Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Apr;107(7-8):2437-2450.
doi: 10.1007/s00253-023-12433-3. Epub 2023 Feb 23.

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

Min Gu #  1   2   3 , Jun Jiao #  1 , Suhan Liu #  1 , Wanchen Zhao  1 , Zhichuang Ge  1 , Kairui Cai  1 , Lijun Xu  1 , Dongchang He  1 , Xinyu Zhang  1 , Xian Qi  4 , Wenming Jiang  5 , Pinghu Zhang  2   3 , Xiaoquan Wang  1   2   3 , Shunlin Hu  1   2   3 , Xiufan Liu  6   7   8
Affiliations

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

Min Gu et al. Appl Microbiol Biotechnol. 2023 Apr.

Abstract

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Keywords: Influenza virus; Linear B-cell epitope; Monoclonal antibody; Nucleoprotein; Universal reactivity.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests

Figures

Fig. 1
Fig. 1
IFA determination of mAb 5F10 targeting NP protein in 293T cells. At 36 h post-transfection with plasmids pHW2000-QD1-NP and pHW2000, 293T cells were fixed and then cultured with primary antibody of mAb 5F10, QD1 virus-positive mouse antiserum, or the mouse (G3A1) mAb IgG1 isotype control. After incubation with the goat anti-mouse FITC-conjugated secondary antibody and then the 4′,6-diamidino-2-phenylindole (DAPI) for nucleus staining, the 293T cells were observed under fluorescence microscopy. The cells treated with the mouse antisera were used for positive control, while the cells transfected with pHW2000 empty vector or incubated with the commercial IgG1 isotype were used for negative control. Representative pictures were taken with the scale bar of 100 µm
Fig. 2
Fig. 2
Western blot analysis of the mAb 5F10 against NP protein. 293T cells transfected with empty pHW2000 vector or recombinant pHW2000-QD1-NP plasmid were lysed at 36 h post-transfection. The mAb 5F10 or IgG1 isotype and HRP-coupled goat anti-mouse IgG were served as primary and secondary antibodies, respectively. Incubation with mAb 5F10 produced a specific immunoreactive band in the lane marked with pHW2000-QD1-NP. The band was localized at about 55 kD, corresponding to the size of IAV NP protein. Treatment with an IgG1 isotype control antibody had no effect in generating positive bands. The housekeeping protein β-Actin was chosen as the internal reference
Fig. 3
Fig. 3
The broad-spectrum reactivity of mAb 5F10 against different hosts and subtypes of IAV-infected MDCK cells analyzed through IFA (A) and western blotting (B). MDCK cells were infected with avian H1~H11 IAV subtypes including H5Nx strains of multiple HA clades and NA subtypes, swine and human H1N1 and H3N2 IAV subtypes, and human type B influenza (FluB) viruses of Victoria and Yamagata lineages, respectively. Cells mock-inoculated with PBS were served as negative control. At 24 h post-infection, the infected cells were all subjected to incubation with mAb 5F10 as the primary antibody. An IgG1 isotype control antibody was simultaneously used for comparison. In the IFA analysis, typical images were taken with the scale bar of 100 µm after treatment with the FITC-conjugated goat anti-mouse IgG secondary antibody. In the western blot analysis, the HRP-conjugated goat anti-mouse IgG secondary antibody was applied to detect the immunoreactive protein bands
Fig. 4
Fig. 4
Conformation and conservation analysis of the epitope recognized by the anti-NP mAb 5F10. A Location of the identified epitope on NP monomer. The NP monomer structure (PDB: 7NT8) was visualized and analyzed with PyMOL software. The epitope recognized by 5F10 was labeled in red, and it locates in a protruding loop region and on the protein surface. B Sequence conservation analysis of the identified epitope. The IAV NP sequences of all sources (n=13,166) and specifically of avian (n=7778), swine (n=838), and human (n=3015) origins were retrieved from GISAID EpiFlu database with the keywords “Type”= A and “Location”= China. The dataset was collected on 7 September 2022. The variation of the epitope was analyzed with WebLogo program, and the conservation degree of each amino acid was reflected by the size of corresponding character. *Total means of the analyzed NP sequences were of all host origins including avian, swine, human, canine, and equine
Fig. 5
Fig. 5
Application of mAb 5F10. A Intracellular localization of IAV NP protein analyzed via confocal microscopy. The CEF cells were infected with a H5N6 or a H3N2 subtype IAV at 1 MOI for 12 h. B Reaction of mAb 5F10 with IAV NP protein by IP assay. MDCK cells were infected with IAV subtypes H3N2 and H5N6 at 1 MOI, and were harvested and lysed after culturing for 24 h. The lysates were then subjected to the IP assay with mAb 5F10. C Detection of IAV antigen in the lung tissue of infected mice through IHC analysis. Six-week-old female BALB/c mice were intranasally infected with a H5N6 or a H3N2 subtype IAV at 106.0EID50, with mock-treated (with PBS) mice served as the negative control. The mice lungs were collected on day 3 post-infection for IHC staining, with mAb 5F10 serving as the primary antibody. Histopathological examination of the HE-stained sections was correspondingly performed, with the H5N6 virus induced severe lung tissue injury and atrophy of the alveoli whereas the H3N2 virus caused a relatively mild lung tissue destruction. Blue arrows indicated representative inflammatory cell infiltration, while brown signals indicated positive IHC staining. Scale bars were shown in the upper-left corner of microscopic images, with 200 µm for core images and 20 µm for zoom-in images
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Anderson TK, Chang J, Arendsee ZW, Venkatesh D, Souza CK, Kimble JB, Lewis NS, Davis CT, Vincent AL. Swine influenza A viruses and the tangled relationship with humans. Cold Spring Harb Perspect Med. 2021;11(3):a038737. doi: 10.1101/cshperspect.a038737. - DOI - PMC - PubMed
    1. Antigua KJC, Choi WS, Baek YH, Song MS. The emergence and decennary distribution of clade 2.3.4.4 HPAI H5Nx. Microorganisms. 2019;7(6):156. doi: 10.3390/microorganisms7060156. - DOI - PMC - PubMed
    1. Asdaq SMB, Rabbani SI, Alkahtani M, Aldohyan MM, Alabdulsalam AM, Alshammari MS, Alajlan SA, Binrokan A, Mohzari Y, Alrashed A, Alshammari MK, Imran M, Nayeem N. A patent review on the therapeutic application of monoclonal antibodies in COVID-19. Int J Mol Sci. 2021;22(21):11953. doi: 10.3390/ijms222111953. - DOI - PMC - PubMed
    1. Bhat S, Bhatia S, Sood R, Bhatnagar H, Pateriya A, Venkatesh G. Production and characterization of monoclonal antibodies against nucleoprotein of avian influenza virus. Monoclon Antib Immunodiagn Immunother. 2013;32(6):413–418. doi: 10.1089/mab.2013.0053. - DOI - PubMed
    1. Bodewes R, Geelhoed-Mieras MM, Wrammert J, Ahmed R, Wilson PC, Fouchier RA, Osterhaus AD, Rimmelzwaan GF. In vitro assessment of the immunological significance of a human monoclonal antibody directed to the influenza a virus nucleoprotein. Clin Vaccine Immunol. 2013;20(8):1333–1337. doi: 10.1128/CVI.00339-13. - DOI - PMC - PubMed