evaluate their efficacy for vaccination [15]. A heterologous Ad26-vectored vaccine

against Zaire Ebola virus was authorized by European Medicines Agency on July 1,

2020 (https://www.ema.europa.eu/en/medicines/human/EPAR/zabdeno). In the race

of COVID-19 vaccines, Janssen used HAd26 as the vector to express stabilized pre-

fusion spike protein of SARS-CoV-2. AstraZeneca selected a chimpanzee AdV

(ChAdY25) as the vector to build ChAdOX1-nCoV.

Despite successful application and safety results of the current AdV vectored

vaccines, enhancement of immunogenicity and long-lasting immunity are still to be

assessed. In the development of vaccines against COVID-19, an intranasal route has

been used for sterilizing immunity in preclinical trials [25]. Intranasal administra-

tion of vaccine can mitigate inactivation of the vectors because of pre-existing

immunity, additionally to the ease of vaccine administration. Genetic modifications

proved to be a promising method to evade pre-existing immunity, and create cell-

specific vectors for immunization enhancement [26,27]. The continuing research on

AdV vectors will hopefully lead to significant improvement of AdV vectored

vaccines by stimulation of strong immune response, while maintaining a high safety

profile with rare side effects contributing to mankind health’s protection.

11.2.1

ADV STRUCTURE AND VECTOR DESIGN

The non-enveloped 90–100 nm icosahedral capsid of AdV is composed of penton

and hexon sub-units [28]. Fiber and knob domains are associated with pentons that

mediate attachment to host cells [29], as shown in Figure 11.1. Affinity of the knob

domain for various cellular receptors varies depending on serotype, with the cox-

sackievirus adenovirus receptor (CAR), CD46, and various integrins used [30].

The viral genome is composed of 26–45 kb of unsegmented, linear, dsDNA,

which is amenable to easy modification. As shown in Figure 11.1, the genome is

organized into five early units (E1, E2a, E2b, E3, and E4), two intermediate units

which are expressed after initiation of viral replication (IVA2, IX), and late units

(L1, L2, L3, L4, and L5), each encoding one or two virus-associated (VA) RNA

controlled by internal polymerase III promoters. Notable non-coding elements of

the genome include the flanking ITR sequences at either end of the genome that

prime DNA replication, the ψ packaging sequence upstream of E1 necessary for the

efficient assembly of mature virions, and various non-coding viral associated RNAs

(VAI, VAII) [17,29,31]. E1A polypeptides can stimulate viral DNA synthesis,

while E1B stops the protein synthesis of host cell proteins and contribute to

transport viral RNA. E2 gene codes for polymerase and DNA-binding proteins

which are essential for viral replication. E3 unit encodes proteins which can guide

virus to escape immunosurveillance. E4 unit is responsible for viral RNA nuclear

export. The IVA2 gene product is essential for AdV assembly and viral DNA

packaging. The IX unit can synthesize a transcriptional transactivator, which can

stabilize the virion. The late unit genes (L1−L5) encode 45 species of RNA, which

play an important role in AdV replication in the early and late phases [3].

First-generation AdV vectors are generated by deleting the E1, and sometimes

also E3, region of the viral genome. Deletion of these genes accomplishes the dual

purpose of ensuring replication incompetence and creating space to accommodate a

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Bioprocessing of Viral Vaccines