Generation Of Novel H7N9 VLP Vaccines Based On The Baculovirus-Insect Cells Expression System

In 2013,human infection with H7N9 subtype avian influenza virus(AIV)first emerged in the Yangtze Delta region in China.H7N9 AIV has already caused five epidemic waves in China so far.As of March 1 2020,a total of 1568 laboratory-confirmed human cases of H7N9 virus infection were reported,including 616 deaths,with the mortality of about 40%.In addition,as of March 2018,11 outbreaks of highly pathogenic H7N9 AIV have been reported in poultry in China,which resulted in the culling of nearly 1 million birds,according to the World Orgnization For Animal Health(OIE).Therefore,H7N9 AIV is a major threat to public health and poultry industry.Vaccination is the most effective means to prevent influenza virus infection.Currently,production of the traditional inactivated vaccine relies on the supply of eggs,with some shortcomings,such as the shortage of eggs during the outbreak of avian influenza,the environmental burden caused by large amount of biohazard waste,and the endogenous virus contamination.In addition,inactivated vaccines can only induce antibody immunity,rather than cellular immunity.Therefore,a safe and efficacious H7N9 avian influenza vaccine needs to be generated using new technologies to meet the needs of disease prevention and control in poultry industry.Virus-like particle(VLP)is a self-assembled noninfectious particle composed of virual structural proteins and antigens,which can simulate the natural structure of virus particles.VLP vaccines can elicite both cellular immune response and humoral immune response,providing a comprehensive immune protection.The Baculovirus Expression System(BES)is widely used to produce influenza VLP vaccines in insect suspension cells in large scale.The BES allows egg-free production of the vaccines,which can lower the cost of good and environmental burden.Based on the significant advantages of the VLP vaccine platform,two H7N9 VLP vaccine candidates based on the HA,neuraminidase(NA)and matrix(M1)protein was generated in the BES.The H7N9 VLP vaccine candidates produced by these two strategies have high antigen yield,good immunogenicity and efficacy,which is as good as the commercial inactivated H7N9 vaccine,and it provides an alternative for the prevention of H7N9 avian influenza in poultry.1.Generation of H7N9 VLP vaccine by co-infection of the recombinant baculoviruses expressing individual HA?NA?M1 geneIn this section,a highly pathogenic avian influenza virus of the H7N9 subtype A/Chicken/Guangdong/GD 15/2016(GD15)was selected as the donor of the target genes.Three recombinant baculoviruses(rBac-HA-GD 15,rBac-NA-GD15,rBac-Ml-GD15)expressing the HA,NA and M1 genes of GD15 were generated by co-transfection of the corresponding transfer plasmids with the linearized Autographa californica multiple Nucleopolyhedrovirus(AcMNPV)genomic DNA into Sf9 cells.The expression of the foreign proteins was detected by IFA.Sf9 cells were co-infected with three recombinant baculoviruses at a MOI ratio of 1:1:1.Transmission electron microscopy revealed the formation of the influenza virus-like particles with a diameter of 80-120 nm without genetic materials inside,indicating that H7N9 VLP(designated as VLP-1)was assembled through co-infection of the recombinant baculoviruses expressing individual HA,NA and M1 genes.The VLP was concentrated by ultrafiltration and subsequently purified by 30-60%sucrose density gradient ultracentrifugation.The highest concentration of the VLP was detected in 40-50%sucrose layer and the HA titer of the VLP sample reached 11 log2.Concentrated and purified VLP samples were harvested to prepare the vaccines.Meanwhile,inactivated vaccine based on the recombinant baculovirus rBac-HA-GD15 expressing the HA protein alone was used as the control.4-week-old SPF chickens were immunized through intramuscular injection and at week 3 post immunization(pi),each vaccine could induce antibody response,and the mean titer of hemagglutination inhibition(HI)antibody was around 6 log2.Compared with the control group,neutralizing antibodies were detected in the serum of all vaccine-immunized chickens,and the average titer of the purified VLP-1 group was 260.The titer of IgY antibody in each vaccine group was above 2560 determined by