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Quantifying low-frequency revertants in oral poliovirus vaccine using next generation sequencing
Eric Sarcey1, Aurélie Serres1, Fabrice Tindy1
1Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France.
Journal of Virological Methods
|May 1, 2017
Summary
Next-generation sequencing (NGS) offers a reliable alternative to the established Mutant Analysis by Polymerase Chain Reaction and Restriction Enzyme Cleavage (MAPREC) method for monitoring poliovirus vaccine safety. This advanced technique accurately quantifies critical revertant mutations in oral poliovirus vaccine serotype 3.
Area of Science:
- Virology
- Vaccine Development
- Molecular Biology
Background:
- Live attenuated oral poliovirus vaccine (OPV) serotype 3 requires monitoring for spontaneous reversion to neurovirulence, primarily due to the 472U>C mutation, to ensure vaccine safety and consistency.
- The World Health Organization-endorsed Mutant Analysis by Polymerase Chain Reaction and Restriction Enzyme Cleavage (MAPREC) is the standard in vitro test but involves radiolabeling, posing challenges for many laboratories.
- There is a need for alternative, modern methods to effectively monitor OPV serotype 3 revertants.
Purpose of the Study:
- To evaluate the performance and suitability of next-generation sequencing (NGS) as a viable alternative to MAPREC for detecting and quantifying OPV serotype 3 revertants.
- To assess the accuracy, reproducibility, and comparability of NGS against the established MAPREC method.
Main Methods:
- Next-generation sequencing (NGS) was employed to analyze oral poliovirus vaccine serotype 3 (OPV3) working seed lots and monovalent bulks (n=21).
- The linearity of NGS was confirmed across a range of 0.25%-1.5% revertant concentrations.
- NGS performance metrics including repeatability, intermediate precision, and reproducibility were assessed across different sequencing platforms and analysis software.
Main Results:
- NGS demonstrated linearity, comparable repeatability and intermediate precision, and high reproducibility across all tested samples and platforms.
- Results from NGS were highly correlated with those obtained from MAPREC for the quantification of 472-C revertants (Pearson correlation coefficient r=0.95585, p<0.0001).
- NGS accurately quantified low-frequency OPV serotype 3 revertants, showing statistically equivalent performance to MAPREC.
Conclusions:
- Next-generation sequencing (NGS) is a suitable and valid alternative to MAPREC for monitoring spontaneous reversion in oral poliovirus vaccine serotype 3.
- NGS provides a modern, non-radiolabeling method that meets the requirements for ensuring vaccine safety and consistency in production.
- The adoption of NGS can facilitate continued robust monitoring of OPV serotype 3 production by laboratories phasing out radiolabeling techniques.
