Molecular Microbiology (1992) 6(2), 133-145

MicroReview Mycobacterial protein antigens: a compiiation D. B. Young,' S. H. E. Kaufmann,^ P. W. M. Hermans^' and J. E. R. Thole** ^MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital, London W12 OHS, UK. ^Department of Immunology. University of (Jim. D-7900 Ulm, Germany. ^National Institute of Public Health and Environmental Protection. 3720 BA Bilthoven, The Netherlands. ''Department of Immunohaematology and Blood Bank, University Hospitai, 2300 RC Leiden, The Netherlands.

system. As an approach to development of improved strategies for mycobacteriai disease controi through vaccination and immunodiagnosis many researchers have tried to identify individual bacterial components involved in interactions with the immune system. In this review, we present a summary of the key features of defined proteins identified as targets of humoral and cell-mediated immune responses in patients and in animal models of mycobacterial infection.

Historical perspective Summary In response to recommendations from the Steering Committees responsible for co-ordination of World Health Organization programmes for research on the immunology of leprosy (IMMLEP) and tuberculosis (IMMTUB), a list was prepared summarizing the properties of mycobacterial proteins currently under investigation with respect to their immunological activities. After consultation with more than 40 laboratories world-wide this list was extended to form the compilation shown below and is intended to provide a comprehensive and convenient reference for future studies in this field. Introduction Mycobacterial pathogens continue to be responsibie for widespread disease in humans and in domestic animais. While improved iiving standards have been accompanied by a progressive decline in cases of tubercuiosis in industrialized countries, the incidence of disease has remained high in most of the developing world. It is estimated that tuberculosis currently accounts for approximately 2.5 million deaths per year, representing as much as 6% of total mortality and 26% of ail avoidable adult deaths worldwide, thus making Mycobacterium tuberculosis probably the largest single infectious cause of human mortality (Murray etai 1990). Pathogenic mycobacteria have the abiiity to survive within host phagocytic ceils, with the outcome of infection — protection or pathoiogy — possibiy being determined by the response of the host immune Received 1 August, 1991; revised 9 October, 1991, *Forcorrespondence. Tel. (71) 261737; Fax (71) 216751,

At the end of the last century, Robert Koch pioneered the approach of using supernatant material from heat-kilied M. tuberculosis cultures ('old tuberculin') to monitor immune responses in skin test assays, and many subsequent attempts have been made to identify the active components from such preparations (Koch, 1890), Removai of the bulk of the carbohydrate materiai by precipitation with ammonium suiphate or trichioroacetic acid resulted in production of 'purified protein derivative' (PPD), a formulation stiil in routine use, Foliowing a series of biochemical fractionation studies (reviewed by Daniel and Janicki, 1978) a reference system for mycobacterial antigens was proposed on the basis of their reactivity with hyperimmune antisera in an immunoelectrophoresis system (Janicki et ai, 1971). Eleven antigens were separated in this US-Japan reference system, Harboe and his colieagues improved the system by the appiication of crossed immunoelectrophoresis techniques (CIE). They identified as many as 50 distinct antigen peaks and established a system of nomenclature based on a CIE reference (Closs etai, 1980; Harboe and Wiker. 1986; Wiker et ai, 1988). In addition, many workers have referred to mycobacterial antigens on the basis of their approximate subunit molecular weights, as judged by sodium dodecyl sulphate/polyacrylamide ge! electrophoresis. This convention has been particularly promoted by studies using murine monoclonal antibodies to identify prominent protein antigens in Western biot experiments (Coates etai, 1981; Engers et ai, 1985; 1986), The deveiopment of recombinant DNA systems for efficient expression of mycobacteriai genes in Escherichia coli represented an important breakthrough in the study of defined antigenic molecules (Young et ai, 1985a,b), with a genetic approach ailowing unequivocal demonstration of

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D. B. Young, S. H. E. Kaufmann, P. W. M. Hermans andJ. E R. Thole

immunotogical activities associated with a particular polypeptide and, through sequence analysis, providing clues as tc the biochemical function of the native protein. Cloning, sequencing and expression of the reievant genes is currentiy seen as an essential step in characterization of mycobacterial protein antigens.

