Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10

Abstract

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.

Figure 1. Prediction of an ensemble of 4E10 GEPs.

Sequences of 4E10 V_L (top line) and V_H (bottom two lines) domains are shown, with CDRs indicated by a blue overscore. Predicted somatic mutations are colored red in the 4E10 sequence, and the corresponding unmutated GEP residues are shown below in grey (unchanged positions are not shown for clarity). All GEP V_L domains are comprised of the IGKV3-20*01/IGKJ1*01 gene segment combination. Each GEP V_H domain comprises the IGHV1-69*06 V gene segment plus one of six D gene segments (listed to the left of the corresponding GEP in the blue boxed field), and either the IGHJ4*02 (resulting sequence differences shown in purple and bolded) or IGHJ1*01 (resulting sequence difference shown in blue and bolded) gene segment, yielding an ensemble of 12 GEPs in toto. GEP shorthand numbering is shown in grey (GEPs 1 to 6) and blue (GEPs 7 to 12) beside the corresponding D plus J gene segment combination sequence differences from 4E10, occurring in HCDR3. Inset (lower right): HCDR3 sequences from candidate 4E10 GEPs, determined through deep sequencing of naïve B cell germline IgH genes from four uninfected individuals , show the degree of variability seen in potential 4E10 precursors present in naïve repertoires. Each germline rearrangement uses the IGHV1-69 and IGJH1 or IGJH4 gene segments. Amino acids in red designate sequence differences between GEP and 4E10. The number of nucleotide changes needed to achieve these somatic mutations is 16 for donor 1, 17 for donor 2 and 18 for donors 3 and 4.

Figure 2. Biophysical and functional characterization of an ensemble of 4E10 GEPs.

(A) CD melting curves are shown for 4E10 and GEPs with T_m values, determined as the inflection point of the sigmoidal melting curve, indicated by a black line (4E10) or a shaded grey box (GEPs). (B) Neutralization potencies are shown for 4E10 IgG (0.2 µM), 4E10 Fv and GEPs (1 µM) against clade A (Q461.d1, Q461.e2) and B (SF162) HIV-1 isolates using standard TZM-bl assays. (C) Double-referenced SPR sensorgrams are shown for the binding of 4E10 and GEPs (300 nM duplicates) interacting with chip-coupled gp140₃. (D) K _Ds for the interaction of 4E10 or GEPs with ES T117 are plotted as k _a vs. k _d, with K _D isotherms indicated by dashed lines and labeled. Due to weak binding and fast kinetics, K _Ds between GEPs and the T72, T344, and T93 ESs could only be analyzed by equilibrium measurements; their values range from 1 to 10 µM, falling within the grey shaded region. The purple triangles show the T117 affinity shift between 4E10 and GEPs, with the sides parallel to the X and Y axes of each triangle highlighting the association and dissociation rate components, respectively.

Figure 3. SPR sensorgrams of the interactions between 4E10 and the indicated ESs are shown.

Time (in seconds) is plotted on the x-axis and SPR response (in RUs) is plotted on the y-axis. Double-referenced binding data are shown in black with corresponding kinetics fits to the data shown in red. Details of the experiments are given in Table 1.

Figure 4. SPR sensorgrams of the interactions between ES T72 and the indicated GEPs (left plots) and the analysis of equilibrium responses versus concentration (right plots).

Details of the experiments are given in Table 1.

Figure 5. SPR sensorgrams of the interactions between ES T93 and the indicated GEPs (left plots) and the analysis of equilibrium responses versus concentration (right plots).

Details of the experiments are given in Table 1.

Figure 6. SPR sensorgrams of the interactions between ES T117 and the indicated GEPs are shown.

Binding data are shown in black with the kinetic fits to the data shown in red. Details of the experiments are given in Table 1.

Figure 7. SPR sensorgrams of the interactions between ES T344 and the indicated GEPs (left plots) and the analysis of equilibrium responses versus concentration (right plots).

Details of the experiments are given in Table 1.

Figure 8. The epitope binding site is conserved between 4E10 and its GEPs.

(A) Residues from the combining sites of 4E10 and GEPs, superimposed based on bound-state structures, contacting the NWFDIT core epitope (shown in a cartoon representation as a grey corkscrew) are shown in a stereo view. 4E10 and GEP residues are shown in licorice stick representation and colored as follows: 4E10 Fv (3LH2.pdb, in complex with ES T88) in purple, 4E10 Fab (2FX7.pdb, in complex with an extended 16-mer NWFDIT-containing peptide) in orange, GEP 1 in yellow, GEP 2 in cyan, and GEP 7 in red. Analyses and depictions were restricted to the core epitope to maximize comparability. Calculated SA buried in the complexes and Sc between core epitope and Ab are: 4E10 Fab/epitope peptide: SA = 355 Ų, Sc = 0.78; 4E10/T88: SA = 367 Ų, Sc = 0.77; GEP 1/T117: SA = 342 Ų, Sc = 0.69; GEP 2/T117: SA = 305 Ų, Sc = 0.74; GEP 7/T117: SA = 343 Ų, Sc = 0.66. (B) The NWFDIT core epitope is shown in a licorice stick representation isolated from the superimposed complex structures, highlighting the high degree of conservation of both the position and conformation of the peptide across 4E10 and GEP complexes.

Figure 9. Interdomain movements within Fv cassettes are limited.

