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. 2020 Oct;84(4):314-318.

A potential approach for assessing the quality of human and nonhuman adenoviral vector preparations

Ekramy E Sayedahmed  1 , Suresh K Mittal  1
Affiliations

A potential approach for assessing the quality of human and nonhuman adenoviral vector preparations

Ekramy E Sayedahmed et al. Can J Vet Res. 2020 Oct.

Abstract
in English, French

Various types of human and nonhuman adenoviral (AdV) vectors are being used as gene delivery vectors in preclinical and clinical investigations. The objective of this study was to determine the ratio between the 2 best assays that would effectively address the variability in the titration of various AdV vectors in different cell lines and help obtain consistent results in preclinical and clinical studies using different AdV vectors. Here, we compared plaque-forming units, tissue culture infectious dose 50, focus-forming units (FFU), virus particle (VP) count, and genome copy number (GCN) of purified preparations of human AdV type C5, bovine AdV type 3, and porcine AdV type 3 to determine a correlation between infectious and noninfectious virus particles. Our results suggest that a VP:FFU or a VP:GCN ratio could accurately reflect the quality of an AdV preparation and could serve as an indicator to control batch-to-batch variability.

Différents types de vecteurs à adénovirus (AdV) humain et non-humain sont utilisés comme vecteurs de livraison de gènes dans des études pré-cliniques et cliniques. L’objectif de la présente étude était de déterminer le ratio entre les deux meilleurs essais qui examinerait la variabilité dans la titration des différents vecteurs AdV dans différentes lignées cellulaires et aiderait à obtenir des résultats fiables dans des études précliniques et cliniques utilisant différents vecteurs AdV. Nous avons comparé les unités formatrices de plaques, la dose infectieuse 50 pour la culture de tissu, les unités formatrices de focus (FFU), le dénombrement des particules virales (VP), et le nombre de copies du génome (GCN) de préparations purifiées d’AdV humain type C5, d’AdV bovin type 3, et d’AdV porcin type 3 afin de déterminer une corrélation entre les particules virales infectieuses et non-infectieuses. Nos résultats suggèrent qu’un ratio VP:FFU ou VP:GCN pourrait refléter avec précision la qualité d’une préparation d’AdV et pourrait servir d’indicateur pour vérifier la variabilité d’une production à l’autre.(Traduit par Docteur Serge Messier).

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Figures

Figure 1
Figure 1
Focus-forming units (FFU) assay for human adenovirus type C5 (HAdV-C5), bovine adenovirus type 3 (BAdV-3), and porcine adenovirus type 3 (PAdV-3). Human embryonic kidney (HEK) 293, Madin-Darby bovine kidney (MDBK), or porcine kidney (PK)-15 cells were infected with serially log-diluted purified preparations of HAdV-C5, BAdV-3, or PAdV-3, respectively.
Figure 2
Figure 2
Titration of human adenovirus type C5 (HAdV-C5), bovine adenovirus type 3 (BAdV-3), and porcine adenovirus type 3 (PAdV-3) by plaque-forming units (PFU), tissue culture infectious dose 50 (TCID50), focus-forming units (FFU), virus particle (VP), and genome copy number (GCN). Titers of A — HAdV-C5, B — BAdV-3, and C — PAdV-3 by various assays and VP ratios with other titration assays for D — HAdV-C5, E — BAdV-3, and F — PAdV-3 are shown. The statistical analysis was done by unpaired t-test with Welch’s correction. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns — not significant at P > 0.05.
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