Establishing colonisation with non-toxigenic Clostridioides difficile in healthy volunteers.

Clostridioides difficile (Cdiff or C.difficile) is the leading cause of
healthcare-associated diarrhoea, with almost 190.000 cases a year and estimated
health-cares costs of 3 billion euros yearly in the European Union. With a
mortality rate of 5% and high relapse rates ranging from 20% after an initial
episode and 60% after multiple prior recurrences the urge for new treatment
strategies has become increasingly important.
The most important advancement in this area has been the prevention of
recurrence by fecal microbiota transplantation (FMT) from a healthy donor. Host
gut microbiota factors are of key importance in the pathology caused by
C.difficile, as disturbances of the microbiota by antibiotics cause C.difficile
to grow out and germinate into toxin-producing cells and thereby causing
disease. Restoring these disturbances of the fecal microbiota with healthy
microbiota through FMT, prevents colonisation of C.difficile. FMT has shown to
have superior efficacy compared to standard treatments in particular for
recurrent disease. However the essential components of FMT, as well as the
specific markers which influence the microbiota susceptibility for colonisation
of C.difficile are unknown. Our understanding of microbiota susceptibility
markers and the components of FMT which effectively provide colonisation
resistance, is urgently needed to find (new) strategies for prevention of
C.difficile infection. Therefore we propose to develop a human model for
colonisation with C.difficile, in which we can investigate colonisation
resistance conferred by the (natural) microbiome. We propose to use a
non-toxigenic strain of C.difficile (NTCD) which lacks the genetic locus for
toxin production (PaLoc) and as such cannot cause C.difficile disease. In the
healthy population asymptomatic colonisation of C.difficile is seen in 4-15%,
of which a varying proportion (30-93%) does not carry toxin genes. Experimental
colonisation with NTCD has been done before in healthy volunteers and was well
tolerated.
In summary, with this trial we aim to investigate the safety, tolerability and
dose needed to obtain colonisation with non-toxigenic C.difficile and
investigate host microbiota factors associated with colonisation.

Primary objective:
- To evaluate the safety and tolerability of colonisation with non-toxigenic C.
difficile.
- To establish the effective protocol to obtain colonisation with non-toxigenic
C. difficile in the majority of subjects.

Secondary objective:
- To determine factors in the host microbiota associated with successful
colonisation.

Exploratory objectives:
- Determine changes in the host microbiota following colonisation.
- Investigate C. difficile in-vivo evolution and adaptation.

This will be an adaptive dose design, randomised double-blind controlled
clinical trial investigating oral exposure to non-toxigenic C.difficile. The
trial will consist of two or, if necessary, three different consecutive
intervention phases. The second and third phase are dependent on results of the
preliminary phases. In every phase one cohort volunteers will be randomised to
different dose levels of NTCD spores or placebo.

Phase 1
In the first phase we will test two dose levels: 5 doses of 10E4 NTCD spores
and 5 doses of 10E7 NTCD spores. These doses will be compared with placebo. 24
volunteers will be randomly divided over 3 groups; A, B and C (as scheduled
below). Group A and B will consist of 10 volunteers each and group C (the
control group) will consist of 4 volunteers. Group A will receive 5 doses of
10E4 NTCD spores on day 0 to day 4, group B will receive 5 doses of 10E7 NTCD
spores on day 0 to day 4, and group C will receive 5 doses of placebo on day 0
to 4.

Phase 1 (N=24):
Group A (N=10): 5 doses 10E4 NTCD spores.
Group B (N=10): 5 doses 10E7 NTCD spores.
Group C (N=4): 5 doses of placebo.

Dose of the second phase will be based upon the colonisation results of the
first phase. See the flow chart below (Figure 1). Progression to the second
phase will be in consultation with the local safety monitor.

Phase 2
Depending on the outcome of phase 1, the dose given in phase 2 will either be
reduced (if colonisation frequency in phase 1 is >60%) or the doses will be
preceded by vancomycin pre-treatment (if the colonisation frequency in phase 1
is <60%) according to predefined criteria. 23 or 26 volunteers will be randomly
divided over 3 groups: D, E and F (as scheduled below). Group D and E will
consist of 10 volunteers each and group F (the control group) will consist of 3
or 6 volunteers. Depending on the results of colonisation of the first phase,
there are three options for dosing schedules in the second phase.

Phase 2 (N=23 or 26)

Option 1 (N=26):
Group D (N=10): 3 doses 10E4 NTCD spores.
Group E (N=10): 1 dose 10E4 NTCD spores, 2 doses placebo.
Group F (N=6): 3 doses placebo.

