Construction And Immunoefficacy Evaluation Of ND-AI-FAdV-4 Tri-chimeric Virus-like Particles

Newcastle disease(ND),H9N2 avian influenza(AI)and Ankara disease are the main animal diseases that threaten the poultry industry in China.ND is a severe avian disease caused by Newcastle disease virus(NDV).It is classified as a ClassⅡanimal disease by the Ministry of Agriculture and Rural Areas of China,and the genotypeⅦNewcastle disease is currently circulating in China.AI is a highly contagious disease caused by avian influenza virus(AIV),H9N2 AIV is the most prevalent genotype,which has caused great harm to poultry industry and public health safety.Avian Ankara disease is a highly fatal infectious disease caused by avian adenovirus groupⅠserotype 4(FAd V-4)and characterized by hydropericardiac-hepatitis syndrome(HHS).Vaccination is the most effective method to prevent ND,AI and avian Ankara disease.However,the vaccines currently used are all traditional all-pathogen vaccines such as inactivated or attenuated vaccines,which have the problem of conflicts between biosafety and immunogenicity,and do not conform to the green and ecological breeding concept.It is an important research direction of national development strategy to develop new multi-linked and multivalent vaccines.The HN,HA and Fiber-2 protein contain major protective antigenic sites,which are the targets for the development of new vaccines against ND,AI and avian Ankara disease.The application of adjuvants or delivery vectors will effectively improve the immune efficaty of candidate vaccines.Virus-like particles(VLPs)are virus-derived structures assembled from one or several viral structural proteins.VLPs are with the similar structure and conformation to natural viruses,and can induce humoral and cellular immune responses.VLPs is an ideal target for the development of new vaccine and carrier platform because it does not contain viral infectious nucleic acid and has high immunogenicity and safety.Newcastle disease virus like particles(ND VLPs)are self-polymerized by NDV M protein,HN protein and F protein,which have strong immune protection and antigen delivery capabilities.Therefore,this study employed ND VLPs carrier platform and insect baculovirus expression system to construct a green and efficient ND-AI-FADV-4 c VLPs novel vaccine candidate that exhibits HN,HA and Fiber-2 proteins on the membrane surface of ND VLPs.In order to improve its immunogenicity,the ND-AI-FADV-4 c VLPs was emulsified with Fredrin adjuvant and tested for its dosage form,centrifugal stability,viscosity,sterility and shelf life.The immunoprotective ability of the vaccines was evaluated by detecting the antibody level,lymphocyte proliferation ability,viral load in vivo and detoxification time in vitro.The main research contents are as follows:1.Construction of recombinant baculovirus expressing FAd V-4 Fiber-2The Fiber-2 gene of FAd V-4 HB 1510 strain(Gen Bank:KU587519.1)was optimized and synthesized according to the codon preference of insect cells.The Fiber-2 gene was ligated with the intracellular domain and transmembrane domain of NDV HN gene and p Fast Bac1 vector by seamless cloning method to construct the recombinant shuttle plasmid rp Fast Bac1-c Fiber2.After Bam HⅠand KpnⅠdouble digestion and bacterial liquid PCR identification,the recombinant baculovirus r Bmid-c Fiber2 was transformed into E.coli DH10Bac competent cells for blue and white spot screening,The correct recombinant bacmid r Bmid-c Fiber2 was extracted and transfected into sf9 cells to rescue the recombinant baculovirus r BV-c Fiber2.The expression of c Fiber2 protein was identified by indirect immunofluorescence and Western blot.The results showed that the size of c Fiber2protein was about 60 k Da,which could be correctly expressed in sf9 cells.In addition,r BV-c Fiber-2 can be stably passed for at least 20 generations,and the virus titer of each generation is above 10~7IFU/m L after 24 months at-80℃.2.Construction of ND-AI-FAd V-4 c VLPs and preparation of vaccineThe r BV-c Fiber2 and r BV-M,r BV-HN and r BV-HA were co-infected with suspension sf9 cells at MOI=5(r BV-M:r BV-HN:r BV-HA:r BV-c Fiber-2=3:1:1:1)to assemble the ND-AI-FAd V-4 c VLPs.The culture supernatant was collected and purified by sucrose gradient ulcentrifugation after 72 hpi.Western blot and transmission electron microscopy were used to detect the protein expression and morphology of ND-AI-FAd V-4 c VLPs,the results showed that all components of ND-AI-FAd V-4 c VLPs were correctly expressed and presented a circular structure about 100 nm with capsule and fibril.To improve the immunogenicity of ND-AI-FAd V-4 c VLPs,the ND-AI-FAd V-4 c VLPs was diluted and emulsified with an equal volume of Fred’s adjuvant(75μg,0.2 m L/aliquot),and the dosage form,centrifugal stability,viscosity,sterility and storage period of the prepared vaccine were tested.The results showed that the vaccine dosage form was water-in-oil type withou microbial contamination such as bacteria and mold,the physical properties were stable,and can be stored at 4℃for more than 12 months.In addition,there was no strong stress reaction after inoculation of test chickens.3.Study on the immune effect of ND-AI-FAd V-4 c VLPsND-AI-FAd V-4 c VLPs vaccine was intramuscularly injected into 7-day-old SPF chickens and booster immunited into 21-day-old SPF at a dose of 0.2 m L/feather(75μg)to evaluate its immune effect.The antibody level induced by ND-AI-FAd V-4 c VLPs was monitored by hemagglutination inhibition test and ELISA.The results showed that the HI antibody titer of ND-AI-FAd V-4 c VLPs immunized group was equivalent to that of NDV attenuated vaccine immunized group and AIV inactivated vaccine immunized group,and it could produce high level of specific antibody against FAd V-4 Fiber2 protein.To evaluate the immunoefficacy of ND-AI-FAd V-4 c VLPs,chickens were challenged with virulent NDV,H9N2 AIV and virulent FAd V-4 for three weeks(42 days of age)after booster immunization.The results of lymphocyte proliferation test showed that the stimulation of Newcastle disease virus,avian influenza virus and serotype 4 fowl adenovirus inactivated virus promoted the activation and proliferation of spleen lymphocytes in ND-AI-FAd V-4c VLPs immunized group.The results of challenge test showed that ND-AI-FAd V-4 c VLPs could provide effective protection against the attack of virulent strains of Newcastle disease virus NA-1 strain,avian influenza virus H9N2 strain and serotype 4 avian adenosis virulent strain.ND-AI-FAd V-4 c VLPs reduce the pathological damage caused by viral infection,and shorten the viral load in vivo(the loading of virus in organs was stopped 9dpi after NDV challenge and 11 dpi after AIV challenge)and the time of virus shedding in vitro(shedding was stopped 7 days after NDV challenge,7 days after AIV challenge,and 9days after FAd V-4 challenge).In summary,this study provides a green and efficient new vaccine candidate strain for promoting the prevention and control of ND,AI and avian Ankara disease,and also enriches the application of ND VLPs as a vector platform.