| The highly pathogenic avian influenza virus(HPAIV)is a primary pathogen that endangers human health,animal husbandry,and public health.HPAIV is classified as a Class B infectious disease by Law of the People’s Republic of China on the prevention and treatment of infectious diseases,although it is managed as a Class A infectious disease.According to the World Health Organization(WHO),from 2003 to March 1,2022,more than 864 cases of avian H5N1 subtype influenza virus infections and 456deaths worldwide had been reported,with a mortality rate of nearly 53%;also,there have been 66 cases of human H5N6 infections,with 29 deaths and a mortality rate of over 43%.The human H5 subtype HPAI is a global problem and an issue of great concern.Fewer studies have investigated human licensed vaccines against H5 avian influenza virus(AIV).Cold-adapted live attenuated influenza vaccines(CAIVs)can be easily administered as a nasal spray to induce mucosal,cellular,and humoral immunity.However,as live viruses,they may reassort and recombine with natural epidemic strains,and thus,their medical application raises safety concerns.Therefore,developing novel attenuated vaccines against the H5 subtype AIV can greatly benefit human health.We constructed novel attenuated influenza vaccine candidates based on a cold-adapted strain of influenza B virus,expressing the surface HA proteins of influenza A viruses H5N1 and H5N6.Also we evaluate the safety,immunogenicity,and protective efficacy in mice and ferrets and provide a new strategy for H5 licensed vaccines.1.The construction and identification of a reverse genetic system of a cold-adapted strain of the influenza B virusReverse genetics is a method for the rapid generation of influenza viruses.In this experiment,an eight-plasmid-based reverse genetic system of the influenza B virus cold-adapted strain B/Vienna/1/1999(abbreviated BV99)was constructed to produce the CAIVs against H5 subtype AIV.Recombinant plasmids for each gene were co-transfected with 293T cells and MDCK cells for viral rescue,and the basic characteristics were performed.The results of PCR amplification and sequencing showed that the eight gene fragments of the BV99 strain were successfully amplified and correctly sequenced.The hemagglutinin titer was 2~6,and influenza virus-like particles were observed under an electron microscope.The rescued virus strain replicated well in the MDCK cells.The results of the virus titers at different temperatures(27°C,33°C,and 39°C)revealed the cold-adapted and temperature-sensitive characteristics of the rescued virus strain.The results of the mouse infection experiments suggested a low virus loads in the lungs.Thus,we successfully constructed a reverse genetic system for the cold-adapted strain of BV99,which was identified through molecular biology techniques and morphology.The rescue virus strain had cold-adapted,temperature-sensitive,and attenuated characteristics,and thus,it was used to produce CAIVs against H5 subtypes.2.The construction and identification of the CAIV candidates for Clade 2.3.2.1H5N1 and Clade 2.3.4.4 H5N6Clade 2.3.2.1 and Clade 2.3.4.4 of H5-subtype AIVs are the primary epidemic Clades in China.One vaccine candidate strain of the AIV Clade 2.3.2.1 H5N1 and four strains of the AIV Clade 2.3.4.4 H5N6 were constructed by inserting the extracellular region of the HA protein of modified H5N1 and H5N6 AIVs into the HA gene of the influenza B virus based on the reverse genetic system of BV99.The basic characteristics were conducted for the rescued vaccine candidate strains.The results of PCR amplification and sequencing showed that eight gene fragments were successfully amplified and correctly sequenced.The hemagglutinin titer was 2~6–2~7,and influenza virus-like particles were observed under an electron microscope.CAIV candidate strains against H5 influenza grew well in the MDCK cells.The results of virus titers at different temperatures(27°C,33°C,and 39°C)showed that the H5 vaccine candidate strains had cold-adapted and temperature-sensitive characteristics.Thus,we successfully constructed CAIV candidates for Clade 2.3.2.1 H5N1 and Clade 2.3.4.4 H5N6 based on the cold-adapted strain of the influenza B virus,which showed