Leprosy

Leprosy remains an important health problem wordlwide. The disease is caused by a chronic granulomatous infection of the skin and peripheral nerves with Mycobacterium leprae. The clinical range from tuberculoid to lepromatous leprosy is a result of variation in the cellular immune response to the mycobacterium. The resulting impaiment of nerve function causes the disabilities associated with leprosy. This review summarises recent advances in understanding of the biology of leprosy, clinical features of the disease, the current diagnostic criteria, and the new approaches to treatment of the infection and the immune-mediated complications. Supervised multi-drug therapy (MDT) for fixed durations is highly effective for all forms of the disease. The widespread implementation of MDT has been associated with a fall in the prevalence of the leprosy but as yet no reduction in the case-detection rate globally. Thus, leprosy control activities must be maintained for decades to interrupt transmission of infection

Comparative protective effects of recombinant DNA and Mycobacterium bovis bacille Calmette-Guérin vaccines against M. avium infection.

A range of strategies are being explored to develop more effective vaccines against mycobacterial infection, including immunization with DNA plasmids encoding single mycobacterial bacterial genes and the use of recombinant live vectors based on the current vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG). We have compared these two approaches using a model of virulent M. avium infection, and the gene for the immunodominant 35 kDa protein which is shared by M. avium and M. leprae, but absent from BCG. Recombinant BCG over-expressing the M. avium 35 kDa protein (BCG-35) induced strong antigen-specific proliferative and interferon-gamma (IFN-gamma)-secreting T cell responses. These were comparable to those induced by a single immunization with a plasmid expressing the same antigen (DNA-35); however, repeat DNA-35 immunization evoked the strongest IFN-gamma release. Immunization with BCG-35 significantly reduced the growth of virulent M. avium, although this effect was similar to that induced by wild-type BCG. Immunization with DNA-35 resulted in significantly greater (2 x log(10)) reduction in the growth of M. avium. Prime-boost strategies combining DNA-35 and BCG-35 increased the protective effect above that achieved by BCG-35, but they were not more protective than DNA-35 alone. Therefore, recombinant BCG-35 and BCG induced similar levels of protection in this model, and maximal protection against M. avium infection was attained by immunization with DNA encoding the 35 kDa protein.

DNA encoding a single mycobacterial antigen protects against leprosy infection.

The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guérin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed. The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine. Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production. DNA-35 immunization induced significant levels of protection against M. leprae footpad infection, comparable to that produced by BCG. Therefore, DNA immunization with the 35 kDa antigen is effective against M. leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.

Vaccination with DNA of the Mycobacterium tuberculosis 85B antigen protects mouse foot pad against infection with M. leprae.

A DNA vaccine composed of the gene for the common mycobacterial secreted protein antigen 85B was demonstrated to protect the mouse foot pad against infection with Mycobacterium leprae. The protective effect was demonstrated by a 61%-88% reduction in the bacterial number, a protective effect less than that of BCG. The same DNA vaccine has been shown to protect mice against M. tuberculosis infection, and the importance of testing other candidate tuberculosis vaccines for their potential to protect against leprosy is discussed.

Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells.

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.

Immunoprophylaxis against Mycobacterium leprae infection with subunit vaccines.

We have investigated the effect of subunit vaccines against infection with Mycobacterium leprae, employing DNA plasmids as the vaccine vectors, and the immunodominant 35 kDa protein of M. leprae as the candidate antigen. A DNA vaccine that expresses the M. leprae 35 kDa protein both stimulated interferon-gamma (IFN gamma)-secreting T cells in mice, and demonstrated protection against M. leprae-infection of mice.

Rapid method for diagnosis of leprosy by measurements of antibodies to the M. leprae 35-kDa protein: comparison with PGL-I antibodies detected by ELISA and "dipstick" methods.

A new rapid immuno-chromatographic test card for the detection of antibodies to the Mycobacterium leprae 35-kD protein is described. The new assay is compared in the same group of subjects with a direct enzyme ELISA method for 35-kD antibodies and with assays for anti-phenolic glycolipid-I (PGL-I) antibodies using a standard ELISA as well as the recently described "dipstick" method. Good concordance was found between the rapid methods and the corresponding ELISA methods. The detection of untreated paucibacillary leprosy by the 35-kD test card was 59% compared with 27% for the PGL-I dipstick; however, the specificity for the 35-kD test card was 90% compared with 100% for the PGL-I dipstick in an endemic population. The potential application of these new, rapid serologic methods for the diagnosis of leprosy under field conditions is discussed.

An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis.

A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.

Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics.

We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively. Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates. Autophosphorylation was augmented by up to 25 mM Ca2+. Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M. leprae Hsp70. Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with those of the DnaK protein of Escherichia coli.

Specific serological diagnosis of leprosy with a recombinant Mycobacterium leprae protein purified from a rapidly growing mycobacterial host.

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the Mycobacterium leprae 35-kDa protein, purified from the rapidly growing host Mycobacterium smegmatis, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein.

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.

Human T-cell clones to the 70-kilodalton heat shock protein of Mycobacterium leprae define mycobacterium-specific epitopes rather than shared epitopes.

