Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles

Abstract

The selection of human monoclonal antibodies (MAbs) specific for human immunodeficiency virus (HIV) type 1 by binding assays may fail to identify Abs to quaternary epitopes on the intact virions. The HIV neutralization assay was used for the selection of human MAb 2909, which potently neutralizes SF162 and recognizes an epitope on the virus surface but not on soluble proteins. Three regions of gp120, the V2 and V3 loops and the CD4 binding domain, contribute to the epitope recognized by MAb 2909. The existence of such a unique MAb, which defines a complex epitope formed by a quaternary structure, suggests that there may be other new neutralizing HIV epitopes to target with vaccines.

FIG. 1.

Neutralization of pseudotyped virus SF162 in the luciferase assay (A) and of parental SF162 virus (B and C) in the GHOST cell neutralization assay and the PHA-stimulated PBMC assay, respectively. MAbs 447-52D (anti-V3), b12 (anti-CD4bd), 2G12 (anti-complex carbohydrate), and 2F5 (anti-gp41) were used as positive controls, given their well-established neutralizing activity, while MAb 1418 (anti-parvovirus B19) served as a negative control. The error bars represent the standard deviations from the means of results from three (A) or five (B) experiments.

FIG. 2.

Reactivities of MAbs in ELISA with detergent-solubilized gp120 (A) and with oligomeric SF162 proteins (B). Detergent-solubilized gp120 was reacted with human MAbs 2909, 447 (anti-V3), b12 (anti-CD4bd), 697 (anti-V2), and 1418 (anti-parvovirus B19). (A) The horizontal line represents the cutoff for the assay (mean optical density for MAb 1418 plus 3 standard deviations). Oligomeric SF162 gp140 (O-SF162gp140), V2 deletion oligomeric gp140 (O-DV2gp140), and a mutant of oligomeric SF162gp140 in which the glycosylation site at position 154 was eliminated (25) (O-GM154gp140) were reacted with human MAb 2909 or murine anti-V2 MAb G3.4 (19).

FIG. 3.

Mapping the 2909 epitope by ELISA. The ability of MAbs to bind intact SF162 virions is shown by the amount of p24 released from virions bound to the same human MAbs used for experiments shown in Fig. 2A. In addition, MAbs 2G12 (anti-carbohydrate complex), 830A (anti-V2), and 670 (anti-C5) were used. The horizontal line indicates the cutoff for the assay (mean level of binding to MAb 1418 plus 3 standard deviations) (A). The ability of various MAbs and sCD4, used at concentration of 20 μg/ml, to block the binding of SF162 virions to MAb 2909 is shown as “% inhibition” in panel B, where the horizontal line indicates the cutoff value (mean inhibition by MAb 1418 plus 3 standard deviations). MAb 2909 serves as its own positive control. MAb 2G12 enhanced the binding of SF162 virions to MAb 2909. (C) A dose-response curve in which various MAbs and sCD4 were used in the competition ELISA at concentrations from 0.001 to 10 μg/ml is shown.

FIG. 4.

Binding of MAb 2909 to wild-type SF162 and its mutants with deletions of the V1 (DV1), V2 (DV2), and V3 (DV3) regions. (A) Binding of 2909 and control MAbs to intact virions was tested using the virus capture assay. The control MAbs were specific to V3 (447 and 2191), V2 (697 and 830A), CD4bd (b12 and 1570), C5 (1331A), gp41 (240 and 246), and parvovirus B19 (1418; negative control). The horizontal line indicates the cutoff (mean binding with MAb 1418 plus 3 standard deviations). (B) Immunoprecipitation analysis of MAb reactivities with wild-type SF162 and its mutants. MAb 2909 (lanes 1, 4, and 7) precipitated SF162 and DV1 but not DV2, while MAb 447-52D (lanes 2, 5, and 8) precipitated all three viruses. Control MAb 1418 (lanes 3, 6, and 9) showed no reactivity with any of the tested viruses.