transgene cassette. AdV vector passages in a cell culture might result in de novo

acquisition of E1 region and replication competent AdV (RCA) [32]. AdV-based

COVID-19 vaccines such as Ad26.COV2-S and Ad5-nCoV use the first-generation

adenovirus with the E1 and E3 genes deleted [1]. Detailed information of AdV-

based COVID-19 vaccines will be further discussed in 11.2.3.3.

Third-generation AdV vectors, or gutless AdV vectors where all viral proteins have

been removed which can significantly reduce the immune response of host cells upon

AdV vector transduction [28] have been mainly used in gene therapy interventions.

11.2.2

PRODUCTION PROCESS OF ADV VECTORS

11.2.2.1

Cell Line Selection

Though many cell lines such as HER-911, HeLa, and HEL-299 are permissive to

AdV infection, the most common cell line used for AdV replication is the Human

Embryo Kidney 293 (HEK-293) cell [33]. The HEK-293 was transformed with

sheared AdV DNA resulting in the incorporation of the gene expressing the HAd5

E1 protein [34,35]. Since the first-generation AdV vectors are replication-defective

due to the loss of E1 region, the HEK-293 cell is used as a complementing cell

line expressing the HAd5 E1 protein, making it possible to propagate the defective

vector in HEK-293 cells. However, HEK-293 cell line incorporates not only the

HAd5 E1 gene, but also viral sequences flanking the E1 region, and thus RCAs can

then be generated through homologous recombination [36]. To overcome this issue,

a PER.C6 cell line, derived from human embryonic retinal cells, was generated,

which contains only E1 elements, such that the effect of homologous recombination

is reduced [37]. For the COVID-19 vaccine developed by CanSino, the recombinant

HAd5 vectored vaccine was amplified by serial passage on HEK293 cells [38].

Though HAd5-based vectors have efficient replication ability in HEK-293 and

PER.C6 cells, some rare serotypes AdV vectors replicate at significantly lower

yields [39]. Research work from Havenga et al. showed that the production of

HAd35 can be significantly enhanced by expressing HAd5 E4-ORF6 protein in

producer cell lines [40]. Comparable yields were observed for other AdV serotypes

such as HAd26, HAd48, and HAd49, using a similar strategy. The E1/E3-deleted

Ad26-vectors adopted by Janssen/Johnson & Johnson (Ad26.COV2-S) were gen-

erated in PER.C6.TetR cells using a plasmid containing a transgene expression

cassette and the genome of Ad26 vector [41]. The production of non-human ser-

otypes AdV vectors is more challenging because the species-specific producer cell

lines may lack the scalability of HEK-293 or PER.C6 cells. The Madin-Darby

Canine Kidney Epithelial (MDCK) cells were engineered to express the CAd2 E1

protein to produce Cad2 [42]. In general, HEK-293 is still an extensively used cell

line for manufacturing the AdVs with the restriction in terms of RCA generation.

The COVID-19 vaccine ChAdOX1-nCoV from Oxford/AstraZeneca used a

ChAdY25 vector. The virus was propagated in T-Rex HEK293 cells [43].

11.2.2.2

Limitations in the Production of AdV

To increase the viral production, the first objective is to maximize the production yield

on a per cell basis. However, one of the critical limitations in the production of AdV is

Vectored vaccines

277