The Pathogenic Characteristics Of Highly Pathogenic H7N9 And The Immune Effect Of Vaccine On Virus

Part 1:The pathogenic characteristics of highly pathogenic H7N9 and its biological characteristicsBackground:The H7N9 virus mutated in 2017,resulting in new cases of highly pathogenic H7N9 avian influenza virus infection.Persistent viremia was found in the highly pathogenic H7N9 infected patient in Guangdong province.In order to further clarify the pathogenic characteristics and biological characteristics of the highly pathogenic H7N9 virus,we conducted the following studies.Methods:Clinical data was collected.The plasma and sputum of highly pathogenic H7N9 patients were collected for virus isolation.The changes of cytokines in the plasma of the highly pathogenic H7N9 patients and the normal patients were tested by liquid phase chip technology.The highly pathogenic H7N9 virus and ZJU01(the H7N9 isolated in 2013)were used to infect the mice and the pathogenic characteristics of the virus were observed.Mice were sacrificed 2 days,4 days and 6 days after infection,serum and organ tissue were collected,and virus was tested and isolated.The highly pathogenic H7N9 virus and ZJU01 were used to infect the A549 cell model,exosomes were extracted and the influenza virus and its genes in the exosomes were tested.Results:Viremia was observed in the patient infected with highly pathogenic H7N9,and the plasma was isolated with live virus.Cytokine storms were also observed in the highly pathogenic H7N9 patient.The expression of IL-lra,IL-6,IP-10,IL-10,IFN-y,MCP-3,IL-18,HGF,MCP-1 was abnormally increased.In animal experiments,the virulence of highly pathogenic H7N9 was significantly stronger than that of ZJU01 and resulted in the death of mice.Virus test was performed on tissues of infected mice.The virus was detected and isolated from several important organs especially the brain.It was further confirmed that the exosomes secreted by virus-infected A549 cells contained the entire viral genome and infectious virus-like particles.Conclusions:Persistent viremia was observed to induce cytokine storm and to injure main organs in patient with highly pathogenic H7N9.Brain infection may be one of the main cause of the patient's death.Exosome encapsulation may be one of the important ways that the virus enters the blood which leads to the infection of the extrapulmonary tissues.Part 2:Stability and immunogenicity of the avian influenza A H 7N9 split vaccine mixed with the MF59 adjuvantBackground:The avian influenza A H7N9 virus caused significant morbidity and mortality in people in China from 2013 to 2018.The H7N9 vaccine was critical to disease prevention.To verify whether the H7N9 split vaccine mixed with the MF59 adjuvant remains stable after long-term storage,we evaluated the stability and immunogenicity of the vaccine.Methods:The vaccine strain was prepared as a monovalent liquid(antigen syringe 0.25 ml/dose and MF59 0.25 ml/vial).Thermal stability testing of the vaccine was performed at 37±2 ? and the hemagglutinin(HA)content was tested at 3,7,14,and 28 days.Accelerated stability test was performed at 25±2? and the HA content was tested at 1,2,3 months.Long-term stability test was performed at 5±3? and the HA content was tested at 3,6,9,12,18,24,30 months.Mice were immunized with vaccines stored after 6,24,30 months.The immunogenic stability of the vaccine was evaluated.The antibody response index was evaluated in terms of the hemagglutination inhibition(HI)and MN(microneutralization)(MN)titers.At the same time,after secondary immunization with MF59 adjuvant vaccine stored after 30 months,the mice were challenged to observe the immune protection effect of MF59 adjuvant vaccineResults:The H7N9 split vaccine mixed with MF59 was stable at 37±2? for 3 days,at 25±2? for 3 months,and at 5±3? for 30 months.Each of these parameters met the criteria for vaccine evaluation in the pharmacopoeia.There were no significant differences in the HI and MN titers over the period to 30 months.The immune mice can effectively resist the infection of H7N9 virus and its pathogenic effect.Conclusions:Our findings demonstrate the high stability of the H7N9 split vaccine mixed with MF59.The vaccine can be stored for more than 30 months at 5±3 ? and suitable for pandemic vaccine stocks.Part 3:The Protective Effects of the A/ZJU01/PR8/2013 Split H7N9 Avian Influenza Vaccine Against Highly Pathogenic H7N9 in BALB/c MiceBackground:Since the first case of novel H7N9 infection was reported,China has experienced five epidemics of H7N9.During the fifth wave,a highly pathogenic H7N9 strain emerged.In order to evaluate whether the H7N9 vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9)was effective in protecting against highly pathogenic H7N9,we conducted this study.Methods:Groups of mice were immunized twice by intraperitoneal injection with 500 ?l of either split vaccine alone(Two doses:2.5?g HA and 7.5?g HA)or MF59-adjuvanted vaccine.Serum was collected 2 weeks after the second vaccine booster.The hemagglutinin inhibition test was conducted on vaccine seed and highly pathogenic H7N9 to evaluate the neutralization of highly pathogenic H7N9.We also immunized mice and challenged them with highly pathogenic H7N9.Mice were observed for illness,weight loss,and death at 1 week and 2 weeks post-infection.Then,the mice were sacrificed and lungs were dissected.Antibody responses were tested and pathological changes in the lung tissue were evaluated.Results:The immune serum can neutralize the highly pathogenic strain,when compared with the ZJU01 strain,the ability of serum to neutralize highly pathogenic H7N9 was reduced.In mice,highly pathogenic H7N9 was more virulent than A/Zhejiang/DTID-ZJU01/2013(H7N9).After challenge with highly pathogenic H7N9,all mice died while mice challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9)all recovered.The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine was able to protect against infection with highly pathogenic H7N9 in mice,with or without MF59.Moreover,H7N9 vaccine adjuvanted with MF59 produced high antibody levels,which lead to better protection.Conclusions:The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9)is effective in protecting against highly pathogenic H7N9.H7N9 vaccine adjuvanted with MF59 offers better protection against infection with highly pathogenic H7N9.In order to make the H7N9 vaccine applicable to humans,further clinical trials are required to evaluate MF59 adjuvanted vaccine.Meanwhile,the vaccine strain should be updated based on the highly pathogenic H7N9 gene sequence.