title: H5N1 influenza A virus with K193E and G225E double mutations in haemagglutinin is attenuated and immunogenic in mice.
abstract: Live-attenuated influenza vaccines (LAIVs) are now available for the prevention of influenza, with LAIV strains generally derived from serial passage in cultures or by reverse genetics (RG). The receptor-binding domain (RBD) in haemagglutinin (HA) of influenza virus is responsible for viral binding to the avian-type 2,3-α-linked or human-type 2,6-α-linked sialic acid receptor; however, the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passage of H5N1 virus A/Vietnam/1194/2004 in Madin-Darby canine kidney cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. RG was used to generate H5N1 viruses containing either single or double substitutions in HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large-plaque morphology, enhanced replication and genetic stability after serial passage, without changing the receptor-binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and haemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against WT H5N1 virus in mice. Taken together, our experiments demonstrated that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor-binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as a novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Peng-Fei
authors.initials: PF
authors.lastname: Han
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Sen
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Yin-Hui
authors.initials: YH
authors.lastname: Yang
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Yu-Chang
authors.initials: YC
authors.lastname: Li
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Kang
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Wu
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Shun-Ya
authors.initials: SY
authors.lastname: Zhu
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Qing-Yu
authors.initials: QY
authors.lastname: Zhu
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Cheng-Feng
authors.initials: CF
authors.lastname: Qin
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D004285:Dogs; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007114:Immunization; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007251:Influenza, Human / Q000276:immunology / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines
chemical_list: D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D014613:Vaccines, Attenuated; D014765:Viral Vaccines