An Elementary Study On Recombinant Attenuated Strain Of SARS-CoV-2S Protein RBD Based On Influenza Viral Vector

COVID-19 is a serious respiratory infection disease caused by the SARS-COV-2 virus,as a novel coronavirus,whose epidemic spreads rapidly around the world during last year.It caused more than 100 million laboratory confirmed cases and more than 2.5million deaths,and it seriously threatened human health and social stability.Since there is no specific effect inhibitors medicine,the vaccines could be most useful and effective methods to prevent and control this COVID-19 pandemic.The live attenuated vaccine（LAV）could be used by intranasal administration,which is simulation of natural infection.Furthermore,the LAV could induce not only nasal immunity,but also humoral and cellular immunity.Additional,LAV is better for the herd immunity barrier construction through limited interpersonal contact.However,the screening of attenuated SARS-CoV-2 candidate vaccine strain by traditional methods need a long period and many complex process,which isn’t suitable for current prevention and control the COVID-19 pandemic.The attenuated A/Yunnan/1/2005（H₃N₂）Vca（Abbreviation as H₃N₂Vca）strain was used in this study.The H₃N₂Vca could be grown in Vero cells by large-scale microcarrier culture,which break the restriction on the source of chicken embryos with normal influenza virus vector.That provide the preparation of large-scale vaccine needs.Importantly,it inserted the RBD gene of SARS-CoV-2 S protein into the NS gene of H₃N₂Vca,then transfected 293T cells with H₃N₂Vca other 7 gene fragements（PB₁,PB₂,PA,NP,M,HA and NA）on the PHW-2000 plasmids,respectively.Finally,the live virus H₃N₂-S-RBDVca could be rescued in 293T cells,which maintained the ca and att phenotype characteristics as same as H₃N₂Vca.Through 5 consecutive passages culture,its HA titter was more than 1:128,which titter could get 2¹⁵,after purification and concentration.It evaluated the production of H₃N₂-S-RBDVca vaccine.Its endotoxin content was 4 EU/m L,its egg albumin content was under 31.3 ng/m L.Furthermore,that H₃N₂-S-RBDVca vaccine still has ts,ca and att phenotypes,confirmed by model animals.After intranasal administration with that vaccine,the mice weight showed a steadily increasing trend,and their neutralizing antibody titter reached 1:16,indicating that the vaccine was safe and effect.In conclusion,this study provided a nove and useful method for the development of live attenuated SARS-CoV-2 vaccine.