View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by LSHTM Research Online Brahmbhatt, S; Hussain, R; Zafar, S; Dawood, G; Ottenhoff, TH; Dri- jfhout, JW; Bothamley, G; Smith, S; Lopez, FV; Dockrell, HM (2002) Human T cell responses to peptides of the Mycobacterium leprae 45- kD serine-rich antigen. Clinical and experimental immunology, 128 (1). pp. 140-8. ISSN 0009-9104 Downloaded from: http://researchonline.lshtm.ac.uk/16453/ Usage Guidelines Please refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alterna- tively contact [email protected]. Available under license: Creative Commons Attribution Non-commercial No Derivatives http://creativecommons.org/licenses/by-nc-nd/2.5/ Clin Exp Immunol 2002; 128:140–148 Human T cell responses to peptides of the Mycobacterium leprae 45-kD serine-rich antigen S. BRAHMBHATT*, R. HUSSAIN†, S. ZAFAR‡, G. DAWOOD§, T. H. M. OTTENHOFF¶, J. W. DRIJFHOUT¶, G. BOTHAMLEY**, S. SMITH*, F. V. LOPEZ†† & H. M. DOCKRELL* *Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK, †Microbiology Department, Aga Khan University, Karachi, Pakistan, ‡Marie Adelaide Leprosy Centre, Mariam Manzil, Karachi, Pakistan, §Masoomeen Hospital, Karachi, Pakistan, ¶Department of Immunohaematology and Bloodtransfusion, Leiden University Medical Centre, Leiden, the Netherlands, **Respiratory Medicine, Homerton Hospital, Homerton Row, London, UK and ††Department of Dermatology, The Middlesex Hospital, London, UK (Accepted for publication 14 December 2001) SUMMARY In order to identify T cell epitopes within the Mycobacterium leprae 45-kD serine-rich antigen, we analysed responses to overlapping 17-mer peptides encompassing the whole antigen in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the United Kingdom and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homo- logue in M. tuberculosis. Human peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-g measured in supernatants by ELISA. Some peptides were recognized more frequently by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential, while other peptides induced greater responses among UK control subjects. Short-term cell lines confirmed that these assays detected specific T cell recognition of these peptides. However, many tuberculosis patients also recognized these potentially specific pep- tides suggesting that there could be a true homologue present in M. tuberculosis. Keywords cell mediated immunity (CMI) diagnosis interferon-gamma (IFN-g) M. leprae peptides INTRODUCTION tistics leprosy still poses some challenges. The biggest challenge for achieving elimination is the fact that even though the preva- Leprosy is a chronic infectious disease caused by Mycobacterium lence of leprosy is decreasing the same is not true for incidence, leprae. Leprosy patients present with a spectrum of clinical which has remained the same [5]. In addition, because of the long disease that depends on the ability of the host to make a cellular incubation period before the disease appears, many more new immune response to the bacterium [1]. At one pole of the spec- leprosy patients will continue to emerge in endemic countries for trum is tuberculoid leprosy (TT), which is associated with strong many years. cell mediated immunity (CMI) and delayed type hypersensitivity What makes the control of leprosy difficult is that no specific (DTH) responses to M. leprae. In contrast, at the other end of the and reliable immunological tool is available to be used for detec- disease spectrum is lepromatous leprosy (LL) where the disease tion of M. leprae exposure. A diagnostic reagent, which would be is disseminated throughout the body. analogous to, but more specific than the Mantoux skin test used Leprosy is still considered a public health problem in 32 coun- for detecting exposure to M. tuberculosis, is required. Although tries; however, 16 of these from the African, Asian and South the Mantoux test can be positive following exposure to non- American continents are responsible for 92% of all registered tuberculous mycobacteria, individuals with skin test indurations patients [2]. Leprosy cases are distributed globally unevenly. The greater than 10 or 15 mm are at higher risk of developing tuber- application of multi-drug therapy (MDT) has changed the picture culosis. Such individuals can then be referred for a chest X-ray of leprosy, with the numbers of registered cases worldwide falling and clinical examination to confirm or exclude tuberculosis. If from 1·3 million in 1995 to 641 091 in 2000 [3,4]. Despite these sta- such a test were to be developed for leprosy it would aid in the Correspondence: Hazel Dockrell, Immunology Unit, Department of understanding of immune responses in healthy M. leprae-exposed Infectious and Tropical Diseases, London School of Hygiene and Tropical individuals and those who are subclinically infected. Such a test Medicine, Keppel Street, London WC1E 7HT, UK. may also be able to identify areas with greater leprosy