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. 2010 Aug 30;207(9):1995-2002.
doi: 10.1084/jem.20101176. Epub 2010 Aug 2.

Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness

John Pietzsch  1 , Johannes F Scheid, Hugo Mouquet, Florian Klein, Michael S Seaman, Mila Jankovic, Davide Corti, Antonio Lanzavecchia, Michel C Nussenzweig
Affiliations

Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness

John Pietzsch et al. J Exp Med. .

Abstract

The identification and characterization of conserved epitopes on the HIV-1 viral spike that are immunogenic in humans and targeted by neutralizing antibodies is an important step in vaccine design. Antibody cloning experiments revealed that 32% of all HIV-neutralizing antibodies expressed by the memory B cells in patients with high titers of broadly neutralizing antibodies recognize one or more "core" epitopes that were not defined. Here, we show that anti-core antibodies recognize a single conserved epitope on the gp120 subunit. Amino acids D474, M475, R476, which are essential for anti-core antibody binding, form an immunodominant triad at the outer domain/inner domain junction of gp120. The mutation of these residues to alanine impairs viral fusion and fitness. Thus, the core epitope, a frequent target of anti-HIV-neutralizing antibodies, including the broadly neutralizing antibody HJ16, is conserved and indispensible for viral infectivity. We conclude that the core epitope should be considered as a target for vaccine design.

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Figures

Figure 1.
Figure 1.
Mapping of the HIV-1 gp120 core epitope. (A) Heat map summarizes the binding of the different anti-core antibodies and b12 to gp120 alanine mutants. Red and orange fields indicate <60% binding, whereas yellow shows no difference compared with the WT gp120 control. The mapping of the anti-core epitope has been confirmed by three independent experiments. (B) Surface diagram of gp120 (PDB ID: 3DNO; Liu et al., 2008) showing the CD4bs (blue), the CD4is (gray), the b12 binding sites (yellow), and the core epitope (green). Residues that distinguished between the anti-CD4bs epitope and anti-core epitope are highlighted with circles. (C) Diagram shows the apparent binding of anti-core antibodies and b12 to mutant gp120D474AM475AR476A relative to gp120 WT in percent. The red star indicates binding to D368A. (D) Venn diagram summarizes the sensitivity for anti-core antibodies binding to D474A, M475A, and R476A. (E) Ribbon diagram of gp120 (PDB ID: 3DNO; Liu et al., 2008) shows the CD4bs (blue), the CD4is (gray), and the core epitope (green).
Figure 2.
Figure 2.
Binding to soluble and cell surface trimeric gp160Δc. (A) Graphs show optical density at 405 nm (OD405nm) for the selected IgG antibodies as measured by capture ELISA with purified BaL gp140 WT and BaL gp140D474A/M475A/R476A. See also Fig. S4. Error bars represent the SD from at least two independent experiments. (B) Graphs show apparent KA (KAapp, M21) for the selected IgG antibodies as measured by surface plasmon resonance (SPR) on chips derivatized with BaL gp140 WT and BaLD474A/M475AR/476A. See also Fig. S5 and Table S2. Error bars represent the SEM from at least two independent experiments. * indicates that no binding to BaL gp140D474A/M475A/R476A was detected. (C) (left) Histogram plots show the binding of the selected antibodies to BaL gp160Δc and BaL gp160ΔcD474A/M475A/R476A expressed on GFP-positive BOSC.23 cells. Controls include mgo53 (Wardemann et al., 2003), mAbs 2–1092 (anti-VL) and b12 (anti-CD4bs). BOSC.23 cells gated on GFPhigh expression. The number of binding events as percentage of the maximum was plotted against APC fluorescence intensity. (right) Graphs show apparent differences in the relative median fluorescence intensity (ΔrMFI) for the selected IgG antibodies between BaL gp160Δc and BaL gp160ΔcD474A/M475A/R476A. Error bars represent the SD from at least two independent experiments.
Figure 3.
Figure 3.
Fusion and infectivity. (A) (left) Histogram plots summarize fusion between BOSC.23 cells expressing GFP alone (w/o) or BaL gp160Δc or BaL gp160ΔcD474A/M475A/R476A with mCherry expressing TZM.bl cells. (right) Fusion events (%) with mean and standard deviation for at least two independent experiments. (B) Image-based analysis of fusion. (left) Single staining compensation controls for BOSC.23 cells expressing BaL gp160Δc and GFP, mCherry expressing TZM.bl cells and CD4-APC stained TZM.bl cells. (middle) An intermediate fusion event. (right) The colocalization of the GFP-mCherry-APC signal after fusion. Experiment has been performed twice. (C) Graphs show the titration curves for TZM.bl cell infection by YU2 and BaL pseudoviruses, which has been performed twice. RLU relative light unit.
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