List of antigens A large number of mycobacterial protein antigens have now been defined by many workers using a variety of approaches based on different methods of biochemical fractionation and on immunological screening with monoclonal antibodies, patients' sera, and human and murine T cells. The key features of each of these antigens are summarized in Tables 1 to 4. Wherever possible, we have tried to inciude information from previous US-Japan, CIE, and subunit molecular weight reference systems in the tables. Genes encoding antigens iisted in Tabies 1 to 3 have already been isolated and characterized tc varying extents, as indicated, while the antigens in Table 4 are currently defined on the basis of /V-terminai amino acid sequencing of purified proteins, A code number has been assigned for each distinct antigen, with an additionai designation (T, L, etc.) being used to distinguish homoiogous genes from different mycobacterial species. It is envisaged that additionai antigens identified in future couid be assigned similar code numbers and that, as studies progress, other antigens wiil join those in Table 1 in having a conventional genetic nomenclature reflecting their functional activity, Detaiis of sequence data base entries ol mycobacterial protein antigens are listed in Table 5.

Selection of antigens It is clear that a large number of mycobacterial proteins are able to induce an immune response, and evaluation of the relative importance of each antigen presents an important challenge tc further progress in this field. It is probable that different antigens wiil be seiected by researchers addressing different aspects of disease control. For immunodiagnosis, for example, antigens inducing species-specific responses are likely to be particularly useful, with a preferential ability to induce antibody or delayed hypersensitivity being important for antigens intended either for seroiogical assays or for skin testing (Ivanyi et ai, 1988). Individual variations in the immune repertoire in outbred populations suggest that more than one defined antigen probabiy wili be required for the design of optimal immunodiagnostic tests (Bothamley et ai, 1989). Selection of antigens for use in potential subunit vaccines presents a more formidable probiem. Recognition of different antigens may be associated with protective and pathologicai aspects of the immune

response tc mycobacteria and testing of this hypothesis provides an important immediate appiication for the panel of defined antigens. Strategies aimed at tackling this question inciude comparison cf immune responses to defined antigens in healthy and diseased populations, and testing of protective efficacy in animal models of infection after immunization either with purified proteins or with recombinant vaccine carriers (Stover et ai, 1991; Aldovini and Young, 1991; Hogen/orst et al.. 1991; Lyons et ai, 1990) expressing individuai antigens. Particuiar antigens may be associated with different forms of immune response either as a consequence of differences in the repertoire of functionaliy distinct iymphocyte subsets, or as a consequence cf the biochemicai properties of the antigen (recognition of a protein secreted from live bacteria may trigger an eariier immune response than recognition of a cytopiasmic antigen, for example). It may be useful, therefore, to consider both immunoiogicai and biochemical properties while pursuing analysis of the defined antigens.

insights into the mycobacteria Several of the mycobacterial antigens have been identified as proteins with important biochemicai functions and regulatory properties, and further study of these proteins and their genes may provide interesting insights into mycobacterial physiology and metaboiism. Proteins impiicated in bacterial responses to stress (heat-shock proteins and superoxide dismutase) are prominent in Table 1, for example, and study of the reguiation of such genes might allow us to understand the way in which mycobacteria adapt to different environments in host tissues during infection. Little is known about molecuiar aspects of nutrient transport in mycobacteria and it is interesting to note that antigen 3 has been identified as a binding protein with a roie in phosphate uptake. Further study of the reguiation and properties of this protein couid uncover fundamentai information about mycobacterial nutrition which may be particularly important with regard to an understanding of the /n vivo metabolism of pathogenic mycobacteria growing inside host cells. Antigen 10 has been shown to bind fibronectin, and detailed analyses of this and related proteins will be important to the study of interactions between mycobacteria and mammalian cells at the molecuiar levei. in addition to its importance in an immunoiogicai context, therefore, the definition of mycobacterial protein antigens also is a significant advance towards our understanding of microbiologicai aspects of mycobacterial disease.

Availabiiity of reagents A significant proportion of the research summarized in

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In response to recommendations from the Steering Committees responsible for co-ordination of World Health Organization programmes for research on the ...
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