found in HIV-1 Env-reactive antibodies (appendix S1) or in human antibody-antigen complexes (fig. S10 and tables S8 and S9) and measured the effect of genomic reversion of these features on affinity for gp120 and neutralization of virus (Fig. 5, A to D, and table S10).

Fig. 5

Unusual VRC01 features. The structure of VRC01 displays a number of unusual features, which if essential for recognition might inhibit the elicitation of VRC01-like antibodies. (A to D) Unusual features of VRC01 are shown structurally (far left), in terms of frequency as a histogram with other antibodies (second from left), and in the context of affinity and neutralization measurements after mutational alteration (second from right and right). Affinity measurements were made by means of enzyme-linked immunosorbent assay to the gp120 construct used in crystallization (93TH057), and neutralization measurements were made with a clade A HIV-1 strain Q842.d12. Additional binding and neutralization experiments are reported in table S10. (A) N-linked glycosylation. The conserved tri-mannose core is shown with observed electron density, along with frequency and effect of removal on affinity and neutralization. (B) Extra disulfide. Variable heavy domains naturally have two Cys, linked by a disulfide; VRC01 has an extra disulfide linking CDR H1 and H3 regions. This occurs rarely in antibodies, but its removal through mutation to Ser/Ala has little effect on affinity or neutralization. (C) CDR L1 deletion. A two-amino-acid deletion in the CDR L1 prevents potential clashes with loop D of gp120. Such deletions are rarely observed; reversion to the longer loop may have a moderate effect on gp120 affinity. (D) Somatically altered contact surface. (Left) The VRC01 light chain is shown in violet, and the heavy chain is in green. Residues altered by affinity maturation are depicted with “balls,” and contacts with HIV-1 gp120 are colored red. Approximately half of the contacts are altered during the maturation process. Analysis of human antibody-protein complexes in PDB shows this degree of contact surface alteration is rare; reversion of each of the contact sites to the genome sequence has little effect (table S10), although in aggregate the effect on affinity is larger.

Higher levels of affinity maturation have been reported for HIV-1–reactive antibodies in general (30) and markedly higher levels for broadly neutralizing ones (31). These maturation levels could be a by-product of the persistent nature of HIV-1 infection and may not represent a functional requirement. Removal of the N-linked glycosylation or the extra disulfide bond, which connects CDR H1 and H3 regions of the heavy chain, had little effect on binding or neutralization (Fig. 5, A and B, and table S10). Insertion of two amino acids to revert the light chain deletion had moderate effects, which were larger for an Ala-Ala insertion (50-fold decrease in binding affinity) versus a Ser-Tyr insertion (fivefold decrease in affinity), which mimics the genomic sequence (Fig. 5C and table S10). Lastly, reversion of the interface was examined with either single-, four-, seven- or 12-mutant reversions. For the single-mutant reversions of the interface to the genomic antibody sequence, all 12 mutations had minor effects [most with a less than twofold effect on the dissociation constant (Kd), with the largest effect for a Gly54Ser change having a Kd of 20.2 nM] (table S10). Larger effects were observed with multiple (four, seven, or 12) changes (Fig. 5D and table S10). Thus, although VRC01 has a number of unusual features, no single alteration to genomic sequence substantially altered binding or neutralization.

Elicitation of VRC01-like antibodies. The probability for elicitation of a particular antibody is a function of each of the three major steps in B cell maturation: (i) recombination to produce nascent antibody heavy and light chains from genomic VH-D-J and Vκ/λ-J precursors, (ii) deletion of auto-reactive antibodies, and (iii) maturation through hypermutation of the variable domains to enhance antigen affinity. For the recombination step, a lack of substantial CDR L3 and H3 contribution to the VRC01-gp120 interface (table S2) indicates that specific Vκ/λ-J or VH(D)J recombination is not required (32) (fig. S11). The majority of recognition occurs with elements encoded in single genomic elements or cassettes, suggesting that specific joining events between them are not required. Within the VH cassette, a number of residues associated with the IGHV1-02*02 precursor of VRC01 interact with gp120; many of these are conserved in related genomic VHs, some of which are of similar genetic distance from VRC01 (fig. S12). These results suggest that appropriate genomic precursors for VRC01 are likely to occur at a reasonable frequency in the human antibody repertoire.

Recombination produces nascent B cell–presented antibodies that have reactivities against both self and nonself antigens. Those with auto-reactivity are removed through clonal deletion. With many of the broadly neutralizing antibodies to HIV-1, such as 2G12 (glycan reactive) (33, 34), 2F5, and 4E10 (membrane reactive) (35, 36), this appears to be a major barrier to elicitation. Although this remains to be characterized for genomic revertants and maturation intermediates, no auto-reactivity has so far been observed with VRC01.

