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Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines.

ABSTRACT: Intrinsic genetic instability of RNA viruses may lead to the accumulation of revertants during manufacture of live viral vaccines, requiring rigorous quality control to ensure vaccine safety. Each lot of oral poliovirus vaccine (OPV) is tested for neurovirulence in animals and also for the presence of neurovirulent revertants. Mutant analysis by PCR and restriction enzyme cleavage (MAPREC) is used to measure the frequency of neurovirulent mutations at the 5' untranslated region (UTR) of the viral genome that correlate with the level of neurovirulence determined by the monkey neurovirulence test. However, MAPREC can only monitor mutations at a few genomic loci and miss mutations at other sites that could adversely affect vaccine quality. Here we propose to use massively parallel sequencing (MPS) for sensitive detection and quantification of all mutations in the entire genome of attenuated viruses. Analysis of vaccine samples and reference preparations demonstrated a perfect agreement with MAPREC results. Quantitative MPS analysis of validated reference preparations tested by MAPREC produced identical results, suggesting that the method could take advantage of the existing reference materials and be used as a replacement for the MAPREC procedure in lot release of OPV. Patterns of mutations present at a low level in vaccine preparations were characteristic of seed viruses used for their manufacture and could be used for identification of individual batches. This approach may represent the ultimate tool for monitoring genetic consistency of live viral vaccines.

SUBMITTER: Neverov A

PROVIDER: S-EPMC2993378 | biostudies-other | 2010 Nov

REPOSITORIES: biostudies-other

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Project description:There have been two major eras in the history of gene discovery. The first was the era of linkage analysis, with approximately 1,300 disease-related genes identified by positional cloning by the turn of the millennium. The second era has been powered by two major breakthroughs: the publication of the human genome and the development of massively parallel sequencing (MPS). MPS has greatly accelerated disease gene identification, such that disease genes that would have taken years to map previously can now be determined in a matter of weeks. Additionally, the number of affected families needed to map a causative gene and the size of such families have fallen: de novo mutations, previously intractable by linkage analysis, can be identified through sequencing of the parent-child trio, and genes for recessive disease can be identified through MPS even of a single affected individual. MPS technologies include whole exome sequencing (WES), whole genome sequencing (WGS), and panel sequencing, each with their strengths. While WES has been responsible for most gene discoveries through MPS, WGS is superior in detecting copy number variants, chromosomal rearrangements, and repeat-rich regions. Panels are commonly used for diagnostic purposes as they are extremely cost-effective and generate manageable quantities of data, with no risk of unexpected findings. However, in instances of diagnostic uncertainty, it can be challenging to choose the right panel, and in these circumstances WES has a higher diagnostic yield. MPS has ethical, social, and legal implications, many of which are common to genetic testing generally but amplified due to the magnitude of data (e.g., relationship misattribution, identification of variants of uncertain significance, and genetic discrimination); others are unique to WES and WGS technologies (e.g., incidental or secondary findings). Nonetheless, MPS is rapidly translating into clinical practice as an extremely useful part of the clinical armamentarium.

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Massively parallel sequencing for monitoring genetic consistency and quality control of live viral vaccines.

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