Protection from Mycobacterium tuberculosis infection: Learning from exposed but uninfected children

BACKGROUND

Tuberculosis (TB) is a major global health problem for children and adults. One of the characteristics of TB is that the bacteria that cause it can stay within the body for many years after infection, and re-activate and cause disease later in life. TB vaccine research is therefore complicated because people need to be followed-up for many years to see whether or not they eventually develop the disease. It's also unclear what sort of immune response is the most protective against TB, and therefore how best to design and test new vaccines.

The World Health Organization is aiming for zero childhood TB deaths and to eliminate the disease by 2050. Identifying an effective immune response and markers in the blood that correspond to protection against TB, would dramatically speed up development and implementation of new vaccines, which is key to achieving these goals.

TB IN CHILDREN

Children are normally exposed to TB bacteria by a coughing infected adult living in their home. Children's responses vary. Some of these children become unwell with TB disease, others are healthy but infected, whilst a third group shows no signs of infection. Children who are healthy but infected are at risk of TB re-activating later in life and causing disease.

We aim to answer the question:

Why do some children exposed to TB show no signs of infection, whilst others in the same household become infected?

To understand the protective mechanisms, this research will compare immune responses in blood from children in the Gambia:

1. Children exposed to TB who are "protected"
2. Siblings exposed to TB who are infected
3. Healthy children from the community

By comparing "protected" children to their infected siblings in the same household and to matched health children from the community, many factors that can influence TB, such as age, nutrition, genetic background, degree of exposure to TB and so on will be taken into account, enabling us to focus on specific questions.

AIM ONE

We will first confirm a previous finding that in laboratory testing, blood from "protected" children is better at killing the bacteria that cause TB. This is important because it will show that there is a test we can carry out in the laboratory that corresponds to the clinical differences between the groups of children. There are several possible explanations for this difference.

AIM TWO

TB infects immune cells and can live within them. It is thought that if infected immune cells die in a carefully regulated process (known as apoptosis), the spread of TB is limited. If the infected TB cell dies in an uncontrolled manner (known as necrosis) the bacteria can multiply more easily and spread rapidly. We will use blood to see if immune cells from "protected" children are more likely to die by apoptosis when infected with TB in the laboratory.

The balance of chemicals that promote or suppress inflammation is an explanation for why some infected cells die by apoptosis or necrosis and potentially why some children are protected against TB infection. We will therefore measure the production of a range of these chemicals in blood in response to infection with TB in the laboratory.

AIM THREE

Having identified inflammatory chemicals that are implicated in protection against TB in children, we can investigate this further by adding specific drugs to blood infected in the laboratory and measure whether it affects killing of bacteria or the manner in which cells die following infection. This will help us to be more certain that what we have seen has a causative impact.

POTENTIAL OUTCOMES

The information gathered from this research project has potential to help test vaccines currently in production, guide development of new vaccines, and see whether addition of medicines that influence inflammation can be helpful in addition to direct anti-TB drugs.

Why do some children exposed to Mycobacterium tuberculosis (M.tb) show no signs of infection, whilst others in the same household become latently infected?

We aim to define protective host immune mechanisms against infection by M.tb through characterizing differences between clinical phenotypes in an established household case-contact platform in The Gambia:

1. TB-exposed children without signs of M.tb infection ("protected"),
2. their latently infected siblings ("susceptible"),
3. age-matched but TB-unexposed community "controls."

We hypothesize that "protected" siblings restrict mycobacterial growth more than their "susceptible" siblings and that this reflects cell death and linked eicosanoid pathways.

Aim 1: To validate pilot data that "protected" children inhibit mycobacterial growth more than "susceptible siblings"

Methods 1: Whole blood reporter-gene tagged M.tb infection with calculation of growth ratios.

Aim 2: To measure cellular and inflammatory differences in responses of blood from "protected" and "susceptible" children to M.tb infection.

