Vijay Mehra

Structure of the Heat Shock Protein Chaperonin-10 of Mycobacterium leprae

Members of the chaperonin-10 (cpn10) protein family, also called heat shock protein 10 and in Escherichia coli GroES, play an important role in ensuring the proper folding of many proteins. The crystal structure of the Mycobacterium leprae cpn10 (Ml-cpn10) oligomer has been elucidated at a resolution of 3.5 angstroms. The architecture of the Ml-cpn10 heptamer resembles a dome with an oculus in its roof. The inner surface of the dome is hydrophilic and highly charged. A flexible region, known to interact with cpn60, extends from the lower rim of the dome. With the structure of a cpn10 heptamer now revealed and the structure of the E. coli GroEL previously known, models of cpn10:cpn60 and GroEL:GroES complexes are proposed.

Human T-cell clones recognize a major M. leprae protein antigen expressed in E. coli

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. As with other intracellular parasites, protective immunity is dependent on T cells and cell-mediated immunity1. In animal models, immunization with killed armadillo-derived M. leprae elicits strong T-cell responses, delayed-type hypersensitivity and protection against viable challenge2–5. We have recently shown that killed M. leprae can induce delayed-type hypersensitivity in healthy human volunteers6. Identification of the M. leprae antigens that are recognized by T cells and may be involved in protection has been hampered by the inability to cultivate the organism in vitro and by difficulties in antigen purification from limited quantities of armadillo-derived bacillus. Because genes for the major protein antigens of M. leprae as seen by mouse monoclonal antibodies have been isolated7,8, it has become possible to test whether these individual antigens are recognized by T cells. We screened crude λ gtll phage lysates of Escherichia coli containing individual M. leprae antigens using M. leprae-specific T-cell clones isolated from M. leprae-vaccinated volunteers. Using this method, we find that nearly half of the M. leprae-specific T-cell clones are stimulated to proliferate by lysates containing an epitope of a M. leprae protein of relative molecular mass 18,000 (18K).

Genes for the protein antigens of the tuberculosis and leprosy bacilli

The λgtl l expression vector permitted us to survey protein antigens ofMycobacterium leprae andMycobacterium tuberculosis expressed inEscherichia coll. Using monocional antibodies, recombinant clones were detected producing three major antigens ofM. tuberculosis and five major protein antigens ofM. leprae. These recombinant antigens produced inE. coli should prove useful for diagnosis, epidemiology and possibly the development of recombinant mycobacterial vaccines.

Mechanisms of Immunological Unresponsiveness in the Spectra of Leprosy and Leishmaniasis

Leprosy and cutaneous leishmaniasis share a number of important characteristics1,2. They are both chronic granulomatous diseases; both affect the skin and both present a spectrum of clinical manifestations. In the case of leprosy, there is a remarkable correlation between the clinical and histopathological spectrum, and cell-mediated immune responsiveness to antigens of M. leprae. At the tuberculoid pole, patients have few lesions which contain rare organisms and are able to mount strong T-cell-mediated immune responses to M. leprae antigens in vitro and in vivo. In contrast, at the lepromatous end of the spectrum, patients have disseminated skin lesions containing large numbers of acid-fast bacilli and are selectively unresponsive to antigens of M. leprae. In American cutaneous leishmaniasis, the spectrum is somewhat less well defined, more variable clinically and less predictable at the histopathologic level. The disease ranges from a single defined lesion containing few amastigotes in localized cutaneous leishmaniasis (LCL) to diffuse cutaneous leishmaniasis (DCL) which, like lepromatous leprosy, is characterized by disseminated granulomata containing macrophages laden with amastigotes and immune unresponsiveness to leishmanial antigens. There are other clinical forms of cutaneous leishmaniasis including the highly destructive mucocutaneous (espundia) form, and a verrucous form, the pathogenesis of which are not entirely predictable from histopathology and molecular immunological data. In addition, in different parts of the world systemic forms of visceral leishmaniasis (kala-azar) occur in Asia, and a mild local form, Oriental Sore, exists in the Middle East primarily. Antibody levels appear to be elevated in both lepromatous leprosy and in diffuse cutaneous leishmaniasis, indicating that antibodies are unlikely to play a major role in protection.

Molecular Approaches to Developing a Vaccine for Leprosy

Leprosy, a chronic infectious disease afflicting 10 million to 15 million people, is caused by the obligate intracellular parasite Mycobacterium leprae. Although M. leprae was the first identified bacterial pathogen of man, it remains one of the few human pathogens that cannot yet be grown in culture. The inability to grow leprosy bacillus in culture has severely limited the understanding of the bacillus and the disease.

Immunological unresponsiveness in leprosy and its relevance to immunoregulation in man

The varied forms of leprosy form a clinical and immunological spectrum which offers extraordinary possibilities for insight into immunoregulatory mechanisms in man. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which ultimately results in killing of bacilli in the tissues, albeit often with damage to nerves. At the lepromatous pole, patients exhibit selective immunological unresponsiveness to antigens ofMycobacterium leprae. Even though all currently known protein species ofMycobacterium leprae and BCG are cross-reactive, lepromatous patients unreactive toMycobacterium leprae antigens frequently respond strongly to tuberculin.In vitro experiments suggest the existence of lepromin-induced suppressor activity, mediated by both monocytes andT cells. TheT suppressor cells have the T8 phenotype of which 50% express the activation markers,Ia and FcR. The one unique species of antigen of the leprosy bacillus is a phenolic glycolipid, and it appears that theTs cells largely recognize the terminal trisaccharide of this unique antigen. Depletion ofTs cells restoresin vitro reactivity of lymphocytes to lepromin in a portion of lepromatous patients, and addition of IL-2 containing supernatants partially restores responsiveness toMycobacterium leprae antigens. Vaccination of lepromatous patients with a mixture ofMycobacterium leprae and live BCG restores cell-mediated immunity in the majority of lepromatous patients, and concomitantly reduces thein vitro suppressor activity and number of activated T8 cells.These experiments suggest the existence of stage-of-disease related suppressor cells in leprosy which appear to block the responsiveness ofTH capable of responding to either specific or cross-reactive mycobacterial antigens. The mode of action of theseTs appears to be the inhibition of production of IL-2 and other lymphokines. Successful immunotherapeutic vaccination appears to overcome this block in the majority of patients.