The mapping and characterization of a novel binding site on HIV-1 gp120 for the broadly neutralizing monoclonal antibody IgG1 b12
HIV infects target cells via fusion events following surface envelope glycoprotein binding to the CD4 receptor and a chemokine co-receptor. Despite the high sequence variability of envelope across and within HIV-1 subtypes, this process requires conserved sequences and structures on gp120, which also represent good targets for HIV-1 neutralizing antibodies. Few examples of HIV-1 broadly neutralizing antibodies exist, but these antibodies may hold the key to a protective HIV-1 vaccine. One such antibody, IgG1 b12 (b12), binds the CD4 binding site on the HIV-1 envelope glycoprotein gp120. To date, no vaccine preparations have been able to elicit a b12-like response. A complete understanding of the mechanism of b12 binding to gp120 is essential to successful design of an b12-like immune response. Until now, strategies to map the b12 binding site on gp120 have utilized indirect techniques and/or core gp120 and have shown that b12 binds to a site on gp120 that overlaps the CD4 binding site. To more directly map the b12 epitope on intact gp120, epitope excision mass spectrometry mapping was carried out in the MALDI QqTOF platform. The putative epitope sequence was confirmed by tandem mass spectrometry sequencing. Epitope mapping revealed a novel binding site for IgG1 b12 at the gp120 amino terminus called Nterm. b12 bound a synthesized peptide of the epitope and the nature of the epitope was explored by ELISA. Although the Nterm epitope is involved in b12-gp120 interactions, ELISAs also show that the epitope does not make up the entire binding site on gp120. Rabbits immunized with a peptide version of the Nterm epitope do express antibodies that bind monomeric gp120, but these antibody responses do not neutralize HIV-1 in vitro. These data indicate that the b12 binding site on gp120 is much more complex than previously thought. The b12 binds the Nterm sequence of gp120, perhaps in conjunction with the CD4 binding site. It has been shown that another HIV-1-neutralizing antibody, 4E10, also binds this novel Nterm epitope, and this may indicate a similar mechanism of action utilized by these two different antibodies. Though not able to elicit neutralizing antibodies on its own, this epitope may be an important element of the neutralizing b12 epitope and an important component of HIV-1 neutralizing antibody responses.