Construction,Identification And Immunogenicity Evaluation Of Recombinant Adenovirus Carrying DsRNA Adjuvant Norovirus VP1

Norovirus(NoV),belonging to the Caliciviridae family and Norovirus genus,is a non-enveloped,single-stranded RNA virus.NoV-induced gastroenteritis is a typical acute disease.In infected populations,healthy adults exhibit self-limiting symptoms,while children and the elderly are at high risk.Due to fecal-oral transmission,large-scale outbreaks often occur in crowded places.Vaccination is an effective approach for preventing and controlling NoV,but since NoV cannot be cultured in vitro,constructing recombinant adenovirus vaccines with adenovirus vectors provides an alternative pathway for NoV vaccine development.Purpose:To use human adenovirus serotype 5 as a vector,we will construct a recombinant adenovirus carrying the VP1 gene of Norovirus.The recombinant adenovirus will be prepared and purified to obtain a replication-deficient recombinant adenovirus vaccine.Simultaneously,we will also construct a recombinant adenovirus containing a dsRNA adjuvant.Preliminary animal immunization evaluations will be performed to investigate the immunogenicity of the recombinant adenovirus and compare the immune responses between the Norovirus VP1 recombinant adenovirus with and without the dsRNA adjuvant.The aim of these experiments is to provide new insights and methods for the development of safer and more effective Norovirus vaccines.Methods:The VP1 sequence(5085 bp-6707 bp)carrying the cleavage site was obtained by PCR amplification through the sequence of G Ⅱ.4 norovirus VP1(Genbank ID:KF306214.1)published according to the NCBI query.VP1 was connected to the shuttle vector pshuttle CMV to construct the pshuttle VP1 recombinant plasmid;Based on literature references,the dsRNA adjuvant lucl sequence was designed and synthesized,and the lucl and VP1 genes were inserted into two polyclonal sites of pcDNA3.0BA,respectively.The expression cassette carrying both luc1 and VP1 genes was obtained through PCR.Connect the expression cassette to the shuttle vector pshuttle-CMV,and construct a double Cistron pshuttle-VP1-lucl with the VP1 sequence of norovirus,.Connect VP1-Iucl double Cistron to shuttle CMV;Linearize the two shuttle vector plasmids pshuttle-VP1 and pshuttle-VP1 luc1,and electroconvert them into BJ5183 cells containing adenovirus skeleton plasmids for homologous recombination.The recombinant adenovirus plasmid is obtained through homologous recombination;Named rAD-VP1 and rAD-VP1 luc1;Transfer the recombinant adenovirus plasmid into HEK293 cell packaging virus particles,.Harvest adenovirus after successful packaging.Extract the genomic DNA of successfully packaged adenoviruses rAD-VP1 and rAD-VP1 lucl,and identify whether the virus genome carries the VP1 gene through PCR.Infect cells with the virus and pass the packaged virus onto the P3 generation;PCR was used to identify the VP1 sequence of the virus genome,and Western blot and immunofluorescence were used to identify the expression of exogenous protein VP1;.The packaged adenovirus was passed to P3 generation in HEK293 cells.After the virus was harvested,it was concentrated about 100 times by ultrafiltration.The recombinant adenovirus was amplified by HEK293 cells,and the adenovirus harvest solution was purified by core700 gel column.The purified solution was centrifuged and concentrated about 100 times by ultrafiltration tube(30K);,After collecting the purified solution,use an adenovirus titer assay kit to determine the titer of the concentrated virus solution;.Animal immune experiments were conducted on 6-8 week old female SPF grade BALB/c mice.rAD-VP1 and rAD-VP1 lucl recombinant adenoviruses were immunized through three immune pathways:intramuscular injection,gavage,and nasal drip.Each mouse was immunized with 1x1010 VP,and PBS intramuscular injection of 100 u1 was used as the control group;Before immunization,14 days and 28 days after immunization,serum was collected to detect norovirus VP1 specific IgG antibody level by ELISA;After 28 days of immunization,the lung homogenate,lung lavage fluid,intestinal homogenate,intestinal lavage fluid and feces of mice were collected respectively,and the level of noro virus VP1 specific IgA antibody was detected by ELISA;Results:The construction of shuttle plasmids pS-VP1 and pS-VP1-luc1 was successful,as confirmed by enzymatic digestion and sequencing.The BJ5183 cells were used for homologous recombination,resulting in the construction of recombinant adenovirus plasmids rAD-VPl and rAD-VP1-lucl,which were also confirmed by enzymatic digestion and sequencing.HEK293 cells were successfully transfected to package the viral particles.After amplification and purification,a high titer of recombinant adenovirus was obtained,with a concentration of 2x1011 copy/ml.In female BALB/c mice,immunization with both rAD-VP1 and rAD-VP1-lucl recombinant adenoviruses induced the production of Norovirus-specific IgG antibodies in the serum compared to the PBS control group.Among the different immunization routes,the intramuscular injection and oral gavage routes showed higher antibody levels,while the oral gavage route in the rAD-VP1-lucl group was significantly higher than other immunization routes.The IgG antibody levels in the rAD-VP1 group were similar and showed no significant differences.Both rAD-VP1 and rAD-VP1-lucl recombinant adenoviruses induced the production of Norovirusspecific IgA antibodies in lung homogenates,intestinal homogenates,and fecal samples.The nasal drop and intramuscular injection routes showed similar levels of IgA antibodies,while the oral gavage route was higher than the other two groups.The rAD-VP1-lucl group had higher levels of IgA antibodies compared to the rAD-VP1 group.Conclusion:Successfully constructed rAD-VP1 and rAD-VP 1-luc1 recombinant adenoviruses,both of which expressed the exogenous proteins efectively in vitro.In animal models,both rAD-VP1 and rAD-VP1-luc1 recombinant adenoviruses demonstrated good immunogenicity,with the oral immunization route showing favorable results for rAD-VP 1-luc 1.This suggests that the dsRNA adjuvant positively influences the immune response of the recombinant adenovirus,particularly when administered via the oral route.Further research is needed to explore the non-specific immunostimulatory effect of luc1.