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New directions in research

Cloning and expression of mycobacterial genes in E. coll. Mycobacterium tuberculosis and Mycobacterium leprae were described as important human pathogens more than 100 years ago but, despite continued research efforts, mycobacterial diseases remain a major source of morbidity and mortality today with particularly devastating effects in developing countries 1'2. Tuberculosis currently afflicts 30 million individuals worldwide with an annual death rate of three million, while leprosy affects 10-15 million people causing debilitating deformities in untreated cases. Vaccination with an attenuated strain of Mycobacterium boris (bacillus Calmette - Guerin, BCG) aided the reduction of tuberculosis in Western Europe 3, but a major W H O trial has shown that BCG vaccine is ineffective in a tuberculosis endemic area of South India 4. Attempts to develop a vaccine against leprosy have been hampered by the failure to culture M. leprae in the laboratory and studies have been limited to the use of bacteria isolated from experimentally infected nine-banded armadillos 5. Highly active multiple drug regimens have been used to treat tuberculosis patients 6 but have not been effective in eradication of the disease from populations living in endemic areas. Attempts to detect and treat the early stages of mycobacterial diseases have been limited by the lack of specific tests for infection. Tuberculin reagents used for skin testing contain a mixture of specific and cross-reactive mycobacterial antigens and the test fails to distinguish active disease from sensitization due simply to exposure to pathogenic or environmental mycobacteria. Some encouraging progress towards the early diagnosis of leprosy has recently been made possible, however, by use of a chemically definedM, leprae -specific glycolipid antigen 7,s. Understanding of the immune response to mycobacterial infection is, therefore, an active area of current research with the goals of producing more specific diagnostic tests and eventually of developing vaccines to protect against mycobacterial infections. Resistance to mycobacteria is mediated by the cellular immune system and many attempts have been made to purify proteins fromM, tuberculosis with the ability to elicit delayed-type hypersensitivity reactions in skin tests 9. These attempts have been limited by the technical difficulties of obtaining large amounts of the slow-growing organisms and of identifying and purifying individual components from the highly complex mixture of proteins, carbohydrates and lipids produced by the bacteria. In the case of M. leprae, few effective studies of this type have been carried out due to the limited quantities of bacteria which have been available. The recent application of monoclonal antibody technology to the study of mycobacteria has allowed the identification of a variety of protein antigens in M. tuberculosis and M. leprae and the presence of species-specific and cross-reaCtive epitopes has been demonstrated ~°-14. While such antibodies provide useful tools for identification and possibly purification of individual antigens by immunoaffinity chromato© 1985, ElsevicrScicnccPublishersB.V.,Amsterdam