Aim 2.1: To compare monocyte cell death modality.

Methods 2.1: M.tb-infected monocytes will be evaluated for apoptosis by (a) Flow cytometry and (b) Cell-death detection ELISAPLUS immunoassay.

Aim 2.2: To use an unbiased lipidomics approach to evaluate lipid mediator profiles.

Methods 2.2: Ultra-performance liquid chromatography with tandem quadrupole mass spectrometry (UPLC-MS/MS) will quantify lipid inflammatory mediators.

Aim 3: Manipulation of lipid mediators of interest in M.tb infection to provide mechanistic insight.

Methods 3: Following analysis of Aim 2.2 data, selected drugs will be tested for impact upon mycobacterial growth, cell death modality and lipid mediators.

OPPORTUNITIES

The protective markers identified will aid vaccine evaluation, whilst the lipidomics opens avenues to investigate immunomodulation in TB.

CHILDREN AT RISK OF TB
Children across the world are the primary intended beneficiaries of this fellowship proposal. There are manifold potential benefits for paediatric health. Markers of protection can provide a fundamental step forward in the development of a better vaccine to prevent infection and disease in TB, thereby helping to meet the goals of zero childhood TB deaths. Understanding the role of inflammation, and using a discovery-generating approach such as lipidomics will further the rapidly burgeoning field of understanding susceptibility to TB. Furthermore treatment of TB may be advanced with immunomodulatory adjunctive treatments to directly anti-tuberculous therapy.

ADULTS AT RISK OF TB
It is not only children who have the potential to benefit from this study, but adults too. Adults with pulmonary TB and a high bacillary burden are the primary mode of transmission to both children and adults. By understanding prevention of infection through this fellowship proposal, vaccines that prevent latent infection, and thereby subsequent reactivation in disease in adult populations will both prevent cases directly, but also prevent transmission, thereby having a dramatic effect upon TB epidemiology.

THE GAMBIAN AND WEST AFRICAN PUBLIC
The people of Gambia and West African region will directly and indirectly benefit from the MRC's investment in resources and people at the MRC Unit in The Gambia. This will come about through the skills development of those involved directly in working on the Fellowship, as well as indirectly through ancillary staff and broader economic impacts upon the community around the Unit. It will also contribute to the excellent track record of high quality research and scientific endeavour in The Gambia and the region as a whole.

THE UK
Particularly in urban areas of the UK, both children and adults are vulnerable to TB and therefore will share in the intended health benefits. Furthermore, the country as a whole will benefit from the high profile investment and collaboration by the MRC into its long-established partnerships with West Africa, promoting the UK as an outward-looking and supportive international leader.

NATIONAL, REGIONAL, AND INTERNATIONAL POLICY-MAKERS
This Fellowship proposal is grounded in the research priorities set by the World Health Organization to meet their goals of zero childhood TB deaths and elimination of the disease by 2050. The research outputs generated will feed back into the policy-makers' arena through driving the research agenda, funding streams, and guiding vaccine development.

INDUSTRY
Translation of markers of protection identified into a format that can be rapidly employed in clinical vaccine trials, and development of novel vaccines guided by protective phenotypes identified will be driven through academic collaboration with industry.

MEDICAL RESEARCH COUNCIL
This Fellowship proposal compliments all four components of the mission of the MRC through aiming to improve human health and wellbeing; training a clinician in scientific research at the highest level; improving the quality of life and economic competitiveness of the UK through protecting children in high prevalence TB urban areas within the UK and the benefits to industry; and promoting dialogue with the public through the communication plan. It also fits with the MRC's Strategic Aim One Research Priority Theme One - Natural Protection within Resilience, Repair and Replacement. It is also in perfect sync with Strategic Aim Three - accelerating progress in international health research. Through the funding of the CTRF and supporting a further collaboration with the existing centre of excellence, The MRC Unit, The Gambia, existing streams of support and investment by the MRC can be strengthened and combined.