The effects of preceding human endotoxemia on the Fluenz-induced immune response, a explorative study

A large proportion of ICU patients suffer from a systemic bacterial infection,
called *sepsis*. This is often complicated by the reactivation of multiple
viruses and secondary respiratory bacterial infections. Recent work has shown
that a immunosuppressive state called *sepsis-induced immunoparalysis* accounts
for this increased vulnerability. This condition renders the host unable to
clear infections and/or increased vulnerability towards secondary infections.
Influenza is one of the respiratory viruses that accounts for secondary
respiratory infections in ICU patients. The Influenza virus is known for its
severe course of infection and systemic effects. Influenza causes >250.000
deaths annually in the Western World with the highest attack rates among
children and young adults.
Previous research in animals and humans has focused on the interaction of
influenza followed by a secondary bacterial agent, showing that influenza
promotes susceptibility for secondary bacterial infections, and thereby
worsening the prognosis.
However, the interaction of bacterial sepsis followed by influenza has only
sparsely been investigated. However, because of the high mortality rates, it is
of main importance to understand this mechanism for the development of putative
preventive and therapeutic interventions in ICU patients.

The live, attenuated, trivalent influenza vaccine (LAIV) for intranasal
administration *Fluenz® (Medimmune Vaccines)* is registered in the European
Union for vaccination against influenza. It contains material from three
different influenza strains recommended by national and international public
health agencies as most likely to be protective against seasonal influenza in
any given year. While inactivated influenza vaccines induce protective levels
of serum antibodies to influenza hemaglutinine (HA) and neuraminidase (NA)
surface proteins, these are strain specific and offer little protection against
heterosubtypic influenza viruses. In contrast, LAIVs like Fluenz®, induce
T-cell responses in addition to antibody responses against HA and NA surface
proteins and vaccination protects against heterosubtypic influenza strains as
well.

The human endotoxemia model consists of administration of lipopolysaccharide
(LPS, a cell-wall compound of gram-negative bacteria) to healthy volunteers. It
therefore represents a model of systemic inflammation caused by bacteria, which
mimics some of the clinical and pathobiological hallmarks of sepsis. Among
other things, we have shown that a second exposure to LPS within one or two
weeks after the first results in a profoundly suppressed state of the immune
system called endotoxin tolerance. This phenomenon, albeit mild, resembles
sepsis-induced immunoparalysis.

In this study, we want to investigate the effects of preceding endotoxemia on
the Fluenz®-induced inflammatory response. In this respect, human endotoxemia
will serve as a model of Gram-negative sepsis and Fluenz® vaccination is used
as a surrogate for an actual influenza infection.
As such, combining the experimental endotoxemia model with the administration
of Fluenz® uniquely enables us to investigate crosstalk between bacterial and
viral infections in humans in vivo. We hypothesize that the LPS-induced
systemic endotoxin tolerance results in an altered inflammatory response,
leading to an increased or decreased viral replication and inflammatory
response upon vaccination with Fluenz®. This study will provide us with unique
in vivo data on mechanistic interactions of systemic LPS followed by mucosal
Fluenz®, which may not only be applicable to influenza infection, but also to
other (respiratory) viruses commonly encountered in septic patients. As such,
it can provide clues regarding the increased vulnerability towards viral
infections in septic patients and open up new avenues to investigate
therapeutic measures to prevent this. Furthermore, it provides important
implications regarding the safety and applicability of the vaccine in
(post)septic or immunocompromised patients.

Our primary objective is to investigate the effects of endotoxin-induced
systemic inflammation and subsequent development of endotoxin tolerance on the
inflammatory response following Fluenz® administration in vivo. To evaluate
whether these effects involve local and/or systemic inflammation, local
inflammatory parameters are measured in nasal wash. Systemic inflammatory
parameters are measured in blood. Furthermore, we want to evaluate whether
preceding endotoxemia influences the viral load of influenza in nasal wash.
Finally, changes in the mucosal microbiome will be assessed. For secondary
objectives see page 14 of the C1 protocol.

A parallel, randomized, open-label trial in which 10 subjects will receive a
intravenous bolus of placebo (NaCl)and 10 subjects LPS (2 ng/kg E.coli
lipopolysaccharide). After one week time, all subjects will be vaccinated with
Fluenz® (spraying 0.1 mL into each nostril in supine position). Nasal wash,
blood samples, temperature and symptom scores will be obtained at various
time-points, and peak expiratory flow will be assessed.

Healthy volunteers who meet all inclusion criteria and none of the exclusion
criteria that have given informed consent to participate in the study will be
randomized to become either administered with an intravenous bolus of endotoxin
(LPS derived from E coli O:113, 2 ng/kg) or placebo. After one week all
subjects will be vaccinated with Fluenz® (spraying 0.1 mL into each nostril in
supine position). Nasal wash, blood samples and symptom scores will be obtained
at various time-points, and peak expiratory flow will be assessed.

A medical interview and physical examination are part of this study. Subjects
have to visit the hospital on a total of 9 occasions; during human endotoxemia
or placebo one whole day, followed by 8 visits of about 10 minutes. During
endotoxemia, volunteers will be monitored on the research unit of our medium
care and receive an arterial line to facilitate blood pressure monitoring and
blood sampling. The arterial line will be placed under local anaesthesia using
2% lidocaïne. Furthermore, a venous cannula will be placed for the
administration of fluids and LPS.
The administration of LPS induces flu-like symptoms for approximately 4-6 hrs.
This model of systemic inflammation has been applied for many years in various
research centers in the world. LPS administration is considered safe and no
long-term effects have ever been documented.
Subjects have to keep a symptoms diary (scoring card) that they fill in at
home. In total, maximally 400 mL of blood will be drawn (on 9 occasions via
venapuncture). This is not associated with side effects (500 mL is also drawn
at the blood bank without any side effects). Furthermore, 6 nasal washes will
be performed, which can lead to slight irritation of the nasal mucosa but is
not associated with risks. Fluenz® is registered for use within the European
Union and has been administered to thousands of subjects. The most common
solicited adverse reactions were mild and included: runny nose or nasal
congestion, headache and sore throat. Large randomized trials have been
performed to test the safety of Fluenz® and no serious adverse reactions have
occurred