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. 2020 Oct 6;11(5):e02315-20.
doi: 10.1128/mBio.02315-20.

Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus

Haihai Jiang #  1   2 , Weiyu Peng #  1   2 , Jianxun Qi  2 , Yan Chai  2 , Hao Song  3 , Yuhai Bi  2   4 , Pramila Rijal  5 , Haiyuan Wang  2 , Babayemi O Oladejo  2 , Jinhua Liu  1 , Yi Shi  2   4 , George F Gao  6   2   3   4 , Alain R Townsend  7 , Yan Wu  3   8
Affiliations

Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus

Haihai Jiang et al. mBio. .

Abstract

Here, we investigate a monoclonal antibody, Z2B3, isolated from an H7N9-infected patient, that exhibited cross-reactivity to both N9 (group 2) and a broad range of seasonal and avian N1 (group 1) proteins but lost activity to the N1 with the substitution K432E. This substitution exists in 99.25% of seasonal influenza strains after 2013. The NA-Z2B3 complex structures indicated that Z2B3 binds within the conserved active site of the neuraminidase (NA) protein. A salt bridge between D102 in Z2B3 and K432 in NA plays an important role in binding. Structure-based modification of Z2B3 with D102R in heavy chain reversed the salt bridge and restored the binding and inhibition of N1 with E432. Furthermore, Z2B3-D102R can protect mice from A/Serbia/NS-601/2014 H1N1 virus (NA contains E432) infection while the wild-type Z2B3 antibody shows no protection. This study demonstrates that a broadly reactive and protective antibody to NA can be in principle edited to restore binding and inhibition to recently drifted N1 NA and regain protection against the variant influenza strain.IMPORTANCE The immune system produces antibodies to protect the human body from harmful invaders. The monoclonal antibody (MAb) is one kind of effective antivirals. In this study, we isolated an antibody (Z2B3) from an H7N9 influenza virus-infected child. It shows cross-reactivity to both group 1 (N1) and group 2 (N9) neuraminidases (NAs) but is sensitive to N1 NA with a K432E substitution. Structural analysis of the NA-antibody fragment antigen-binding (Fab) complex provides a clue for antibody modification, and the modified antibody restored binding and inhibition to recently drifted N1 NA and regained protection against the variant influenza strain. This finding suggests that antibodies to NA may be a useful therapy and can be in principle edited to defeat drifted influenza virus.

Keywords: influenza; neuraminidase; neutralizing antibody; structure-based modification.

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Figures

FIG 1
FIG 1
Binding profile of Z2B3 to N1 and N9. (A to C) Gel filtration analysis of Z2B3 Fab survive with 18N1 (A), AH-N9 (B), and Serbia N1 (C). Z2B3 (magenta), NA (cyan), or Z2B3-NA (blue) mixtures were analyzed by a Superdex 200 gel filtration column. The elution fractions were pooled and analyzed by SDS-PAGE. (D to F) An SPR assay characterizing the specific binding between Z2B3 and specific NAs, including 18N1 (D), AH-N9 (E), and Serbia N1 (F). The binding affinity (KD) values were calculated using a 1:1 Langmuir model produced with BIAcore 3000 analysis software (BIAevaluation version 4.1).
FIG 2
FIG 2
Molecular determinants of Z2B3 with 18N1 and AH-N9. (A) Overall structure of Z2B3/18N1 complex, with NA in green and the heavy chain (HC) and light chain (LC) of one Fab in magenta and in yellow, respectively. Each NA monomer binds to one Z2B3 Fab. (B) The side view of the Z2B3/18N1 complex structure. The HC and LC of Z2B3 are colored in magenta and yellow, respectively. Residues of HC and LC involved in the interactions are colored in cyan and blue, respectively. The epitopes of Z2B3 are colored in wheat. (C and D) The footprints of Z2B3 on 18N1 (C) and AH-N9 (D). NAs are represented in the gray surface. Residues interacting with FR3, HCDR2, and HCDR3 are colored in wheat, magenta, and cyan, respectively. Residues that interact with both HCDR1 and HCDR2 are colored in orange. Residues that interact with both HCDR2 and HCDR3 are colored in yellow. Residues that interact with HCDR1, HCDR2, and HCDR3 are colored in green. Residues interacting with LCDR1 or LCDR3 are colored in blue. Residues contacted less than 4.5 Å are involved. (E and F) The interactions between residues in HCDR3 of Z2B3 and the key residues in the active site of 18N1 (E) or AH-N9 (F). The Z2B3 HCDR3 and NA are shown in orange and green, respectively, in the cartoon representation.
FIG 3
FIG 3
Residue 432 on NA plays a key role in the binding to Z2B3. (A) Sequence alignment of 430-loop among three NAs. Residue 432 is highlighted with a green star. (B) The polymorphisms of residue 432 in N1 from H1N1 seasonal influenza viruses pre- (left) and post (right)-2013 are shown in pie charts. There are 45,999 NA sequences of human H1N1 strains available in the GISAID database (from 1 March 2009 to 31 December 2019), including 13,828 sequences pre-2013 and 32,171 sequences post-2013. The pie charts show the percentage of H1N1 viruses that carry lysine (blue), glutamic acid (pink), or other amino acid residues (gray) at position 432. (C and D) The salt bridge between K432 in 18N1 (B)/AH-N9 (C) and D102 in Z2B3 Fab. The distance is labeled accordingly.
FIG 4
FIG 4
A D102R substitution in Z2B3 can restore the reactivity to Serbia N1. (A and D) Gel filtration survival analysis of Z2B3-D102R Fab with Serbia N1 (A) and 18N1 (D). Z2B3-D102R (magenta), NA (cyan), or Z2B3-D102R-NA (blue) mixtures were analyzed by a Superdex 200 gel filtration column. The elution fractions were pooled and analyzed by SDS-PAGE. (B and E) An SPR assay characterizing the specific binding between Z2B3-D102R and specific NAs, including Serbia N1 (B) and 18N1 (E). The binding affinity (KD) values were calculated using a 1:1 Langmuir model produced with BIAcore 3000 analysis software (BIAevaluation version 4.1). (C and F) NA inhibition activity of Z2B3 and Z2B3-D102R for Serbia N1 (C) and 18N1 proteins (F).
FIG 5
FIG 5
Protection efficacy of Z2B3 or Z2B3-D102R against lethal infection with influenza viruses in mice. (A, C, E, and G) The prophylactic efficacy of Z2B3 against mouse-adapted A/California/07/2009 (H1N1) (A), A/Puerto Rico/8/1934 (H1N1) (C), A/Vietnam/1194/2004 (H5N1) (E), and A/Anhui/1/2013 (H7N9) (G) infection were tested in BALB/c mice (n = 5 per group). (B, D, F, and H) The therapeutic efficacy of Z2B3 against mouse-adapted A/California/07/2009 (H1N1) (B), A/Puerto Rico/8/1934 (H1N1) (D), A/Vietnam/1194/2004/2004 (H5N1) (F), and A/Anhui/1/2013 (H7N9) (H) infection were tested in BALB/c or DBA/2 mice (n = 5 per group). (I and J) The prophylactic (I) and therapeutic (J) efficacy of Z2B3 or Z2B3-D102R against A/Serbia/NS-601/2014 (H1N1) infection were tested in DBA/2 mice (n = 5 per group). Mouse survival rate and body weight were recorded daily. The dose of antibody is labeled accordingly. 13C6 and PBS are set as the negative controls. The survival curves and body weight loss curves were generated using GraphPad Prism 5. Values are mean ± SEM.
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