Construction And Immunogenicity Of Replicating Recombinant Human Adenovirus Serotype 4 Expressing S Protein Of Chinese PEDV Variant

Porcine epidemic diarrhea(PED)is an acute and highly infectious intestinal disease,caused by porcine epidemic diarrhea virus(PEDV),is characterized by watery diarrhea,vomiting,dehydration,and high mortality in nursery piglets.Pigs at all ages are susceptible to PEDV.The presence of PED in China was confirmed in 1984.The PEDV killed or attenuated vaccines have been used in China since then.The outbreak of PED in China is sporadic.However,outbreaks of PED caused by PEDV variant have reemerged in the major pigproducing areas of China since December 2010,resulting in major losses of suckling piglets.The major differences between the PEDV variant and classical vaccine strain were located at the N-terminus of spike(S)protein.The inactivated vaccine and the attenuated vaccine based on classical vaccine strain can not provide enough immunity to fight the infection of the PEDV variant.Anti-PEDV Mucosal immunity plays important roles in controlling PEDV infection.Currently high-quantity cultivation of PEDV variant is still difficult.Therefore,it is imperative to explore new ways to develop new and efficient vaccines which can induce mucosal immune responses for controlling PEDV variant.Adenovirus vector can infect a wide range of cells,effectively induce humoral,cellular and mucosal immune responses,and has been widely used in human and animal gene therapy and vaccine research and development.The replicating human adenovirus serotype 4(HAd V-4)vaccine strain has been used in the field of prevention and treatment of human acute respiratory disease for many years and has been shown to be safe and effective.The replicating HAd V-4 can mimic the natural virus infection in host cells,and induce all kind of immune responses including mucosal immunity at all levels.In this study,the virus vector system based on HAd V-4 vaccine strain was constructed.In order to lay a foundation for the development of PED vaccine and for more effective control of PED,the N-terminal(1 ~ 380aa)of PEDV S gene was inserted into E3 region of HAd V-4 genome,and a recombinant adenovirus capable of expressing PEDV variant S protein was constructed.The immunogenicity of recombinant virus was studied in mouse model.The thesis consists of the following three parts:1.Construction of replicating HAd V-4 vector system and investigation of growth characteristics of recombinant adenovirusd in porcine cell lineIn this section,a recombinant HAd V-4 vector system based on the HAd V-4 vaccine strain was constructed.The system is composed of the virus genome backbone vector p Ad4 FAST and the shuttle vector p Ad4FAST-shuttle.Homologous recombination was carried out in BJ5183 host.Recombinant adenovirus infectious clones were identified,extracted and the full-length infectious clones were digested with restriction enzyme Pac I and transfected into 293 cells to rescue the recombinant virus.In order to verify the feasibility of the system,EGFP was used as a marker gene to construct a recombinant adenovirus r Ad4△E3-EGFP capable of expressing green fluorescent protein.Different pig cell lines were infected with recombinant virus r Ad4△E3-EGFP.Typical cytopathic effect and green fluorescence were observed in recombinant virus r Ad4△E3-EGFP infected cells.The results provide a tool and theoretical basis for the development of pig vaccines using HAd V-4 vaccine strains as vectors.2.Construction and identification of replicating recombinant HAd V-4-expressing S gene of PEDV Chinese variantIn this section,the N-terminal of S gene(AA1~380)of PEDV Chinese variant was used as the target gene,and the recombinant HAd V-4 infectious clones was prepared by inserting the gene into the genome of type 4 adenovirus using the HAd V-4 vector system constructed in Experiment 1.The infectious clone DNA were transfected into 293 cells to rescue the recombinant virus r Ad4△E3-S.Western blot showed that the recombinant PEDV S protein was expressed successfully.The results provided basic experimental materials for further evaluation of the immunogenicity of the recombinant virus,and laid the foundation for the development of PEDV live vector vaccine.3、Evaluation of the immunogenicity of the replicating recombinant r Ad4△E3-S in mice In order to evaluate the immunogenicity of the recombinant HAd V-4 expressing S gene of PEDV Chinese variant.We choose mouse as experimental animal model.After the first immunization,the recombinant virus stimulated mice to produce S-specific antibody.After the second immunization,antibody levels were significantly increased and maintained for a longer period of time.The results of the virus neutralization test showed that the anti-S protein antibody produced by the recombinant virus r Ad4△E3-S immunized mice had neutralizing effects on the mutant strain CH / ZMDZY / 11 and the vaccine strain CV777,but the neutralizing activity on the mutant strain was significantly higher than that on the classical vaccine strain.The average neutralization titer of Anti-S protein antibody that inhibited the infection of mutant strain CH / ZMDZY / 11 in Vero cells was 1:68,but the average antibody titer in the vaccine strain CV777 was only 1:8.The T lymphocyte responses were detected by in vitro lymphocyte transformation assay and flow cytometry.The proliferation of lymphocytes in the r Ad4△E3-S-immunized group was significantly higher than that in the empty vector and DMEM-immunized control group(P<0.05).The levels of PEDV S-specific IFN-γ,IL-2 and TNF-α in CD4+ and CD8+T lymphocytes in r Ad4△E3-S-immunized mice were higher than those in the other groups,suggesting that the recombinant virus could stimulate the mice to produce antigen-specific cellular immune responses.The results provide a theoretical basis for further study on the immunogenicity of recombinant virus in swine,and also provide a theoretical basis for the development of PEDV live vector vaccine.