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Functional interrogation and mining of natively paired human VH:VL antibody repertoires

Nature Biotechnology volume 36, pages 152–155 (2018)Cite this article

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Abstract

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

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Figure 1: High-throughput cloning, yeast display, and functional analysis of the human natively paired VH:VL antibody repertoire.
Figure 2: Examples of natively paired antibody repertoire analysis and functional characterization.

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GenBank/EMBL/DDBJ

Sequence Read Archive

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Acknowledgements

We gratefully acknowledge B. Hartman for assistance with figures, N. Doria-Rose, G. Ippolito, and M. Kanekiyo for advice and guidance, S. Lucas, and K. Zhou for help with experiments, S. Darko for assistance with data processing, and E. Shusta and S. Harrison for kindly sharing reagents and research tools. This work was funded in part by the intramural research program of the Vaccine Research Center, NIAID, NIH, NIH grant DP5OD023118-01 to B.J.D., NIH grant 5R21CA191239-01 to A.D.E., Leidos Biomedical Research Inc. contract 15X219, DTRA contract HDTRA1-12-C-0105, and NIH grant 1R56AI106006 to G.G.

Author information

Author notes

  1. Bo Wang and Brandon J DeKosky: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA

    Bo Wang, Jiwon Lee, Jonathan R McDaniel, George Delidakis, William N Voss & George Georgiou

  2. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA

    Brandon J DeKosky, Morgan R Timm, Erica Normandin, John Misasi, Rui Kong, Kendra E Leigh, Thomas Niezold, Chang W Choi, Elise G Viox, Alberto Cagigi, Aurélie Ploquin, Kwanyee Leung, Eun Sung Yang, Wing-Pui Kong, David R Ambrozak, Amy R Henry, Farida Laboune, Julie E Ledgerwood, Barney S Graham, Daniel C Douek, Nancy J Sullivan & John R Mascola

  3. Department of Chemical & Petroleum Engineering, The University of Kansas, Lawrence, Kansas, USA

    Brandon J DeKosky

  4. Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas, USA

    Brandon J DeKosky & Ahmed Fahad

  5. Laboratory of Molecular Medicine, Boston Children′s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Aaron G Schmidt

  6. Duke Human Vaccine Institute, Duke University Medical School, Durham, North Carolina, USA

    M Anthony Moody

  7. Department of Pediatrics, Duke University Medical School, Durham, North Carolina, USA

    M Anthony Moody

  8. National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA

    Mark Connors

  9. Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, USA

    Andrew D Ellington & George Georgiou

  10. Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA

    Andrew D Ellington & George Georgiou

  11. Department of Bioengineering, The University of Texas at Austin, Austin, Texas, USA

    George Georgiou

Authors
  1. Bo Wang
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  2. Brandon J DeKosky
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  3. Morgan R Timm
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  4. Jiwon Lee
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  28. Mark Connors
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  33. George Georgiou
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Contributions

B.W., B.J.D., J.R. Mascola, and G.G. conceived the study and designed the experiments. B.W. and B.J.D. conducted the experiments with help from M.R.T., J.L., E.N., J.M., R.K., J.R. McDaniel, G.D., K.E.L., T.N., C.W.C., E.G.V., A.F., A.C., A.P., K.L., E.S.Y., W.-P.K., W.N.V., A.G.S., M.A.M., D.R.A., A.R.H., F.L., J.E.L., B.S.G., M.C., and D.C.D. N.J.S, A.D.E., J.R. Mascola, and G.G. supervised the study. B.W., B.J.D., J.R. Mascola, and G.G. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to John R Mascola or George Georgiou.

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Competing interests

Competing financial interests statement G.G., B.J.D., and A.D.E. declare competing financial interests in the form of a patent filed by the University of Texas at Austin.

Integrated supplementary information

Supplementary Figure 1 Map of pCT-VHVL-K1 native VH:VL display vector.

Natively-paired VH:VL sequences are cloned en masse into this vector for human antibody repertoire mining, and their corresponding Fabs are expressed on the yeast cell surface via galactose induction.

