Summary:

The efficacy of BCG vaccination is doubtful and significant problems with resistance against anti-tuberculosis drugs have emerged. These problems could be fuelled by the selection of more adapted variants of Mycobacterium tuberculosis. This project aims to further the hypothesis on the selection of M.tuberculosis genotypes by the measures used to control the tuberculosis(TB) epidemic, anti-TB treatment and BCG vaccination. This will be carried out to:

1. Study the dynamics of the population structure of M. tuberculosis in various regions in relation to drug resistance and BCG vaccination
2. Identify genetic factors affecting bacterial adaptability
3. Study the virulence of strains of emerging genotypes and the level of BCG protection against these strains in vivo
4. Gain insights into the adaptation of M. tuberculosis in response to control efforts should allow improved strategies for treatment and control and possibly reveal new targets for intervention.

The sequence of selection followed by adaptation may lead to the selection of particularly adaptable strains. Although potentially a serious threat, detailed knowledge of the adaptation process in M. tuberculosis should ultimately allow these pathways to be targeted. This is because, although the accumulation of mutations is a random and unpredictable process, bacteria have specific mechanisms of repairing mutations in DNA. Thus, inhibition of these pathways should greatly reduce the probability of bacteria escaping selective pressure by becoming resistant to anti-TB drugs or becoming escape variants of BCGinduced immunity.

Background:

TB remains a major public health threat, still emerging in some areas of the world. The major tools to control the TB epidemic are worldwide implementation of the directly observed therapy short course (DOTS) programme and BCG vaccination. The protection offered by the BCG vaccine against lung infection is questioned and at best limited. It is therefore suspected that certain emerging strains may have adapted to evade the current vaccine. Despite the availability of effective treatment, the emergence of bacteriaresistant to the anti-TB drugs used is a constant threat. This reservoir of resistant, infectious, pathogenic and probably highly adaptable strains is likely to spread.

So far, the research on the spread of particular M. tuberculosis strains has been focused on strains of the Beijing genotype family. A recent worldwide survey on the distribution of these strains and their associations with drug resistance, covering data on over 29,000 patients in 35 countries, was finalised (Figure 1). This study showed that the Beijing genotype is emerging and is, sometimes in high levels, associated with drug resistance. These strains have also been associated with vaccine escape. Additionally, genetic changes have been identified in putative mutator genes of these strains. The exact phenotypic consequences of this genetic variation in M. tuberculosis (if any) are unknown but it is predicted that these genes are involved in adaptation of the bacteria in the host.

The sequence of selection followed by adaptation may lead to the selection of particularly adaptable strains. Although potentially a serious threat, detailed knowledge of the adaptation process in M. tuberculosis should ultimately allow these pathways to be targeted. This is because, although the accumulation of mutations is a random and unpredictable process, bacteria have specific mechanisms of repairing mutations in DNA. Thus, inhibition of these pathways should greatly reduce the probability of bacteria escaping selective pressure by becoming resistant to anti-TB drugs or becoming escape variants of BCGinduced immunity.

Aim:

The objective of the project is to further the hypothesis that the current TB epidemic is fuelled by the selection of more adapted variants of M. tuberculosis by the measures used to control the epidemic. These selection mechanisms will be investigated by studying the population structure of M. tuberculosis in various regions by using DNA fingerprint tools. Trends in the dynamics of the population structure (emerging genotypes) will be identified. The association of the various genotypes with drug resistance or possible vaccine escape will be studied.

Furthermore, the influence of genetic variability among M. tuberculosis strains on the virulence and immune response induced by these strains and the protection of various vaccines against these strains will be studied in a BALB/c mouse model for pulmonary tuberculosis. In addition, the effect of genetic variation, in the M. tuberculosis genome on the mechanisms of bacterial adaptability and response to stress will be studied. This will involve in vitro studies of the phenotypic effect of specific drug resistance and other mutations, with an emphasis on mutations in (putative) DNA repair genes.

Expected results:

The project will obtain insight in the population structure of M. tuberculosis in various areas, including high- and low-TB incidence countries and multidrug resistant (MDR) TB hot-spot regions. Emerging genotypes will be identified and it will be investigated whether the emergence of certain genotypes is associated with BCG vaccination and/or drug resistance.

