Epitope Mapping of Conformational V2-specific Anti-HIV Human Monoclonal Antibodies Reveals an Immunodominant Site in V2

<div><p>In the case-control study of the RV144 vaccine trial, the levels of antibodies to the V1V2 region of the gp120 envelope glycoprotein were found to correlate inversely with risk of HIV infection. This recent demonstration of the potential role of V1V2 as a vaccine target has catapulted this region into the focus of HIV-1 research. We previously described seven human monoclonal antibodies (mAbs) derived from HIV-infected individuals that are directed against conformational epitopes in the V1V2 domain. In this study, using lysates of SF162 pseudoviruses carrying V1V2 mutations, we mapped the epitopes of these seven mAbs. All tested mAbs demonstrated a similar binding pattern in which three mutations (F176A, Y177T, and D180L) abrogated binding of at least six of the seven mAbs to ≤15% of SF162 wildtype binding. Binding of six or all of the mAbs was reduced to ≤50% of wildtype by single substitutions at seven positions (168, 180, 181, 183, 184, 191, and 193), while one change, V181I, increased the binding of all mAbs. When mapped onto a model of V2, our results suggest that the epitope of the conformational V2 mAbs is located mostly in the disordered region of the available crystal structure of V1V2, overlapping and surrounding the α4β7 binding site on V2.</p></div

Binding of conformational V2 mAbs to wildtype and mutant SF162 pseudovirus lysates.

<p>Binding of (A) mAb 697, (B) mAb 830A, (C) mAb 1357D, (D) mAb 1361, (E) mAb 1393A, (F) mAb 2158, and (G) mAb 2297 to wildtype and 22 mutant SF162 pseudovirus lysates. The residue at each position in SF162 and the amino acid to which it was mutated is shown for each pseudovirus on the y-axis. Binding levels are shown on the x-axis; they are normalized to CD4-IgG2 binding (as described in Methods) and expressed as percentages of SF162 wildtype binding (black; 100%). The means of 3–5 experiments are shown with standard error of the mean.</p

Additional file 7: Table S5. of Validation of a high resolution NGS method for detecting spinal muscular atrophy carriers among phase 3 participants in the 1000 Genomes Project

Results for all volunteer and Coriell samples. (XLSX 49 kb

Mutations in the V2 region of SF162 used in epitope mapping studies.

<p>(A) The SF162 V2 wildtype sequence and a list of the mutations that were introduced at 13 different positions. The ß-strands (B, C, and D; purple) with the disordered region between the C and D β-strands (grey) are illustrated as arrows above the sequence. (B) The V1V2 domain of SF162, modeled after the structure published by McLellan et al.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070859#pone.0070859-McLellan1" target="_blank">[4]</a>, indicating in red the amino acid where mutations were introduced.</p

Additional file 11: Figure S5. of Validation of a high resolution NGS method for detecting spinal muscular atrophy carriers among phase 3 participants in the 1000 Genomes Project

95 % Posterior (credible) intervals for π are plotted for each 1000 Genomes Project subject. Samples are colored and symbolized as in Fig. 5. (PDF 62 kb

Comparison of the epitope surface areas of PG9 and the conformational V2 mAbs.

<p>Epitopes mapped onto the SF162 model of V1V2 for (A) PG9 (epitope shown in brown), (B) 697, 830A, 1361, 1393A, 1357D, 2158 and 2297 (epitope shown in red), (C) CH58/CH59 (epitope shown in green).</p

Additional file 10: Figure S4. of Validation of a high resolution NGS method for detecting spinal muscular atrophy carriers among phase 3 participants in the 1000 Genomes Project

A plot of the scaled proportion of reads aligning to SMN1 versus their frequency for (a) the volunteer and Coriell subjects and (b) the 1000 Genomes subjects. In both datasets, most individuals have an estimate of π to the right of the line at 0.38; it is unlikely they are carriers. (PDF 114 kb

Summary of binding of conformational V2 mAbs to wildtype and mutant SF162 pseudovirus lysates.

*<p>The values are normalized to CD4-IgG2 (as described in Methods) and expressed as percentages of SF162 wildtype binding (100%). The means of 3–5 experiments are shown.</p

Location of conformational V2 epitopes and the effects of mutations.

<p>The V1V2 region of SF162 is illustrated as (A) a schematic diagram, and as (B) a 3D model where the effect of amino acid substitutions on binding is indicated and coded by color: Red: residues where binding of at least six conformational V2 mAbs was reduced to ≤15% of binding to wildtype; Orange: binding of at least six conformational V2 mAbs where binding was reduced to ≤50% of wildtype; Dark grey: effect dependent on amino acid substitution. Dashed lines outline the surface area of the LDV integrin binding site.</p

Thermodynamic Signatures of the Antigen Binding Site of mAb 447–52D Targeting the Third Variable Region of HIV‑1 gp120

The third variable region (V3) of HIV-1 gp120 plays a key role in viral entry into host cells; thus, it is a potential target for vaccine design. Human monoclonal antibody (mAb) 447–52D is one of the most broadly and potently neutralizing anti-V3 mAbs. We further characterized the 447–52D epitope by determining a high-resolution crystal structure of the Fab fragment in complex with a cyclic V3 and interrogated the antigen–antibody interaction by a combination of site-specific mutagenesis, isothermal titration calorimetry (ITC) and neutralization assays. We found that 447–52D’s neutralization capability is correlated with its binding affinity and at 25 °C the Gibbs free binding energy is composed of a large enthalpic component and a small favorable entropic component. The large enthalpic contribution is due to (i) an extensive hydrogen bond network, (ii) a π–cation sandwiching the V3 crown apex residue Arg³¹⁵, and (iii) a salt bridge between the 447–52D heavy chain residue Asp^H95 and Arg³¹⁵. Arg³¹⁵ is often harbored by clade B viruses; thus, our data explained why 447–52D preferentially neutralizes clade B viruses. Interrogation of the thermodynamic signatures of residues at the antigen binding interface gives key insights into their contributions in the antigen–antibody interaction