[12] TOWARDS A MOLECULAR UNDERSTANDING OF THE BROAD NEUTRALIZING ACTIVITY OF THE HUMAN ANTI-GP120 ANTIBODY B12

1st International AIDS Society Conference on HIV Pathogenesis and Treatment

Buenos Aires, Argentina - July 8-11, 2001

[TITLE:] TOWARDS A MOLECULAR UNDERSTANDING OF THE BROAD NEUTRALIZING ACTIVITY OF THE HUMAN ANTI-GP120 ANTIBODY B12

[AUTHOR(S):] Zwick M, Ollmann Saphire E, Pantophlet R, Dawson P, Wilson I, Parren P, Burton D
The Scripps Research Institute, La Jolla, CA, USA

IAS Conf HIV Pathog Treat 2001 Jul 8-11;1st: Abstract No. 12

[ABSTRACT:] Background: The human monoclonal antibody (mAb) b12 potently neutralizes a broad range of primary isolates of HIV-1. Despite years of screening efforts by our laboratory and others, no other mAb directed to the CD4-binding site (CD4bs) of gp120 neutralizes HIV-1 as broadly and potently as b12. We sought to better understand this unique property of b12 using structural analysis, mutagenesis, and fine epitope mapping studies

Methods: The X-ray crystal structure of the whole IgG1 b12 was solved and used in docking experiments with the core of gp120. A peptide corresponding to the third hypervariable region of the heavy chain (H3) of b12 was synthesized, conjugated to BSA and used in neutralization assays. Based on the docking experiments, four different positions in the H3 of b12 were targeted for mutagenesis, and the resulting mutants compared to wild-type b12 for binding gp120. For epitope mapping, various anti-gp120 mAbs were used in competition ELISA with b12, and a panel of gp120 mutants were tested against wild-type b12The X-ray crystal structure of the whole IgG1 b12 was solved and used in docking experiments with the core of gp120. A peptide corresponding to the third hypervariable region of the heavy chain (H3) of b12 was synthesized, conjugated to BSA and used in neutralization assays. Based on the docking experiments, four different positions in the H3 of b12 were targeted for mutagenesis, and the resulting mutants compared to wild-type b12 for binding gp120. For epitope mapping, various anti-gp120 mAbs were used in competition ELISA with b12, and a panel of gp120 mutants were tested against wild-type b12The X-ray crystal structure of the whole IgG1 b12 was solved and used in docking experiments with the core of gp120. A peptide corresponding to the third hypervariable region of the heavy chain (H3) of b12 was synthesized, conjugated to BSA and used in neutralization assays. Based on the docking experiments, four different positions in the H3 of b12 were targeted for mutagenesis, and the resulting mutants compared to wild-type b12 for binding gp120. For epitope mapping, various anti-gp120 mAbs were used in competition ELISA with b12, and a panel of gp120 mutants were tested against wild-type b12

Results: In the X-ray crystal structure of IgG1 b12, the long (18 aa) H3 loop protrudes strikingly from the antigen binding surface of b12. Attempts to dock b12 onto the gp120 core suggest Trp100 of the H3 of b12 may bury in the hydrophobic pocket of gp120 in much the same way as Phe43 of CD4. The reactivity of b12 with a panel of gp120 mutants supports this analysis. A synthetic H3 peptide-conjugate, designed on the basis of the crystal structure of IgG1 b12, neutralizes lab-adapted strains of HIV-1, albeit at higher concentrations than IgG1 b12. Moreover, mutations of residues within the H3 of b12 support the conclusion that the H3 plays a key role in HIV-1 recognition In the X-ray crystal structure of IgG1 b12, the long (18 aa) H3 loop protrudes strikingly from the antigen binding surface of b12. Attempts to dock b12 onto the gp120 core suggest Trp100 of the H3 of b12 may bury in the hydrophobic pocket of gp120 in much the same way as Phe43 of CD4. The reactivity of b12 with a panel of gp120 mutants supports this analysis. A synthetic H3 peptide-conjugate, designed on the basis of the crystal structure of IgG1 b12, neutralizes lab-adapted strains of HIV-1, albeit at higher concentrations than IgG1 b12. Moreover, mutations of residues within the H3 of b12 support the conclusion that the H3 plays a key role in HIV-1 recognition In the X-ray crystal structure of IgG1 b12, the long (18 aa) H3 loop protrudes strikingly from the antigen binding surface of b12. Attempts to dock b12 onto the gp120 core suggest Trp100 of the H3 of b12 may bury in the hydrophobic pocket of gp120 in much the same way as Phe43 of CD4. The reactivity of b12 with a panel of gp120 mutants supports this analysis. A synthetic H3 peptide-conjugate, designed on the basis of the crystal structure of IgG1 b12, neutralizes lab-adapted strains of HIV-1, albeit at higher concentrations than IgG1 b12. Moreover, mutations of residues within the H3 of b12 support the conclusion that the H3 plays a key role in HIV-1 recognition In the X-ray crystal structure of IgG1 b12, the long (18 aa) H3 loop protrudes strikingly from the antigen binding surface of b12. Attempts to dock b12 onto the gp120 core suggest Trp100 of the H3 of b12 may bury in the hydrophobic pocket of gp120 in much the same way as Phe43 of CD4. The reactivity of b12 with a panel of gp120 mutants supports this analysis. A synthetic H3 peptide-conjugate, designed on the basis of the crystal structure of IgG1 b12, neutralizes lab-adapted strains of HIV-1, albeit at higher concentrations than IgG1 b12. Moreover, mutations of residues within the H3 of b12 support the conclusion that the H3 plays a key role in HIV-1 recognition

Conclusions: Our data identify, in fine structural and functional detail, unique features of the b12 paratope. These findings have important implications for HIV-1 vaccine design.

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