Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

Abstract

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 ؋ 10 12 vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

Human amniocyte‐derived cells are a promising cell host for adenoviral vector production under serum‐free conditions

“…The PER.C6® cell line has been widely adopted by the industry for AdV production, mainly due to its improved documentation of history and properties [5]. However, in two publications regarding PER.C6® cells, the generation of unusual vector recombinants has been observed, resulting in vector specimens carrying and expressing E1 functions [8, 9]. Moreover, PER.C6® prices and licensing are also prohibitive for academic institutions, limiting the progress of R&D for new clinical gene therapy and vaccine application using these cells.…”

Adenoviral Vector Vaccines Antigen Transgene

“…PER.C6 cells offer an alternative 27 ; they only carry the E1 domain of HAdV-5, which reduces the chance of insertion of E1 into the genome of HAdV-5 vectors. 28 For other serotypes, packaging cells have to be constructed typically by stable E1 transfection of a cell line that is readily infected with adenoviruses.…”

Adenovirus as a carrier for the development of influenza virus-free avian influenza vaccines

“…The PER.C6 cell line contains only Ad5 nucleotides 459–3510, which precludes double-crossover-type homologous recombination between the PER.C6 genome and a matched Ad5 vector with this E1 subfragment deleted. However, a helper-dependent E1-positive particle (HDEP) occurs at an extremely low frequency (not more than one HDEP in 7.5 × 10 12 Ad5 particles) during production of Ad5 vectors in PER.C6 cells [77]. Unlike RCA, HDEP contains E1 derived from the PER.C6 genome but are devoid of E2 , E3 , E4 and late genes and, hence, cannot replicate independently [77].…”

“…However, a helper-dependent E1-positive particle (HDEP) occurs at an extremely low frequency (not more than one HDEP in 7.5 × 10 12 Ad5 particles) during production of Ad5 vectors in PER.C6 cells [77]. Unlike RCA, HDEP contains E1 derived from the PER.C6 genome but are devoid of E2 , E3 , E4 and late genes and, hence, cannot replicate independently [77]. In contrast to RCA that outgrows E1 -defective recombinant Ad5 vector in culture [76], HDEP could be diluted out over time under low multiplicity of infection (MOI) due to its replicative dis advantage.…”