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graphy, the supply of purified antigens remains limited particularly in the case ofM. leprae. It is in this light that the recent reports 15'isof the cloning and expression of M. tuberculosis and M. leprae genes in Escherichia coli are seen as a major breakthrough in mycobacterial research. Young et al. 15have cloned DNA from M. leprae and from a virulent strain of M. tuberculosis in the phage expression vector Igtl 1. Mycobacterial D N A fragments (1-8 kilobases, kb) were produced by mechanical shearing and libraries each consisting of 4 x 106 individual recombinant phages were generated. The mycobacterial D N A was placed alongside a gene for//-galactosidase production, and switching,0 n transcription of this gene by a chemical inducer allowed the possible production of large amounts of the mycobacterial protein coded by the D N A insert. A specific mutation in a protease of the host E. coli prevented degradation of the accumulated foreign protein. The tgtl 1 system can conveniently be screened for antigen production by immunoblotting of proteins in phage plaques onto nitrocellulose membranes. The membranes are then screened using specific antibodies with secondary antibody - immunoperoxidase detection. A zone of antibody reactivity is seen over plaques formed by phages containing the appropriate mycobacterial DNA. Using this system my colleagues and I have shown expression of 3 out of 4 mycobacterial proteins recognized by a panel of monoclonal antibodies to M. tuberculosis antigens originally developed by workers in London 1°. Screening of the M. leprae library demonstrated expression of antigens recognised by 12 monoclonal antibodies directed towards different epitopes on 5 separate proteins. The 65 kDa protein antigen ofM. leprae was analysed in detail using 6 different monoclonal antibodies and 17 recombinant D N A clones. A preliminary restriction map was built up indicating that the antigen, which appears with several lower molecular weight forms during gel electrophoresis, is coded by a single gene. Using an alternative approach, Clark-Curtiss et al. 17 have constructed gene libraries with D N A from a variety of mycobacteria including M. leprae. The cosmid cloning vector used in this system (pHC79) contained significantly larger D N A inserts (40-45 kb) than the ;{gtl 1 vector and screening of clones by enzyme complementation assays failed to show evidence of functional mycobacterial proteins. Subcloning of smaller D N A fragments (1-10 kb) into a plasmid vector (pYA626) containing the Streptococcus mutants asd promoter did however, result in synthesis of M. leprae polypeptides in a minicell assay. No recognition of mycobacterial antigen was observed on screening of clones using several monoclonal antibodies or polyclonal anti-M, leprae antiserum. The ~tgtll system appears to have an advantage as a sensitive screening method for assaying large numbers of clones for antigen production. The success of this system

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in screening for expression of single epitopes recognised by monoclonal antibodies is particularly striking and such a combination of monoclonal antibody and recombinant D N A technologies represents a potent approach to further analysis of mycobacterial antigens. The asd promoter in the plasmid vector ofClark-Curtiss et al. may be essential for enhancement of expression of particular proteins. These authors found no evidence for expression of recombinant genes in the absence of a strong nonmycobacterial promoter but, while the 3~gtl1 vector does contain a/3-galactosidase promoter, preliminary results suggest that expression of some mycobacterial antigens is independent of the//-galactosidase gene (D.B. Young, unpublished). Further study of the antigens coded by the recombinant mycobacterial D N A can proceed by a variety of strategies. In some cases the antigen may he present as a fusion protein linked to fl-galactosidase and purification by immunoaffinity chromatography using anti-fl-galactosidase antibody may be possible. In other cases, where E. coli recognizes mycobacterial transcription or ribosome binding sequences, the antigen may be synthesized as a free protein identical to that found in the native mycobacteria and immunoaffinity chromatography using monoclonal antibodies will be most productive. Alternatively, subcloning of restriction fragments may be used to identify segments of D N A coding for particular epitopes and synthetic peptides based on nucleotide sequences may be produced without recourse to protein purification. With the application of these novel developments in antigen production and purification, detailed investigation of the possible role of individual antigens in stimulation or suppression of cellular immunity can be under-

taken. Isolated proteins or peptides can be tested for their ability to induce T-cell proliferation and delayed-type hypersensitivity reactions, and possible modulation of the course of infection in experimental animals following vaccination with purified components can be assessed. It is hoped that such studies will lead to the development of improved diagnostic reagents for early detection of leprosy and tuberculosis and, in the longer term, to the production of effective anti-mycobacterial vaccines. ~-1 References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

