TESTING THE VIRUS
It is important to note that, as well as the requirements to grow
the virus under cGMP conditions, a number of tests must be performed before it can be used to infect human subjects in a challenge trial. These are similar to those involved in a vaccine testing
program: for example, the presence of any adventitious agents
must be excluded, which can be carried out by further incubation
in embryonated eggs, in mouse studies and potentially in other
cell lines that support contaminant human pathogens.
Such studies should show up whether any other infectious
agents are present, which may include human viruses such as
HIV, herpes and hepatitis and also, if it has been grown in eggs,
the avian leucosis virus. Other pathogens that might be present
include spiroplasma and mycoplasma, which are a type of bacterium without a cell wall that are common contaminants in cell
lines. Toxin tests should also be performed to rule out bacterial,
fungal and other toxins that may have leached into the product.
Genetic testing is employed to ensure there is nothing present other than challenge virus DNA. Next generation sequencing
(NGS) can confirm the purity of the virus and assist in determining its homology to WT. Even with 100% homology with the
original candidate virus on sequencing, there is always a chance
that fragments of DNA from the egg or from adventitious agents
may be present.
Before the virus can be given to humans (first-in-human or
FIH studies), it must undergo animal testing to check for unusual,
unexpected or unwanted pathological changes, as well as the severity of the disease induced. Influenza animal studies usually
involve female ferrets, which represent a good model for human
influenza. The ferrets are given the virus via intranasal inoculation and the course of the resultant disease is monitored.
Warning signals of unusual pathogenicity in mammals may
include a very high temperature, vomiting, diarrhea, decreased
or altered visual acuity and systemic problems such as falling
blood pressure. The ideal challenge agent should only cause limited symptoms, akin to a bad cold or a mild dose of influenza,
with a zero or vanishingly small mortality rate, even in vulnerable populations. If nothing severe or worrying is seen, the ferrets
are sacrificed after eight or 10 days and pathological sections are
taken to examine for virus in the lung, brain, heart and other major organs. For the virus to be deemed fit for use in human studies
it should not spread widely from the nose and throat and cause
respiratory illness only, with no dissemination into bodily organs.
Under normal circumstances, if the challenge virus being
tested is a seasonal H3N2 or H1N1 strain, there should be no
problems with only low-grade infection likely to develop. In con-
trast, if the virus is a wild type H1N1, pandemic strain, such as
the H1N1/2009pdm, ferrets and humans may develop lung le-
sions and a profound lower respiratory tract infection. Such com-
plications rarely occur with normal, circulating H3N2 and H1N1
strains. Unattenuated, pandemic H1N1 or H3N2 strains or po-
tentially pandemic H5, H7 and H9 should not be used as chal-
lenge agents due to the risk of triggering fatal cytokine events.
The recently-developed SGS 3C H2N2 challenge agent (A/
Belgium/4217/2015) has passed all of the above criteria following extensive testing and its profile appears to match up to the
ideal virus. A/Belgium does not cause lower lung lesions or other
adverse events, and gives a classic influenza-like illness, with a
raised temperature, shivering, decreased movement, and reduced
appetite in ferrets. On pathological examination of animals, there
were no lesions other than those usually associated with a moderate upper respiratory tract infection.
With the evidence from the initial work in cell lines, the pilot and
scaled-up manufacture in eggs (or a cell line), the testing for adventitious agents and other contaminants and the animal model
safety test, a dossier can then be submitted to the regulatory authorities for its use in human subjects.
Viral challenge testing in humans is a new way to obtain
proof-of-concept for the effectiveness of an anti-viral drug or
vaccine early in Phase I, after initial safety and PK/PD assessments, and can assist in faster go-no-go decision making. Viral
challenge testing clinical trials must obviously be conducted according to the Good Clinical Practices (GCP) within a dedicated
clinical pharmacology unit which has hospitalization beds within
a biosafety level 2 Q compliant quarantine facility.
The final part of the testing process is a titration study in human subjects to determine the titer that gives the optimum attack
rate and symptomologies. This characterization study typically
involves three different titers being dosed intranasally to cohorts
of between 12 and 24 subjects in three arms to determine the optimal dosage level. The consistency of viral shedding is assessed
via nasopharyngeal swabs taken twice a day, and symptoms that
develop are recorded using a symptom scorecard (SSC).
The optimal symptomology reflects the usual influenza cycle
of nasal congestion, coughing, raised temperature and lethargy,
none of which should be too severe. If all of these develop and
there is a consistent viral shedding profile after inoculation (with
a good area under the curve) this indicates an effective infection.
A consistent shedding pattern with few ‘blips’ and a high peak
titer allows the effect of a drug or vaccine against the virus to be
assessed in subsequent challenge trials.
There are ongoing discussions about how symptom scores
should be taken and interpreted, and it is likely that there will be
some changes in human challenge trial endpoints and outcomes in
the near future. But, however the studies are performed, and regardless of the way the results are assessed, the ultimate aim is to emulate the infection that happens in the community, so the challenge
virus can be used to estimate the effects of novel interventions. CP
“Before the virus can be given to humans it must undergo animal testing to
check for unusual, unexpected or unwanted pathological changes, as well as
the severity of the disease induced.”