Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries.

Chao-Ping Tung,Shiou-Hwa Ma,Jia-Tsrong Jan,Chung-Ming Yu,Yu-Ching Lee,Hung-Pin Peng,Ing-Chien Chen,An-Suei Yang,Jhih-Wei Jian

doi:10.1038/srep15053

Abstract

Broadly neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information.

Highlights

Neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood
CR6261 in complex with SC1918/H1 HA or Viet04/H5 HA interacts with two patches of epitope on HA: the membrane-distal patch composed of hydrophobic side chains from C-terminal end of helix A in HA2 and adjacent hydrophobic side chains from N- and C-terminal ends of HA1 interacts with Pro[28] and Phe[29] in complementarity determining region H1 (CDR-H1) and Phe[74] in framework region 3 (FR3); the membrane-proximal patch composed of highly conserved Gly[20], Trp[21], Tyr[22], and Ile/Val[45] from HA2 with adjacent conserved His[18] and His[38] from HA1 interacts with IGHV1–69-encoded Ile[53] and Phe[54] in CDR-H2 and Tyr[98] in CDR-H3 (HA residue numbers throughout this work are based on the numbering of the HA structure in 3GBN4)
F10 – another IGHV1–69-bnAb of the group 1 type A influenza viruses – recognizes the common IGHV1–69-bnAb epitope with similar CDR-H1~3 loop conformations: while the highly conserved membrane-proximal epitope patch is recognized with the paratope configuration similar to that of CR6261, the membrane-distal epitope patch is recognized with CDR-H1 loop adapting the type 1 canonical structure where the germline-encoded Phe[29] is not accessible for antigen-binding and with the key aromatic side chain of Phe[74] in FR3 replaced by small hydrophilic side chain of Ser[6]

Summary

Introduction

Neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. As few as 2 ~ 3 additional CDR-H1 and CDR-H2 mutations (Ser30Arg – Pro52AAla, or Thr28Pro – Ser30Ile – Ile53Val) in the IGHV1–69-encoded precursor IgG enable hetero-subtypic neutralizing activity, and additional favorable mutations can substitute the key roles of the aromatic side chains of Phe[54] and Tyr[98], suggesting that the affinity maturation pathways of the IGHV1–69-bnAbs could be redundant[1] All these results support the speculation that IGHV1–69 germline gene has been optimized by Darwinian evolution co-existed with the ubiquitous influenza virus infections in humans[2]. It is reasonable to anticipate that the antibody library developed could be applicable to some portion of H1 HA strains

Methods

Results

Conclusion