![]() ![]() ![]() For adenoviral constructs, this often means replacement of the E1 gene, possibly the E3 or other genes with the genetic payload of choice ( Farina et al., 2001 Tatsis et al., 2006). Recent successes using these constructs include modified chimpanzee adenovirus type 3 ebolavirus vaccine (cAd3-EBO) ( Stanley et al., 2014 Ledgerwood et al., 2017) and replication-deficient simian adenoviral vectored vaccine ChAdOx1 nCoV-19 against SARS-CoV-2 ( Folegatti et al., 2020 Ewer et al., 2021 Putter, 2021).įundamentally, recombinant viral vectors have a genomic composition different than native virus. Non-human primate adenoviral vectors are attractive therapeutic vectors as they share similar beneficial characteristics of human adenovirus like robust antigen expression, but with reduced levels of neutralizing antibodies ( Capone et al., 2013 Cheng et al., 2015 Zhang et al., 2017 Zhao et al., 2018). In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. ![]()
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