The third step influencing the elicitation of VRC01-like antibodies is affinity maturation, which is a process that involves the hypermutation of variable domains combined with affinity-based selection that occurs during B cell maturation in germinal centers (37). In the case of VRC01, 41 residue alterations were observed from the genomic VH gene and 25 alterations from the Vκ gene (including a deletion of two residues) (fig. S13) (38). To investigate the effect of affinity maturation on HIV-1 gp120 recognition, we reverted the VH and Vκ regions of VRC01, either individually or together, to the sequences of their genomic precursors. We tested the affinity and neutralization of these reverted antibodies (Fig. 6A) and combined these data with the genomic reversion data obtained while querying the unusual molecular features of VRC01 (previous section) (Fig. 6B).

Fig. 6

Somatic maturation and VRC01 affinity. Hypermutation of the variable domain during B cell maturation allows for the evolution of high-affinity antibodies. With VRC01, this enhancement to affinity occurs principally through the alteration of noncontact residues, which appear to reform the genomic contact surface from affinity too low to measure to a tight (nanomolar) interaction. (A) Effect of genomic reversions. The VH- and Vκ-derived regions of VRC01 were reverted to the sequences of their closest genomic precursors, expressed as immunoglobulins, and tested for binding as VH- and Vκ-revertants (gHgL), as a VH-only revertant (gH), or as a Vκ-only revertant (gL) to the gp120 construct used in crystallization (93TH057) or to a stabilized HXBc2 core (13). These constructs were also tested for neutralization of a clade A HIV-1 strain Q842.d12. Additional neutralization experiments with clade B and C viruses are reported in (15). (B) Maturation of VRC01 and correlation with binding and neutralization. Affinity and neutralization measurements for the 19 VRC01 mutants created during the structure-function analysis of VRC01 were analyzed in the context of their degree of affinity maturation. Significant correlations were observed, with extrapolation to VH- and Vκ-genomic precursors suggesting greatly reduced affinity of the initial genomic arrangement for gp120.

No antibodies containing VH and Vκ regions, which were fully reverted to their genomic precursors, bound gp120 or neutralized virus (39). Binding affinity and neutralization showed significant correlations with the number of affinity-matured residues (P < 0.0001). Binding to stabilized gp120 did not correlate well with other types of gp120 or to neutralization (table S11), which is related in part to greater retention of binding to VRC01 variants with genomically reverted Vκ regions. Extrapolation of the correlation to the putative genomic V gene sequences predicted binding affinities of 0.7 ± 0.4 μM Kd for gp120 stabilized in the CD4-bound conformation and substantially weaker affinites for nonstabilized gp120s (Fig. 6B and fig. S14).

No single affinity maturation alteration appeared to affect affinity by more than tenfold, suggesting that affinity maturation occurs in multiple small steps, which collectively enable tight binding to HIV-1 gp120. When the effects of VRC01 affinity maturation reversions are mapped to the structure of the VRC01-gp120 complex, they are broadly distributed throughout the VRC01 variable domains rather than focused on the VRC01-gp120 interface. Noncontact residues therefore appear to influence the interface with gp120 through indirect protein-folding effects. Thus, for VRC01 the process of affinity maturation entails incremental changes of the nascent genomic precursors to obtain high-affinity interaction with the HIV-1 Env surface.

Receptor mimicry and affinity maturation. The possibility that antibodies use conserved sites of receptor recognition to neutralize viruses effectively has been pursued for several decades. The recessed canyon on rhinovirus that recognizes the unpaired terminal immunoglobulin domains of intercellular adhesion molecule–1 highlights the role that a narrow canyon entrance may play in such occlusion of bivalent antibody-combining regions (40), although framework recognition can in some instances permit entry (41). Partial solutions such as those presented by antibody b12 (neutralization of ~40% of circulating isolates) (12, 13) or by antibody HJ16 (neutralization of ~30% of circulating isolates) (42), a recently identified CD4-binding-site antibody, may allow recognition of some HIV-1 isolates.

With VRC01, the potency and breadth of neutralization (over 90%) suggest a more general solution. It remains to be seen how difficult it will be to guide the elicitation of VRC01-like antibodies from genomic rearrangement, through affinity maturation, to broad and potent neutralization of HIV-1. Accumulating evidence suggests that the VRC01-defined mode of recognition is used by other antibodies (12). These findings suggest that VRC01 is not an isolated example and probably provides a template for a general mode of recognition. The structure-function insights of VRC01 described here thus provide a foundation for rational vaccine design that is based not only on the particular mode of antibody-antigen interaction but also on defined relationships between genomic antibody precursors, somatic hypermutation, and interface-recognition elements.

