Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01

Abstract

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.

Figure 1. Structure of antibody VRC01 in complex with HIV-1 gp120

Atomic-level details for effective 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 interactive surface is provided by the CDR H2, with the CDR L1 and L3 and the CDR H1 and H3 providing additional contacts.

Figure 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 the VRC01 complex (right) and the CD4 complex with the lowest gp120 RMSD (left) (Table S3). The CD4 complex (3JWD) (14) is colored 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,C) Interface details for CD4 (B) and VRC01 (C). Close-ups are shown of critical interactions between the CD4-binding loop (purple) and the C” strand as well as between Asp368_gp120 and either Arg59_CD4 or Arg71_VRC01. Hydrogen bonds with good geometry are depicted by blue dotted lines, and those with poor geometry 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, though 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.

Figure 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 (e.g. A, B and C) and recombinants (e.g. CDR02_AG) labeled. The strains are colored by their neutralization sensitivity to VRC01 (left) or CD4 (right). VRC01 neutralizes 72% of the tested HIV-1 isolates with an IC₈₀ of less than 1 ug/ml; by contrast, CD4 neutralizes 30% of the tested HIV-1 isolates with an IC₈₀ of less than 1 ug/ml (Table S13). (B) Molecular footprints of VRC01 and CD4 on gp120. The molecular surface of HIV-1 gp120 has been colored according to its underlying domain substructure: red for the conformationally invariant outer domain, grey for the inner domain and blue for the highly mobile bridging sheet. Regions of the gp120 surface that interact with VRC01 or CD4 have been colored in green and yellow respectively. (C) Neutralization of viruses with altered sampling of the CD4-bound state. Mutant S375W_gp120 favors the CD4-bound state, whereas mutants H66A_gp120 and W69L_gp120 disfavor this state. Neutralization by VRC01 (left) is similar for wild-type (WT) and all three mutant viruses, whereas neutralization by CD4 (right) correlates with the degree to which gp120 in the mutant viruses favors the CD4-bound state. (D) Comparison of binding affinities. Binding affinities (K_Ds) for VRC01 and various other gp120-reactive ligands as determined by surface-plasmon resonance are shown on a bar graph. White bars represent affinities for gp120 restrained from assuming the CD4-bound state (21) and black bars represent affinities for gp120 fixed in the CD4-bound state (24). Binding too weak to be measured accurately is shown as with an asterisk and bar at 10^-5 M K_D.

Figure 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) VRC01 recognition and target site of vulnerability. The CD4-define site of vulnerability is the initial contact surface of the outer domain of gp120 for CD4 and comprises only 2/3 of the contact surface of gp120 for CD4 (22). The molecular surface of gp120 in the VRC01 bound conformation is colored according to its domain substructure as in Fig. 3B, with the interactive footprint of VRC01 shown in green and the CD4-defined site of vulnerability outlined in yellow. (B) Antigenic variation. The polypeptide backbone of gp120 is colored according to sequence conservation, blue if conservation is high and red if conservation is low. (C) VRC01-resistant HIV-1 isolates. The 17 isolates of HIV-1 that resist VRC01 neutralization are displayed in ribbon and stick representation after threading onto the structure of gp120 in the VRC01-bound conformation. Side chains that clashed with VRC01 are highlighted in red. (D) Sequence in V5 region for 17 HIV-1 isolates that resist neutralization by VRC01. (E) HIV-1 clashes with VRC01. A close-up of threaded, resistant isolates is shown along with the molecular surface of VRC01, colored light blue for the light chain and green for the heavy chain. Clashes predicted to interfere with VRC01-gp120 interactions are highlighted in red. (F) 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.

Figure 5. Unusual VRC01 features

The structure of VRC01 displays a number of unusual features, which if essential for recognition and difficult to elicit, might inhibit the elicitation of VRC01-like antibodies. (A) VRC01 sequence and extent of affinity maturation. The sequence of VRC01 is shown along with nearest V_H- and V_Κ/λ-genomic precursors for heavy and light chain, respectively. Affinity maturation changes are indicated in green, with residues involved in interaction with HIV-1 gp120 highlighted by “●”, if involved in both main- and side-chain interactions, by “○” if main chain-only, and by “☼” if side chain-only. “” marks a site of N-linked glycosylation, “” for cysteine residues involved in a non-canonical disulfide, and “” if the residue has been deleted during affinity maturation. In B-E, unusual features of VRC01 are shown structurally (far left panel), in terms of frequency as a histogram with other antibodies (second panel from left), and in the context of affinity meaurements after mutational alteration (right two panels). Affinity measurements were made by ELISA to the gp120 construct used in crystallization (93TH057) and to a disulfide stabilized HXBc2 core (22). (B) N-linked glycosylation. The conserved tri-mannose core is shown with observed electron density, along with frequency and effect of removal on affinity. (C) 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 by mutation to Ser/Ala has little effect on affinity. (D) 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 10-100-fold effect on gp120 affinity. (E) Somatically altered contact surface. The far left panel shows the VRC01 light chain in violet and heavy chain in green. Residues altered by affinity maturation are depicted with “balls” and contacts with HIV-1 gp120 are colored red. About half the contacts are altered during the maturation process. Analysis of human antibody-protein complexes in the protein-data bank shows this degree of contact surface alteration is rare; reversion of each of the contact site to genome has little effect (Table S12), though in aggregate the effect on affinity is larger.

Figure 6. Somatic maturation and VRC01 affinity

Hypermutation of the variable domain during B cell maturation allows for the evolution of high affinity antibodies. Interestingly, this enhancement to affinity occurs principally through the alteration of non-contact residues, which appear to reform the genomic contact surface from affinity too low to measure to a tight (nM) interaction. (A) Effect of genomic reversions. The V_H- and V_Κ-derived regions of VRC01 were reverted to the sequences of their closest genomic precursors, expressed as immunoglobulins and tested for binding as V_H- and V_Κ-revertants (gHgL), as a V_H-only revertant (gH), or as a V_Κ-only revertant (gL) to either the gp120 construct used in crystallization (93TH057) or to a stabilized HXBc2 core (22). (B) Maturation of VRC01 and correlation with binding. Affinity measurements for the 19 VRC01 mutants created during the structure-function analysis of VRC were analyzed in the context of their degree of affinity maturation. Significant correlations were observed, with extrapolation to V_H- and V_Κ-revertants suggesting greatly reduced affinity for gp120. (C) Maturation of VRC01 and effect on HIV-1 gp120 affinity. Cryo-electron microscopy density (pink) corresponding to the unliganded state of the trimeric HIV-1 viral spike (50) is shown in complex with the antigen-binding fragment of VRC01. The Cα-backbone ribbon is displayed for gp120s (red) and VRC01 grey. With VRC01, the molecular surface is shown for residues altered from the closest V_H- and V_Κ-genomic precursor sequences and colored gray to green depending on the effect of the reversion on gp120 affinity, with gray indicating small effects and green larger ones.