(A) Superpositions of the V_H domains from two 4E10 ligand-bound structures (2FX7.pdb, 3LH2.pdb), unbound 4E10 (4LLV.pdb), ligand-bound GEP 1 (4M8Q.pdb), unbound GEP 1 (4LRN.pdb), ligand-bound GEP 2 (4M62.pdb), ligand-bound GEP 7 (4ODX.pdb), and unbound GEP 7 (4OB5.pdb) are shown in Cα backbone representations, colored as indicated. Residue P14H in each Fv, chosen as a reference point to illustrate interdomain movement upon binding between 4E10 and GEPs, is shown as a sphere and colored to match the corresponding backbone. When isolated V_H domains are superimposed, the P14H spheres are nearly coincident, indicating that the V_H domain structure is highly conserved among these Abs. (B) Fv cassettes of 4E10 and GEPs, colored as in (A), superimposed only on V_L domains (oriented on the right side of the panel), are shown as represented in (A). In this view, interdomain movements can be visualized as the relative movement of V_H domains, particularly at the P14H reference point. (C) Orthogonal view of the P14H spheres excerpted from (B) illustrating the pattern and degree of interdomain movements.

Figure 10. CDR restructuring.

Views from the side (A) and below (B) are shown of superpositions of isolated V_H domains, in cartoon representations, with FRW regions colored grey and β-strands indicated as arrows. HCDRs are shown in a B-factor putty representation (tube width correlates with crystallographic Debye-Waller, or B, factor), colored by Ab. On the left in both frames is shown the superposition of V_H domains from ligand-bound 4E10 (3LH2.pdb, with green HCDRs) vs. unbound 4E10 (4LLV.pdb, with blue HCDRs), and on the right in both frames is shown the superposition of ligand-bound structures of GEP 1 (4M8Q.pdb), GEP 2 (4M62.pdb), and GEP 7 (4ODX.pdb), all with pink HCDRs, vs. unbound GEP 1 (4LRN.pdb) and GEP 7 (4OB5.pdb), both with orange HCDRs. (C) RMSD values for V_H, V_L, or Fv superpositions, calculated with or without CDR residues, are plotted for Cα atoms only (left) or all atoms (right).

Figure 11. GEPs binding surfaces on T117.

(A) The complex of GEP 2 (orange) bound to ES T117 (red) is shown in molecular surface representations. Non-epitope scaffold contacts are highlighted in grey. (B) The interaction partners were separated as indicated in (A) to reveal details of the interface. T117, on the left, is shown in a red semi-transparent molecular surface representation with the NWFDIT epitope, in a licorice-stick representation, visible within the surface. The GEP 2 Fv structure is shown on the right, colored as in (A). The NWFDIT epitope, excised from the T117 structure, is shown as bound for reference. The footprint of GEP 2 (non-epitope contacts) on T117 is colored dark grey; the footprint of T117 to non-epitope residues on GEP 2 is colored light grey. The position of the side-chain of F54H is indicated.

Figure 12. Peptidome binding results.

(A) PhIP-Seq results are plotted as −Log₁₀ P-values, one replicate on the abscissa, the other on the ordinate, colored by Ab as indicated; note the discontinuity in axis scales. The top scoring 4E10 peptide derived from IP₃R is highlighted with a red arrow; one peptide, derived from the zinc finger Ran-binding domain-containing protein 3, which bound to both GEP 2 and GEP 4, is highlighted with purple arrows. Proximity to the diagonal indicates good replicate concordance; peptides with highly discordant replicate values, falling along the axes, were discarded from the analysis. Overall library scoring statistics are: 4E10, average = 0.32, σ = 0.35; GEP 2, average = 0.22, σ = 0.44; GEP 4, average = 0.25, σ = 0.52; 1C6, average = 0.27, σ = 0.50. (B) The molecular surfaces of the Fv domains of 1C6 (4LCI.pdb), unbound 4E10 (4LLV.pdb) and unbound GEP 7 (4OB5.pdb) are shown, oriented with V_H domains at left and the V_L domains at right. The surface is colored to show hydrophobic patches, defined by the program HotPatch ; patches are colored in descending order of total area (red, orange, yellow, …). The total surface area for red and orange hydrophobic patches is shown. The crystal structure of GEP 7 is partially disordered in HCDR1 and 3 and so patch area is underrepresented in the calculation.

Figure 13. Effects of 4E10/gp140₃ pre-binding on 447-52D and b12 binding.

Double-corrected sensorgrams of the SPR response of 10 nM analytes of Fab b12 (red and green traces) or Fab 447-52D (blue and black traces) to chip-coupled SF162 gp140₃ in the absence (red and black traces) or presence (green and blue traces) of pre-bound Fv 4E10 are shown. Fv 4E10, at a concentration of 3 µM, was flowed over the chip starting 5 minutes before the 447 or b12 injections and continued throughout the experiment. The black and blue 447-52D traces were completely superimposed, showing no effect of 4E10 pre-binding on 447-52D binding. While the dissociation phases of the b12 traces are nearly parallel, showing no minimal effect of 4E10 pre-binding on the dissociation of b12 from the bound state, the association phases are clearly not parallel, resulting in a lower peak response in the presence of 4E10 pre-binding, showing a modest, but important, reduction in the association rate. This result suggests that the CD4 binding site adopted a distinct conformational state prior to b12 binding as a consequence of 4E10 binding at the MPER.