Construction And Immunoprotective Evaluation Of Recombinant Adenovirus Vaccine Expressing AIV Conserved Antigens NP And M2e

Avian influenza virus（AIV）is a disease that significantly impacts the world’s poultry production and is a severe threat to human health,so it is critical to develop an effective vaccine to prevent avian influenza.Nucleoprotein（NP）and matrix protein（M2）of influenza virus,conservatism between these antigens from different subtypes of influenza virus are generally high compared to viral external glycoproteins,and adenovirus vector vaccination expressing influenza virus NP induces NP-specific T cell responses and better protection is observed after a heterologous virus attack.Adenoviral vectors offer many advantages over other viral vectors:First,the genome is structured and easily manipulated.Therefore,it is relatively easy to insert foreign genes.This method can quickly achieve the desired gene transfer or antigen expression.Secondly,it can widely infect dividing and non-dividing mammalian cells and induce high levels of exogenous protein gene expression;When recombinant adenovirus vaccines pass through the mucosal route,it is beneficial to stimulate the mucosal response.Therefore,the adenovirus vector vaccine is vital for future vaccine research.The human adenovirus type 5 vector has been widely used in vaccine development and design and is a safe and effective live virus vector.Because of its genome deletion and few E1regions,recombinant adenovirus cannot replicate autonomously,has high safety,high transduction efficiency,relatively large transgenic capacity,and high titer generation,etc.,it is widely used as a gene delivery vehicle in basic research of gene therapy and gene function analysis.Therefore,in this experiment,a multi-copy influenza virus conserved antigen NP,and M2e recombinant adenovirus shuttle plasmid were successfully constructed,and two strains of recombinant adenovirus were successfully packaged.In addition,mice were immunized by nasal immunization to evaluate the immune and protective effects of multi-copy influenza virus conserved antigens NP and M2e recombinant adenovirus on mice.The research contents of this thesis are as follows:（1）Preparation of recombinant adenovirus expressing AIV conservative antigenGenBank’s gene sequence encoding AIV NP and M2e protein was queried and cloned to the shuttle vector pDC316-mCMV.The recombinant shuttle plasmid was transferred to the DH-5αcompetent state.After double digestion verification and plasmid sequencing,it was confirmed that the recombinant shuttle plasmid was successfully constructed,PCR amplified GFP.The GFP gene was further cloned in the recombinant shuttle plasmid,and the recombinant shuttle plasmid was constructed by double digestion and plasmid sequencing.The successfully constructed recombinant shuttle plasmid and skeleton plasmid were co-transfected into HEK-293A cells with Lipofectamine 3000.After the two recombinant adenoviruses were successfully packaged,recombinant adenoviruses expressing multiple copies of influenza virus conservative antigens NP and M2e were named rAd-NP-M2e-GFP.The packaged empty vector adenovirus was named rAd-GFP,and the successful expression of multi-copy influenza virus conserved western blot test results demonstrated antigens NP and M2e proteins.（2）Study on the expression of AIV conservative antigen prokaryotic and the immune effect of recombinant adenovirus miceMulti-copy influenza conserved antigen NP,M2e,was cloned on the pET-28a vector,the recombinant plasmid was transferred to the BL21 competent state,and the recombinant granules were successfully constructed by double digestion verification and plasmid sequencing.The recombinant E.coli expression protein was first induced with IPTG,followed by mass purification.C57BL/6J mice were divided into 4 groups,which were grouped as follows:PBS group,rAd-GFP group,influenza virus vaccine group as a control group,and rAd-NP-M2e-GFP as the experimental group（n=15）.The nasal immunological dose of each group was 50μL,the nasal drop dose of the rAd-NP-M2e-GFP group and rAd-GFP group contained 5.0×10^8.5CFU recombinant adenovirus,and the positive control group immunized influenza vaccine containing polydic adjuvant influenza vaccine with a final concentration of 125ug/m L 50ul.After the primary immunization,a booster was given for 14 days,and samples were tested on day 28.Flow cytometry detected cytokine and granulase（IFN-γ,TNF-α,and Gr B）secreted by CD4⁺T cells and CD8⁺T cells in mouse lungs and spleen.The results showed that rAd-NP-M2e-GFP could promote the enhancement of specific T cell response in mouse lungs and spleen,antigen-specific CD4⁺IFN-γ⁺（or CD8⁺IFN-γ⁺）,CD4⁺TNF-α⁺（or