Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning.


Abstract

Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA) are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies) which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1)pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu), 2009 H1N1 pandemic (swine flu) and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our deep sequence analysis predicted that binding to the emerging H1N1 strain (A/Christchurch/16/2010) carrying the HA2-E47K mutation would not affect binding was confirmed experimentally. This demonstrates yeast display, in combination with deep sequencing, may be able to predict antibody reactivity to emerging influenza strains so assisting in the preparation for future influenza pandemics.

Submission Details

ID: Yia94mWW4

Submitter: Wesley Field

Submission Date: June 27, 2018, 3:41 p.m.

Version: 1

Publication Details
Gaiotto T;Hufton SE,PLoS One (2016) Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning. PMID:27741319
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5VLI 2017-09-27 1.8 Computationally designed inhibitor peptide HB1.6928.2.3 in complex with influenza hemagglutinin (A/PuertoRico/8/1934)
4GXX 2012-12-19 1.8 Crystal structure of the 'avianized' 1918 influenza virus hemagglutinin
1WBX 2005-01-19 1.9 CRYSTAL STRUCTURES OF MURINE MHC CLASS I H-2 Db AND Kb MOLECULES IN COMPLEX WITH CTL EPITOPES FROM INFLUENZA A VIRUS: IMPLICATIONS FOR TCR REPERTOIRE SELECTION AND IMMUNODOMINANCE
6ONA 2019-05-01 1.95 Crystal structure of Influenza hemagglutinin from strain A/Hickox/JY2/1940
5VMC 2017-11-08 2.15 Influenza hemagglutinin H1 mutant DH1 in complex with 6'SLN
1RVX 2004-03-30 2.2 1934 H1 Hemagglutinin in complex with LSTA
3GBN 2009-03-10 2.2 Crystal Structure of Fab CR6261 in Complex with the 1918 H1N1 influenza virus hemagglutinin
1RVZ 2004-03-30 2.25 1934 H1 Hemagglutinin in complex with LSTC
5W5S 2017-10-04 2.28 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141019 (P5)
1RU7 2004-03-30 2.3 1934 Human H1 Hemagglutinin
5VMF 2017-11-08 2.35 Influenza hemagglutinin H1 mutant DH1D in complex with 6'SLN
5VMG 2017-11-08 2.45 Influenza hemagglutinin H1 mutant DH1E in complex with 6'SLN
5W5U 2017-10-04 2.46 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141037 (P4)
5W6T 2017-10-04 2.59 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP151070 (P7)
4EEF 2012-06-27 2.7 Crystal structure of the designed inhibitor protein F-HB80.4 in complex with the 1918 influenza virus hemagglutinin.
4JUG 2013-05-01 2.7 Crystal structure of 1918 pandemic influenza virus hemagglutinin mutant D225G
1RUY 2004-03-30 2.7 1930 Swine H1 Hemagglutinin
5W6R 2017-10-04 2.73 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141099 (P6)
3LZF 2010-04-07 2.8 Crystal Structure of Fab 2D1 in Complex with the 1918 Influenza Virus Hemagglutinin
4JUH 2013-05-01 2.81 Crystal structure of 1918 pandemic influenza virus hemagglutinin mutant D225G complexed with avian receptor analogue LSTa
5W6U 2017-10-04 2.88 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP121068 (P2)
1RUZ 2004-03-30 2.9 1918 H1 Hemagglutinin
4PY8 2014-05-21 2.91 Crystal structure of Fab 3.1 in complex with the 1918 influenza virus hemagglutinin
5VMJ 2017-11-08 2.95 Influenza hemagglutinin H1 mutant DH1E in complex with 3'SLN
1RD8 2004-03-23 3.0 Crystal Structure of the 1918 Human H1 Hemagglutinin Precursor (HA0)
2WRH 2009-09-29 3.0 structure of H1 duck albert hemagglutinin with human receptor
2WRG 2009-09-29 3.0 structure of H1 1918 hemagglutinin with human receptor
4JUJ 2013-05-01 3.01 Crystal structure of 1918 pandemic influenza virus hemagglutinin mutnat D225G complexed with human receptor analogue LSTc
5W6I 2017-10-04 3.1 Crystal structure of the A/Puerto Rico/8/1934 (H1N1) influenza virus hemagglutinin in complex with cyclic peptide CP141046 (P3)
3R2X 2011-05-11 3.1 Crystal structure of the de novo designed binding protein HB36.3 in complex the the 1918 influenza virus hemagglutinin
4GXU 2012-12-19 3.29 Crystal structure of antibody 1F1 bound to the 1918 influenza hemagglutinin
6HJN 2018-09-26 3.3 Structure of Influenza Hemagglutinin ectodomain (A/duck/Alberta/35/76)
6HJP 2018-09-26 3.3 Structure of Influenza Hemagglutinin ectodomain (A/duck/Alberta/35/76) in complex with FISW84 Fab Fragment
6HJQ 2018-09-26 4.1 Structure of Full-length Influenza Hemagglutinin (A/duck/Alberta/35/76) in complex with FISW84 Fab Fragment
6HJR 2018-09-26 4.2 Structure of full-length Influenza Hemagglutinin with tilted transmembrane (A/duck/Alberta/35/76[H1N1])

