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. 2020 Feb 20:7:100806.
doi: 10.1016/j.mex.2020.100806. eCollection 2020.

Titration methods for rVSV-based vaccine manufacturing

Jean-François Gélinas  1 , Sascha Kiesslich  1 , Rénald Gilbert  1   2 , Amine A Kamen  1
Affiliations

Titration methods for rVSV-based vaccine manufacturing

Jean-François Gélinas et al. MethodsX. .

Abstract

The recombinant Vesicular Stomatitis Virus (rVSV) is an emerging platform for viral vector-based vaccines. Promising results have been reported in clinical trials for the rVSV-ZEBOV vaccine for Ebola virus disease prevention. In this study, we describe the titration tools elaborated to assess the titre of rVSV-ZEBOV productions. • A streamlined Median Tissue Culture Infectious Dose (TCID50) assay to determine the infectious titer of this vaccine was established. • A digital polymerase chain reaction (dPCR) assay to assess the total number of viral particles present in cell-free culture supernatants of rVSV productions was developed. • These assays are used to titre rVSV-ZEBOV samples and characterize the ratio of total particles to infectious units for monitoring process robustness and product quality attributes and can be used to titre samples generated in the production of further rVSV vectors.

Keywords: Ebola; HIV; TCID50; dPCR; rVSV-HIV; rVSV-ZEBOV.

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Figures

Fig 1
Fig. 1
Production and titration variability using TCID50. Functional titres were measured by TCID50. Bars represent the mean of the twelve samples ± standard deviation. A) Titration repeatability. Independent titration by TCID50 in 12 replicates on the same day of a single production sample. B) Titration intermediate precision. Independent titrations by TCID50 repeated on 12 separate days for aliquots of the same production. C) Production repeatability. Production yields for 12 independent infections with rVSV-ZEBOV at MOI 0.001 of two 6 well plates containing 1 × 106 cells/mL in 2 mL per well.
Fig 2
Fig. 2
Histogram of dPCR analysis of a dilution series of cDNA extracted from rVSV-ZEBOV. The extracted cDNA was diluted in a 1:2 dilution series starting from 1:100 (sample F02) to 1:102,400 sample (H03). Sample A04 consisted of a non-template control. Channel 1 amplitude is given in arbitrary fluorescent units. Positive events are marked in blue, negative events in gray.
Fig 3
Fig. 3
Histogram of dPCR analysis of rVSV-ZEBOV. This exemplary histogram shows the data of an rVSV-ZEBOV sample at a cDNA dilution resulting in a clear peak resolution of dPCR events. Channel 1 amplitude is given in arbitrary fluorescent units. Positive events (blue) typically peaked around 20,000 to 25,000 and negative events (gray) peaked between 5000 and 8000.
Fig 4
Fig. 4
Titration variability of rVSV-ZEBOV using dPCR. Viral genome copy number was measured by dPCR. Bars represent the mean of the twelve samples ± standard deviation. A) Total assay variability. dPCR analysis of the same sample with 12 independent RNA extractions. B) Combined reverse transcription and dPCR variability. dPCR analysis of the same RNA extract with 12 independent reverse transcriptions. C) dPCR variability. dPCR analysis was performed 12 times of the same cDNA.
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