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Rapid conformational epitope mapping of anti-gp120 antibodies with a designed mutant panel displayed on yeast.

ABSTRACT: gp120 is a substrate for protein engineering both for human immunodeficiency virus (HIV) immunogen design and as a bait for isolating anti-HIV antibodies from patient samples. In this work, we describe the display of a stripped core gp120 on the yeast cell surface. Validation against a panel of neutralizing antibodies confirms that yeast-displayed gp120 presents the CD4 binding site in the correct conformation. We map the epitope of the broadly neutralizing anti-gp120 antibody VRC01 using both a random mutagenesis library and a defined mutant panel and find that the resultant epitope maps are consistent with one another and with the crystallographically identified contact residues. Mapping the VRC01-competitive antibodies b12 and b13 reveals energetic differences in their epitopes that are not obvious from existing crystal structures. These data suggest mutation sets that abrogate binding to broadly neutralizing antibodies with greater specificity than the canonical mutation D368R, useful in rapidly assessing the nature of a vaccine response.

SUBMITTER: Mata-Fink J

PROVIDER: S-EPMC3785227 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Rapid conformational epitope mapping of anti-gp120 antibodies with a designed mutant panel displayed on yeast.

Mata-Fink Jordi J Kriegsman Barry B Yu Hui Xin HX Zhu Hanna H Hanson Melissa C MC Irvine Darrell J DJ Wittrup K Dane KD

Journal of molecular biology 20121115 2

gp120 is a substrate for protein engineering both for human immunodeficiency virus (HIV) immunogen design and as a bait for isolating anti-HIV antibodies from patient samples. In this work, we describe the display of a stripped core gp120 on the yeast cell surface. Validation against a panel of neutralizing antibodies confirms that yeast-displayed gp120 presents the CD4 binding site in the correct conformation. We map the epitope of the broadly neutralizing anti-gp120 antibody VRC01 using both a ...[more]

PMID: 23159556

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Project description:Nerve growth factor (NGF) plays a central role in multiple chronic pain conditions. As such, anti-NGF monoclonal antibodies (mAbs) that function by antagonizing NGF downstream signaling are leading drug candidates for non-opioid pain relief. To evaluate anti-canine NGF (cNGF) mAbs we sought a yeast surface display platform of cNGF. Both mature cNGF and pro-cNGF displayed on the yeast surface but bound conformationally sensitive mAbs at most 2.5-fold in mean fluorescence intensity above background, suggesting that cNGF was mostly misfolded. To improve the amount of folded, displayed cNGF, we used comprehensive mutagenesis, FACS, and deep sequencing to identify point mutants in the pro-region of canine NGF that properly enhance the folded protein displayed on the yeast surface. Out of 1,737 tested single point mutants in the pro region, 49 increased the amount of NGF recognized by conformationally sensitive mAbs. These gain-of-function mutations cluster around residues A-61-P-26. Gain-of-function mutants were additive, and a construct containing three mutations increased amount of folded cNGF to 23-fold above background. Using this new cNGF construct, fine conformational epitopes for tanezumab and three anti-cNGF mAbs were evaluated. The epitope revealed by the yeast experiments largely overlapped with the tanezumab epitope previously determined by X-ray crystallography. The other mAbs showed site-specific differences with tanezumab. As the number of binding epitopes of functionally neutralizing anti-NGF mAbs on NGF are limited, subtle differences in the individual interacting residues on NGF that bind each mAb contribute to the understanding of each antibody and variations in its neutralizing activity. These results demonstrate the potential of deep sequencing-guided protein engineering to improve the production of folded surface-displayed protein, and the resulting cNGF construct provides a platform to map conformational epitopes for other anti-neurotrophin mAbs.

Dataset Information

Rapid conformational epitope mapping of anti-gp120 antibodies with a designed mutant panel displayed on yeast.

Publications

Rapid conformational epitope mapping of anti-gp120 antibodies with a designed mutant panel displayed on yeast.

Similar Datasets

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