Risk Wise

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Cell culture (Vero) derived

pandemic influenza vaccines

P. Noel Barrett, Baxter Bioscience, Biomedical Research Centre,

Orth/Donau and Hartmut J. Ehrlich, Baxter Bioscience, Vienna

fficient vaccine production requires the growth of large

quantities of virus produced with high yields from a reli-

able, available, safe host system. Conventional methods

for producing influenza vaccines are based on the growth of the

viruses in embryonated chicken eggs. This is a cumbersome

process in which each egg must be sterilized, candled, inocu-

lated with virus and incubated before harvesting small volumes

of allantoic fluid from each egg and pooling before purification.

Between one and two eggs are typically required for one dose of

seasonal trivalent influenza vaccine and the supply of eggs for

seasonal influenza vaccine production requires up to six months

to scale up. There is, therefore, a concern that there could be a

major shortfall in the case of a major pandemic, when there

would be the need for large quantities of vaccine to be produced

rapidly, possibly in a situation where chicken flocks have been

depleted by infection with highly pathogenic influenza virus.

There is also evidence that selection of human influenza viruses for

high yield growth in eggs is associated with the selection of antigen

variants, which may be sub-optimal for inducing protective anti-

bodies to wild type virus circulating in humans. In contrast to

influenza viruses grown in eggs, virus propagated exclusively in

mammalian-derived tissue culture has been reported to be repre-

sentative of the natural virus. Studies in ferrets have also

demonstrated that an inactivated influenza vaccine grown in Maden-

Darby canine kidney cells (MDCK) induced higher mean serum

haemagglutination inhibition and neutralizing antibody titres than

did egg-grown vaccine, and induced superior protection against

subsequent challenge with infectious virus grown in either cell type.

These reports demonstrated the possible superiority of a

mammalian cell-derived vaccine and emphasise the necessity for a

mammalian cell line that could be used to replace chicken eggs in the

production of influenza vaccines. Consequently, the development of

inactivated, cell-derived influenza vaccines grown at an industrial

scale using a variety of continuous cell lines (CCLs), specifically,

Vero, MDCK and PER.C6 cells, is well advanced.

Although a number of CCLs are being considered for production

of influenza and other viral vaccines, Vero cells are the most widely

accepted CCL by regulatory authorities for manufacture of viral

vaccines. Vero cells were first used for human vaccines with the

production of IPV by Montagnon and colleagues at the Institute

Merieux, Lyon, France in the early 1980s, and this was followed by

its use for an inactivated rabies vaccine. Vero cells have also been

used for many years for the production of live oral poliovirus vaccine.

As such there is over 25 years experience with Vero-

derived human vaccines with hundreds of millions of

vaccine doses being distributed worldwide.

This expe-

rience has provided substantial evidence supporting the

safety of this cell substrate and has provided encour-

agement to further explore the use of this cell line for a

range of different viral vaccines including influenza.

Another major advantage of the Vero cell line for

vaccine production is that it can be grown and infected

on microcarrier beads and cultivated in fermenters to

allow the large-scale production of vaccines. These

developments were pioneered by Anton Van Wezel, who

first demonstrated the high-density cell growth on

microbeads for the production of polio and rabies virus

vaccines. This microcarrier technology has been further

developed to allow large-scale production of a number

of vaccines using a serum-free medium. Such processes

have been developed to allow amplification of a single

one-millilitre ampoule of cells to achieve a fully conflu-

ent microcarrier culture at a 6,000-litre scale within

eight weeks. This upscaling can be carried out without

loss of cell productivity or viability with extremely

consistent results.

Advantages associated with cell culture derived

pandemic influenza vaccines

Most pandemic H5N1 vaccine candidates tested to date

were manufactured using attenuated reassortant viruses.

These reassortants are generated using the haemagglu-

tinin (HA) and the neuraminidase (NA) genes of the

circulating wild-type (WT) virus and the six remaining

genes of the H1N1 influenza strain A/PR/8/34 (6:2 reas-

sortants), which usually confer high growth properties

in embryonated hens’ eggs. This reassortant virus is also

attenuated by removal of the polybasic cleavage site of

the HA which is associated with high pathogenicity.

These reverse genetics (RG)-derived reassortants are

then subjected to extensive safety testing before distri-

bution to the influenza vaccine manufacturers. This

procedure is essential to allow use of the virus under the

biosafety level two enhanced, which is the highest safety

level available in egg-based manufacturing facilities, and

to generate the potential high-growth phenotype

required for adequate vaccine antigen yield. However,