Evaluation and validation of next-generation sequencing to support lot release for a novel type 2 oral poliovirus vaccine

Abstract

Highlights Genetic variants were evaluated to assess which were important to ensure nOPV2 quality. The cDNA preparation and NGS method was validated through evaluating mixtures of Sabin-2 and nOPV2. Pre-specified validation criteria for linearity and precision were met at all positions. The method was assessed to be fit-for-purpose for vaccine lot release. Understanding the co-location of genetic variants was important to interpret NGS results.

Evaluating stability of attenuated Sabin and two novel type 2 oral poliovirus vaccines in children

“…These are potentially relevant to neurovirulence and viral fitness, though largely anticipated from prior work studying the evolution of the Sabin-2 and nOPV2 strains. Some mutations previously identified in the nOPV2-c1 vaccine lots and characterized phenotypically 16 showed anticipated behavior upon replication in vivo. For example, VP1-E295K—which causes a temperature-sensitive defect limiting replication at 37 °C—was selected against unless compensated for by the presence of a VP1-N171D mutation.…”

“…Mutations associated with amino acid changes and meeting reporting requirements are summarized in Supplementary Table 4 . Some of these (VP3-E234K, VP1-N171D and VP1-E295K) are present at moderate levels in the vaccine lots and thus are expected to appear in shed virus 16 . For example, the level of VP3-E234K in the lot used in this study was 45%.…”

“…Polymorphisms (present at ≥1%) were reported as SNPs with the impact on amino acid coding of the SNP noted per SNP. Re-analysis of select FASTQ files was conducted in Geneious Prime 2020.0.5 (Biomatters) to assess the possible co-location of VP1-143, VP1-N171D and VP1-E295K in nOPV2-c1 shed virus samples 16 . In brief, for these analyses, reads were merged and mapped against reference sequences from nucleotides 2969-3054 for 143/171 or 3052-3426 for 171/295, requiring the mapped reads to cover the full reference.…”

High throughput AS LNA qPCR method for the detection of a specific mutation in poliovirus vaccine strains

“…When using attenuated strains as an alternative in IPV manufacturing, this biosafety hazard was considered significantly lower when compared with using wild-type stains. However, attenuated Sabin poliovirus strains may revert to neurovirulence before inactivation in rare cases (5′UTR nucleotide 481-G as key neurovirulence marker in Sabin PV2 [sPV2]) [15] and therefore still present a potential biosafety hazard [16] should they escape during the manufacturing process. Current manufacturing processes that use attenuated Sabin strains during vaccine production (sIPV) allow for process modernization and optimization, thereby increasing containment safety and reducing costs [17] .…”

Next-Generation Sequencing: A Promising Tool for Vaccines and Other Biological Products

“…It is the speed and accuracy of the NGS that allows scientists to sequence multiple genomic variants of target viruses and develop potential vaccines [ 3 ]. Additionally, NGS can ensure that genetically modified regions of the vaccine virus genome remain as designed and, in this context, a recent study confirmed NGS to be suitable for vaccine lot release [ 4 ]. NGS has proven to be indispensable during the recent COVID-19 pandemic where the progress from pathogen discovery to novel vaccine development was made in record time.…”