Protective Ffficacy Ot The Cold Adapted,Attenuated Recombinant Influenza Vaccine Carrying HAdV Epitopes

Human adenovirus(HAdVs)is one of the main pathogens of respiratory tract disease,it is known to be the largest and one of the most complex non enveloped DNA virus and distribute extensive in nature.About 5%respiratory diseases in children under 5 years old caused by adenovirus type 3 and 7.In 2004 and 2005,adenovirus type 3 epidemic outbreaked in several regions including Jiangsu,Beijing,Guangzhou and Hubei in China.And the outbreaks of adenovirus type 7 happened in Shanxi Province in 2009.A number of countries and regions reported outbreaks of different adenovirus type worldwide.Adenovirus threatened to human health.Despite the cases of adenoviral infections in children is increasing,there are few specific treatment or curative drugs.Therefore,the prevention of adenovirus is particularly important.With the development of adenovirus vaccine,several adenovirus vaccines have not been large-scale development.Therefore,the research and development of novel adenovirus vaccines is an important issue that need to be solved.To construct a influenza chimeric vaccine expressing protective antigens of adenovirus,we explored influenza virus as vaccine vector which the protective antigen gene were inserted into specific genes of influenza virus genome.The advantages of intranasal immunization of these vaccines include antigen conservation,similarity of natural infection,specific immune protection against protective antigens.This dissertation consists of two parts:Part one:Rescue of the cold-adapted,attenuated recombinant vaccine rgFlu/HAdV-CaObjective:Reverse genetics technologies were utilized to construct and rescue recombinant rgFlu/HAdV-Ca vaccines based on the influenza virus vector carrying hexon protein antigenic epitopes from HAdV.Methods:Recombinant plasmid Hexon-NA-Hexon was constructed,in which two copies of the hexon protein antigenic epitopes was introduced into the NA genes of A/Califomia/07/2009 virus at a specific position,six internal genes(PB2,PB1,PA,NP,M,NS)of A/Ann Arbor/6/60 ca influenza virus(H2N2)and HA gene of A/Califomia/07/2009 were constructed to bi-directional expression vector pAD3000 and co-transfected into COS1/MDCK cells.Recombinant rgFlu/HAdV-Ca vaccine was identified by HA?RT-PCR?WB.Results:The gene sequencing analysis of the recombinant rgFlu/HAdV-Ca vaccine showed that the reassorted vaccine strain was same as we expected.The recombinant rgFlu/HAdV-Ca vaccine could passage in chicken embryos.The expression of HAdV-specific protein,its particle characteristics and size distribution were identified.These data displayed that the recombinant vaccine rgFlu/HAdV-Ca were rescued successfully.Part two:Protective efficacy of the cold-adapted attenuated vaccine recombinant vaccine rgFlu/HAdV-CaObjective:To evaluate the immunogenicity and protective efficacy of the cold adapted attenuated vaccine rgFlu/HAdV-Ca,these data will provide supports for the development of the adenovirus vaccine candidates.Methods:Six-week-old female BALB/c mice were divieded into four groups and PBS acted as control group.Mice were immunized intranasally(i.n.)twice in a twenty-first interval.The specific antibody titers(IgG;IgGl and IgG2a)against HAdV were measured in serum samples.Meanwhile,the sIgA titers in lung and nasal lavage fluids were examined to evaluate the mucosal immunity responses.In addition,we detected the cytokines levels of IL-4 and IFN-y against HAdV in spleen cell suspension on two weeks after boost.To further evaluate the protetive efficacy of rgFlu/HAdV-Ca against HAdV infection,mice were challenged with wild-type HAdV-3 and HAdV-7 virus.Results:BALB/c mice immunized intranasally(i.n.)with 105,106 or 107TCID50 rgHAdV-Flu ca,respectively,results showed that specific robust antibody response against influenza and HAdV in dose-dependent manner.More importantly,potent humoral,mucosal and cellular immune responses could protect against subsequent wild-type HAdV-3 or HAdV-7 challenges,as determined by a significant decreases in viral titers and a noticeable alleviation of histopathological alterations in the lung tissue of challenged mice.Conclusion:We generated the recombinant rgFlu/HAdV-Ca vaccine by RG.The recombinant rgFlu/HAdV-Ca vaccine could replicate effectively in vitro.The reassorted viruses could induce robust humoral,cellular and mucosal immune response against HAdV-3 and HAdV-7 virus.RgFlu/HAdV-Ca virus could confer efficient protection against wt BJ809 or BJ506 in mice.Thus,our data demonstrated that rgFlu/HAdV-Ca vaccine warrant as a potential candidate vaccine against HAdV infection.