Construction,Selection And Immunogenicity Of Recombinant Vaccinia Virus Vector Vaccine Against SARS-CoV-2

New Coronavirus pneumonia(Corona Virus Disease 2019,COVID-19)is a severe acute respiratory syndrome caused by severe acute respiratory syndrome coronavirus 2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2).SARS-CoV-2 belongs to the ?-type coronavirus in the genus coronavirus of coronaviridae.It is a single positive strand RNA virus with envelope.SARS-CoV-2 consists of four major structural proteins(spike glycoprotein S,envelope protein E,membrane protein M,capsid protein N)and 16 non structural proteins(nsp1-nsp16).Among them,structural protein S is the most important,which contains S1 and S2 subunits.S1 subunit contains receptor domain RBD(receptor binding domain),which can make angiotensin converting enzyme 2(ACE2)as a receptor cell to bind to virus.Because of its biological advantages,vaccinia virus is often used as a vaccine vector for disease treatment.However,vaccinia virus also has some defects,and the side effects become an obstacle in the research process,so the construction of gene deletion vaccinia virus is more and more widely.In this study,Tiantan vaccinia virus was used as the vector to reduce the virulence and host range of the virus,The E3 L gene of Tiantan vaccinia virus was knocked out by Cre/loxp system,and the recombinant vaccinia virus rVTT?E3L-RBDEPI was constructed by homologous recombination with SARS-CoV-2 RBDEPI gene.Firstly,the proliferation inhibition of CCK-8,crystal violet staining and one-step growth curve were used to detect the diffusion ability and replication ability of the deletion vaccinia virus.BALB/c mice were infected by intranasal inoculation to observe the effect of the deletion vaccinia virus on the weight of mice and analyze its virulence level.Secondly,two mice were injected intramuscularly into the quadriceps femoris of BALB/c mice.At the same time,the same dose of VTT and PBS were injected as control.Twenty one days after the first immunization,the booster immunization was carried out.Every week after immunization,the blood was collected from the tail vein and the serum was separated.The specific antibody level was detected by ELISA kit.Two weeks after booster immunization,splenic lymphocytes were isolated from mice,and the expression levels of IL-4 and IFN-? were detected by ELISPOT kit to analyze the immunogenicity of recombinant vaccinia virus;On the first,seventh and fourteenth day after booster immunization,the mice were dissected to detect virus residues in heart,liver,spleen,lung,kidney,brain and muscle,and the safety of recombinant vaccinia virus was analyzed.It was found that the SARS-CoV-2 recombinant vaccinia virus vector vaccine with gene deletion was successfully constructed,and the virulence of the virus was significantly lower than that of the wild virus VTT.There was no wild virus in 40 generations of continuous passage,which proved that the genetic stability was good.After immunizing mice,it can cause obvious immune reaction,and its immunogenicity is good.No obvious residual amount of virus was found in organs,which proves that the constructed recombinant vaccinia virus vector vaccine of SARS-CoV-2 is safe.