Increased Epitope Complexity Correlated with Antibody Affinity Maturation and a Novel Binding Mode Revealed by Structures of Rabbit Antibodies against the Third Variable Loop (V3) of HIV-1 gp120

Abstract

The third variable (V3) loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (MAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these MAbs bound to target epitopes were determined. 10A3 binds to the V3 crown (³⁰³TRKSIHIGPGRAF³¹⁷) using the cradle binding mode, similar to human V3 MAbs encoded by IGHV5-51 germ line genes, and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region (³⁰⁸HIGPGRAFYTTGEI³²³). Unexpectedly, the ³¹⁵RAFYTT³²⁰ portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity to human antibody germ line genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.IMPORTANCE The apex crown of V3 of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein, and many MAbs targeting this region have been developed. Structural understanding of V3 crown MAbs not only can help understand how antibody responses target this unique region but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 MAbs, 10A3 and 10A37, developed from a rabbit immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of the V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates.

Keywords: HIV-1; V3; V3 loop; gp120; neutralizing antibody.

FIG 1

Structure of Fab 10A3 in complex with V3_ConB. (A and B) Ribbon representations of the Fab 10A3 in complex with V3_ConB from two different perspectives. The light and heavy chains are colored cyan and green, respectively, while the epitope is colored magenta (the coloring scheme is the same for all figures, except where indicated otherwise). (C) The antigen binding site of 10A3, with the CDR loops labeled and colored differently from the rest of Fab. (D) Electrostatic potential surface of the antigen binding site of 10A3, with red color indicating negatively charged while blue indicates positively charged. (Inset) The sequence of the peptide used for crystallization is shown, and residues observed in the electron density map are shown in magenta.

FIG 2

Antigen-antibody interactions of 10A3/V3_ConB. (A) Hydrophilic interactions between the side chains of the epitope and residues of 10A3. (B) Hydrophilic interactions between the main chain of the epitope and residues of 10A3. (C) Schematic illustration of antigen-antibody interactions. Hydrogen binding interactions are indicated by dashed lines between the residues, while van der Waals contacts are indicated by eyelashes. Residues in solid ovals contribute to the interactions by their main-chain atoms, and those in dashed ovals contribute to their side-chain atoms.

FIG 3

Structure of Fab 10A37 in complex with V3_JR-FL. (A and B) Ribbon representations of the Fab 10A37 in complex with V3_JR-FL from two different perspectives. (C) The antigen binding site of 10A37 with the CDR loops labeled and colored differently from the rest of Fab. (D) Electrostatic potential surface of the antigen binding site of 10A37. (Inset) The sequence of the peptide used for crystallization is shown, and residues observed in the electron density map are shown in magenta.

FIG 4

Antigen-antibody interactions of 10A37/V3_JR-FL. (A) Hydrophilic interactions between the side chains of epitope and residues of 10A37. (B) Hydrophilic interactions between the main chains of the epitope and residues of 10A37. (C) Schematic illustration of antigen-antibody interactions. The helix in the epitope is indicated by pink shading.

FIG 5

Structural comparison of rabbit V3 MAb complexes 10A3/V3_ConB with R56/V3_JR-FL. (A) Superimposition of the light-chain variable-domain structures of 10A3/V3_ConB and R56/V3_JR-FL (Cα RMSD, 0.32Å). 10A3 and its epitope are colored as described for Fig. 1. The light and heavy chains of R56 are colored gray, and its epitope is colored orange. (B) Superimposition of the backbone structures of V3 epitopes bound to antibodies 10A3 and R56 with the side chains shown as sticks (Cα RMSD, 0.92 Å). (C) Sequence alignment of the heavy (top) and light (bottom) chains of 10A3 and R56 and their germ lines. CDR1, CDR2, and CDR3 are indicated. Critical residues are indicated as shown in the key.

FIG 6

Structural comparison of rabbit V3 MAb complex 10A3/V3_ConB with human V3 MAb complex and others encoded by IGHV5-51 germ line genes. (A) Superimposition of the backbone structures of 10A3/V3_ConB and 2557/V3_NY5 (Cα RMSD, 0.94 Å). V3_ConB and V3_NY5 peptides are shown in magenta and red, respectively. 10A3 VL and VH are colored cyan and green, while those of 2557 are colored gray. Note that the V3 epitope bound in different orientations relative to the MAbs. (B) Superimposition of the peptides bound to 10A3 and 2557 with the side chains shown as sticks (Cα RMSD, 1.58 Å). (C) Comparison of the V3 epitopes of rabbit and human MAbs. Plot showing the distribution and number of contacts formed with the V3 loop by different MAbs. Squares containing letters indicate peptide differences from that complexed with 10A3, the sequence of which is shown above the grid.