Supplementary Figure 2 Flow cytometry analysis of a panel of human anti-HA antibodies before and after display optimization, and of the 2 anti-EBOV antibodies and 1 anti-HIV-1 bNAb in the optimized system.

(a) Display of the six anti-HA antibodies listed in Figure 1c that did not functionally display in EBY100 (upper) and in AWY101 with LZ-forced Fab dimerization (lower). (b) Anti-EBOV antibodies c13c6 and KZ52 and anti-HIV-1 bNAb VRC34.01 displayed in the optimized system. For anti-HA antibodies, 100nM recombinant A/California/07/2009 HA was used to stain D1 H1-2, D1 H1-3/H3-3, D1 H1-53, D1 H1-12, and D1 H1-17/H3-14, and 100nM recombinant B/Brisbane/60/2008 HA was used to stain D1 Vic-8/Yama-20. 23 nM GPΔmuc-APC was used to stain c13c6 and KZ52; 50nM VRC34-epitope scaffold-FP-APC was used to stain VRC34.01. A representative profile from 5 (a) or 3 (b) independent experiments for each antibody is shown.

Supplementary Figure 3 Representative FACS gating strategy for EBOV library sorts.

Yeast cells were stained with 2 μg/ml anti-FLAG-FITC and 23 nM GPΔmuc-APC.

Supplementary Figure 4 Flow cytometry antigen binding profiles of monoclonal yeast populations expressing EBOV.YD.09-EBOV.YD.11, which were identified by single colony picking.

Yeast cells were stained with 2 μg/ml anti-FLAG-FITC and 23 nM GPΔmuc-APC.

Supplementary Figure 5 Biolayer interferometry response curves for human anti-EBOV antibodies from the plasmablast cognate VH:VL repertoire.

Binding was assessed against GPΔmuc. Global analyses were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations used in each experiment.

Supplementary Figure 6 Neutralization of EBOV GP pseudotype infection by human anti-EBOV antibodies.

% Infection is shown relative to the negative control antibody VRC01. Data are reported as average ± standard deviation for three technical replicates.

Supplementary Figure 7 Representative FACS gating strategy for HIV-1 library sorts.

Yeast cells were stained with 2 μg/ml anti-FLAG-FITC, 50 nM VRC34-epitope scaffold-FP-APC, and 50 nM VRC34-epitope scaffold-KO-PE for the isolation of HIV-1 fusion peptide-specific antibodies.

Supplementary Figure 8 Biolayer interferometry response curves for HIV-1 FP-specific antibodies from the B cell repertoire of an HIV-1 slow progressor.

The FP-scaffold protein was immobilized on the biosensor chip. Global analyses were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations used in each experiment.

Supplementary Figure 9 HIV-1 neutralization IC50 potency for natively paired VRC34 family antibodies discovered via yeast display.

Neutralization was determined against a panel of 22 viruses.

Supplementary Figure 10 Sequence alignments of HIV-1 FP-specific antibodies from the peripheral B cell repertoire of donor N123.

Mutations are colored in red.

Supplementary Figure 11 Representative FACS gating strategy for flu library screening.

Yeast cells were stained with 2 μg/ml anti-FLAG-FITC, and either 40nM A/Solomon Islands/3/2006 H1 HA (upper panel) or 40nM A/Wisconsin/67/2005 H3 HA (lower panel) for the isolation of H1 and H3-specific antibodies, respectively.

Supplementary Figure 12 Surface plasmon resonance binding curves for anti-HA antibodies from the B cell cognate VH:VL repertoire.

Representative binding curves from 3 independent experiments for each antibody are shown.

Supplementary Figure 13 Neutralization profiles of anti-HA antibodies isolated via yeast display.

CR9114 and CR6261 were included as positive and negative controls, respectively. Data are reported as average ± standard deviation from three technical replicates.

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Wang, B., DeKosky, B., Timm, M. et al. Functional interrogation and mining of natively paired human VH:VL antibody repertoires. Nat Biotechnol 36, 152–155 (2018). https://doi.org/10.1038/nbt.4052

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