CD4 binding site antibodies inhibit human immunodeficiency virus gp120 envelope glycoprotein interaction with CCR5

Abstract

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.

FIG. 1.

Inhibition of variable loop-deleted gp120 glycoprotein binding to CCR5 by CD4BS antibodies. Radiolabeled ΔV1/V2 gp120 glycoproteins from the ADA strain (A) or JR-FL strain (B) of HIV-1 were incubated at 37°C with Cf2Th cells expressing CCR5 in the presence of the indicated amounts of CD4BS antibodies (F105, IgG1b12, and 15e) or a control antibody, C11. The amount of the ΔV1/V2 gp120 glycoproteins bound to the Cf2Th-CCR5 cells is presented as the percentage of the amount bound in the absence of antibody. The experiment was performed twice with comparable results.

FIG. 2.

Fab fragment and antibody inhibition of variable loop-deleted gp120 binding to CCR5. Radiolabeled ΔV1/V2 gp120 glycoproteins from the ADA strain (A) or JR-FL strain (B) were incubated with Cf2Th cells expressing CCR5 (Cf2Th-CCR5) in the presence of the indicated amounts of antibodies (left panels) or Fab fragments (right panels). The amount of the ΔV1/V2 gp120 glycoprotein bound to the Cf2Th-CCR5 cells is presented as the percentage of the amount bound in the absence of added antibody or Fab. The experiment was performed twice with comparable results.

FIG. 3.

Fab fragment and antibody inhibition of CD4-independent syncytium formation. 293T cells expressing the ΔV1/V2 envelope glycoproteins from the ADA HIV-1 strain were cocultivated with Cf2Th cells expressing CCR5 in the presence of antibodies (A) or Fab fragments (B). IgG1b12, F105, and F91 are CD4BS antibodies, whereas 2G12 recognizes a distinct, carbohydrate-dependent gp120 epitope (25). The percentage of syncytia observed, compared with the number seen in the absence of added antibody or Fab, is reported. The experiment was performed twice with comparable results.

FIG. 4.

Model of the mechanism of neutralization by CD4BS antibodies. Free gp120 may sample multiple conformations but is locked into the CD4-bound conformation by CD4 (17). This gp120 conformation is competent for chemokine receptor binding and virus entry. Because CD4BS antibodies prefer to bind other conformations of gp120 (33), they prevent gp120 from assuming the CD4-bound conformation and from binding the chemokine receptor.