Study On The Immunogen Preparation Of H7N9 Subtype Influenza Virus-Like Particles And Refined Immunoglobulin

H7N9 is a new subtype of influenza virus,which was firstly found in Shanghai and Anhui on March 2013.Up to February 22,2017,there were 1230 human cases of infection reported worldwide,including 428 deaths,with a mortality rate of 34.80%.At present,many countries have begun to carry out research and development of products used for diagnosis and prevention of H7N9 influenza virus.Vaccination is an effective means of prevention for high-risk populations,while passive immunization is an effective means of emergency prevention and treatment for susceptible populations and infected patients.Therefore,it is necessary to develop a safe,effective,economical and practical influenza vaccine and therapeutic antibody.The pathogenicity of influenza virus is mainly depended on the molecular characteristics of virus and host species differences.It will help us to prevent and treat the disease by constructing animal infection model of influenza virus and studying pathogenic molecular mechanism.Palladino et al treated immunodeficient mice infected influenza A virus by the neutralizing monoclonal antibody,showing a good therapeutic effect and suggesting that antibody therapy may be one of the effective means to combat H7N9 influenza in the future.Although human antibody is the most ideal therapeutic antibody,it is difficult to obtain a large amount of human serum in the short period.Therefore,heterologous antibody(animal-derived immunoglobulin)becomes one of the most important alternatives for antibody therapy at present.In a variety of animals,the production and application horse anti-serum products have 100 years of history,which has the advantages of large-scaled preparation,good specificity,and a relatively short cycle of production and has played an important role in response to the traditional diseases and new or emerging infectious diseases.The aim of this study was to develop a refined equine immunoglobulin against H7N9 influenza.First of all,a mouse model of influenza H7N9 virus infection was established to provide an evaluation platform for the research of H7N9 influenza therapeutic antibody.Secondly,the virus-like particles(VLPs)containing the HA,NA,and M1 proteins of A/Shanghai/2/2013(H7N9)strain were packed utilizing the insectcell-baculovirus expression system and the in vitro biological activity of VLPs and its immunogenicity in mice were evaluated.Finally,by optimizing the production process,the hyperimmune serum from horses immunized with VLPs as immunogen was further refined to obtain the immunoglobulin F(ab?)2 and the effect of prevention and treatment s of F(ab?)2 was evaluated.1.Construction of infection mouse model and study on the molecular basis of pathogenicity of influenza H7N9 subtype virusThe mouse-adapted strains,P10M1,P10M2,and P10M3 from A/Shanghai/2/2013(H7N9),were obtained by serial passages in BALB/c mouse lungs.The mouse model of H7N9 influenza virus infection was successfully established by evaluating indicators of half lethal dose,mortality rate,proliferation efficiency,and tissue tropism,which provides a platform for evaluating the efficacy of drugs and vaccines against influenza H7N9 virus.Genomic sequence analysis revealed that there were several variant sites on PA,HA,NA,M,and NP genes,which may be molecular markers to be pathogenic to mice.It is noteworthy that PA-T97 I might be one of the molecular bases for the enhanced pathogenicity of influenza H7N9 virus in mammals.2.Preparation of H7N9 influenza VLPsThe VLPs containing the HA,NA,and M1 proteins of A/shanghai/2/2013(H7N9)was constructed using the insect cell-baculovirus expression system.The spherical shape influenza VLPs can be observed by the transmission electron microscope,with typical spikes on their surfaces,and a diameter of approximately100 nm.The results of PCR,indirect immunofluorescence and western blot showed that the VLPs were correctly constructed.The hemagglutination titer of H7N9 influenza VLPs was 27~28.The BALB/c mice were immunized with 10 ?g VLPs by intramuscular injection or intranasal administration,and boosted after two weeks later.Two weeks after the booster immunization,all the vaccinated mice survived from the challenge of 10 MLD50 mouse-adapted influenza H7N9 virus strain.As a result,the H7N9 VLPs have excellent immunogenicity,which lays a foundation for the preparation of influenza H7N9 virus immunoglobulin.3.Study on the refined immunoglobulin from influenza H7N9 virusHyperimmune serum of HI antibody titer of 1:10240 was obtained after horses were immunized by VLPs as immunogen for five times.Each group of mouse model was intraperitoneally injected with 0.2 ml of hyperimmune serum 36 h before or 24 hafter challenge with 5 MLD50 by intranasal administration.The survival rates for groups of mice were 100%,but all those of the control group died.The results showed that the hyperimmune serum prepared from horse against influenza H7N9 virus has good effects of emergency prevention and treatment.Based on the traditional purification process of horse serum,improvement and optimization of preparation were applied mainly by the enzyme digestion purification process to remove the Fc fragment of antibody and then by the affinity chromatography to purify immunoglobulin F(ab?)2.The results of SDS-PAGE and TLC scanning showed that the purity of F(ab?)2 was above 93%.The neutralizing antibody titer was 1:5120 based on the fixed virus-diluted immunoglobulin method.The mouse models were injected 0.2 mg of F(ab?)2 before 4h or after 8h challenge with 5 MLD50 by intranasal administration.The survival rates of mice were up to100%.And there were 75% survival rates in those mice given a dose of 0.4 mg 24 h after infection.The results showed that the refined immunoglobulin F(ab?)2 has a good therapeutic effect on influenza H7N9 virus infection.In summary,H7N9 subtype influenza VLPs has a good immunogenic and protective effect,demonstrated by the established mouse model of H7N9 subtype influenza virus infection.The refined immunoglobulin F(ab?)2 obtained from horse hyperimmune serum against influenza H7N9 VLPs has potential application value in the prevention and treatment of H7N9 influenza.It provides scientific and technological supports for the development of drugs and vaccines against H7N9 subtype influenza virus.