MTB-Exposure: The role of trained versus adaptive immunity following exposure to Mycobacterium tuberculosis

Tuberculosis is caused by infection with Mycobacterium
tuberculosis.
M. tuberculosis is transmitted by patients with infectious TB who spread the
disease to others by coughing or sneezing droplets containing M. tuberculosis.
Individuals who inhale these airborne droplet nuclei can become infected. A
susceptible person needs to be exposed to an infectious TB patient to be become
infected and may later develop the disease. With an estimated one-fourth of
the world population infected and approximately 1.7 millions deaths in 2006
attributed to TB world-wide, TB remains a major public health concern today.
Although the Netherlands belongs to the countries with the lowest rates of TB,
it had not yet been eliminated in the Netherlands, with approximately 1000 new
patients cases occurring every year. TB treatment requires long-term therapy,
faithful and a combination of (toxics) antibiotics, with a change of lever
disease. A new therapy or medicine is therefore urgently needed.
Despite extensive efforts immunological protection against TB has not been
characterized completely. Recently, our lab has employed a functional assay to
measure the capacity of the immune system to control mycobacterial outgrowth.
Samples from individuals that show very strong growth inhibition in this assay
will be very instrumental to decipher the immunological process responsible for
this. Our initial studies applying these functional assays (submitted) revealed
that very strong control of mycobacterial outgrowth was present in samples from
individuals recently exposed to Mtb. These were mostly individuals that were
included from contact investigations, but not those with long-term established
latent TB infection (LTBI). Here we aim to decipher the functional mechanism
behind this enhanced growth control and to identify (immunological) mechanisms
responsible for growth control. We hypothesize that training of the innate
immune system is involved in mediating mycobacterial growth control and
therefore we think it is important to collect samples as early as possible
after Mtb exposure.
Previous data from our group suggest that specific monocyte populations are
involved in the mycobacterial growth reduction. We have identified chemokines
CXCL9 and CXCL10 as molecules involved in this process. Here we want to assess
in great detail the cells and pathways involved to understand the mechanisms of
mycobacterial growth control. Understanding growth inhibition will be essential
for development of novel vaccines against tuberculosis.
Intriguingly, a considerable proportion of exposed individuals within the
contact investigations did not convert their TST skin test or IGRA, however
almost all of them controlled BCG outgrowth in the MGIA. Therefore we
hypothesize that Mtb exposure results in temporary trained innate immunity, but
this does not necessarily activate adaptive immunity. In the current project we
aim to unravel in more detail the induction of trained innate immunity, but
also correlate this to the induction of adaptive immunity later in time.

2.1 Hypothesis
Primary infection with Mtb induces temporal changes in the innate immune
system, known trained innate (aangeboren) immunity, and the extent of trained
innate immunity influences subsequent activation of adaptive (verworven)
immunity.
2.2 AIM
Investigate the relationship between magnitude and duration of trained innate
immunity and the subsequent activation of adaptive immunity using peripheral
blood samples of individuals with recent exposure to active pulmonary TB
patients.

Contact investigations are regularly executed in the Netherlands following
diagnosis of sputum positive pulmonary TB patients. Since trained innate
immunity occurs in the early period after Mtb exposure we plan to recruit
recently exposed individuals participating in contact investigations. In
addition to the earliest possible time point, ie at the stage of contact
investigation, we will also ask the volunteers to return for 2 additional blood
drawings at week 6 and month 6 to allow longitudinal analysis of trained innate
immunity as well as to follow activation of the adaptive immune system.
Municipal health authorities will be approached to notify the study team when
performing contact investigations of sputum 3+, 4+ or 5+ TB index cases.
Previous studies have shown that contagious TB patients with these high
bacterial burdens in their sputum have the highest transmission rates within
the Dutch population (ref =EM Lohmann et al, INT J TUBERC LUNG DIS 16(11):1477-
1484, 2012). Since we want to study individuals with recent infection as a
result of exposure we want to select for the contact investigations with the
highest risk of transmission..
We will approach all individuals in first and possibly in second rings of the
contact investigation as set up by the municipal health service according to
national guidelines that are above 18 years of age. Individuals with known
immuno-deficiencies will be excluded from study participation (including HIV
infected individuals, individuals with immune modulating therapies). All
participants will be informed on study purposes and asked to voluntarily
participate. Participants will sign informed consent forms before blood samples
are drawn and will have the possibility to refrain from further participation
at any stage.
National guidelines instruct Tuberculin Skin Testing (TST) as screening method
for Mtb infection in contact settings. These will be performed by local
municipal health services according to their standard care, but with permission
in the informed consent, results will be shared with the study team.
A second step to determine Mtb infection according to national guidelines is to
measure IFNγ release in response to specific Mtb antigens using eg the
Quantiferon TB Gold assay, an IFNgamma release assay (IGRA). For our research
purposes these IGRA results are essential and therefore we will run the
Quantiferon TB Gold on all samples that we will collect. We are happy to share
these results, in real-time, with the municipal health system consulting the TB
contact. Thus, if any benefit to the contacts from participating in our study
should be indicated, it will be the availability of a second independent
measure of Mtb infection at multiple time points post exposure.
Care and counselling of all exposed individuals will remain with the local
municipal health care team. Decision to participate in our observational study
will at no point influence the decision to treat the potential latent TB
infection. The decision to treat will be taken by local physicians in consult
with the exposed or infected individuals based on clinical and personal
circumstances and will follow national guidelines and does not involve the
study team.
However, the study team will ask specific permission to be notified on the
decision to treat, including the dates of treatment, treatment duration and
type of treatment received since this may significantly influence the immune
responses measured in vitro.
Participants in our study will be asked to donate blood samples for research
purposes. Part of that blood will be used to measure adaptive immunity in the
diagnostic IGRA, the Quantiferon TB Gold (QFT), the remainder of the blood will
be used in exploratory immunological assays. Blood samples will be processed to
store plasma, peripheral blood mononuclear cells (PBMCs), DNA and RNA.
Laboratory measurements may include mycobacterial growth inhibition assays
(MGIA), measurements of soluble molecules such as cytokines and chemokines,
stimulation with mycobacterial antigens or live mycobacteria, extensive
phenotyping of blood cells including monocytes by advanced flow cytometry
technologies, measurement of RNA expression profiles, determination of DNA
methylation profiles and any other immunological or molecular technologies
required to understand innate and adaptive immune responses towards Mtb.
A second and third blood sample will be collected 6 weeks and 6 months later to
assess induction of adaptive immunological memory and determine kinetics of
trained innate immunity. Also the QFT will be repeated to assess late
conversions. The same number of tubes will be collected and the same assays
will be run.

No risks are associated with participation in this study other than those of
routine collection of blood.
There is no direct anticipated benefit expected for the volunteers by
participating in the study. Indirectly, access to QFT results for the physician
may comfort the participants.