Structural insights into antibody recognition of mycobacterial polysaccharides.

Mycobacteria are major human pathogens responsible for such serious and widespread diseases as tuberculosis and leprosy. Among the evolutionary adaptations essential for pathogenicity in mycobacteria is a complex carbohydrate-rich cell-wall structure that contains as a major immunomodulatory molecule the polysaccharide lipoarabinomannan (LAM). We report here crystal structures of three fragments from the non-reducing termini of LAM in complex with a murine antibody Fab fragment (CS-35Fab). These structures reveal for the first time the three-dimensional structures of key components of LAM and the molecular basis of LAM recognition at between 1.8- and 2.0-A resolution. The antigen-binding site of CS-35Fab forms three binding pockets that show a high degree of complementarity to the reducing end, the branch point and one of the non-reducing ends of the Y-shaped hexasaccharide moiety found at most of the non-reducing termini of LAM. Structures of CS-35Fab bound to two additional tetrasaccharides confirm the general mode of binding seen in the hexasaccharide and indicate how different parts of LAM are recognized. Altogether, these structures provide a rational basis for understanding the overall architecture of LAM and identify the key elements of an epitope that may be exploited for the development of novel and more effective anti-mycobacterial vaccines. Moreover, this study represents the first high-resolution X-ray crystallographic investigation of oligofuranoside-protein recognition.

IgG subclass distribution of antibody responses to protein and polysaccharide mycobacterial antigens in leprosy and tuberculosis patients.

Immunoenzymatic assays were developed for the measurement of antibodies against mycobacterial lipoarabinomannan (LAM), a cell-free proteic extract (CFX) of Mycobacterium leprae, and the 38-kD protein antigen of M. tuberculosis. Sera from 108 leprosy patients, belonging to all clinical-immunological forms of the spectrum, and 81 patients with localized or disseminated tuberculosis (TB) were tested for antibodies of the four IgG subclasses. Standard calibration curves were used to allow comparisons between results of different isotypes and specificities. Mean concentrations of total IgG antibodies were higher in the overall leprosy population than in TB patients. In leprosy, levels of anti-CFX increased from tuberculoid toward lepromatous forms, with a clear switch from IgG1 to IgG2 subclass predominance. A similar IgG1 to IgG2 conversion was observed in anti-LAM antibodies, although total levels of anti-LAM were similar in patients with tuberculoid and lepromatous forms. In TB, antibodies against polysaccharide and protein antigens were both predominantly of IgG1 subclass, whatever the patient's clinical status, although lower in disseminated forms, probably due to concomitant HIV infection. A hypergammaglobulinaemia was also found in most leprosy and TB patients. In TB this was due to increased IgG1 and IgG3, especially in HIV co-infected patients. Based on the current knowledge of the influence of T cell-secreted cytokines on human immunoglobulin isotype expression, these results do not fit with a putative role of Th1 (such as found in TB and tuberculoid leprosy (TT)) and Th2 (such as found in leprosy lepromatous (LL) leprosy) environment in the isotypy of antibody responses in mycobacterial infections. Nor do variations of isotypy according to pathological conditions seem to be related to the biochemical nature of antigens, since antibodies to LAM and protein antigens had comparable evolutions of their subclass distribution. Other factors are to be investigated in order to understand better the significance and possible roles of antibodies in mycobacterial diseases.

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture.

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.

Most Mycobacterium leprae carbohydrate-reactive monoclonal antibodies are directed to lipoarabinomannan.

Each of more than 30 monoclonal antibodies that had been raised against Mycobacterium leprae and previously classified as reactive with carbohydrate was shown to be directed against lipoarabinomannan, a prominent, highly pervasive, myo-inositol-phosphate-containing, cross-reactive antigen within the leprosy bacillus. Some of the antibodies preferentially bound to the lipopolysaccharide of M. leprae rather than to that of Mycobacterium tuberculosis, suggesting the presence of distinguishing structural features. The presence of alkali-labile inositol 1-phosphate in the lipopolysaccharide from M. tuberculosis and its apparent absence from the M. leprae product may account for the difference.

Mycobacterium leprae antigen-induced suppression of T cell proliferation in vitro.

