Molecular approaches to distinguish between latent and active tuberculosis and identify factors involved in protection.

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Tuberculosis is caused by an infection with a microorganism called Mycobacterium tuberculosis (Mtb) and is responsible for nearly 2 million deaths worldwide per year. Approximately one third of the world is exposed to the infection but only a proportion will develop active symptomatic disease. The people who do not develop active disease are thought to have a protective immune response and are termed "latent", but the nature of this protective response is not understood. The BCG vaccine, although used widely to protect against tuberculosis, has variable efficacy in protecting individuals against tuberculosis disease. Because the body's defensive response to tuberculosis has not yet been fully worked out it is difficult to create a successful protective vaccine as it is not known how to assess if a vaccine is effective. In addition, it is not known what part of the immune system is required to be stimulated and how, in order to provide protection.

This study aims to search for differences in the body's response between asymptomatic latent individuals and infected individuals who develop active tuberculosis (pre and post-treatment). We will do this by looking at differences in the activity of all the genes in the blood of the infected individuals, those with and without disease, compared to healthy uninfected people. To ensure the gene activity is detectable in all the infected individuals, we will cause an exaggerated response by adding Mtb specific proteins before measuring the gene activity and we will also measure the gene activity at different times to see if there is a difference in gene expression over time. If a set of gene activities and/or proteins is present in the blood of asymptomatic latent individuals but missing in the blood of active tuberculosis patients after treatment this may represent a protective signature or fingerprint that keeps the latent individuals from becoming ill. Such a gene activity signature or fingerprint may be helpful in monitoring the efficacy of vaccines in the future. In addition, comparison of this protective gene activity in latent individuals with the gene activity in BCG vaccinated individuals, may reveal a unique gene fingerprint in the blood of latent individuals and provide information on a gene activity signature that may protect against adult tuberculosis, which the BCG vaccine does not always achieve.

Once we have identified the differences in gene activity it may be possible to use this information to help monitor the host response during vaccination and so develop better vaccines to protect others from developing tuberculosis, if exposed to Mtb.

Technical Summary

Mycobacterium tuberculosis (Mtb) is estimated to infect a third of the world's population, yet only 10% develop active tuberculosis (TB),causing 1.8 million deaths annually. The immune factors protecting the remaining 90% of latent individuals who do not develop disease are as yet unclear. It has already been shown that there is a specific transcriptional signature from mycobacterial antigen-stimulated blood that is present in healthy vaccinated, latent and active TB individuals, but absent in unvaccinated healthy individuals.

Aim: To determine whether there is a specific transcriptional signature in Mtb and Mtb-specific antigen-stimulated blood cells of latent TB and BCG vaccinated individuals but absent in active TB patients, before and after treatment.

Objectives: Define and compare the transcriptional signature by microarray analysis, and cytokine and chemokine profiles by immunoassay, after different stimulation times with Mtb and mycobacterial antigens of whole blood or peripheral blood mononuclear cells (PBMC) from: healthy non-BCG vaccinated, healthy BCG vaccinated, individuals with latent TB and those with active TB (pre and post-treatment).
Design and Methods: 4 cohorts of individuals as described above will be enrolled. Samples of their whole blood/PMBCs will be stimulated in vitro with Mtb, Mtb antigens (ESAT6, Ag85, PPD), SEB (positive control) and TLR-2 ligand PAM3Cys. Blood and PBMC stimulated for different time points will be frozen, RNA will be isolated and microarray analysis undertaken to identify signatures of protection or pathogenesis. Supernatants will be collected at the same time points for immunoassay.

Scientific and medical opportunities: The identification of a specific transcriptional signature which differentiates between latent and active TB will help identify factors involved in protective innate and adaptive immunity to help design future vaccines as well as identifying biomarkers for use in monitoring response to vaccines.

