tetano
Editor, Senior Moderator
Int J Biol Macromol
. 2026 Jul 11:153483.
doi: 10.1016/j.ijbiomac.2026.153483. Online ahead of print.
A nanobody against haemagglutinin of H9N2 avian influenza virus shows efficacy in vitro and in vivo
Zhengyang Geng[SUP] 1 [/SUP], Qin Fang[SUP] 1 [/SUP], Mengran Zhang[SUP] 1 [/SUP], Zhiyu Fan[SUP] 2 [/SUP], Jingfeng Zhang[SUP] 2 [/SUP], Jinping Fu[SUP] 2 [/SUP], Huawei Sun[SUP] 2 [/SUP], Xiaoxi Zhang[SUP] 2 [/SUP], Sha Zhao[SUP] 2 [/SUP], Yuyang Cao[SUP] 2 [/SUP], Weidong Sun[SUP] 3 [/SUP], Linzhu Ren[SUP] 4 [/SUP], Jing Qian[SUP] 5 [/SUP]
Affiliations
H9N2 subtype avian influenza virus (AIV) facilitates viral adaptative evolution and cross-species transmission, result in inflicting severe economic losses on the poultry industry and posing significant public health threats. Currently, conventional vaccine-based prophylactic strategies exhibit certain limitations, highlighting an urgent need for the development of novel prevention and control measures. In this study, using phage display technology, we screened 37 potential binding sequences from the PBMCs of an alpaca immunized with the H9N2 virus and its HA protein. AlphaFold3 further predicted seven nanobody candidates (Nb1, Nb2, Nb7, Nb13, Nb24, Nb26 and Nb34) with potential neutralizing activity. Subsequently, we successfully expressed these recombinant nanobodies in the Pichia pastoris system and tested the HI activity of the six candidates (excluding Nb24). While all nanobodies exhibited binding activity in ELISA and IFA, several also showed viral neutralizing activity in cells and embryonated chicken eggs. Notably, Nb13 exhibited broad inhibitory activity against the early h9.4.2.1 (JS2002) strain and two temporally distinct h9.4.2.5 strains (DC17 and NJ150), with IC[SUB]50[/SUB] values of 14.38 ± 1.01, 1.20 ± 0.20, and 1.14 ± 0.14 μg/mL, respectively. In chick challenge models, intratracheal administration of Nb13 provided robust protection. Both prophylactic and therapeutic regimens dose-dependently reduced the titer and duration of oropharyngeal viral shedding, and effectively alleviated tracheal pathological lesions. Mechanistically, Nb13 neutralizes the virus by blocking viral entry into host cells, which structural predictions suggest is mediated by binding to the RBS. Overall, this study provides a highly promising novel candidate for the clinical prevention of H9N2 infections.
Keywords: Avian influenza viruses; H9N2; HA protein; Nanobody; Neutralization activity.
. 2026 Jul 11:153483.
doi: 10.1016/j.ijbiomac.2026.153483. Online ahead of print.
A nanobody against haemagglutinin of H9N2 avian influenza virus shows efficacy in vitro and in vivo
Zhengyang Geng[SUP] 1 [/SUP], Qin Fang[SUP] 1 [/SUP], Mengran Zhang[SUP] 1 [/SUP], Zhiyu Fan[SUP] 2 [/SUP], Jingfeng Zhang[SUP] 2 [/SUP], Jinping Fu[SUP] 2 [/SUP], Huawei Sun[SUP] 2 [/SUP], Xiaoxi Zhang[SUP] 2 [/SUP], Sha Zhao[SUP] 2 [/SUP], Yuyang Cao[SUP] 2 [/SUP], Weidong Sun[SUP] 3 [/SUP], Linzhu Ren[SUP] 4 [/SUP], Jing Qian[SUP] 5 [/SUP]
Affiliations
- PMID: 42435874
- DOI: 10.1016/j.ijbiomac.2026.153483
H9N2 subtype avian influenza virus (AIV) facilitates viral adaptative evolution and cross-species transmission, result in inflicting severe economic losses on the poultry industry and posing significant public health threats. Currently, conventional vaccine-based prophylactic strategies exhibit certain limitations, highlighting an urgent need for the development of novel prevention and control measures. In this study, using phage display technology, we screened 37 potential binding sequences from the PBMCs of an alpaca immunized with the H9N2 virus and its HA protein. AlphaFold3 further predicted seven nanobody candidates (Nb1, Nb2, Nb7, Nb13, Nb24, Nb26 and Nb34) with potential neutralizing activity. Subsequently, we successfully expressed these recombinant nanobodies in the Pichia pastoris system and tested the HI activity of the six candidates (excluding Nb24). While all nanobodies exhibited binding activity in ELISA and IFA, several also showed viral neutralizing activity in cells and embryonated chicken eggs. Notably, Nb13 exhibited broad inhibitory activity against the early h9.4.2.1 (JS2002) strain and two temporally distinct h9.4.2.5 strains (DC17 and NJ150), with IC[SUB]50[/SUB] values of 14.38 ± 1.01, 1.20 ± 0.20, and 1.14 ± 0.14 μg/mL, respectively. In chick challenge models, intratracheal administration of Nb13 provided robust protection. Both prophylactic and therapeutic regimens dose-dependently reduced the titer and duration of oropharyngeal viral shedding, and effectively alleviated tracheal pathological lesions. Mechanistically, Nb13 neutralizes the virus by blocking viral entry into host cells, which structural predictions suggest is mediated by binding to the RBS. Overall, this study provides a highly promising novel candidate for the clinical prevention of H9N2 infections.
Keywords: Avian influenza viruses; H9N2; HA protein; Nanobody; Neutralization activity.