A Human IgG1 (b12) Specific for the CD4 Binding Site of HIV-1 Neutralizes by Inhibiting the Virus Fusion Entry Process, but b12 Fab Neutralizes by Inhibiting a Postfusion Event

Abstract

The human b12 IgG1, specific for the CD4 binding site of the gp120 of HIV-1, was prepared by recombinant DNA technology. It had a high neutralization rate constant (-3.5 x 10(5) M(-1) sec(-1)), although this is about 10-fold less than the values for the best poliovirus or influenza A virus MAbs. The recombinant b12 Fab neutralized well, with about one-tenth of the activity of b12 IgG. The mechanisms by which b12 IgG1 and its Fab neutralize HIV-1 IIIB on C8166 cells have been investigated. Neither inhibited attachment of virus to the target cell as judged by FACS, immunofluorescence, and ELISA data. This was controlled using MAb F105, another human IgG1, that did neutralize by inhibiting attachment under our conditions. The interactions of b12 IgG- and Fab-neutralized virions with target cells were compared with those of nonneutralized virus using a number of different techniques (fluorescence dequenching of R18-labeled virions, immunofluorescence of virion gp41 and p24 antigens, and acquisition of resistance to removal of virions from the cell by protease). These and the inhibition of HIV-1-mediated cell-cell fusion all demonstrated that b12 IgG neutralized by inhibiting the primary fusion-uncoating mechanism. However, the interactions of b12 Fab-neutralized and nonneutralized virions with C8166 cells were indistinguishable. Thus b12 Fab did not inhibit fusion uncoating, and by inference inhibited a stage of infection that occurs after the entry of the virion core into the cytoplasm. It is therefore possible that b12 IgG kills HIV-1 twice over, by fusion-inhibition and by inhibiting the postentry event proposed for the Fab. The mechanism of neutralization of b12 Fab and of other MAbs that neutralize in a similar way and why b12 Fab and IgG neutralize by different mechanisms are discussed.

Antibody-Mediated Neutralization of Primary Human Immunodeficiency Virus Type 1 Isolates: Investigation of the Mechanism of Inhibition

“…For the well-known MAb IgG1b12, directed against the CD4bs, conflicting results have been published. Ugolini et al reported inhibition of attachment (51), whereas McInerney et al showed inhibition of fusion by the whole antibody and an effect at an unidentified postfusion step for its Fab (26). In our hands, this antibody did not inhibit adsorption for either HIV-1 MN or PI.…”

“…The effect of such target cell interaction in the neutralization mechanism has been described for many viruses (13). For HIV-1, interaction with heparan sulfates has been reported to be involved in attachment (26). Although this nonspecific attachment has been well documented for some cell lines, such as MT-4 (37,40), no such heparan binding phenomena have been detected for PBMC and X4-or R5-type virus interactions (18).…”

“…For HIV, contradictory results have been obtained concerning the way in which antibodies inhibit infection. Postattachment neutralization phenomena, such as the inhibition of fusion, have been described (1,2,26). In such cases, the neutralizing antibodies react with the virus after it has become attached to the cell but before it is internalized.…”

An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

“…2G12 recognizes a posttranslational glycan epitope on gp120 (2,3). CH103-CH106 (4), b12 (5,6), VRC01 (7), and numerous other mAbs with VRC01-like characteristics, such as VRC03, VRC-PG04, CH30-CH34, and the HAAD motif antibodies (7)(8)(9), recognize the CD4 binding site (CD4bs). HJ16 binds to an epitope near the CD4bs (10).…”

Susceptibility of Recently Transmitted Subtype B Human Immunodeficiency Virus Type 1 Variants to Broadly Neutralizing Antibodies

“…The four most broadly neutralizing MAbs were isolated from HIV-infected individuals, which has led to the hypothesis that it may be possible to elicit these antibodies of similar specificity and breadth by vaccination. MAbs b12, 2G12, 2F5, and 4E10 generally exhibit broad cross-clade neutralization of HIV-1 in vitro (3,5,22,27,38), although a small number of viruses have been identified that are not neutralized by any of the four MAbs at the concentrations tested (5). All four MAbs have been shown to protect against viral challenge in vivo in animal models when administered alone or in combination (1,15,17,25,26,35,43,50).…”