Construction And Evaluation Of Multi-epitope Influenza Nano-vaccine Based On Ferritin

Influenza is an acute respiratory disease caused by influenza virus(?)[1].Influenza outbreaks are mainly caused by subtypes of influenza A virus,and some subtypes have been spread among people.Influenza pandemic is caused by recombinant human avian influenza virus,among which influenza A H1N1,H3N2 and B viruses infect more people,showing seasonal epidemic every year,often accompanied by cough,high fever,runny nose,loss of appetite and muscle soreness.At present,avian influenza viruses H5N1 and H7N9 have broken through the interspecific barrier and have the characteristics of zoonosis,which may cause a pandemic[2].Influenza virus is prone to antigenic drift or antigenic transformation,which leads to the coexistence of multiple subtypes of influenza virus,and the cross protection between different subtypes is not good.The current influenza vaccine can not provide effective protection against the original variant influenza virus strains,so it is urgent to develop a vaccine that can prevent multiple subtypes of influenza virus at the same time.In this study,the conserved antigens HA2 and M2e of influenza virus were inserted into the N-terminal of Ferritin Nanoparticles by using the self-assembly and modifiable properties of Ferritin Nanoparticles.The multi-epitope influenza nanoparticles based on Ferritin were expressed by prokaryotic expression system and insect baculovirus expression system.Adjuvants(Alum,MF59,CpG,Poly:IC,MF59+CpG,MF59+Poly:IC).They were mixed with multi-epitope influenza nanoparticles to prepare vaccine and immunize mice.Through the immune effect,the adjuvant suitable for multi epitope influenza nano-vaccine was selected.The protective effect of each group of vaccines was evaluated by challenge experiment,which laid a foundation for the successful development of universal influenza nano vaccine.1.Prokaryotic expression and identification of multi-epitope influenza Nanovaccine based on FerritinHA2-M2e-Ferritin gene(HM2e-Ferritin for short)was formed by concatenating HA2 and M2e conservative antigen genes of H1/H3/B subtype influenza virus.The amino acid sequence of HM2e-Ferritin was optimized and synthesized according to the common codons of Escherichia coli.Meanwhile,Ferritin gene was amplified and cloned into prokaryotic expression vector pET-3 2a.The recombinant plasmids were identified by restriction enzyme digestion and sequencing.The positive plasmids were transferred into competent cells BL21(DE3)and induced by IPTG.By optimizing the induction conditions,induction temperature and inducer concentration,the best protein expression conditions were obtained.The recombinant protein was purified by nickel column and identified by SDS-PAGE.The results showed that HM2e-Ferritin protein and Ferritin protein were soluble in the protein supernatant.WB analysis showed that HM2e-Ferritin and Ferritin protein had immunoreactivity,and HM2e-Ferritin protein had HM2e and Ferritin activity,respectively.Electron transmission microscopy and nanoparticle size analyzer were used to analyze the recombinant protein.The results showed that the two proteins could form nanoparticle structure.The particle size of ferritin protein was 12?20nm,and that of HM2e-Ferritin protein was 20?70nm,which showed narrow size distribution of nano cage structure.The results showed that HM2e-Ferritin protein with nanoparticle structure was obtained by E.coli expression system.The protein has biological activity,which provides a theoretical basis for the development of new nano-vaccines and disease diagnosis and treatment.2.Baculovirus expression and identification of the multi-epitope influenza Nano-vaccine based on FerritinAccording to the whole genome sequence of a/Jilin/jyt-01/2018(H1N1)strain,HA2 gene and HM2e Ferritin gene were synthesized according to codon preference.The recombinant shuttle plasmid PFBD-HA2-HM2e-Ferritin was constructed by inserting HA2 gene and HM2e-Ferritin gene into PFastBacDual vector respectively.The recombinant baculovirus was successfully rescued through the transformation of recombinant shuttle plasmid,selection of baculovirus from blue and white spots,and transfection of recombinant baculovirus into Sf9 cells.The recombinant baculovirus named rbv-HA2-HM2e-Ferritin was successfully rescued by enzyme digestion,PCR,WB and IFA.Sf9 cells were infected with recombinant baculovirus,and the cell suspension was collected.The recombinant protein was obtained by ultracentrifugation and sucrose density gradient centrifugation.The recombinant protein was analyzed by electron microscope,and the virus like particles of about 20 nm could be seen under TEM.The results showed that the two-component multi epitope influenza nanoparticles were obtained by baculovirus system,which provided a new idea for the development of broad-spectrum influenza virus vaccine.3.Selection and evaluation of adjuvants for multi-epitope influenza NanovaccineThe purified HM2e-Ferritin protein expressed in prokaryotic cells was used as antigen.MF59,CpG,Poly:IC,Alum,MF59+CpG and MF59+Poly:IC.The adjuvants were mixed in a certain proportion to make the vaccine.Five week old female C57BL/6 mice were immunized with the prepared vaccine at a dose of 50 ?g each time,once every three weeks for three times.The positive and negative control group,simple antigen control group(hm2e ferritin protein)and adjuvant control group were set up.For humoral immune response,the blood of mice in each group was collected from orbit every week,and the serum was separated for IgG antibody typing and specific IgG level detection.The results showed that all the vaccine groups(including HM2e-Ferritin group)could induce strong serum antibody IgG,IgGland IgG2a responses,with significant difference compared with the control group(P<0.001).The results of antibody titer showed that Alum and MF59+CpG vaccine groups could significantly improve the production of specific antibody,and the highest antibody titer was more than 1:8×105,which was significantly different from the control group(P<0.01).For cellular immune response,the mice immunized for one week were randomly killed,and the lymphocytes were isolated from the spleen for flow cytometry analysis and lymphocyte proliferation test.The results showed that HM2e-Ferritin could induce T lymphocyte immune response.The results of cytokine assay showed that the IL-4 and IFNy responses induced by alum,CpG and MF59+CpG vaccine groups were significantly different from those induced by PBS group(P<0.01).The lung pathological changes,body weight changes and survival rate of mice after challenge showed that MF59+CpG and Poly:IC vaccine group The lungs of mice had no significant difference;the weight of all groups decreased,and the weight of PBS group decreased the fastest and recovered slowly.On the 7 th to 9 th day,the weight of mice in the vaccine group gradually recovered,and the survival rate of mice in MF59 and MF59+CpG vaccine groups reached 100%.In conclusion,the multi epitope influenza nano vaccine can induce strong cellular and humoral immunity in mice,which can effectively resist the infection of H1N1 influenza virus.