Planned Impact

Tuberculosis (TB) is caused by an infection with a microorganism called Mycobacterium tuberculosis (Mtb) and is responsible for nearly 2 million deaths worldwide per year. Approximately one third of the world is exposed to the infection but only a proportion (10%) will develop active symptomatic TB disease. The people who do not develop active disease are thought to have a protective immune response and are termed "latent", but the nature of this protective response is not understood. The BCG vaccine, although used widely to protect against tuberculosis, has variable efficacy in protecting individuals against tuberculosis disease. Moreover what constitutes a protective response to tuberculosis is not understood. Hence, it is difficult to create a successful protective vaccine since it is not known how to assess if a vaccine is effective. In addition, it is not known what part of the immune system is required to be stimulated and how, in order to provide protection. TB has a considerable impact in terms of morbidity and mortality, as well as socio-economic cost and it is one of the leading infectious causes of death in the world. The ultimate goal is to develop a successful vaccine against tuberculosis, and eliminate the disease from our society. This project proposes to identify a protective RNA transcriptional signature that is in the blood of latent individuals, that is not detected in the blood of active TB patients, pre or post-treatment.

The currently used TB vaccine, called BCG, affords only variable protection against adult pulmonary TB, and a new vaccine is needed. Future vaccines are in development however the assessment of potential protection uses readouts focusing predominately on IFN-Gamma and CD4+ T cell responses, which may not always correlate with protection. Vaccine development is therefore limited as the immune factors involved in protection against disease and the immune responses to disease are not fully understood. Hence biomarkers that correlate with successful vaccination are much needed.

The impact of this study will be in the identification of potential biomarkers reflecting protective innate and adaptive immune responses in people who have been exposed to Mtb and have no clinical signs of disease (i.e. latent) and those with active TB disease (pre and post-treatment). The ultimate aim of this research is to identify biomarkers which correlate with protection. If successful this will have a major impact on one of the worlds leading infectious causes of death by enabling the design and testing of an effective vaccine.

The time-scale whereby results from this proposal could provide new information with respect to protective biomarkers against TB, would be within the range of two years after initiation of the study. This information, would be made available to the research community and public by publication, and would then be invaluable to clinicians and researchers evaluating vaccines in ongoing clinical trials. Ultimately this research has the potential to impact on preventative measures agains TB, thereby reducing the morbidity, mortality and socio-economic cost that this disease currently has.

There is also a potential economic impact as a patent has been filed on "The identification by microarray analysis of blood of a distinct and reciprocal immune signature in Latent versus Active tuberculosis (TB) patients, as compared to healthy controls" as a result of a study led by Dr. O'Garra.
The co-inventors are: Jacques Banchereau, Damien Chaussabel, Baylor Institute for ImmunologyResearch, Dallas, Texas, USA (40%).Anne O'Garra, Matthew Berry, MRC National Institute for Medical Research, Mill Hill, London, UK.(40%) Onn Min Kon, Imperial College Healthcare NHS Trust, London, UK. (20%). Additional results and discoveries related to potential biomarkers of protection, useable in vaccine development will be patented.

Publications

10 25 50
 
Title Publicly available whole genome expression data 
Description Gene Expression Omnibus (GEO) is a database repository of high throughput gene expression data and hybridization arrays, chips, microarrays. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Our own publication PLoS One. 2014 May 19;9(5):e97702. doi: 10.1371/journal.pone.0097702. eCollection 2014. Identification of the key differential transcriptional responses of human whole blood following TLR2 or TLR4 ligation in-vitro. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55375
 
Description Clinical Collaborators for project 
Organisation Guy's and St Thomas' NHS Foundation Trust
Department Respiratory Department
Country United Kingdom 
Sector Hospitals 
PI Contribution Collecting of clinical samples for the research project
Collaborator Contribution Collecting of clinical samples for the research project
Impact None as yet
Start Year 2009
 
Description Clinical Collaborators for project 
Organisation Imperial College Healthcare NHS Trust
Department Respiratory Medicine
Country United Kingdom 
Sector Hospitals 
PI Contribution Collecting of clinical samples for the research project
Collaborator Contribution Collecting of clinical samples for the research project
Impact None as yet
Start Year 2009
 
Description Clinical Collaborators for project 
Organisation Royal Free London NHS Foundation Trust
Department Department of Thoracic Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Collecting of clinical samples for the research project
Collaborator Contribution Collecting of clinical samples for the research project
Impact None as yet
Start Year 2009
 
Description NIMR open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Approximately 200 of the public attended for an Open day at MRC-NIMR. I presented a poster of the work that I am undertaking.

It seemed to be well recieved
Year(s) Of Engagement Activity 2013
 
Description Schools visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Poster and small informal session talking about project

Well recieved open day - with high satisfaction reported by participants.
Year(s) Of Engagement Activity 2014