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J Virol Methods. 2017 Aug;246:75-80. doi: 10.1016/j.jviromet.2017.04.016. Epub 2017 Apr 26.

Quantifying low-frequency revertants in oral poliovirus vaccine using next generation sequencing.

Author information

1
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Eric.Sarcey@sanofi.com.
2
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Aurelie.Serres@sanofi.com.
3
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Fabrice.Tindy@sanofi.com.
4
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Audrey.Chareyre@sanofi.com.
5
Sanofi Pasteur, Microbiology & Virology Platform, Department of Analytical Research & Development North America, 1755 Steeles Avenue West, Toronto, Ontario M2R 3T4, Canada. Electronic address: Siemon.Ng@sanofi.com.
6
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Marine.Nicolas@sanofi.com.
7
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Emmanuelle.Vetter@sanofi.com.
8
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Thierry.Bonnevay@sanofi.com.
9
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Eric.Abachin@sanofi.com.
10
Sanofi Pasteur, Analytical Research and Development Department EU, Campus Mérieux-1541, Avenue Marcel Mérieux, 69280, Marcy L'Etoile, France. Electronic address: Laurent.Mallet@sanofi.com.

Abstract

Spontaneous reversion to neurovirulence of live attenuated oral poliovirus vaccine (OPV) serotype 3 (chiefly involving the n.472U>C mutation), must be monitored during production to ensure vaccine safety and consistency. Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has long been endorsed by the World Health Organization as the preferred in vitro test for this purpose; however, it requires radiolabeling, which is no longer supported by many laboratories. We evaluated the performance and suitability of next generation sequencing (NGS) as an alternative to MAPREC. The linearity of NGS was demonstrated at revertant concentrations equivalent to the study range of 0.25%-1.5%. NGS repeatability and intermediate precision were comparable across all tested samples, and NGS was highly reproducible, irrespective of sequencing platform or analysis software used. NGS was performed on OPV serotype 3 working seed lots and monovalent bulks (n=21) that were previously tested using MAPREC, and which covered the representative range of vaccine production. Percentages of 472-C revertants identified by NGS and MAPREC were comparable and highly correlated (r≥0.80), with a Pearson correlation coefficient of 0.95585 (p<0.0001). NGS demonstrated statistically equivalent performance to that of MAPREC for quantifying low-frequency OPV serotype 3 revertants, and offers a valid alternative to MAPREC.

PMID:
28456668
DOI:
10.1016/j.jviromet.2017.04.016
[Indexed for MEDLINE]
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