cold-adapted,temperature-sensitive,and attenuated characteristics.These CAIV candidates could be used to evaluate animal immunity.3.Immunization studies of CAIV candidates against Clade 2.3.2.1 H5N1Evaluating the immunogenicity of vaccines and their ability to protect animals is necessary for developing vaccines.Thus,the immunogenicity against toxic attacks after immunization of Clade 2.3.2.1 H5N1 CAIV candidate strains were evaluated in mice.The systemic immunity of the immunized mice in response to vaccine candidate strains were comprehensively evaluated through one-dose and two-dose immunizations.The protection efficacy was evaluated after the immunized mice were challenged with Clade2.3.2.1 H5N1 wild-type virus.The results demonstrated that the mice infected with the H5N1 CAIV candidate vaccine showed low viral load in the lungs.After immunizations,HI antibodies,neutralizing antibodies,and serum Ig G antibodies were detected in the serum of mice,and the mice with two-dose immunization showed higher levels of antibodies than those with one-dose immunization;mucosal Ig A antibodies were also detected in the lung lavage fluid of immunized mice.Besides,immunized mice showed higher production of intracellular cytokines such as IFN-γsecreted by CD4~+T cells and CD8~+T cells in splenocytes.Thus,the immunization induced specific cellular immune responses.Furthermore,the survival rate of the immunized mice was 100%after being challenged with the homologous Clade 2.3.2.1 H5N1 wild-type virus.The above results suggested that the CAIV candidates were safe and increased humoral,mucosal,and cellular immune responses in mice and protected against the homologous Clade 2.3.2.1H5N1 wild-type virus.4.The immunization studies of CAIV candidates against Clade 2.3.4.4 H5N6The ferrets an ideal experimental animal recommended by the WHO for evaluating influenza vaccines.Thus,we evaluated immunogenicity efficacy after immunization of four CAIV candidate strains of Clade 2.3.4.4 H5N6 in mice and ferret.The systemic immunity of the immunized mice in response to the vaccine candidate strains were comprehensively evaluated through one-dose and two-dose immunizations.The cross-protection was evaluated after immunization.Representative Clade 2.3.4.4b and Clade2.3.4.4h H5N6 CAIV candidates were selected for immunization studies in ferret and cross-protection after immunization.The results indicated that the four candidate strains of Clade 2.3.4.4 H5N6 CAIV were safe for administration and increased the levels of HI antibodies,neutralizing antibodies,and Ig G antibodies in the serum of immunized mice,with higher antibody levels in mice with two-dose immunization than that in mice with one-dose immunization.Specific mucosal Ig A antibodies were detected in the lung lavage fluid of immunized mice,along with higher production of intracellular cytokines IFN-γsecreted by CD4~+T cells and CD8~+T cells in splenocytes;thus,the virus candidates could induce specific cellular immune responses in mice.The results of the cross-protection studies showed that immunized mice received cross-protection against four strains of Clade 2.3.4.4 H5N6 wild-type influenza virus,where two H5N6 vaccine candidates(Clade 2.3.4.4b and Clade 2.3.4.4h)were safe and showed low pathogenicity in ferret.After immunization,HI antibodies,neutralizing antibodies,and Ig G antibodies were detected in the serum of ferret,accompanied by an increase in the secretion of IFN-γcytokine in splenocytes,and cross-protection after being challenged with the wild-type virus strain.The results of the immunized ferret were consistent with those of the immunized mice,except that only two strains of 2.3.4.4b and 2.3.4.4h H5N6 vaccines were safe for the ferret.The attenuated vaccine strains of H5N1 and H5N6 avian influenza viruses for human use based on the cold adapted backbone strain of influenza B virus constructed in this study have the advantage of not reassorting with H5 circulating strains in nature and being able to induce a variety of immune responses,which provides a foundation for the development of novel human vaccines for highly pathogenic H5 subtype avian influenza virus. |
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