A Pilot Study of Immunomodulatory effects of Inulin-type fructans in a hepatitis B vaccination study in seniors

Inulin-type fructans (ITFs) are dietary fibres, which have beneficial effects
on general health, including the immune system. These fibres are part of our
daily diet and can, for instance, be found in chicory roots or sugar beets.
ITFs are digested in different chain lengths and have been described to
stimulate the bacteria in our gut (microbiota) and to modulate the immune
system. How this beneficial effect of ITFs occurs is still not well-known. We
recently showed that beneficial health effects may be related to effects of
ITFs on so-called pattern recognition receptors (PRRs) on immune cells. These
PRRs are sensors of the immune system and determine whether a stimulation or
inhibition of the immune system occurs when they bind to molecules such as
fibres. We found that the effect of ITFs on PRRs was dependent on the chain
length of the ITFs with a more stimulating effect of long chain ITFs and a more
regulatory (or inhibiting) effect with short chain ITFs. To confirm that this
also occurs in vivo, we performed a pilot study with ITF fibre supplementation
around an already existing hepatitis B vaccination program to determine whether
ITF intake improved immune function after the first vaccine injection (METc:
NL41644.042.13). The hepatitis B vaccination was chosen, since this is a
relative ineffective vaccine, as the vaccination protocol requires more than
one boosts in order to reach safe antibody titers. Indeed, we found that long
chain ITFs (DP10-60) increased the specific antibody titre against hepatitis B
after the first vaccine injection (placebo: [0.52, 3.72 IU/mL] vs. long chain
ITF: [0.97, 8.05 IU/mL]). Additionally, we found 17% responders in the long
chain ITF groups as compared with 0% responders in the control group after the
first vaccine injection. Also we found that only the long chain ITFs
significantly increased the total Th1 cell population at day 35 as compared to
day 0 after the first injection of the vaccine. With ageing (from an age of 40
years) the number of non-responders to hepatitis B vaccination is known to
increase. Seniors may therefore benefit more from the immune stimulating effect
of the long chain inulin. Therefore, we would like to confirm the immune
stimulating effect of the long chain ITF in a similar study but with senior
people and at the same time incorporate additional parameters to investigate
the underlying mechanism. To avoid interference of fluctuating levels of sex
hormones due to menopause in women, we would only like to include individuals
from an age of 55 and older. We would like to investigate, in a pilot study,
whether administration of long chain ITFs (DP10-60) to seniors during a
hepatitis B vaccination protocol results in a better protection and more
responders of the senior people. Beside the effect of the fibre on the antibody
titre after the first shot of Hepatitis B, we would like to investigate the
effect after the second shot (1 month after the first shot) and the third shot
(6 months after the first shot), as we expect these to be increased by the
fibre as well. Our main focus will be the antibody titre after the second shot,
as the fibre may increase the vaccine-specific antibody titer after this shot
and therefore might make the third shot redundant. In our previous study we
found that the antibody titre against hepatitis B increased till 4 weeks after
vaccination (highest level). Therefore, we would like to administer the fibre
for the whole period in which the immune response is developing instead of 14
days around vaccination. So, we would like to extend the administration of the
fibre to a daily intake until 4 weeks after the second vaccination (day 63), to
maximise the immune stimulating effect.

Primary Objective:
The primary objective is to study the effects of long chain ITF supplementation
on hepatitis B vaccination efficacy in seniors after the second shot of
hepatitis B vaccination. This will be measured as the hepatitis B specific
antibody titer 4 weeks after the second shot of hepatitis B vaccination.

Secondary Objective(s):
The second objective is to study underlying and concomitant mechanisms of long
chain ITF supplementation on improved hepatitis B vaccination efficacy. To this
end, modification of various immune cell populations will be evaluated. To
study whether the effect of ITFs is induced by changing the microbiota, we will
study microbiota composition, Short Chain Fatty Acid levels, and levels of IgA
in the feces.