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
549.6 B,D,F,H,J,L Hemagglutinin B4URD6 HEMA_I06A0
552.6 B,D,F,H,J,L Hemagglutinin Q0HD60 HEMA_I40A0
542.4 B,D,F,H,J,L Hemagglutinin Q9WCD9 HEMA_I30A0
559.8 B,D,F,H,J,L Hemagglutinin A4K143 HEMA_I54A2
556.2 B,D,F,H,J,L Hemagglutinin A4GCK8 HEMA_I43A0
549.6 B,D,F,H,J,L Hemagglutinin A3DRP0 HEMA_I96A2
556.2 B,D,F,H,J,L Hemagglutinin A4U6V2 HEMA_I45A0
559.8 B,D,F,H,J,L Hemagglutinin P03454 HEMA_I33A0
559.8 B,D,F,H,J,L Hemagglutinin A8C8J4 HEMA_I07A0
546.0 B,D,F,H,J,L Hemagglutinin Q9WCE3 HEMA_I80A1
559.8 B,D,F,H,J,L Hemagglutinin Q07FI5 HEMA_I96A3
562.8 B,D,F,H,J,L Hemagglutinin A4GCH5 HEMA_I83A1
546.0 B,D,F,H,J,L Hemagglutinin Q9WCE1 HEMA_I81A3
562.8 B,D,F,H,J,L Hemagglutinin A4GCJ7 HEMA_I80AA
562.8 B,D,F,H,J,L Hemagglutinin P18875 HEMA_I79A4
562.8 B,D,F,H,J,L Hemagglutinin P03453 HEMA_I77AB
562.8 B,D,F,H,J,L Hemagglutinin A4GBX7 HEMA_I77AA
562.8 B,D,F,H,J,L Hemagglutinin A8C8W3 HEMA_I67A2
562.8 B,D,F,H,J,L Hemagglutinin Q9WCD8 HEMA_I61A1
562.8 B,D,F,H,J,L Hemagglutinin P18876 HEMA_I54A0
562.8 B,D,F,H,J,L Hemagglutinin A4U7A6 HEMA_I51A0
559.8 B,D,F,H,J,L Hemagglutinin P03452 HEMA_I34A1
562.8 B,D,F,H,J,L Hemagglutinin Q289M7 HEMA_I00A1
549.6 B,D,F,H,J,L Hemagglutinin P26562 HEMA_I76A4
562.8 B,D,F,H,J,L Hemagglutinin A4GCI6 HEMA_I36A0
562.8 B,D,F,H,J,L Hemagglutinin A4GCL9 HEMA_I35A3
559.8 B,D,F,H,J,L Hemagglutinin Q9WFX3 HEMA_I18A0
566.4 B,D,F,H,J,L Hemagglutinin P03455 HEMA_I76AI
1114.2 A,B,C,D,E,F,G,H,I,J,K,L Hemagglutinin P26140 HEMA_I88A6