View All Abstracts for Session 24
127 Mapping of the Epitopes on HIV-1 gp120 Recognized by Neutralizing Human Monoclonal Antibodies by Alanine-Scanning Mutagenesis
R. Pantophlet*, E. Ollmann Saphire, I. A. Wilson, P. W. H. I. Parren, and D. R. Burton
The Scripps Res. Inst., La Jolla, CA, USA
Background: Recently we solved the structure of IgGb12, a broadly neutralizing monoclonal antibody (mAb) against HIV-1 primary isolates. Docking models of b12 and the gp120HxBc2 core structure gave insight into how this antibody might bind gp120 and neutralize HIV-1. Here, alanine-scanning mutagenesis was used to identify residues on gp120 that modulate b12-binding, in comparison to b6, which only neutralizes T-cell line-adapted isolates.
Methods: Mutants containing alanine point mutations and variable loop-deleted mutants were generated in pSVIIIenv-JR-CSF and used to transfect 293T cells together with pNL4.3LucR-E-. After 48 h, recombinant virions were lysed, added to microtiter plate wells coated with anti-gp120 antibody D7324, and subsequently incubated with serial dilutions of mAb. Bound antibodies were detected with HRP-conjugated goat anti-human F(ab)2 and TMB-peroxidase substrate. Apparent affinities were calculated as the antibody concentration at 50% maximal binding. Changes in relative antibody affinity were expressed as [apparent affinity (wild type)/apparent affinity (mutant)] ( 100%.
Results: Reduced affinity was observed with all variable-loop deleted mutants and many point mutations affected b6 and b12 similarly, but there were also differences. b12 was more affected by changes at the inside face of the V1/V2-loop stem (e.g., T123), whereas b6-binding was more affected by changes at the “tip” of the stem (e.g., V127).
Conclusions: b6 and b12 binding is affected by many of the same residues. The main difference between the 2 mAbs may result from their angle of interaction with gp120: b12 binding at the inside face of the stem at an angle that would orientate it similar to CD4 (i.e., away from the trimer interface), which is also supported by the docking model. Antibody b6 probably requires additional residues at the distal end of the stem for binding. This may tilt it toward the trimer interface, slightly overlapping the nonneutralizing face of gp120, which is not accessible on the trimer.
Contact Author about this Abstract
