Construction,Characterization And Immunogenicity Study Of Recombinant Adenovirus Expressing SARS-CoV-2 RBD And Influenza H1N1 NP Proteins

[Objective]:To construct and characterize recombinant adenovirus rAd-RBD expressing S protein receptor binding domain(RBD)of SARS-CoV-2 B.1.618 mutant strain and recombinant adenovirus rAd-NP expressing Influenza A virus(IAV)H1N1 nucleoprotein(NP)protein.The immunogenicity of the two recombinant adenoviruses was evaluated by animal experiments and pseudovirus neutralization experiments,and whether there is immune synergism between rAd-NP and rAd-RBD by comparing different immunization protocols of the two recombinant adenoviruses alone and in combination with immunization.[Methods]:1.Construction and identification of recombinant adenovirus rAd-RBD and rAd-NP:The RBD sequence of SARS-CoV-2 B.1.618 mutant strain,IAV H1N1 NP sequence and its upstream and downstream primers were synthesized,and the PCR products of RBD sequence and NP sequence were double digested and ligated to pShuttle-CMV shuttle vector,and the positive clones were screened for recombinant shuttle After screening for positive clones of the recombinant shuttle plasmid pShuttle-NP and pShuttle-RBD,PCR and target gene sequencing were performed.The linearized recombinant shuttle vector plasmids carrying the target genes were electrotransformed into BJ5183 receptor cells containing the adenovirus backbone plasmid pAdEasy-1 for homologous recombination,and the prAd-RBD and prAd-NP recombinant adenovirus plasmids carrying the target genes were screened.The recombinant prAd-RBD and prAd-NP were identified by PacI digestion and sequencing.The identified prAd-RBD and prAd-NP plasmids were linearized and purified by PacI and transfected into HEK293 cells to package the recombinant adenovirus.The genomic DNA of the recombinant adenovirus was extracted and the target genes were identified by PCR and agarose electrophoresis.After confirming that the target gene had been recombined into the adenovirus genome,the recombinant adenovirus was infected with cells,and total cellular RNA was extracted 48h after infection,and whether the target gene in the recombinant adenovirus could be transcribed normally in the cells by RT-PCR.After confirming the normal transcription of the target genes,the expression of RBD and NP proteins was further characterized by Western Blot(WB)and immunofluorescence.2.Amplification and purification of recombinant adenovirus:HEK293 cells were amplified with recombinant adenovirus rAd-RBD and rAd-NP,and the harvested solution of rAd-RBD and rAd-NP viruses were concentrated by centrifugation in ultrafiltration tubes and purified with Core 700 chromatography columns were used for purification.The purified products were identified as recombinant adenovirus rAd-RBD and rAd-NP by WB,transmission electron microscopy and PCR.3.Recombinant adenovirus immunogenicity study:20 BALB/c female mice aged 6-8 weeks were randomly divided into four groups:negative control group,rAd-NP group,rAd-RBD group and rAd-NP,rAd-RBD combined immunization group.The mice were immunized with the same dose of PBS buffer,rAd-NP,rAd-RBD,rAd-NP and rAd-RBD by intramuscular injection in the inner hind limbs.immunization doses were 1 dose of immunization,and sera were collected 1 day before,14 days after and 21 days after immunization,respectively,and evaluated by ELISA potency assay and SARS-CoV-2 Delta strain and Omicron strain Pseudovirus neutralization assay was performed to evaluate the IgG and neutralizing antibody levels after vaccination.Spleen cells from mice at 21 days post-immunization were collected.for flow cytometry to evaluate the level of cellular immunity of the vaccine in mice,and finally to complete the assessment of the immunogenicity of recombinant adenovirus rAd-RBD and rAd-NP and the comparison of the effects between different immunization regimens.[Results]:1.Construction and characterization of recombinant adenovirus rAd-RBD and rAd-NP:PCR and sequencing results showed that the recombinant shuttle vector plasmids pShuttle-NP and pShuttle-RBD were successfully constructed.pShuttle-NP and pShuttle-RBD were successfully recombined with the adenovirus backbone plasmid pAdeasy-1 and successfully constructed.After transfection of HEK293 cells with the recombinant adenovirus backbone plasmid for 48h,HEK293 cells showed a significant cytopathic effect(CPE),and after 72h when all the cells were diseased,the viral fluid and cell precipitation were harvested and identified by the viral genome DNAPCR.The target gene RBD sequence and NP sequence could be replicated,transcribed,translated and expressed normally in the host cells after the identification of viral genome DNAPCR,total RNART-PCR of HEK293 cells after infection,WB identification of total protein of HEK293 cells after infection and immunofluorescence identification of MA104 cells after infection.The recombinant adenovirus rAd-RBD and rAd-NP were successfully constructed.2.Amplification and purification of recombinant adenovirus:rAd-RBD and rAd-NP were amplified and harvested by 1L each,concentrated to about 50mL each with ultrafiltration tubes,and purified by Core 700 chromatography column to collect the rAd-RBD and rAd-NP viruses monitored by LP Data View software in real time.The first flow-through peak of the concentrated solution after sampling,the purified product was identified as recombinant adenovirus carrying the target gene by WB method,transmission electron microscopy observation and PCR method.The titers of rAd-RBD and rAd-NP concentrated and purified by qPCR were 1.5 × 1011 Copies/mL and 4.7×1012 Copies/mL,respectively.3.Immunogenicity study of recombinant adenovirus:ELISA potency assay results showed that both rAd-NP and rAd-RBD induced the production of antibodies to the target protein RBD/NP IgG in mice.In flow cytometry experiments,rAd-NP was able to induce a better T-cell immune response in mice while rAd-RBD was not.immunization was found to be superior to the rAd-RBD immunization group in terms of antibody levels against the target protein RBD/NP IgG,neutralizing antibody levels against the pseudovirus,total T cell count,CD4+cell count,and CD8+cell count detected by flow cytometry.[Conclusion]:1.The recombinant adenovirus plasmids carrying SARS-CoV-2 B.1.618 mutant strain RBD and IAV H1N1 NP genes were successfully constructed;2.The constructed recombinant adenovirus plasmids were able to successfully package recombinant adenovirus rAd-RBD and rAd-NP in 293 cells;3.The recombinant adenovirus rAd-RBD and rAd-NP carried in the packaged target The recombinant adenoviruses rAd-RBD and rAd-NP were able to induce humoral and cellular immune responses in mice after immunization:both rAd-NP and rAd-RBD induced elevated levels of serum anti-RBD/NP IgG antibodies in mice;the neutralizing antibodies produced after immunization with rAd-RBD were able to neutralize Delta and Omicron strains of pseudoviruses.rAd-RBD immunized mice produced neutralizing antibodies that could neutralize Delta and Omicron strains of pseudovirus,and the neutralizing effect on Omicron strain of pseudovirus was better than that of Delta strain;rAd-NP could induce a better T-cell immune response in mice,while rAd-RBD could not.5.The immunization results of rAd-RBD alone,rAd-NP alone and rAd-RBD and rAd-NP combined showed that the immunization effect of rAd-NP on The immunization effect of rAd-RBD was immunosynergistic and bystander effect.