Preparation And Immunogenicity Of Clade 2.3.4.4 H5 Subtype Avian Influenza Virus-like Particles

H5 subtype highly pathogenic avian influenza?HPAI?poses serious threat to the poultry industry and public health in China and other countries around the world.Currently,clade 2.3.4.4 H5 subtype HPAI viruses are dominantly prevalent in China.The prevention of HPAI mainly relies on vaccination and biosafety management.At present,the widely used vaccines against H5 subtype HPAI are inactivated vaccines that were developed using reverse genetics and produced by means of SPF chicken embryos.This approach of vaccine production has a number of weaknesses,such as strict requirement for operating environment,limited source of chicken embryos,relatively poor biological safety,and so on.In this study,we sought to generate clade 2.3.4.4 H5subtype HPAI VLP using baculovirus-insect cell expression system and to evaluate the immunogenicity of the VLP in order to provide a theoretical basis for the research and development of virus-like particles vaccine.1.Construct of THX4#-HA,THX4#-NA,THX4#-M1 recombinant baculovirus DNAs.Three gene fragments bearing HA,NA and M1 of H5 HPAI virus were amplified by RT-PCR employing specific primers designed,and subsequently,the three fragments were cloned into the vector pFastBac1,respectively;PFastBac1-THX4#-HA,pFastBac1-THX4#-NA,and pFastBac1-THX4#-M1 was successfully obtained by double enzyme digestion and sequencing,then,we obteined recombinant baculovirus vector plasmid by transformed them into DH10Bac cells,respectly.2.Identification of the VLPs generated by packaging of THX4#-HA,THX4#-NA,THX4#-M1 proteins.The positive recombinant baculovirus DNAs were co-transfected into sf9 insect cells in an appropriate proportion.The transfectant supernatant was harvested at 5 days post-transfection,centrifuged at low-speed centrifugation,and then purified by sucrose density gradient centrifugation.The expression of the HA,NA and M1 protein was detected by hemagglutination?HA?assay,transmission electron microscopy?TEM?,SDS-PAGE and Western blot.Our results indicated that the three target genes were successfully cloned into the pFastBac1 vector.On day 5,cytopathic effect?CPE?was observed in the transfected cells,including wrinkled,deformed,fused and vesicular changes in morphology.The HA titer reached 6Log2 in the 3rd passage.SDS-PAGE and Western blot verified successful expression of the three proteins.The typical morphology of avian influenza VLPs with a diameter of 80¹20 nm were observed under electron microscope.3.Immunogenicity of VLP.The 6-week-old SPF chickens were primarily immunized with VLPs generated via route of subcutaneous injection,followed by intramuscular injection for boosting vaccination 15 days later.Serum was collected while conducting boosting immunization and 15 days after boosting immunization.The titer of antibody in the serum was detected by hemagglutination inhibition?HI?test and the presence of IgG1,IL-2,IL-4,IL-10,IFN-?were dectected by ELISA.The level of antibody induced by VLPs was detected by neutralization test.Our results showed that the HI titer in the serum collected at 15 days after boosting immunization reached10Log2.The neutralizing antibody titer of VLP immunized serum to the same branch strain was 2^-7.15,and the neutralizing antibody titer of 2^-6.85.VLP vaccine immunized group was significantly higher than that of negative control group?P<0.05?.The content of IgG1,IL-2 were higher than negative control group.In conclusion,VLP assembled by 2.3.4.4 branch H5 subtypes HPAIV HA,NA and M1 protein was successfully constructed and had good immunogenicity.This study will lay a solid foundation for the research and development of a new candidate vaccine for2.3.4.4 branch H5 avian influenza and the effective prevention and control of the disease,and provide conditions for ensuring the safe production of poultry.