Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4-CCR5 complexes

Abstract

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor, typically CCR5. Here we provide evidence that purified gp120(JR-FL)-CD4-CCR5 complexes exhibit an epitope recognized by a Fab (X5) obtained by selection of a phage display library from a seropositive donor with a relatively high broadly neutralizing serum antibody titer against an immobilized form of the trimolecular complex. X5 bound with high (nM) affinity to a variety of Envs, including primary isolates from different clades and Envs with deleted variable loops (V1, -2, -3). Its binding was significantly increased by CD4 and slightly enhanced by CCR5. X5 inhibited infection of peripheral blood mononuclear cells by a selection of representative HIV-1 primary isolates from clades A, B, C, D, E, F, and G with an efficiency comparable to that of the broadly neutralizing antibody IgG1 b12. Furthermore, X5 inhibited cell fusion mediated by Envs from R5, X4, and R5X4 viruses. Of the five broadly cross-reactive HIV-1-neutralizing human monoclonal antibodies known to date, X5 is the only one that exhibits increased binding to gp120 complexed with receptors. These findings suggest that X5 could possibly be used as entry inhibitor alone or in combination with other antiretroviral drugs for the treatment of HIV-1-infected individuals, provide evidence for the existence of conserved receptor-inducible gp120 epitopes that can serve as targets for potent broadly cross-reactive neutralizing antibodies in HIV-1-infected patients, and have important conceptual and practical implications for the development of vaccines and inhibitors.

Figure 1

Binding isotherms of X5 to gp120 and gp120–sCD4. Gp120 and gp120–sCD4 complexes were coated directly on 96-well plates and washed, and X5 was added at the indicated concentrations. Bound X5 was detected by anti-human F(ab′)₂-HRP and represented as optical density at 405 nm. The background was estimated by the amount of X5 bound to BSA and subtracted. The data were fitted to the Langmuir adsorption isotherm [B/B_max = X5/(K_d + X5), where B is the amount of bound X5, B_max is the maximal amount of bound X5, X5 is its bulk concentration, and K_d is the equilibrium dissociation constant]. The continuous lines represent fitting of the data for X5 binding to gp120 [data represented by empty squares (JR-FL) and diamonds (89.6)] and sCD4–gp120 (data represented by solid symbols).

Figure 2

Binding of X5 to gp140 (120)s lacking variable loops and disulfide bond stabilized gp140. Wild-type gp120_JR-FL, gp120 with deleted V3 loop (gp120_JR-FLΔV3), and gp140 with deleted V1 and V2 loops (gp140_JR-FLΔV1V2*) or deleted V1, V2, and V3 loops (gp140_JR-FLΔV1V2*V3) (15), as well as cleaved disulfide bond stabilized gp140_HXB (SOS gp140_HXB2) (16), were captured by the antibody D7324 to 96-well plates and X5 added at the indicated concentrations in the absence (A) or presence (B) of sCD4 (2 μg/ml). The amount of bound X5 was detected by Fab-specific alkaline phosphatase conjugate and represented as optical density at 490 nm.

Figure 3

Binding of X5, Fab b12, and 17b to cell-surface-associated oligomeric gp120-gp41. The T-cell line H9 was infected with the TCLA X4 HIV-1_MN isolate for 10 days. At this time postinfection there was no detectable CD4 remaining at the cell surface, no syncytium formation in the culture, but strong Env expression detected using gp120-specific mAbs and flow cytometry. These H9 cells were preincubated with sCD4 (20 μg/ml) or buffer alone for 1 h at 20°C, then incubated with Fab X5, 17b, or Fab b12 at the indicated concentrations. The amount of bound antibodies was measured by flow cytometry and represented in arbitrary units. The continuous lines represent fitting of the binding data by using the Langmuir adsorption isotherm as described in the legend of Fig. 1.