The effects of BCG-vaccination on the innate immune response and immunoparalysis in healthy volunteers. Two proof-of-principle studies.

Sepsis is a major medical challenge associated with a high mortality rate.
Release of pro-inflammatory mediators can result in hemodynamic instability,
coagulation abnormalities and end-organ dysfunction. Previous strategies have
aimed to treat sepsis by inhibition of pro-inflammatory mediators, however,
most of these approaches have failed. This might be due to the fact that the
majority of septic patients do not succumb to the initial pro-inflammatory
*hit*, but die at a later time-point in a pronounced immunosuppressive state.
This so-called *immunoparalysis*, which renders patients extremely vulnerable
to secondary infections, results from the triggering of counter-regulatory
anti-inflammatory pathways along with the pro-inflammatory response, already
starting in the beginning of sepsis. Immunoparalysis is increasingly being
recognized as the overriding immune dysfunction during sepsis. As a
consequence, reconstitution of immunocompetence is now emerging as a new and
promising therapeutic target to improve outcome in sepsis patients.
Bacille Calmette-Guérin (BCG) is one of the most commonly administered vaccines
worldwide. In addition to protection against tuberculosis, evidence suggests
that BCG immunization has a number of additional beneficial non-specific
immunological effects, hereby protecting against infections with pathogens
other than tuberculosis. The underlying immunologic mechanisms are not fully
elucidated. Recently it was demonstrated that monocytes can be functionally
reprogrammed to an enhanced and lasting phenotype after vaccination with BCG.
Production of pro-inflammatory cytokines by monocytes isolated from volunteers
after BCG vaccination, was found to be enhanced upon ex vivo stimulation with
non-related pathogens, even months after BCG vaccination. The observed effects
are proposed to be due to modulation of the innate immune system in a process
called *trained immunity*. Upon stimulation with a pathogen, the innate immune
system becomes primed and is able to react faster and more efficient to a
secondary (and non-related) stimulus, even months later. Monocyte *training*
was shown to rely on epigenetic reprogramming, namely increased methylation of
histone 3 at lysine 4 (H3K4me3) at the level of cytokine and TLR4 gene promoter
regions.

Considering these potentiating effects of BCG on innate host defense, it could
be a viable treatment option for sepsis-induced immunoparalysis. However, the
effects of BCG vaccination on the innate immune response in humans have
hitherto only been shown ex vivo. It has yet to be established whether these
findings can be extrapolated to the human in vivo situation, because previous
data from our group indicates that ex vivo measurements do not accurately
reflect the in vivo situation. The human endotoxemia model, in which healthy
volunteers receive lipopolysaccharide (LPS) derived from Escherichia coli, is
widely used to study the effects of systemic inflammation in humans in vivo and
is considered a safe and highly reproducible method to investigate the innate
immune response. Furthermore, LPS administration results in a hyporesponsive
state towards a second LPS administration called *endotoxin tolerance*, which
resembles sepsis-induced immunoparalysis, and can thus be used as a model to
investigate therapeutic interventions to reverse this condition.

The intended target group for this novel therapy, sepsis patients, are
immunocompromised. Therefore, use of a live attenuated vaccine such as BCG
could present a risk of disseminated mycobacterial infection. Therefore, we
will use γ-irradiated (inactivated) BCG vaccine in this study. Recent, yet
unpublished results of the group of Prof. Netea have shown that the effects of
γ-irradiated BCG on monocyte training are comparable to those of the live
vaccine.

In the present study, we first want to investigate whether BCG-vaccination
enhances the innate immune response in humans in vivo during human endotoxemia.
In the second experiment we want to investigate whether BCG-vaccination can
reverse the tolerant state observed upon a second LPS administration. Our goal
is to ultimately translate our results into clinic applications to reverse for
example sepsis-induced immunoparalysis.

Primary objective:
1. Single endotoxemia
To determine the effects of γ-irradiated BCG-vaccination on the in vivo innate
immune responses induced by human endotoxemia. This will be determined by
measuring plasma levels of various pro- and anti-inflammatory cytokines and
assessing the difference in the Lipopolysacharide (LPS)-induced cytokine
response between γ-irradiated BCG-vaccined subjects and placebo-treated control
subjects.

2. Repeated endotoxemia
To determine the effects of γ-irradiated BCG-vaccination on endotoxin tolerance
induced by human endotoxemia. This will be determined by measuring plasma
levels of various pro- and anti-inflammatory cytokines and assessing the
difference in the LPS-induced cytokine response following the first and second
endotoxemia, between γ-irradiated BCG-vaccined and placebo-treated control
subjects.

Secondary Objective(s): There are 5 secondary objectives:
1. To determine the effects of γ-irradiated BCG-vaccination on ex vivo
responsiveness of leukocytes to various inflammatory stimuli.

2. To determine the effects of γ-irradiated BCG-vaccination on the phenotype of
circulating monocytes (e.g. expression pattern of cell-surface receptors by use
of flow cytometry).

3. To determine the effects of γ-irradiated BCG-vaccination on inflammatory
transcriptional pathways (by use of qPCR/microarrays).

