Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody

Abstract

Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121-125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.

Copyright © 2010 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Inhibition of representative HIV-1 isolates by ibalizumab IgG and Fab

HIV-1 primary isolates, Du156.12 (clade C), QH0692.42 (clade B), ZM53M.PB12 (clade C) and Q769.d22 (clade A) were analyzed for inhibition by the anti-CD4 monoclonal antibody ibalizumab using a luciferase-based virus neutralization assay with TZM.bl cells. Serial dilutions of ibalizumab in the bivalent IgG form (filled square) or the monovalent Fab form (open square) were tested for neutralization against each virus. 50% of inhibition is indicated by a dashed, horizontal line. IC50 values and MPI (maximum percent inhibition) are summarized in Table 1. The experiments were repeated twice with similar results. See also Figure S1 and Table S2.

Figure 2. Structure of the complex of 2 domain CD4 and the Fab fragment of ibalizumab

(A) Top and side views of the overall structure of 2D CD4 in complex with ibalizumab Fab, shown in ribbon representation. CD4 is in green (lighter green for D2 and darker green for D1), the heavy chain of ibalizumab in red, and the light chain in magenta. The BC-loop in the second domain of CD4, which constitutes the core epitope, and the CDR loops of ibalizumab are all indicated. (B) Surface representation of the complex. CD4 and ibalizumab are shown in the same color scheme as in (A). See also Figure S2 and Figure S4, and Table S1.

Figure 3. Close-up of major contacts between CD4 and ibalizumab

(A) Residues making direct contacts between the heavy- and light- chains of ibalizumab and the BC-loop (residues 121-125) of D2 in CD4. The heavy and light chains of ibalizumab are shown by surface representation in red and magenta, respectively, and the residues that make contacts with CD4 are also shown by stick model in the same color scheme. The BC-loop of CD4 is in green in ribbon diagram and the residues interacting with ibalizumab are shown by stick model with carbon atoms in yellow, nitrogen in blue and oxygen in red. (B) Contacting residues between the FG loop (residues 164, 165) of D2 in CD4 and the ibalizumab heavy-chain. The residues that make direct contacts with the FG loop of CD4 in the heavy chain are shown by stick model in red. The FG loop of CD4 is shown in ribbon diagram in green with residues 164 and 165 in stick model in the same color scheme as in (A). The conformation of the two residues in the unbound CD4 is also shown in cyan; a flip of the loop upon binding to ibalizumab is evident. See also Figure S3 and Table S1.

Figure 4. Impact of flexibility in the CD4 ectodomain on HIV-1 inhibition by ibalizumab

To introduce flexibility in the ectodomain of CD4, four variants with its D3-D4 (residues 179-365) substituted by a flexible linker, GGGGS (CD4-L1), GGGGSGGGGSGGGGS (CD4-L2), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (CD4-L3) and GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (CD4-L4) were generated. Two additional CD4 variants were also constructed with either a 5-residue (residues 367-371; TPVQP) deletion (CD4-Δ5) or the entire D3-D4 and TM (residues 179-433) replaced by a human CD8α hinge region and TM (residues 115-214) (CD4-CD8TM). ACTOne CCR5 cells were transfected with wildtype CD4 and six variants. Surface expression levels of CD4 and CCR5 were detected by fluorescence-activated cell sorting (FACS) analyses and the mean fluorescence intensity (MFI) values are summarized. The relative infectivity of these target cells by a recombinant HIV-1 Env pseudovirus, SC422661.8, expressing the firefly luciferase reporter gene is shown by relative luminescence units (RLU). Inhibition of this HIV-1 Env pseudovirus by ibalizumab IgG are reported as IC50 values and MPI (maximum percent inhibition). The experiments were repeated twice with similar results and one set of data is shown. See also Figures S8 and S9.