Phenotypic and genetic characterization of a next generation live-attenuated yellow fever vaccine candidate.

We assessed the genetic and phenotypic characteristics of a yellow fever vaccine candidate, which was cloned from a YF-VAX substrain selected for growth in Vero cells (vYF-247), during the manufacturing process from the master seed lot (MSL) and working seed lot (WSL) through to the drug substance (DS) stage. There were nine minor nucleotide variants observed from the MSL to the DS stage, of which five led to amino acid changes. The variant positions were, however, not known risks for any virulence modification. vYF-247 exhibits a homogenous plaque size profile (as expected for a cloned vaccine candidate) composed of small plaques (<1 mm) that remained consistent throughout the manufacturing process. In addition, there was no change in the viral replication rate. Of note, the DS sequences across the two manufacturing campaigns (2018 and 2019) were very similar suggesting a high batch-to-batch consistency. All MSL, WSL and DS batches exhibited similar neurovirulence profiles in mice and had a more attenuated neurovirulence phenotype than the YF-VAX (egg-based vaccine) comparator. Overall, the neurovirulence phenotype of vYF-247 does not change from MSL, WSL to DS. These data collectively support the safety and genetic stability of vYF-247 during the production process.

Multiplex reverse transcriptase droplet digital PCR for the simultaneous quantification of four dengue serotypes: Proof of concept study.

During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.

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

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.