Fine Mapping of the Interaction of Neutralizing and Nonneutralizing Monoclonal Antibodies with the CD4 Binding Site of Human Immunodeficiency Virus Type 1 gp120

Abstract

Alanine scanning mutagenesis was performed on monomeric gp120 of human immunodeficiency virus type 1 to systematically identify residues important for gp120 recognition by neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the CD4 binding site (CD4bs). Substitutions that affected the binding of broadly neutralizing antibody b12 were compared to substitutions that affected the binding of CD4 and of two nonneutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gp120 comparable to that of b12. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the MAbs and for CD4. However, in contrast to what was seen for the MAbs, no enhancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gp120-CD4 interaction. Although the epitope maps of the MAbs overlapped, a number of key differences between b12 and the other two antibodies were observed. These differences may explain why b12, in contrast to nonneutralizing antibodies, is able to interact not only with monomeric gp120 but also with functional oligomeric gp120 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants mostly showed a reasonable correlation between the effects of the mutations on b12 binding to monomeric gp120 and neutralization efficacy. However, some mutations produced an effect on b12 neutralization counter to that predicted from gp120 binding data. It appears that these mutations have different effects on the b12 epitope on monomeric gp120 and functional oligomeric gp120. To determine whether monomeric gp120 can be engineered to preferentially bind MAb b12, recombinant gp120s were generated containing combinations of alanine substitutions shown to uniquely enhance b12 binding. Whereas b12 binding was maintained or increased, binding by five nonneutralizing anti-CD4bs MAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. These reengineered gp120s are prospective immunogens that may prove capable of eliciting broadly neutralizing antibodies.

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

“…8A). There was an ϳ50% increase in b12 binding, in agreement with previous studies showing that glycans, in particular the one at position 386, can restrict access to the b12 epitope (74,75). Deglycosylation had no effect on basal or sCD4-induced interactions between the trimers and the coreceptor binding site-directed antibody 17b.…”

Rapid development of glycan-specific, broad, and potent anti–HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque

“…Pseudoviruses incorporating single-alanine substitutions were generated by transfection of 293T cells with an Env-expressing plasmid and an Env-deficient genomic backbone plasmid (pSG3ΔEnv), as described previously (23). For generation of NB-DNJ-treated pseudoviruses, 2 mM of the glycosidase inhibitor was added at the time of transfection (17).…”

The Orientation of HIV-1 gp120 Binding to the CD4 Receptor Differentially Modulates CD4+ T Cell Activation

“…ICs were prepared by incubating mammalian cell-derived recombinant HIV-1 gp120 JR-FL (endotoxin level , 1 endotoxin units/mg; Progenics Pharmaceuticals, Tarrytown, NY) or gp120 LAI together with non-CD4BS anti-gp120 mAbs [clone 2G12 (63) and clone 1-79 (64)] or with CD4BS anti-gp120 mAbs [clone b6 (65) and b12 (66)] at a molar gp120/anti-gp120 mAb ratio of ∼4:5 (final concentration of gp120 [f.c. gp120 ] 5 mg/ml) to prepare gp120-anti-gp120 IC and gp120-anti-gp120-CD4BS control IC, respectively.…”