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

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.

Antibody 27F3 Broadly Targets Influenza A Group 1 and 2 Hemagglutinins through a Further Variation in VH1-69 Antibody Orientation on the HA Stem

“…Such overall similarities appear to allow these two antibodies to accommodate the glycosylated Asn38 in group 2 HAs and attain greater breadth. In summary, the previous finding that the IFY motif of V H 1-69 antibodies is essential for their development into broadly neutralizing anti HA-stem antibodies is strongly reinforced here in this study ((Pappas et al, 2014; Tung et al, 2015). Furthermore, the similarities and differences observed in the evolution of these antibodies broaden our understanding of how the V H 1-69 class of anti-stem human antibodies are selected and evolve that should accelerate development of immunogens to elicit immune responses against the HA stem.…”

Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design

“…Allelic polymorphism (i.e., the rearrangement of VDJ gene segments) and somatic mutations are required for V H 1-69 generation (28). Further, HA is capable of activating B cell receptors encoded with the germ line V H sequence (IgM form), but only 2 mutations in the complementarity-determining region H1 and 5 mutations in the framework region 3 are required for the affinity maturation of precursor IgG, as well as for fully mature bNAb activity (29). However, it remains unclear how the stem helix A epitope contributes to bNAb development.…”

High throughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries

“…1 ); most of the synthetic scFv variants can be prepared as soluble scFv molecules with more than 1 µg/mL (or 33 nM) in crude E. coli culture in 96-well deep well plate overnight, feasible for many high throughput functional assays 31 . By contrast to the unpredictable expression of the scFv molecules from conventional phage-displayed scFv libraries constructed from natural antibody gene repertories, where the scFv molecules are frequently unable to be expressed in soluble form with sufficient concentration, the stable expression of functional synthetic scFvs enables a high throughput screening procedure effectively compatible with the high throughput micro-neutralization assay used in screening neutralizing antibodies against IAV infection 32 . The overall schematic high throughput procedure enabled by the phage-displayed synthetic antibody libraries is summarized in Fig.…”

“…But the utility of the GH-based technological platform had been limited to one synthetic antibody library design (GH2) and for immunotoxin discovery against cancer cells 29 , 31 . In a previous work on neutralizing antibody discovery against IAV infection 32 , 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 had been systematically explored, and 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 32 . But this approach necessitated known antibody-antigen recognition information specific for the parent antibody (F10 in this case) to design and construct phage-displayed antibody libraries, aiming to discover more potent neutralizing antibodies in comparison with the parent antibody.…”