OR

Option 2 (N=26):
Group D (N=10): 3 doses 10E7 NTCD spores.
Group E (N=10): 1 dose 10E7 NTCD spores, 2 doses placebo.
Group F (N=5): 3 doses placebo

OR

Option 3 (N-23):
Day -7: one day of vancomycin (4 times a day 250mg).
Group D (N=10): 5 doses 10E4 NTCD spores.
Group E (N=10): 5 doses 10E7 NTCD spores.
Group F (N=3): 5 doses placebo.

Escalation to the third phase will only be done if option 3 is selected in
phase 2. Dosing of the third phase will be based upon the colonisation results
of the second phase. Progression to the third phase will be in consultation
with the local safety monitor.

Phase 3
Depending on the outcome of phase 2, the dose given in phase 3 will either be
reduced (if colonisation frequency in phase 2 is >60%) or the vancomycin
pre-treatment will be extended to five days (if the colonisation frequency in
phase 2 is <60%) according to predefined criteria. 23 volunteers will be
randomly divided over 3 groups: G, H and I (as scheduled below). Group G and H
will consist of 10 volunteers each and group F (the control group) will consist
of 3 volunteers. Depending on the results of colonisation of option 3 of the
second phase, there are three options for dosing schedules in the second phase,
which is scheduled below.

Phase 3 (N=23)

Option 1:
Day -7: 1 day vancomycin (4 times a day 250mg).
Group G (N=10): 3 doses 10E4 NTCD spores.
Group H (N=10): 1 dose 10E4 NTCD spores, 2 doses placebo.
Group I (N=3): 3 doses of placebo.

OR

Option 2:
Day -7: 1 day vancomycin (4 times a day 250mg).
Group G (N=10): 3 doses 10E7 NTCD spores.
Group H (N=10): 1 dose 10E7 NTCD spores, 2 doses placebo.
Group I (N=3): 3 doses of placebo.

OR

Option 3:
Day -11: 5 days vancomycin (4 times a day 250mg).
Group G (N=10): 5 doses 10E4 NTCD spores.
Group H (N=10): 5 doses 10E7 NTCD spores.
Group I (N=3): 5 doses of placebo.

All volunteers in all phases will visit the trial centre on the days of spores
or placebo ingestion, with collection of feces prior to the ingestion. If phase
3 of the trial is needed, volunteers will also visit the trial centre twice
between intake of vancomycin and spores/placebo ingestion for fecal sample
collection. During the four follow-up weeks (after spores/placebo ingestion)
volunteers will visit the trial centre three times a week for fecal sample
collection, with a weekly follow-up visit for adverse events (AEs) registration
on day 7, 14, 21 and 28. Safety blood tests will be performed on day 14 and 28.
After three months (day 84) there will be a (final) follow-up visit, with
collection of fecal samples and AEs. Should a volunteer still be positive for
C.difficile at the three month timepoint, the volunteer is asked to return for
follow-up every one to two months for fecal sample collection until the sample
is negative for C.difficile, up till a maximum of one year after the start of
the trial.

Different dose levels (10E4 or 10E7) of NTCD spores or placebo for 1 or 5 days
with optional vancomycin pre-treatment.

There is no direct benefit to the volunteers taking part. Since the strain used
for colonisation is a non-toxigenic strain, it is not expected to cause any
symptoms. In a previous double-blind placebo
controlled trial using a non-toxigenic strain gastro-intestinal complaints such
as nausea, flatulence and cramping have been described. These AEs were all
classified as mild and were comparable to those experienced by the placebo
group. Because colonisation with NTCD is very common in the general population,
NTCD colonisation will not be terminated with antibiotics. Rescue treatment for
NTCD is available in case of unexpected adverse events. Antibiotics can be used
to clear the NTCD. Additionally, FMT (using the fecal microbiota composition of
a healthy donor) is available which can be used as a rescue treatment in case
of persistent disturbances to the host microbiota. Based on prior published
studies administering NTCD to healthy volunteers, the need for rescue treatment
is not expected.
If volunteers in phase 2 or phase 3 will be pre-treated with oral vancomycin,
they may experience gastro-intestinal side effects of vancomycin (i.e.
diarrhoea).
In order to limit the risk of transmission of the NTCD strain to household
members, volunteers will be instructed to practice proper hygiene measures
during the course of the trial and volunteers living with household member who
are considered *vulnerable* will be excluded. Since non-toxigenic strains are
present in the natural environment and asymptomatic colonisation with NTCD
occurs in an estimated 4-15% of the healthy population, household members will
experience a minimal added risk compared to the daily natural risk of exposure
to NTCD.