ELISA.Additionally,HI antibody levels induced by the VLP vaccine at 15 ?g were higher than that induced by the vaccine at 7.5 ?g.Three weeks pi,all the chickens were challenged with H7N9 GD15 strain at a dose of 106.0 EID50 through the intraocular and intranasal routes.The non-immunized birds developed severe clinical symptoms post challenge(pc),and all died within 5 days.All vaccine-immunized chickens showed no symptoms and no death was seen within 14 days pc,indicating that the H7N9 VLP-1 vaccine provided 100%clinical protection against H7N9 virus challenge.At day 3,5,7 and 9 pc,laryngotracheal and cloacal swabs were collected for monitoring virus shedding.The results showed that the highest rate of virus shedding from the chickens pc was 30%and the amount of virus shedding was low.Virus shedding was only detected at day 3 pc for the purified VLP-1 group.Histopathological analysis showed that the VLP-1 vaccine administered at 15 ?g dramatically suppressed the pulmonary lesions caused by H7N9 virus infection.These results indicate that the H7N9 VLP can be successfully assembled through co-infection of the recombinant baculoviruses expressing individual HA,NA and M1 genes,and the VLP-1 vaccine is highly immunohenic and efficacious in chickens against H7N9 AIV.2.Generation of H7N9 VLP vaccine by the recombinant baculovirus expressing tandem HA?NA?M1 genesIn this section,the HA,NA and M1 genes of a highly pathogenic H7N9 avian influenza virus A/Chicken/Guangdong/GD15/2016(GD15)were cloned as a tandem into the shuttle vector(pVL1393-NA-M1-HA).The recombinant shuttle plasmid was co-transfected with the linearized AcMNPV genomic DNA into Sf9 cells for VLP assembly.The expression of the HA,NA and M1 proteins in the recombinant baculovirus was detected by IFA.Influenza virus-like particles with a diameter of about 100 nm were observed by transmission electron microscopy,indicating that H7N9 VLP(designated as VLP-2)was successfully generated.Sf9 cells were inoculated with the recombinant baculovirus to prepare VLP,which was concentrated by ultrafiltration and purified by ultra centrifugation.The HA titer of the purified VLP sample was significantly increased(6 log2 to 11 log2).The vaccines based on VLP-1 and VLP-2 as well as the commercial inactivated H7N9 vaccine were used for animal immunization and challenge experiments.At week 3 pi,all the vaccines induced high HI antibody response with the mean HI titer of 6 log2,and HI titer induced by the commercial vaccine and VLP-1 vaccine reached as high as?10 log2.Chicken antisera had good cross-reactivity with H7N9 field strains isolated from 2013 to 2018.Virus neutralizing antibodies were detected in immunized chickens and neutralizing antibody titers induced by the VLP vaccines were slightly lower than that induced by the inactivated H7N9 vaccine.All the tested vaccines elicited high levels of IgY antibody(titer 2560-5120)as measured by ELISA and there was no significant difference between the VLP vaccines and inactivated vaccine.When the VLP-1 and VLP-2 vaccine were immunized at the same dose of 15 ?g,the VLP-1 vaccine induced higher antibody levels.At week 3 pi,chickens were challenged with 106.0 EID50 of highly pathogenic H7N9 avian influenza virus GD15 strain.Within 14 days pc,no obvious clinical symptoms and death were observed for each vaccine-immunized group,indicating that both the VLP vaccines and commercial vaccine provided complete protection against H7N9 virus challenge.Virus shedding was detected in 50%of the chickens immunized with the commercial vaccine,and the corresponding rates for two VLP vaccines were 30%and 40%,respectively,and there were no significant differences among these groups.No virus was detected in the organs of chickens immunized with the VLP vaccines and commercial vaccine,indicating that all the vaccines effectively inhibited replication of the challenge virus.These results indicate that the H7N9 VLP-2 vaccine generated based on the tandem expression of the HA?NA?Ml genes can induce a strong antibody immune response in SPF chickens,provide complete clinical protection against H7N9 virus challenge and significantly inhibit virus shedding and replication.The immunogenicity and efficacy of the VLP-2 vaccine are comparable to that of the VLP-1 vaccine and commercial inactivated vaccine.