4. To determine the effects of γ-irradiated BCG-vaccination on epigenetic
changes, including H3K4 trimethylation, in circulating immune cells.

5. To determine the effects of γ-irradiated BCG-vaccination on LPS-induced
clinical symptoms (illness score) and hemodynamic/temperature changes.

Study design
1. Single endotoxemia
A randomized double-blind placebo-controlled pilot study in healthy human
volunteers during experimental endotoxemia.
In this pilot study, we will enrol 20 subjects. On day 1, 10 subjects will
receive γ-irradiated BCG-vaccination and 10 subjects will receive placebo. On
day 6, all subjects will undergo experimental endotoxemia.

2. Repeated endotoxemia
A randomized double-blind placebo-controlled pilot study in healthy human
volunteers during repeated experimental endotoxemia. This study will be
performed after study 1 has been completed, and the results of study 1 have
been discussed with the ethical committee. In this pilot study, we will enrol
16 subjects. All subjects will undergo experimental endotoxemia on day 1. On
day 3, 8 subjects will receive γ-irradiated BCG-vaccination and 8 subjects will
receive placebo. On day 8, all subjects will undergo experimental endotoxemia
for the second time.

Single endotoxemia:
all subjects (n=20) are double-blind randomized to receive either γ-irradiated
BCG-vaccination (n=10) or placebo (NaCl 0.9% subcutaneously, n=10) on day 1. On
day 6, all subjects will undergo experimental endotoxemia (LPS derived from E
coli O:113, 1 ng/kg).

Repeated endotoxemia:
all subjects (n=16) will undergo experimental endotoxemia on day 1 (LPS derived
from E coli O:113, 2 ng/kg). On day 3, subjects will receive either γ-
irradiated BCG-vaccine (n=8) or placebo (NaCl 0.9% subcutaneously, n=8) in a
randomized, double-blind manner. On day 8, all subjects will undergo
experimental endotoxemia for the second time (LPS derived from E coli O:113, 2
ng/kg).

All subjects will visit the hospital for a screening visit in which a medical
interview and physical examination will be carried out (30 minutes).

BCG is one of the world*s most widely used vaccines and is well-tolerated in
healthy volunteers. Local side-effects are frequently seen but do not cause
serious harm. To avoid serious systemic adverse events, ie disseminated
Mycobacterium Bovis infection, we choose to use γ-irradiated (inactivated) BCG
substrate. Local side-effects are expect to be less pronounced compared to live
vaccine. Seroconversion and thus positivity to Mantoux testing, has been
described in 41.8% after vaccination with alive BCG, reduced to only 21.2%
after 10 years. In case of serconversion, Mantoux cannot be used as a
diagnosticum for tuberculosis any longer. However, other diagnostics such as
Quantiferon and chest x-ray can still be used.

During endotoxemia, volunteers will be monitored on the research unit of our
intensive care and receive an arterial line to facilitate blood pressure
monitoring and blood sampling. The arterial line will be placed ultrasound
guided and 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 10 years in our department
and thousands of subjects in various research centres in the world have
participated in experimental endotoxemia trials. LPS administration is
considered safe and no long-term effects have ever been documented.
Lipopolysacharide 2 ng/kg is the most widely used dose in human endotoxemia
research, although higher doses (for example 4 ng/kg) have also been used with
no documented long term effects. In 18 human endotoxemia trials performed in
our centre to date (see IMPD), we have used 2 ng/kg. In one trial (CMO
2006/166), we administered increasing LPS dosages on 5 consecutive days (0.2,
0.5, 1, 2, and 2 ng/kg). Because we hypothesize that BCG-vaccination will
potentiate the immune response following human endotoxemia, we will administer
only half the dose of LPS normally used, namely 1 ng/kg, in our first,
single-endotoxemia study. In the second study evaluating immunoparalysis, 2
ng/kg will be used, since treatment with BCG will take place after the first
LPS administration, which will result in a blunted response upon a second LPS
administration. It is to be expected that BCG will at most restore the response
to a second LPS administration to levels comparable with those observed during
the first LPS administration, but not potentiate it further. 2 ng/kg LPS was
also used in a previous trial (CMO 2011/105) in which LPS was administered
twice, where very potent compounds (IFN-γ and GM-CSF) were used to reverse the
tolerant state after the first LPS administration. In this trial, IFN-γ was
most effective in restoring the immune response upon a second LPS
administration but did not potentiate it beyond the level observed after the
first LPS administration.

At the Radboud University Medical Centre, over 370 volunteers have received
more than 445 injections of LPS. Therefore, there is sufficient experience
with this model at this centre. LPS administrations will be carried out in a
consecutive manner. Furthermore, randomization will be carried out in a manner
guaranteeing that LPS will not be administered to more than one person with the
same intervention (BCG-vaccination or placebo) on one day. A physician or nurse
will be present during the LPS experiment at all times, and subjects will be
continuously monitored (heart rate, blood pressure, oxygen saturation). In
total, a maximum of 600 ml blood will be drawn during the study, which is
comparable to previous studies and never resulted in adverse events. Subjects
will not benefit directly from participation to the study. A subject fee is
provided.