the currently circulating strains. Ideally, the challenge agent will
be derived from a pediatric source, as the patient will be less likely to be co-infected with other viruses and will have a clear and
simple medical history. The agent should also show a strong and
consistent symptom profile and not cause any serious adverse
events (sequelae). Another important characteristic of the challenge agent is that it should have a epidemic strain in its lineage,
in order to confer some cross-protection should that season’s
strain become epidemic too.
The manufacturing process for challenge agents, as for vaccines, is heavily regulated. Production of live virus must be carried out in line with cGMP guidelines, within accredited premises, and using accredited laboratory testing, compliant with
Good Laboratory Practice (GLP). Although there are currently no
specific guidelines for developing challenge agents, possibilities
to lighten the burden of proofs for safety are under discussion, in
the hope that it may be possible to develop new challenge agents
faster and at a lower cost.
THE MANUFACTURING PROCESS
The first step in the process for identifying a suitable challenge
virus (i.e. a virus causing acute disease with a low mortality rate,
limited morbidity and little or no evidence for chronicity, sequelae or serious long term disease, for example many respiratory
viruses) is to isolate and identify a candidate strain from the community, preferably in a pediatric patient with a well-documented
medical history. Preferably the candidate virus is then sequenced
to ensure it is representative of circulating strains, and to look
for resistance to available therapeutics. Pilot tests may be undertaken on a range of candidate viruses to see how well each grows
on an appropriate cell line, such as Madin-Darby Canine Kidney
(MDCK), and to check whether the virus can infect the cells easily, without losing infectivity or showing signs of attenuation, and
if it may easily be grown on, or amplified in, other cell lines.
At this stage it may also be prudent to check the antigenic
properties, comparing them for similarities (homology) to current
With a suitable viral candidate identified, further pilot studies
may be performed to determine whether the virus can multiply
successfully in the Working Cell Bank or the chosen egg stock during the manufacturing process. Pilot studies are used to identify
the relative suitability of differing cell-lines and embryonated eggs
from differing specific-pathogen free (SPF) chicken flocks by investigating dosing titers and incubation temperatures and times.
Cell lines are increasingly being preferred to egg stock in the light
of egg adaptation and the increase of egg albumin allergies. If eggs
are ultimately the medium of choice, it is advisable to test out more
than one supplier of eggs to check that there is no“egg effect”; it is
not unknown for eggs to be resistant to a particular virus.
The optimal titer for inoculating the medium in the manufacturing process is important. Too many rounds of amplification is
not ideal, as the virus will be more likely to become adapted to
its new host medium and thus become less effective at infecting
humans. So-called egg or cell-line adapted virus will have a reduced capacity for causing symptomatic disease and may exhibit
truncated or decreased shedding, thus compromising its usefulness as a successful challenge agent.
Once it is certain that the candidate agent grows well in pilot studies, the viral seed-stock can be produced. Seed-stock or
the starting inoculum to grow on into the final product is grown
either in high-yield cell lines or a small number of embryonated
eggs from SPF flocks. Virus is injected or inoculated at a dilution
established during the pilot study as optimal for rapid expansion:
this might be 1: 100, 1: 10 or even neat. Seed virus is harvested after a minimal number of amplification rounds (2-3), centrifuged,
filtered to remove cellular material and any extraneous contaminants and frozen down in aliquots.
Small quantities of harvested seed-stock are used to inoculate the manufacturing cell-line (which may differ from the seed-stock cell-line) or a large number of embryonated eggs. To obtain
a reasonable volume of virus it is typical to utilize up to 300-400
eggs, or to propagate the virus in a small bioreactor dosed with
cells from the cell bank.
Inoculated eggs are typically incubated at 35°C for three
days before being harvested and centrifuged down. Virus suspended in the supernatant is further filtered at 0.4µm and
subsequently through a 0.22 µm filter to remove the majority
of the remaining egg residue, proteinaceous clumps and any
bacteria, leaving the supernatant containing only challenge
virus. Finally, virus is aliquoted out into 2ml vials before snap
freezing in liquid nitrogen.
A manufacturing process as described should generate sufficient virus to fill about 2,500 vials; these are stored at – 80°C to
ensure long-term viability.
The entire manufacturing process is designed to ensure the
minimal number of passages that yields the highest possible
viral titer. As the virus has not been passaged or amplified multiple times, the final agent should remain highly infectious to
humans, and not have undergone attenuation. The ideal challenge agent should be very close to the wild type (WT) virus, as
the aim of a human challenge trial is to reflect real-life infections in the community.
“The manufacturing process for
challenge agents, as for vaccines,
is heavily regulated. Production of
live virus must be carried out in
line with cGMP guidelines, within
accredited premises, and using
accredited laboratory testing,
compliant with Good Laboratory