| Objective To evaluate the immunogenicity and protective effects of different SARS-CoV-2 VOC and IAV bivalent DNA vaccines,and to evaluate the immunogenicity and protection of different dual quadrivalent DNA vaccine matching regimens.Methods1.Construction and effect evaluation of bivalent DNA vaccines based on SARSCoV-2 Omicron BA.1 variant and IAV H1N1/Victoria/2019:Based on the previous structure of the SARS-CoV-2 and Influenza A virus bivalent DNA vaccine,pVRC-BS1-2A-HA3,we selected the S1 antigen of the currently prevalent Omicron BA.1 variant as the target protein,and added the T4 motif trimer motif at the end of S1,replacing the S1 sequence of the Beta variant in the original structure.At the same time,according to the latest recommended components of influenza vaccine in the northern hemisphere of 2021-2022 by WHO,the source of HA antigen in DNA was replaced by H1N1/Victoria/2019 HA antigen from H3N2/Cambodia/e0816360.The designed fragments were deeply optimized for human sources,synthesized and inserted into the pVRC plasmid vector named pVRC-OS1T-2A-HA1,and the expression of DNA was verified in 293 T cells by WB and indirect immunofluorescence.After verifying the expression,DH5α E.coli was used to amplify the DNA vaccine,and the protein expression of S1 and HA was evaluated after transient transfection into 293 T cells,and BALB/c mice were selected as animal research models for DNA vaccine immunization,and the injection method was intramuscular injection + Electroporation(IM+EP),30μg each time,and DNA vaccine immunization was performed according to the Prime+Boost immunization protocol.We detected the humoral immune response 2 weeks after primary immunization(Day14),2 weeks after booster immunization(Day35)and before challenge(Day69)by ELISA,hemagglutination inhibition experiment and pseudovirus neutralization experiment,and used ELISPOT method to detect the specific IFN-γ secretion of the sensitive peptide pool of Wuhan original strain S protein and the sensitive peptide pool of Omicron variant S protein,and evaluated the specific cellular immune response of different peptide pools at different times.The protective effect of the vaccine was evaluated by the challenge experiment of novel coronavirus and influenza A virus through the nose on Day69,and the mice were sacrificed on the 3rd day after the challenge,and the mouse lung tissue was titrated with MDCK cells to determine the influenza viral load of mouse lung tissue,and Vero cells were used to titrate the viral load of mouse lung tissue.Lung tissue lesions were evaluated by lung tissue sections and H&E staining.2.Construction of bivalent DNA vaccines based on SARS-CoV-2 VOC and influenza A and evaluation of the effect of dual quadrivalent vaccines:Under the condition that pVRC-OS1T-2A-HA1 has a good effect on the attack,we further explore the structure of bivalent DNA vaccine,swap the S1 and HA positions back and forth,replace S1 with the structure of the new crown Delta variant S1,and synthesize two new DNA fragments with H3N2/Cambodia/e0816360 and H1N1/Victoria/2019,respectively,and synthesize and insert pVRC after deep human sequence optimization The DNA vectors are named pVRC-HA1-2A-DS1 T,pVRC-HA3-2A-DS1 T.The four DNA structures were grouped into a: pVRC-BS1-2A-HA3,b: pVRC-OS1T-2A-HA1,c:pVRC-HA1-2A-DS1 T,d: pVRC-HA3-2A-DS1 T.WB and introductory immunofluorescence were used to verify DNA expression at the 293 T cell level;After verification,three different immune groups of a+b,a+c,b+d were mixed in 1:1 equal ratio(15μg+15μg)according to the following matching scheme,and the immune experiment was carried out in the mock group 30μg,and the immunization strategy used IM+EP and Prime + Boost methods.IgG antibody titers were detected by indirect ELISA method 2 weeks after primary immunization(Day14)and 2 weeks after booster immunization(Day35),hemagglutination inhibitory antibody titers were evaluated by hemagglutination inhibition assay and cellular immune responses of Day14 and Day35 were evaluated by ELISPOT.A virus H1N1 PR8 challenge experiment was performed on Day42 3 weeks after the booster immunization,and some mice were sacrificed on the 7th day after the challenge to evaluate lung tissue virus RNAcopies,virus titer,and lung histopathology,and continuously monitored the weight change for 14 days to calculate the survival rate.At Day70 hours,the novel coronavirus challenge experiment was carried out,and mice were sacrificed on the third day after the challenge,and lung tissue virus titers,RNAcopies and lung tissue H&E staining were detected to evaluate lung histopathology.Results1.Based on SARS-CoV-2 Omicron BA.1 variant and IAV H1N1/Victoria/2019 bivalent DNA vaccine pVRC-OS1T-2A-HA1 is well expressed on 293 T cells through WB and indirect immunofluorescence,because 2A self-shearing peptide action can independently express two different antigens.After booster immunization,a good immune response was induced on BALB/c mouse models,and its pseudovirus neutralizing antibodies could produce a certain cross-neutralization effect against new crown variants such as WT,Delta,Beta and Omicron.Hemagglutination inhibitory antibody titers can reach 1:49 in Day35 and stabilize at 1:45 on Day69.The attack against the new crown Omicron BA.2 variant has played a certain protective role,and the challenge against the influenza A virus H1N1 PR8 mouse lung adapted type also has a protective effect.2.Based on the two DNA structures of pVRC-BS1-2A-HA3 and pVRC-OS1T-2AHA1,the anterior and posterior positions of S1 and HA were further replaced,and S1 was replaced with S1 of the Delta variant,and two new structures,pVRC-HA1-2A-DS1 T and pVRC-HA3-2A-DS1 T,were obtained,and the two different proteins of S1 and HA were verified by WB and indirect immunofluorescence to verify that both structures can independently express S1 and HA.The four DNA structures were mixed in equal proportions while ensuring that there were both HA1 and HA3 in the three groups,and three matching schemes of a+b,a+c and b+d were obtained,all three structures could induce the immune response of good specific IgG antibodies,and the new coronavirus neutralizing antibody titer detection a+b matching scheme had good neutralizing antibody titers for a variety of pseudoviruses.The results of hemagglutination inhibition showed that all three combinations could produce good hemagglutination inhibitory antibodies against H1N1 and H3N2.The three groups could induce good cellular immune response after booster immunization,among which the combination of a+b and a+c had a better effect than the b+d group,the results of influenza A virus challenge showed that the three matching regimens provided good protective effect against H1N1 PR8 and H3N2 X31,the lung tissue virus RNAcopies,viral titer and lung histopathology all suggested the protective effect,and the survival rate calculated by monitoring the weight change for 14 days suggested that the a+b combination could achieve the most balanced protective effect.After the novel coronavirus attack,all immune groups could effectively reduce the viral load of lung tissue and improve lung tissue lesions.Among them,the comprehensive protection effect of A+B structure is the best.Conclusion:1.In this study,bivalent DNA vaccines based on SARS-COV-2 Omicron BA.1 and IAV H1N1/Victoria/2019 were successfully constructed and had good protective effect.2.Through the exploration of different structures,the construction strategy of bivalent DNA epidemic with novel coronavirus S1 and influenza A virus HA as target antigens was determined.3.Through the verification of different matching schemes and vaccine protection,a new bivalent matching strategy is obtained,which has a better protection effect than a single bivalent vaccine. |
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