The format of the present intervention study is a double blind randomized
placebo-controlled trial. Both female and male subjects will be recruited in
the ages >55 years. Equal numbers of subjects will be at random allocated into
the following groups: I) receiving 8g/d long chain ITFs (Frutafit®TEX!) + 2g/d
glucose or II) receiving 5g/d of glucose, which serves as a placebo and is
accepted by the scientific community as an appropriate control. A small amount
of glucose is added to the long chain inulin to mask the taste so that it
cannot be deducted whether the placebo or the fibers were ingested. All
supplements will be dissolved by the subjects in a glass of tea (flavour chosen
by subjects) in hot water, before administration to standardize the way of
intake. In each group, the subjects will receive a vaccination against
hepatitis B on day 7, day 35 and day 175 after the start of supplementation,
and supplementation of fibres will continue for 56 days (thus 63 consecutive
days in total).
Blood samples for measuring antibody titre and immune cell populations will be
taken at days 0, 7, 35, 63 and 203 (on each time point 10 ml of heparinized
blood will be collected per subject). Blood sampling will be performed by
trained professionals from the department of Laboratory Medicine of the UMCG.
Antibody levels in blood samples will be analysed using ELISA microtiter
plates. To study peripheral immune cell populations, leukocytes in whole blood
will be stained for relevant cell markers to distinguish different populations
using flow cytometry. Additionally, hepatitis B specific memory cells will be
measured using ELISPOT and cytokine production will be measured after ex vivo
stimulation of blood cells with the hepatitis B vaccine using Luminex. Faecal
samples also will be collected at days 0, 7, 35, 63, 91, 147 and 203 to analyse
the microbiota composition, short chain fatty acid profile and IgA levels in
the feces. Microbiota produce breakdown products, such as SCFAs, when
fermenting fibres. We will analyse faecal samples for the presence of both
whole inulin type fructans and the their breakdown products such as SCFA, which
can also be used as a measurement for supplement compliance. As the microbiota
composition in an individual can change rapidly, multiple time-point for the
collection of feces were chosen, to make sure that all changes in the
microbiota composition will be measured. Finally, the study subjects are asked
to fill in a nutrition diary at the start and the end of the study to enable
the investigators to make an estimation of the total dietary fibre consumption,
and the study subjects are asked to refrain from intake of pre-, and probiotic
nutritional supplements outside of the specified study supplements.

The subjects in this study will be treated with an ITF formulation or a placebo
(glucose) derived from products in the food industry and which do not have any
negative side effects. 8g of fibres per day will be administered to healthy
subjects, which according to the literature is an effective dose. Our previous
hepatitis B study, in which students consumed exactly the same amounts of
fibres, demonstrated that the subjects did not have difficulties consuming this
amount of fibres. Glucose is a safe food grade ingredient, which does not have
adverse side effects, provided it is consumed in normal amounts (up to 50g/day,
Voedingscentrum), related to the advised daily intake of calories. For
comparison, the dose we propose for use as a placebo (5g/d) does not exceed the
amount of glucose in one average sized banana, which is ca. 9.8g.

Intake of fibres or placebo (8g/d or 5g/d for 63 days), and collection of fecal
samples (7 times over a period of 203 days) does not carry any risks for the
study subjects. It involves fibers and a placebo, which are all food grade and
already applied in commercial products. Blood sampling will be performed by
trained professionals from the department of Laboratory Medicine of the UMCG
and will comprise 10 ml of blood per person per sampling time point. Blood will
be drawn at days 0, 7, 35, 63 and 203. Thus in total, 50 ml of blood will be
taken, spread out over a period of 203 days. This blood sampling may cause a
small hematoma (*blauwe plek*) at the inside of the elbow in some persons, but
in general if pressure is put on the site of sampling just after sampling this
does not occur. The study subjects are asked to fill in a nutrition diary, and
to refrain from intake of fibre supplements and probiotic supplements besides
the specified study supplements. The hepatitis B vaccination takes place at day
7, day 35 and day 175. The hepatitis B vaccination is part of the vaccination
program from the *rijksinstituut voor volksgezondheid en milieu* (RIVM) and is
therefore a safe and approved vaccination. The vaccination will be given to the
subjects by their own general practitioner. The mild side-effects of this
vaccination protocol include injection site reactions (pain, soreness, redness,
swelling), fever, headache, fatigue, tiredness, irritability, sore throat,
nausea, vomiting, abdominal pain, diarrhea, loss of appetite, dizziness, muscle
pain, and flu-like symptoms.