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. 2020 Aug 20;10(1):151.
doi: 10.1186/s13568-020-01086-4.

Generation of a recombinant chickenized monoclonal antibody against the neuraminidase of H9N2 avian influenza virus

Fei Wang  1   2 , Yajuan Wang  1   2 , Zhimin Wan  1   2 , Hongxia Shao  1   2   3 , Kun Qian  1   2   3 , Jianqiang Ye  4   5   6 , Aijian Qin  7   8   9
Affiliations

Generation of a recombinant chickenized monoclonal antibody against the neuraminidase of H9N2 avian influenza virus

Fei Wang et al. AMB Express. .

Abstract

We previously reported a monoclonal antibody (mAb), 1G8, against the neuraminidase (NA) of H9N2 avian influenza virus (AIV) with significant NA inhibitory activity. To generate a recombinant chickenized mAb (RCmAb) against the NA of H9N2 AIV for passive immunization in poultry, the gene of the fragment of antigen binding (Fab) of mAb 1G8 was cloned and fused with the fragment crystallizable (Fc) gene of chicken IgY. The RCmAb 1G8 was expressed in COS-1 cells and could be detected in cell culture supernatant. The results of NA inhibitory activity tests of the RCmAb 1G8 in an enzyme-linked lectin assay (ELLA) and a microneutralization (MN) assay showed that the RCmAb 1G8 maintained significant NA inhibitory activity and neutralizing ability. This is the first chickenized antibody against AIV, which would be a good candidate for passive immunization in poultry.

Keywords: H9N2 AIV NA; NA inhibitory activity; Neutralizing ability; Recombinant chickenized monoclonal antibody.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Sequences of the 1G8 variable region and 3-D structure model of the 1G8 Fab. The variable region sequences of the light chain (a) and heavy chain (b) are shown with underlined CDRs. The image of the Fab structure was constructed with DeepView software (Swiss PDB Viewer). The model is displayed in top view (c) and side view (d) with carton and surface rendering. The light chain is shown in white, with CDR1 marked in blue, CDR2 marked in cyan and CDR3 marked in green. The heavy chain is shown in grey, with CDR1 marked in red, CDR2 marked with purple and CDR3 marked with yellow
Fig. 2
Fig. 2
Production of RCmAb 1G8 against the NA of X1 virus. a Western blot analysis of the RCmAb 1G8 in the supernatant. Non-reducing sample (DTT−) and reducing sample (DTT+) of RCmAb 1G8 were tested. b IFA result of the RCmAb 1G8 reacting with X1 virus-infected MDCK cells. The supernatant of COS-1 cells transfected with pCAGGS was used as primary antibody in negative control. c IFA result of the RCmAb 1G8 reacting with pCAGGS-NA (X1) plasmid-transfected COS-1 cells. The supernatant of COS-1 cells transfected with pCAGGS was used as primary antibody in negative control
Fig. 3
Fig. 3
Inhibitory effect and neutralizing ability of the RCmAb 1G8 against NA. a Inhibition of NA activity measured via ELLA. RCmAb 1G8 was diluted from 2− 1 to 2− 10and incubated with X1 virus. Purified mAb 1G8 at initial concentration of 20 µg/mL was used as positive control. The supernatant of COS-1 cells transfected with pCAGGS was used as negative control. b HA titer and c TCID50 of X1 virus in MN assay incubated with RCmAb 1G8. mAb 1G8 at concentration of 5 µg/mL was used as positive control. The supernatant of COS-1 cells transfected with pCAGGS was used as negative control
See this image and copyright information in PMC

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