A Method to Generate and Rescue Recombinant Adenovirus Devoid of Replication-Competent Particles in Animal-Origin-Free Culture Medium

Abstract

Adenoviruses are promising vectors for vaccine production and gene therapy. Despite all the efforts in removing animal-derived components such as fetal bovine serum (FBS) during the production of adenovirus vector (AdV), FBS is still frequently employed in the early stages of production. Conventionally, first-generation AdVs (E1 deleted) are generated in different variants of adherent HEK293 cells, and plaque purification (if needed) is performed in adherent cell lines in the presence of FBS. In this study, we generated an AdV stock in SF-BMAdR (A549 cells adapted to suspension culture in serum-free medium). We also developed a limiting dilution method using the same cell line to replace the plaque purification assay. By combining these two technologies, we were able to completely remove the need for FBS from the process of generating and producing AdVs. In addition, we demonstrated that the purified AdV stock is free of any replication-competent adenovirus (RCA). Furthermore, we demonstrated that our limiting dilution method could effectively rescue an AdV from a stock that is highly contaminated with RCA.

Keywords: adenovirus vector; animal-origin-free culture media; limiting dilution; replication-competent adenovirus.

Figure 1

Workflow for rescuing recombinant adenoviruses from stock highly contaminated with RCA via limiting dilution and characterization of AdV clones. This protocol consists of three steps. (I) Limiting dilution step in which 48 wells were infected with 6 serial log₂ dilutions (5 to 0.16 ifu) per well of the AdV/RCA mixed stock. (II) Detection step in which positive wells were identified by immunostaining using HEK293A cells. (III) Differentiation step in which, after one additional round of clone amplification in SF-BMAdR cells, each clone infects HEK293A and A549 cells in parallel to differentiate whether they are rAdV or RCA.

Figure 2

RCA detection in A549 cells. (A) Presence of RCA was evaluated in purified virus stock by two successive passages in A549 cells followed by the immunostaining over the third passage. In the second passage, the dishes in the control group showed CPE since 6 dpi (dish infected with 3 IU of wild-type shown) but no CPE was detected in the eight test dishes until the end of the experiment. (B) For immunostaining, the A549 cells were infected with different dilutions of cell lysis harvested from the second amplification. Only one photo from each virus dilution is shown. The first row shows the control group in the first passage co-infected with 3 IU of wild-type adenovirus. The second to fourth rows show three out of eight test dishes infected with only E1-deleted AdV at the first passage. The last row shows noninfected cells.