The mycobacterial 70-kDa heat shock protein (Hsp70) is a dominant antigen during the human T-cell response to mycobacterial infection despite the conserved sequence with the human homolog. To determine whether this response is pathogen specific, CD4+ T-cell clones were isolated from Mycobacterium leprae Hsp70-reactive individuals. The cytokine profile of the clones was mixed, with all of the clones releasing interferon gamma and half releasing interleukin-4 on stimulation, while six demonstrated cytolytic activity. Five clones reacted with the N-terminal half of the molecule, and the epitopes identified were mycobacterium specific. Residues 241 to 260 were identified by three clones, one of which was restricted by HLA-DR7 (DR7), while a DR1-restricted clone identified residues 71 to 90 and residues 261 to 280 were recognized in the context of DR3. The remaining five T-cell clones reacted with the C-terminal half of the molecule, and the precise position of these epitopes was mapped with 12-mer peptides overlapping by 11 residues. Two of these clones identified overlapping epitopes from residues 411 to 425 and 412 to 428, the latter restricted by DR3. Further epitopes were mapped to residues 298 to 313 restricted by DRw53, residues 388 to 406 restricted by DRw52 or DQ2, and residues 471 to 486 restricted by DR1. The sequences of three epitopes, residues 411 to 425, 412 to 428, and 471 to 486, showed significant identity with the equivalent regions of the prototype human Hsp70. However, when amino acid substitutions that made the sequence more like the human sequence were introduced, the changes were tolerated poorly as measured by proliferation, cytokine production, and cytotoxic potential. Therefore, T-cell recognition of the M. leprae Hsp70 antigen occurs in the context of multiple HLA-DR phenotypes and is exquisitely species specific.

A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae.

The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.

Drug resistance in Nepali leprosy patients.

Although multidrug therapy (MDT) was introduced into Nepal in 1983, the MDT coverage only recently exceeded 67%. In view of the large number of patients who were still receiving dapsone monotherapy, it is relevant to investigate the current levels of dapsone and rifampin resistance. The study was undertaken at a leprosy referral hospital near Kathmandu. Over a 5 1/2-year period, 157 leprosy patients with a bacterial index (BI) > or = 2.0 were investigated for drug resistance according to the method of Rees. Among previously untreated cases, 6% of 88 isolates showed low-dose dapsone resistance; among previously treated patients with a presumed relapse, 47% of 34 isolates demonstrated dapsone resistance. In the remaining 35 cases there was no growth in control mice. Rifampin resistance was not confirmed in any case.

Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae.

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.

The immune response to mycobacterial 70-kDa heat shock proteins frequently involves autoreactive T cells and is quantitatively disregulated in multiple sclerosis.

Heat shock proteins (HSP) are the most conserved molecules known to date that may also function as immune targets during infection. Hence, theoretically there is a high chance of cross-reactive responses to epitopes shared by host and microbe HSP. If not properly regulated, these responses may contribute to the pathogenesis of autoimmune disease. To determine if immune responses to HSP could contribute to the pathogenesis of multiple sclerosis, we raised T lymphocyte lines specific for the purified protein derivative of Mycobacterium tuberculosis (PPD) from patients with multiple sclerosis, patients with tuberculosis and from healthy individuals. These lines were then screened for their proliferative response to a M. tuberculosis 70-kDa heat shock protein (M.tb.HSP70). The relative frequency of the recognition of highly conserved sequences of M.tb.HSP70 compared to variable ones was also assessed by mapping experiments on those PPD specific T lymphocyte lines which also recognized the mycobacterial 70-kDa heat shock protein. In patients with multiple sclerosis, we observed a significantly higher estimated frequency of PPD-specific T lines responding to M.tb.HSP70 compared to healthy individuals and patients with tuberculosis. Furthermore, mapping experiments using recombinant proteins representing mycobacterial and human HSP70 sequences and a panel of synthetic peptides encompassing the whole sequence of Mycobacterium leprae HSP70, showed that the response to conserved epitopes of HSP70 is a frequent event in each of the three conditions studied, often leading to the cross-recognition of microbial and human sequences. These findings implicate the 70-kDa heat shock proteins as potential autoantigens in multiple sclerosis.

T cell clones from a non-leprosy exposed subject recognize the Mycobacterium leprae 18-kD protein.

Although Mycobacterium leprae shares many protein antigens with other mycobacterial species, there is a degree of specificity in the T cell response to the organism. This is evident in the failure of cross-protection between mycobacterial species and the specific unresponsiveness to M. leprae in lepromatous leprosy patients. The antigenic basis of this specificity is unresolved, but the M. leprae 18-kD protein is one candidate because of its restricted distribution and the isolation of M. leprae-specific T cell clones reactive with the protein from M. leprae-vaccinated subjects. In the course of analysing the human T cell repertoire to mycobacteria we have isolated further CD4+ T cell clones reactive with this protein from a subject who had never been exposed to M. leprae. These clones did not respond to other mycobacteria, including M. tuberculosis and M. bovis (BCG). In addition, they were unreactive with the M. tuberculosis 16-kD protein which has recently been shown to have limited amino acid identity with the M. leprae 18-kD protein. Both clones reacted with peptide 38-50 from the M. leprae 18-kD protein, the T cell response to which is restricted by HLA-DR4. Although homologues for the gene encoding the M. leprae 18-kD antigen have been identified in M. avium and M. intracellulare, the clones failed to respond to preparations of M. avium. Both clones secreted interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) and were cytolytic against autologous targets pulsed with peptide 38-50 or the 18-kD protein. The nature of the antigen which stimulates this apparently 'M. leprae-specific' response is unknown. Nevertheless the recognition of the 18-kD protein by individuals not exposed to leprosy indicates that this protein may not be suitable as a reagent to distinguish between infection with M. leprae and other pathogenic mycobacteria.