Comparative in vitro studies of the virulence and immune response induced by representative strains of different M. tuberculosis genotype families will improve the understanding of the mechanisms of success of the major M. tuberculosis strains causing the current worldwide tuberculosis epidemic. The in vitro studies of the effect of drug resistance and DNA repair mutations will enhance the understanding of the impact of these genes on drug resistance acquisition

Potential applications:

Various strain collections will be established, including strains from high MDRTB prevalence countries, strains of well-characterised epidemic M. tuberculosis genotypes, and laboratory mutants with modified DNA repair genes. These strains are likely to be used in further studies.

Characterisation of M. tuberculosis strains that are emerging because they have a higher ability to resist the anti-TB drugs and/or BCG vaccination will contribute to the research on the development of new tuberculosis vaccines. If differences in susceptibility between M. tuberculosis strains to BCG-induced immunity are found this would emphasise that new anti-TB vaccines should be tested for efficacy against a variety of circulating M. tuberculosis strains. If different levels of association with drug resistance or BCG vaccination exist between the closely related strains of a certain genotype this may accelerate the research on the development of drug resistance and the preparation of a new anti-TB vaccine, by studying the (limited) genetic differences between these strains. The study of genetically closely related, more and less successful strains of a certain genotype may thus indirectly aid the development of an effective anti-TB vaccine.

Coordinator:

Partners:

Nº	Principal Scientific Participants	Official Address	Other Information
2	Richard M. Anthony	Royal Tropical Institute (KIT) Biomedical Research P.O. box 95001 1090 HA Amsterdam The Netherlands	Tel: +31-20-5665449 Fax: +31-20-6971841 E-mail: r.anthon@Kit.nl Website:www.kit.nl
3	Rogelio Hernandez Pando	National Institute of Medical Sciences and Nutrition “Salvador Zubiran” (NIMSN) Experimental Pathology Section Department of Pathology Vasco de Quiroga no. 15, col. seccion XVI 14000 Mexico D.F. - Mexico	Tel: +52-5554870900 Fax: +52-5554853491 Website:www.innsz.mx
4	Gunnar A. Bjune	University of Oslo Department for General Practice and Community Medicine Ullevaalsalleen 1130 Blindern / 0317 Oslo Norway	Tel: +47-22850640 Fax: +47-2285672 Email: g.a.bjune@medisin.uio.no Website:www.uio.no
5	Afranio L. Kritski	Federal University of Rio de Janeiro Department of Clinical Medicine Tuberculosis Research Unit Av. Brigadeiro Trompowsky s/n Ilha do Fundao-Predio Hospital Universitario 4 Andar, Unidade Pesq TB Rio de Janeiro Brazil	Tel: +55-21-25622426 Fax: +55-21-25506903 Email: kritskia@gmail.com Website:www.ufrj.br
6	Paul D. van Helden	Stellenbosch University Faculty of Health Sciences Department of Medical Biochemistry P.O. box 19063 7505 Tygerberg South Africa	Tel: +27-21-9389124 Fax: +27-21-9389476 E-mail: pvh@sun.ac.za Website: http://academic.sun.ac.za/med _biochem/index.html
7	Brigitte Gicquel	Institut Pasteur 28, Rue du dr. Roux 75015 Paris Cedex 15 France	Tel: +33-1-45-688828 Fax: +33-1-45-688843 E-mail: bgicquel@pasteur.fr Website:http://www.pasteur.fr
8	Abigail Wright	World Health Organisation (WHO) Stop TB department 20 Avenue Appia 1211 Geneva Switzerland	Tel: +41-22-7914975 E-mail: wrighta@who.int Website:http://www.who.int
9	Martien.W. Borgdorff	KNCV Tuberculosis Foundation P.O. box 146 2501 CC Den Haag The Netherlands	Tel: +31-70-416722 Fax: +31-70-3584004 E-mail: borgdorffm@kncvtbc.nl Website:www.tuberculose.nl
10	T. Trao Vu	National Institute of Hygiene and Epidemiology (NIHE) 1 Yersin street Hanoi Viet Nam	Tel: +84-4-9715470 Fax: +84-4-9715470 E-mail: trao@hn.vnn.vn
11	Tone Tønjum	Rikshospitalet- Radiumhospitalet Health Authority Centre for Molecular Biology and Neuroscience Institute of Medical Microbiology Sognsvannsvn. 20 NO-0027 Oslo Norway	Tel: +47-23-074065 Fax: +47-23-074061 Email: tone.tonjum@medisin.uio.no Website:www.cmbn.no