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Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral
K M Chumakov1, L B Powers, K E Noonan
1Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.
Abstract:
Production of live attenuated oral poliomyelitis vaccine (OPV) requires rigorous neurovirulence safety testing of each vaccine lot, currently carried out in monkeys. It has been reported that a change from 472-U to 472-C in the type 3 OPV RNA is associated with an increased histologic lesion score produced upon intraspinal inoculation of the mutant virus in monkeys. We have developed a method, based on polymerase chain reaction, for measuring the relative abundance of these mutant sequences directly in vaccine preparations and used this method to evaluate the proportion of 472-C in 40 different lots of type 3 OPV. Six vaccine lots that had failed the intraspinal monkey neurovirulence test contained a higher proportion of 472-C than all other lots that had passed this test. OPV type 3 virus containing 472-C was rapidly selected during serial passages in African green monkey kidney cells that are used for manufacturing of the vaccine. We have also found that the wild-type poliovirus type 3 strain Leon/37, from which the vaccine strain was originally derived, contained a mixture of 472-U and 472-C sequences. No other mutations in OPV type 3 RNA have been detected by similar assays at position 2034, also associated with attenuation, or at several other positions reported to be altered in some vaccine preparations. Our results suggest that molecular diagnostics may provide a supplement or a potential alternative to animal testing of live attenuated vaccines.
Insights
A new molecular diagnostic test can detect a specific mutation (472-C) in oral poliovirus vaccine (OPV) type 3. This mutation is linked to neurovirulence, potentially offering an alternative to animal safety testing.
Area of Science:
- Virology
- Vaccinology
- Molecular Diagnostics
Background:
- Live attenuated oral poliomyelitis vaccine (OPV) production necessitates rigorous neurovirulence safety testing.
- Current safety testing relies on animal models, specifically monkeys, which is time-consuming and resource-intensive.
- A specific RNA mutation (472-U to 472-C) in type 3 OPV has been linked to increased neurovirulence in monkeys.
Purpose of the Study:
- To develop and validate a molecular diagnostic method for quantifying the 472-C mutation in type 3 OPV.
- To assess the correlation between the proportion of 472-C and the results of the monkey neurovirulence test.
- To investigate the selection of the 472-C mutant during vaccine manufacturing and its presence in the original wild-type strain.
Main Methods:
- Development of a polymerase chain reaction (PCR)-based assay to measure the relative abundance of 472-C sequences.
- Analysis of 40 different lots of type 3 OPV using the developed PCR assay.
- Serial passage of OPV type 3 in African green monkey kidney cells to observe viral evolution.
- Sequencing assays to detect other potential mutations at specific RNA positions.
Main Results:
- Six vaccine lots that failed the monkey neurovirulence test showed a higher proportion of 472-C compared to lots that passed.
- The 472-C mutant was rapidly selected during serial passages in cell cultures used for vaccine manufacturing.
- The original wild-type poliovirus type 3 strain (Leon/37) contained a mixture of 472-U and 472-C sequences.
- No other significant mutations at tested positions (e.g., 2034) were detected by the molecular assays.
Conclusions:
- The proportion of 472-C in type 3 OPV is a potential indicator of neurovirulence.
- Molecular diagnostics, such as the developed PCR assay, could supplement or replace animal testing for OPV safety.
- Understanding viral evolution during manufacturing is crucial for vaccine safety and quality control.