endemic- E-mail: [email protected] ity within a country. © 2002 Blackwell Science 140 Human T cell responses to peptides of M. leprae 45-kD serine-rich antigen 141 Skin tests using Lepromin and Leprosin have been used, order to measure IFN-g, which is an important cytokine in the which are prepared from whole autoclaved M. leprae and soluble protective immune response against M. leprae. Skin lesions from antigens from fractionated M. leprae, respectively. Lepromin is a tuberculoid leprosy patients contain the type 1 cytokine IFN-g biphasic test developed by Mitsuda called the Fernandez reaction mRNA, which is not found in lesions from lepromatous leprosy [6] with reactions read after 48–72 h and also read at 4 weeks, the patients [29]. Lepromatous leprosy patients were not included in Mitsuda reaction. Lepromin and Leprosin are not diagnostic this study as they are unable to make M. leprae-specific immune reagents for leprosy because they lack the required specificity responses resulting in the uncontrolled growth of the organism in and have been shown to induce positive skin test reactions in vivo [30,31]; however, they produce immune responses to cross- the majority of healthy endemic and non-endemic controls. The reactive antigens such as PPD. In a study by Kaplan et al. Lepromin test is better used for classifying leprosy patients across bacterial loads were reduced significantly within the lesions of the spectrum of disease. lepromatous leprosy patients when injected with recombinant In order to develop a specific immunological tool for the IFN-g [32]. The aim of our study was to identify immunodomi- purpose of diagnosing M. leprae exposure, one approach is to nant T cell determinants within the 45-kD antigen and to assess identify T cell epitopes within an antigen that are recognized if there were differences in the IFN-g responses from peripheral specifically by individuals exposed to M. leprae and not other blood mononuclear cells of tuberculoid leprosy patients, healthy mycobacteria. A number of antigens have been identified that UK controls and tuberculosis patients to the peptides. Our study induce T cell responses from tuberculoid patients and leprosy excluded LL leprosy patients, as they have depressed type 1 patient contacts in vitro: for example, the 70-kD, 65-kD, 35-kD, immune responses and defective CMI. In initial studies, all the 30/31-kD, 18-kD and 10-kD antigens [7–12]. However, the major- peptides encompassing the 45-kD antigen were tested; further ity of these antigens induce cross-reactive immune responses experiments involved testing a few peptides in additional UK as there are homologues present in other mycobacterial species controls and leprosy patients and tuberculosis patients from the [13]. Previous studies using M. leprae fractionated antigens endemic country to evaluate specificity. Multi-drug therapy has on nitrocellulose indicated that many additional proteins might been highly successful in the treatment of leprosy; however, the be recognized as antigens [14–17]; however, there are problems identification of T cell epitopes that are specific to M. leprae could with the specificity issue when considering such fractions as still prove of major importance in the context of identifying antigens. people who are exposed and possibly subclinically infected with A number of other studies have demonstrated the usefulness M. leprae. of in vitro assays for T cell proliferation or IFN-g secretion as good correlates of skin test responsiveness in humans [18] [19]. MATERIALS AND METHODS To our knowledge, no M. leprae peptides to date have been used as diagnostic reagents in humans under in vivo conditions, Patients and controls although it should be possible to elicit DTH responses to such In the United Kingdom, 40 heparinized buffy coat blood bank peptides in man, as shown for two human immunodeficiency samples (North London Blood Transfusion Service, Colindale, virus peptides in a study on human volunteers [20]. Previous UK and South London Blood Transfusion Service, Tooting, UK) studies have demonstrated skin test responses to peptides of were used as a source of PBMC from leprosy-unexposed individ- M. tuberculosis 19-kD in mice [21] or M. leprae 65-kD, 28-kD, uals. As the blood bank will not accept blood donations from 18-kD peptides in guinea pigs [22] and M. tuberculosis and M. volunteers who may have lived or spent long periods in the intracellulare 19-kD peptides in guinea pigs [23]. Recently a tropics, the blood used was derived from individuals who would study on bovine tuberculosis demonstrated the importance of not have been exposed to M. leprae. Blood samples from six antigen-specific cytokine readout systems for the early identifi- pulmonary tuberculosis patients in the United Kingdom were cation of M. bovis infection in cattle and stressed the potential obtained with informed consent, recruited at the Homerton of using antigen cocktails for immunodiagnosis [24].