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. 2010 Aug 13;329(5993):856-61.
doi: 10.1126/science.1187659. Epub 2010 Jul 8.

Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1

Xueling Wu  1 , Zhi-Yong Yang, Yuxing Li, Carl-Magnus Hogerkorp, William R Schief, Michael S Seaman, Tongqing Zhou, Stephen D Schmidt, Lan Wu, Ling Xu, Nancy S Longo, Krisha McKee, Sijy O'Dell, Mark K Louder, Diane L Wycuff, Yu Feng, Martha Nason, Nicole Doria-Rose, Mark Connors, Peter D Kwong, Mario Roederer, Richard T Wyatt, Gary J Nabel, John R Mascola
Affiliations

Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1

Xueling Wu et al. Science. .

Abstract

Cross-reactive neutralizing antibodies (NAbs) are found in the sera of many HIV-1-infected individuals, but the virologic basis of their neutralization remains poorly understood. We used knowledge of HIV-1 envelope structure to develop antigenically resurfaced glycoproteins specific for the structurally conserved site of initial CD4 receptor binding. These probes were used to identify sera with NAbs to the CD4-binding site (CD4bs) and to isolate individual B cells from such an HIV-1-infected donor. By expressing immunoglobulin genes from individual cells, we identified three monoclonal antibodies, including a pair of somatic variants that neutralized over 90% of circulating HIV-1 isolates. Exceptionally broad HIV-1 neutralization can be achieved with individual antibodies targeted to the functionally conserved CD4bs of glycoprotein 120, an important insight for future HIV-1 vaccine design.

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Figures

Fig. 1
Fig. 1
Design and antigenic profile of RSC3 and analysis of epitope-specific neutralization. (A) Surface structure model of the RSC3. The outer domain contact site for CD4 is highlighted in yellow. Regions highlighted in red are antigenically resurfaced areas, shown on both the inner (left) and outer (right) faces of the core protein. Glycans are shown in light blue. (B) Antigenicity of the RSC3 protein based on ELISA using the neutralizing CD4bs mAb b12 and CD4-Ig fusion protein. mAb 2G12 was used to confirm the structural integrity of the protein. OD indicates optical density. IgG, irrelevant IgG. (C) mAb b12 was immobilized on the sensor chip for SPR kinetic binding analysis with the proteins shown. RU, resonance units. (D) RSC3 blockade of HIV-1 viral strain HXB2 neutralization by the broadly neutralizing CD4bs mAb b12 but not mAb F105, which recognizes the CD4bs differently and has limited neutralization breadth. The V3-neutralizing mAb 447-52D is shown as a control. (E) Analysis of serum 45 neutralization of a panel of 17 viruses, using RSC3 and ΔRSC3 to block neutralization activity. The percent reduction in the serum 50% inhibitory dilution (ID50) caused by competition with RSC3 or ΔRSC3 is shown on the y axis (±SEM of three independent experiments). Viral strains and clades are shown on the x axis. Values less than 20% are not considered significant in this assay.
Fig. 2
Fig. 2
Isolation of individual CD4bs-directed memory B cells by cell sorting and binding characterization of isolated mAbs. (A) Twenty-five million PBMC from donor 45 were incubated with biotin-labeled RSC3 and ΔRSC3 that were complexed with SA-APC and SA-PE, respectively. Memory B cells were selected on the basis of the presented gating strategy. Twenty-nine B cells that reacted with RSC3 and not ΔRSC3 (representing 0.05% of all memory B cells) were sorted into individual wells of a 96-well plate containing lysis buffer. FITC, fluorescein isothiocyanate. FSC-H, forward scatter height; FSC-A, forward scatter area; and SSC, side scatter area. (B) ELISA antigen binding profile of three isolated mAbs, VRC01, VRC02, and VRC03. Solid symbols show mAb binding to RSC3 (left) and YU2 gp120 (right). Open symbols indicate binding to ΔRSC3 (left) or to the CD4bs knockout mutant of gp120, D368R (right). WT, wild type. (C) SPR binding analysis of VRC01 reacted with RSC3 and ΔRSC3. VRC01 was captured with an antibody against human IgG-Fc that was immobilized on the sensor chip. The SPR and ELISA data shown are from a representative experiment; several additional assays produced similar data.
Fig. 3
Fig. 3
Antigenic and biophysical characterization of novel CD4bs-directed mAbs. (A) Competition ELISA performed with a single concentration of biotin-labeled VRC01 (left) or the co-receptor binding site mAb 17b (right). The mAbs indicated near each line were titrated into the ELISA at increasing concentrations to evaluate the effect on VRC01 and 17b binding, respectively. The results shown are from a representative experiment; two additional assays produced similar data. (B) ITC to assess the change in enthalpy (ΔH) and entropy (–TΔS) upon binding of mAbs to YU2 gp120. Each measured value is shown ±SEM.
Fig. 4
Fig. 4
Analysis of neutralization by mAbs VRC01 and b12 against a panel of 190 Env pseudoviruses representing all major circulating clades of HIV-1. Dendrograms, made by the neighbor-joining method, show the protein distance of gp160 sequences from 190 HIV-1 primary isolates. The clade B reference strain HXB2 was used to root the tree, and the amino acid distance scale is indicated with a value of 1% distance as shown. The clades of HIV-1 main group, including circulating recombinant forms (CRFs), are indicated. Neutralization potency of VRC01 and b12 is indicated by the color of the branch for each virus. The data under the dendrograms show the percent of viruses neutralized with a 50% inhibitory concentration (IC50)<50 μg/ml and <1 μg/ml and the geometric mean IC50 value for viruses neutralized with an IC50 <50 μg/ml.
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