World Health Organization. (1983) Bull. WHO 61, 779-785 Bloom, B. R. and Godal, T. (1983) Rev. Infect. DIS. 5, 765-780 Medical Research Council. (1972) Bull. WHO 46, 371-376 World Health Organization. (1980) WHO TechnicalReport Series, No. 651 Kirchheimer, W. F. and Storrs, E. E. (1971) Int. J. Lepr. 39, 693-702 Fox, W. (1980) in Recent Advances in Respiratory Medicine (D. C. Flenley ed.) p. 183, Churchill-Livingstone, Edinburgh and London Young, D. B. and Buchanan, T. M. (1983) Science 221, 1057-1059 Cho, S. N., Fujiwara, T., Hunter, S. W., Rea, T. H., Gelber, R. H. and Brennan, P. J.. (1984)J. Infect. Dis. 150, 311-322 Ma, Y. and Daniel, T. M. (1983)3. Infect. Dis. 148, 500-509 Coates, A. R. M., Allen, B. W., Hewitt, J., Mitchison, D. A. and Ivanyi, J. (1981) Lancet ii, 167-169 Gillis, T. P. and Buchanan, T. M. (1982) Infect. Immun. 37, 172-178 Ivanyi, J., Sinha, S., Aston, R., Cussel, D., Keen, M, and Sengupta, U. (1983) Clin. Exp. Immunol. 52, 528-536 Young, D. B., Fohn, M . J . , Khanolkar, S. R. and Buchanan, T. M. (1985) Clin. Exp. Immunol. 60, 546-552 Engers, H. D. et al. (1985). Infect. Immun. 48, 603-605 Young, R. A., Bloom, B. R., Grosskinsky, C. M., Ivanyi, J. and Davis, R. W. (1985)Proc. NatlAcad Sci. USA 82, 2583-2587 Young, R. A., Mehra, V., Sweetser, D., Buchanan, T., Clark-Curtiss, J,, Davis, R. W. and Bloom, B. R. (1985) Nature (London) 316, 450-452 Clark-Curtiss, J. E., Jacobs, W. R., Docherty, M. A., Ritchie, L. R. and Curtiss, R. (1985)J. Bacteriol. 161, 1093-1102 DOUGLAS B. YOUNG

M R C Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Road, London W12 OHS, UK.

Immunoregulation of MHC antigen expression It is now apparent that the expression of major histocompatibility complex (MHC) antigens on the cell surface is not constant but a variable phenomenon which may depend on the immune status of the individual. It is often stated that class II M H C antigens can be present constitutively (as on B cells, activated T cells, dendritic cells and macrophages) or they may be induced. In rats, epidermal keratinocytes and intestinal epidermal cells, normally negative for class II antigens, become positive during a graft versus host response 1'2. Similarly, in rats and mice class II antigens are induced on vascular endothelial cells in transplanted hearC and skin ~. Studies in vitro have demonstrated that gamma interferon (IFN-lt) is a potent inducer of class II antigens on human endothelial cells 5 and on a number of human 6 and murine cell lines 7. Supernatant from phytohaemagglutinin (PHA)- or alloantigen-activated T cells causes induction of class II antigens on human umbilical vein 5 presumably by the action of IFN-),, but the possibility that other lymphokines cause M H C induction cannot be excluded. Detailed studies of the distribution of class II antigens in human organs are possible using frozen sections, monoclonal antibodies and immunofluorescent 0 or immunoenzymatic 9'1° techniques. Such studies have

demonstrated considerable variation in antigen expression within organs and between individuals. For example, in a study of 46 normal kidneys, all expressed class II antigen on the endothelium of glomeruli, but in only 60% of the kidneys was tubular epithelium class II positive 9. Similarly, in another investigation 1° vascular endothelium from capillaries was found to be consistently class II positive in all organs (except brain, testis and placenta) but large blood vessels showed variable expression within an organ. The possibility therefore arises that some of this variation in 'normal tissue' is due to immune stimulation of unknown origin and some of it may be caused by the observer, depending on whether the procedures for sampling material is traumatic1°. A recent paper suggests this is the case for expression of class II antigens on canine vascular endothelium 11. The authors treated normal dogs with large doses of cyclosporin A and found that such treatment abrogated the expression of class II antigens on endothelial cells of kidney and skin. The dog resembles humans i n ' normally' expressing class II antigens on the capillary endothelial cells of skin and the glomerular capillary endothelium of the kidney. The exact mode of action of cyclosporin A is unknown, but one © 1985,ElsevierSciencePublishersB.V., Amsterdam 0167 4919/85/$02.00

Cloning and expression of mycobacterial genes in E. coli.

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