systems seeded with HGRs in comparison to wild-type viruses [23]. HGR gen-

eration, performed only by a few laboratories in the world, consists of coinfecting

the same egg with both viruses and screening the viral progeny for a reassortant

with the desired characteristics, a process that takes between 4–6 weeks to be

completed [17,18]. To reduce the time required for HGR generation and increase

responsiveness to potential pandemics, the use of reverse genetics for viral seed

stock production have been studied. This technique consists of generating live

viruses by co-transfecting cells with a set of plasmid-cloned cDNA encoding the

influenza viral genome [24]. Rapid production of influenza virus seed stocks using

reverse genetics have been demonstrated for different cell lines [25–27].

9.4.1

INACTIVATED INFLUENZA VACCINE (IIV)

IIVs, which represent almost 90% of influenza vaccines produced globally, may be

composed of virus sub-unit, whole or split virus. Inactivated vaccines are safe, owing

to the inability of the virus to replicate, and are highly effective, promoting mostly

humoral immune response [16,28]. Cellular immune response may be increased if

viral structures are successfully preserved during the inactivation process. While virus

inactivation may be achieved through chemical (formaldehyde, β-propiolactone) or

physical processes (UV, gamma irradiation, USP laser), each technique results in a

different product and, consequently, may trigger slightly different immunological

responses [29].

The majority of IIVs, around 80%, is produced using embryonated hens’ eggs, a

technology established in 1940s where fertilized eggs are used for virus propagation

[17]. Yet, driven by advances in large-scale cell culture for recombinant protein pro-

duction [30,31], the last decades have seen an increase in the use of cell culture plat-

forms as a faster alternative for IIV production [32–37]. In short, cells are cultivated

until sufficiently high cell densities are achieved, followed by infection with the desired

virus strain. Currently, five IIVs produced using cell-culture systems are approved for

use, and only two of them are in production for commercial use (Table 9.3).

Advantages of cell-culture−based systems over traditional egg production include: i)

scalability, notably with the use of suspension cell lines [30]; ii) greater process control,

which results in a more reliable product [38]; iii) a better match between vaccine and

circulating strains, as egg-growth adaptation may generate undesirable antigenic

modification [39,40]; iv) shorter production cycles, and consequently faster responses to

TABLE 9.2

Approved antiviral drugs for the influenza disease

Mechanism of action

Drug

Neuraminidase inhibitor (blocks particle

release)

Rapivab (Peramivir), Relenza (Zanamivir), Tamiflu

(Oseltamivir phosphate)

Inhibitor of viral polymerase activity (inhibits

viral replication)

Xofluza (Baloxavir marboxil)

Manufacturing of influenza vaccines

229