Supporting Online Material

Materials and Methods

Figs. S1 to S16

Tables S1 to S14


Appendix S1

References and Notes

  1. Antibodies 2F5 and 4E10 require nonspecific membrane interaction (35, 36), antibody 2G12 recognizes carbohydrate and is domain swapped (33, 34), and antibody b12 was originally derived by means of phage display and has heavy-chain-only recognition (13).
  2. The Env components of this strain derive from Clade E HIV-1 lineages (43).
  3. Materials and methods are available as supporting material on Science Online.
  4. The four independent copies of the VRC01-gp120 complex in the asymmetric unit resembled each other closely for the antibody variable domain–gp120 components, with an Cα-root-mean-square deviation of less than 0.2 Å. Elbow variation, however, between variable and constant domains was apparent, and we found one copy (molecule 1) to be more ordered than the others. In the figures, we display molecule 1; see fig. S15 for a comparison of all of the molecules in the asymmetric unit.
  5. Surface areas of interaction reported in this paper were determined with the program PISA, as implemented in CCP4 (44). Values were about 20% higher than those reported previously for the gp120-CD4 complex (20), which were obtained using the program MS (45).
  6. The overlap of molecular volumes was calculated by comparing the separate volumes of interacting domains with the combined volume of these domains after gp120 superposition.
  7. The relative orientation of the light-chain variable domain and the heavy-chain variable domain of VRC01 is similar to that of other antibodies (fig. S16).
  8. In a companion paper (12), we show that VRC01 binding induces 17b and CCR5 binding in the context of monomeric gp120 with an unusual entropy signature characteristic of transiting to the CD4-bound state (46); neutralization data, however, show that VRC01 does not induce 17b or CCR5 binding in the context of the viral spike. This difference probably arises from the more constrained gp120 conformation in the trimeric spike. Thus, although VRC01 is able to induce large conformational changes in monomeric HIV-1 gp120 that resemble those induced by CD4, VRC01 interaction with gp120 does not depend on these conformational changes.
  9. Endo H treatment of gp120 for crystallization removed all but the protein-proximal N-acetyl-glucosamine and potential 1,6-fucose from sites of N-linked glycosylation. Examination of the VRC01 interactions with the N-acetyl-glycosamine at residue 276gp120 shows that both 1,4 additions and 1,6 additions are tolerated.
  10. Four residues are provided by the CDR H3, Asp99VRC01 to Trp100BVRC01, with a combined interaction surface of 123 Å2 (tables S3 and S12). These four residues are probably contributed by the D segment (IGHD3-16*02), and none of them appears critical to VRC01 recognition because changes are observed in two of these residues in the closely related broadly neutralizing antibody VRC03, which was one of two antibodies we isolated along with VRC01 (12). Meanwhile, three residues are provided by the CDR L3—Tyr91VRC01, Glu96VRC01, and Phe97VRC01—with a combined interaction surface of 190 Å2 (tables S3 and S13). These three residues lie at the junction between V and J genes. They make important hydrophobic interactions with loop D of gp120, and two of them are conserved between VRC01 and VRC03. Although it is difficult to know how precisely the CDR L3 needs to be aligned, with only three contact residues variation at the Vκ-J gene junction should provide sufficient diversity for it to be represented in the repertoire.
  11. Analysis of the HIV-1 Env-reactive antibody repertoire from infected individuals shows increased levels of affinity maturation (30). Analysis of a subset of this data (appendix S1) containing 147 heavy and 147 light chains from HIV-1 Env-reactive antibodies reveals an average of 15 alterations (30 maximum) for the heavy chain and an average of 8.6 alterations (22 maximum) for the light chain (fig. S13). In terms of the subset of HIV-1 Env-reactive antibodies that are broadly neutralizing (such as 2G12, 2F5, 4E10, and b12), antibodies b12 and 2G12 have 45 and 51 changes, respectively, relative to nearest genomic precursors in their VH and J segments of the heavy chain (31).
  12. Similar significant reductions in affinity have been observed with reversion of other broadly neutralizing antibodies to HIV-1 to putative genomic sequences (4749); these observations have led to the suggestion that the dramatically reduced germline affinity for gp120 might hinder the initiation of affinity maturation of these antibodies (50). That is, if the affinity for gp120 of the genomic precursor of a broadly neutralizing antibody were below the threshold required for the nascent B cell to mature, then maturation would either not occur or would need to occur in response to a different immunogen. This lack of guided initiation of the maturation process may provide an explanation for the absence of such broadly neutralizing antibodies in the first few years of infection. Conversely, the introduction of modified gp120s with affinity to genomic precursors and affinity maturation intermediates could provide a mechanism by which to elicit antibodies like VRC01.
  13. T.Z., I.G., Z.Y., J. S., L.S., G.J.N., J.R.M., and P.D.K. designed the research; T.Z., I.G., X.W., Z.Y., K.D. A.F., W.S., L.X., Y.Y., and J.Z. performed the research; X.W., Y.K., J.F.S., M.C.N., and J.R.M. contributed new reagents or reference data; T.Z., I.G., X.W., J.S., L.S., G.J.N., J.R.M., and P.D.K. analyzed the data; and T.Z., I.G., J. S., L.S., G.J.N., J.R.M., and P.D.K. wrote the paper, on which all authors commented. We thank I. A. Wilson and members of the Structural Biology Section and Structural Bioinformatics Core, Vaccine Research Center, for discussions and comments on the manuscript; the staff of sector 22 for assistance with data collection; and J. Stuckey for assistance with figures. Support for this work was provided by the Intramural Research Program of NIH and by grants from NIH and from the International AIDS Vaccine Initiative’s Neutralizing Antibody Consortium. Use of sector 22 (Southeast Region Collaborative Access team) at the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract W-31-109-Eng-38. Coordinates and structure factors for the VRC01-gp120 complex have been deposited with the PDB under accession code 3NGB.
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