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This article is part of the supplement: AIDS Vaccine 2012

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Biophysical dissection of the antigen-antibody interaction of the broadly reactive anti-V3 human mAb 447-52D

A Killikelly1*, H Zhang1, B Spurrier1, C Williams2, MK Gorny2, S Zolla-Pazner2 and X Kong1

  • * Corresponding author: A Killikelly

Author Affiliations

1 New York University Medical Center, New York, NY, USA

2 Veterans Affairs New York Harbor Healthcare System, New York, NY, USA

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Retrovirology 2012, 9(Suppl 2):P67  doi:10.1186/1742-4690-9-S2-P67

The electronic version of this article is the complete one and can be found online at:

Published:13 September 2012

© 2012 Killikelly et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The immunogenic third variable region (V3) of HIV-1 gp120 is a target for AIDS vaccines. V3 is recognized by mAb 447-52D, known for its ability to neutralize viruses with a GPGR beta turn motif at the apex of V3, which is characteristic of clade B viruses. Interestingly, 447-52D can also bind non-clade B V3 peptides containing a GPGQ motif. A detailed biochemical and biophysical dissection of the antigen-antibody interaction of 447-52D was undertaken to understand this disparity.


We cloned and produced large amounts of the Fv fragment of 447-52D and a panel of mutations. We then measured their epitope binding characteristics by Isothermal Titration Calorimetry (ITC).


We assessed the Fv-V3 binding by ITC for the following mutations in residues of the mAb that are thought to mediate three key interactions: (i) YH100j of the heavy chain (H) to T (YH100jT) or YH33A. These two aromatic residues form a pi-cation interaction, sandwiching the side chain of R315 of the GPGR motif in the V3-peptide. These mutations reduce binding affinity by 56 and 171-fold, respectively. (ii) WL91 of the light chain (L) to A (WL91A). This residue packs against P313 of the V3 GPGR turn. This mutation reduces binding 230-fold. (iii) DH95R of the heavy chain or R315Q of the epitope. These two residues form a salt bridge between the antigen and the antibody. These mutations reduce binding by 224 and 171-fold, respectively. These data suggest a hierarchy of interactions and the salt bridge plays an important role in the affinity.


mAb 447-52D binds non-clade B peptides with the R315Q variation with much less affinity, explaining why it cannot neutralize non-clade B viruses. Through probing specific contributions of individual residues by mutagenesis and ITC, we were able to fully characterize the interactions between V3 and 447-52D.