CD8⁺TNF-α⁺）and CD4⁺Gr B⁺（or CD8⁺Gr B⁺）frequency increased significantly（P<0.05）.Flow cytometry detected follicular helper T cells（Tfh）cells in mouse lungs.The results showed that the number of Tfh cells in the lungs of mice with rAd-NP-M2e-GFP and rAd-GFP immune group increased significantly（P<0.05）.The germinal center B cells in the mediastinal lymph nodes of mice were further detected by flow cytometry.The results showed that rAd-NP-M2e-GFP and rAd-GFP promoted a significant increase in germinal center B cells in mice’s lung mediastinal lymph nodes（P<0.01）.The changes in the number of B220⁺IgA⁺B cells and CD138⁺IgA⁺plasma cells in the lungs of mice after booster immunization were detected using immunofluorescence technology,and the results showed that the rAd-NP-M2e-GFP group and rAd-GFP group were significantly different from the PBS group compared with the B220⁺IgA⁺B cells（P<0.05）.In addition,CD138⁺IgA⁺plasma cells differed considerably between the rAd-NP-M2e-GFP group,rAd-GFP group,and vaccine group compared with the PBS group（P<0.01）.The changes of specific IgG antibody in serum and specific SIgA antibody levels in alveolar lavage solution before and two weeks after ELISA detected immunization,and it was found that rAd-NP-M2e-GFP could promote a very significant increase in the level of specific SIgA in mouse alveolar lavage fluid and the level of specific IgG in serum（P<0.01）.The results above show that nasal immunoassay rAd-NP-M2e-GFP can induce strong cellular,humoral,and mucosal immunity in mice.（3）Evaluation of the immunoprotective effect of recombinant adenovirus on miceTo study the protective effect of rAd-NP-M2e-GFP on mice infected with influenza subype H1N1,mice’s body weight and survival were monitored for two consecutive weeks.It was found that the PBS group lost 25%of their body weight,and all died from an infection on day 6.However,weight in the rAd-NP-M2e-GFP group and rAd-GFP gradually recovered after day 9.In addition,the survival rate of mice in the rAd-NP-M2e-GFP and rAd-GFP groups was significantly higher than that in the PBS and vaccine groups（P<0.05）.In summary,rAd-NP-M2e-GFP and rAd-GFP protected mice against the H1N1 subtype influenza infection.Flow cytometry was used to detect cytokines and granzyme（IFN-γ,TNF-α,and Gr B）secreted by CD4⁺T and CD8⁺T cells after challenge,and the results showed that rAd-NP-M2e-GFP could promote the enhancement of specific T cell response in the lungs of mice after H1N1 challenge.The antigen-specific CD4⁺IFN-γ⁺,CD4⁺TNF-α⁺,and CD4⁺Gr B⁺frequency were significantly increased（P<0.05）.However,the cytokine secretion level of CD8⁺T cells did not differ significantly between the groups,and the protective effect of rAd-NP-M2e-GFP on mice was evaluated by indirect immunofluorescence and pathological tissue sectioning.It was found that the rAd-NP-M2e-GFP group reduced the replication of viruses in the lungs of mice after the challenge and alleviated the pathological damage of the lungs.Therefore,the rAd-NP-M2e-GFP group can help mice resist H1N1 influenza virus infection after the booster immunization,and it is worth noting that the survival rate of mice in the rAd-GFP group has also reached half,and the body’s non-characteristic immunity may be at work.To study whether the recombinant adenovirus can be protected against avian influenza subtype H9N2 by giving only primary immunization to mice,we monitored the changes in body weight,survival rate,and pathological changes in mice.We found that only a single immunization dose could not protect mice against the avian influenza subtype H9N2 virus.The dose of recombinant adenovirus and the immune agent are critical to the resistance of mice to viral infection.Another possibility is that the stimulation of mice in the process of nasal immunization may cause choking,leading to insufficient doses of the virus and poor immune effect.To investigate whether single immunization of mice rAd-NP-M2e-GFP against AIV subtype H9N2in mice,we monitored the weight change,survival rate,and pathological changes of mice,and found that only a single dose of immunized mice did not protect mice against AIV subtype H9N2,indicating that the dose of recombinant adenovirus and the immune program was essential for mice to resist viral infection.In summary,two strains of recombinant adenovirus rAd-NP-M2e-GFP and rAd-GFP were successfully packaged and obtained,which could induce a mucosal immune response and systemic immune response in mice after nasal booster rAd-NP-M2e-GFP.Also,they protected against H1N1 subtype influenza virus infection.It lays the foundation for developing and utilizing new oral and other mucosal vaccines against influenza viruses in the future.