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@#ObjectiveTo construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N. The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV. The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging. The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay. 12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5 × 109copies per mouse. Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells. Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.
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@#Objective To analyze the correlation of different methods for the detection of antibody titer after immunization with severe acute respiratory symptom corona virus 2(SARS-CoV-2) vaccine,and provide a methodological basis for the specific antibody detection.Methods The seroconversion rate of IgG antibody and neutralizing antibody titer of SARS-CoV-2 inactivated vaccine clinical trial serum samples were detected by micro-cell neutralization assay and three ELISA methods(using S protein,N protein and inactivated whole virus particles as antigens respectively),and the correlation among the methods was analyzed.Results The seroconversion rates detected by neutralizing antibody,S antibody,N antibody and whole virus antibody were 84.3%,91.3%,65.2% and 46.6%,and the geometric mean titers were 16.6,945.9,72.7 and 18.8,respectively.The correlation coefficients(r) between the results of three ELISA methods(using S protein,N protein and inactivated whole virus particles as antigens) and micro-cell neutralization assay were 0.494,0.371 and 0.181respectively.Conclusion Detection of SARS-CoV-2 S antibody level reflected the activation of the humoral immune response characterized by elevated antibody level to a great extent.
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@#Objective To compare the sensitivity(dilution) of antigen-detecting rapid diagnostic cards for severe acute respiratory symptom coronavirus 2(SARS-CoV-2) at home and abroad to different strains.Methods Vaccine bulks of four SARS-CoV-2 strains(original strain,Beta,Delta and Omicron) produced by Wuhan Institute of Biological Products Co.,Ltd.were used as the sample panel for sensitivity assessment,of which a series of diluted samples were detected by using 21 batches of SARS-CoV-2 antigen-detecting rapid diagnostic cards from 17 domestic and foreign manufacturers and SARS-CoV-2 nucleic acid detection reagent from Shanghai GeneoDx Biotech Co.,Ltd,respectively.The sensitivity of antigendetecting rapid diagnostic cards and nucleic acid detection reagent was evaluated according to the dilutions.The results of SARS-CoV-2 antigen-detecting rapid diagnostic reagents and nucleic acid detection reagent were compared to determine the nucleic acid detection Ct value corresponding to the group of antigen-detecting rapid diagnostic reagent with the highest dilution,namely the highest sensitivity.Results The sensitivity of antigen detection cards for the vaccine bulks of original strain,Beta,Delta and Omicron was 1:10～1:8 × 10~4.1:10~3～1:2 × 10~5,1:10~2～1:4 × 10~4,and 1:10～1:4 × 10~5,respectively;The sensitivity of nucleic acid detection cards was 10~(-6),10~(-5),10~(-4) and 10~(-7),respectively.The Ct values of N gene which were reached by high sensitivity antigen-detecting rapid diagnostic cards were as follows:original strain(10~(-4)) of more than 31,Beta variant(10~(-5)) of more than 36,Delta variant(10~(-4)) of more than 34,Omicron variant(10~(-5)) of more than 33,meeting the requirements of domestic and European Union for SARS-CoV-2 antigen-detecting rapid diagnostic cards.Conclusion All the antigen-detecting rapid diagnostic cards detected the four virus strains,while the sensitivity of different reagents to different variants varies to some extent,among which the sensitivity to Omicron variant varies the most.
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@#Objective To optimize the culture conditions of four vaccine candidates of severe acute respiratory symptom coronavirus 2(SARS-CoV-2) Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells.Methods The harvest time(24,48,72 and 96 h) and MOI(0.01,0.001,0.0001 and 0.000 01) of four Omicron variants cultured in Vero cells were optimized by using cytopathic effect(CPE),viral nucleic acid copy number and viral titer as evaluation indexes.Results The optimum harvest time of the four Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells was 72 h,and the optimum MOI was 0.001～0.000 01,0.001～0.000 01,0.01～0.000 01 and 0.01～0.000 01,respectively.Conclusion The culture conditions of four Omicron variants in Vero cells were optimized,which laid a foundation of the development of SARS-CoV-2 Omicron variant inactivated vaccine based on Vero cells.
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@#ObjectiveTo construct self-amplifying RNA(saRNA)vaccine of severe acute respiratory syndrome coronavirus2(SARS-CoV-2)Delta mutant strain(B.1.617.2)based on Coxsackievirus-A5(CV-A5)replicon and evaluate its immunogenicity.MethodsThe recombinant plasmids pDelta-S10,pDelta-S5 and pDelta-S1(10,5 and 1 amino acid residues at the upstream of S-VP1/2A cleavage site of the fusion polyprotein respectively)were constructed by In-fusion cloning of the plasmids containing the full-length genome sequence of CV-A5 and substituting the S protein gene of SARS-CoV-2 Delta mutant for the P1 structural protein gene of CV-A5 with different lengths.Three RNA molecules,Delta-S10,Delta-S5 and Delta-S1,were obtained by in vitro transcription of linearized recombinant plasmids and transfected into HEK-293T cells respectively,which were analyzed for the expression of S protein by Western blot.The RNA molecule with the highest expression of S protein was screened out and detected for the self-amplification in HEK-293T cells by qPCR.BALB/c mice(female,6 ～ 8 weeks old and five for each group)were immunized i.m.with two doses(0.5 and 2.5 μg)of the screened Delta-S packaged with lipid nanoparticles for once on day 1 and day 14 seperately.Blood samples were collected on days 14and 28,detected for serum binding antibody titers by ELISA,and detected for neutralizing antibody titers by micro neutralization method.The spleens were harvested on day 42 and detected for the level of IFNγ secreted by mouse spleen cells by enzyme linked enzyme linked immunospot assay(ELISPOT).ResultsThe recombinant RNA molecule Delta-S10showed the highest expression of S protein and self-amplified in HEK-293T cells,which of both high and low doses induced specific binding antibody against SARS-CoV-2 Delta S1 protein in mice with obvious dose effect and enhanced immune effect;The high dose of Delta-S10 induced neutralizing antibodies and cellular immune responses in mice.ConclusionThe SARS-CoV-2 Delta mutant(B.1.617.2)saRNA vaccine Delta-S10 based on CV-A5 replicon was successfully constructed,which induced humoral and cellular immune responses in mice,laying a foundation of the further study of the construction of SARS-CoV-2 saRNA vaccine by enterovirus replication elements.