Establishment and validation of pseudovirus-based neutralization assay for in vivo potency of SARS-CoV-2 mRNA vaccine
Objective To establish and verify a stable pseudovirus-based neutralization assay(PBNA)for the detection of the potency in vivo of SARS-CoV-2 mRNA vaccine so as to use the method for the quality control of SARS-CoV-2 mRNA vaccines.Methods Three different immunization schedules of BALB/c mice(half male and half female)were set up,and each immunization schedule had two doses of vaccine.The titer of neutralizing antibody(Nab)in serum was detected by PBNA,and the appropriate immunization schedule was screened.After the immunization schedule was determined,the mice were immunized at the dose in the range of 0.5-6 µg/mouse to further determine the linear range between serum Nab titer and immunization dose.The PBNA method for detecting the potency of SARS-CoV-2 mRNA vaccine in vivo was established,and the relative accuracy,intermediate precision,linearity and range of the method were verified.In addition,the potency in vivo of three batches of Omicron XBB.1.5 variant SARS-CoV-2 mRNA vaccine was detected by using the established method,and the results were compared with those of the original detection method of the enterprise.Results The immunization schedule of SARS-CoV-2 mRNA vaccine was determined as follows:the blood samples were taken 7 d after the booster immunization which was conducted 14 d after the primary immunization,the immune dose was 2 μg/mouse,with the linear dose range of 0.5-3 µg/mouse.A PBNA method for detecting the potency in vivo of SARS-CoV-2 mRNA vaccine in mice was established.The results of methodological verification showed that the relative bias(RB)of each titer level detected by the method was within±20％,meeting the requirements of relative accuracy.The geometric coefficient of variation(GCV)of each titer level measured six times was less than 50％,meeting the intermediate precision requirements.The linear regres-sion equation was fitted as y=1.003 4 x-0.011 7,r=0.887 0,and the linear regression was significant(P=9.67 × 10-15),in accord with the linear acceptability criteria.The titer level ranged from 25％to 150％,which met the quality standard range of in vivo potency of common vaccines.The method was used to detect three batches of Omicron XBB.1.5 variant SARS-CoV-2 mRNA vaccine,and the results were consistent with those of the original method,indicating that the method could be used for in vivo potency detection.Conclusion A stable PBNA method was established to detect the potency of SARS-CoV-2 mRNA vaccine in vivo,and can be applied to the quality control of SARS-CoV-2 mRNA vaccines.
SARS-CoV-2mRNA vaccinePotency in vivoPseudovirus-based neutralization assay(PBNA)
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