The extent to which M. leprae and its products induced suppression of T lymphocyte proliferation in vitro was evaluated. M. leprae antigens suppressed T cell proliferation in response to mitogens and antigens in both lepromatous and tuberculoid patients, as well as controls never exposed to M. leprae or M. leprae endemic areas. Both soluble and particulate fractions of M. leprae were found to suppress proliferation in a dose-dependent manner. The extent of suppression was inversely related to the proliferative response of the donors mononuclear cells to M. leprae. Evidence indicates that M. leprae contains both stimulatory and suppressive molecules for T cells. One such suppressive antigen, Lipoarabinomannan (LAM)-B of M. leprae, also suppressed the proliferative response of tuberculoid patients. Suppression was also observed with the LAM-B of M. tuberculosis. The suppressive effects observed were not due to the toxicity of the antigen. Some of the suppressive activity was mediated by T8+ suppressor cells and was expressed in both lepromatous and tuberculoid patients. We suggest that previous sensitization to M. leprae and other cross-reactive mycobacterial antigens determines the sensitivity of T cells to the suppressive effects of M. leprae antigens.

Separate antigenic determinants on cell wall associated carbohydrate antigens of Mycobacterium leprae defined with monoclonal antibodies.

Monoclonal antibodies (Mabs) raised against Mycobacterium leprae sonicate defined two different determinants on related, cell-wall-associated, carbohydrate antigens common to M. leprae, M. bovis (BCG), and M. tuberculosis. Antigen inhibition ELISA and antigen capture assays demonstrated that the two antigens were present in a cell-wall fraction, M. leprae resonicate. There was species variation in the distribution of the antigens; the 4.5-6 kD antigen was more abundant in M. tuberculosis and M. bovis, while the 30-40 kD antigen was more concentrated in M. leprae preparations. Although both were present in the cell wall, only determinants on the 30-40 kD antigen were accessible on intact bacilli. The results from capture assays and Mab affinity chromatography with both L9 and L4 indicated that the 4.5-6 kD antigen was probably a fragment of the larger molecule. Both antigens are significant immunogens in the human B-cell response to M. leprae.

Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria.

Leprosy-derived corynebacteria (LDC) are non-acid-fast organisms isolated from leprosy lesions in humans. In this study 20 antigens of native LDC cytoplasm were identified by immunoelectrophoresis, and autoclaving yielded the M1 component, which strongly cross-reacted with antigen 60 of Mycobacterium bovis BCG (bacille Calmette-Guérin) and antigen 7 of Mycobacterium leprae. The polysaccharide moiety of M1 was immunologically related to the LDC cell wall polysaccharide previously characterized as arabinogalactomannan. The latter polysaccharide competitively inhibited the formation of immune complexes by labeled M1 and antisera to the LDC cell wall; cytoplasm and wall polysaccharides from other bacteria produced lower-level inhibition. In a radioimmunoassay with 125I-labeled antigen 7 of M. leprae, sera from patients with leprosy and antisera to the LDC cell wall yielded overlapping curves. Sera from patients with tuberculoid leprosy and those from patients with lepromatous leprosy afforded different levels of inhibition in this radioimmunoassay; this result indicated a difference in antibody specificity in the two forms of leprosy. In conclusion, the cell wall polysaccharide of LDC corresponds to the main thermostable cytoplasmic antigen M1, which strongly crossreacts with sera from patients with leprosy and, more specifically, with antigen 7 of M. leprae.

Immunological unresponsiveness in leprosy.

The various 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 of M. leprae. Even though all currently known protein species of M. leprae and BCG are cross-reactive, lepromatous patients unreactive to M. leprae antigens frequently respond strongly to tuberculin. In vitro experiments suggest the existence of lepromin-induced suppressor activity, mediated by both monocytes and T cells. The T 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 the TS cells largely recognize the terminal trisaccharide of this unique antigen. Depletion of the TS cells restores in vitro reactivity of lymphocytes to lepromin in a portion of lepromatous patients, and addition of IL-2-containing supernatants partially restores responsiveness to M. leprae antigens. Vaccination of lepromatous patients with a mixture of M. leprae and live BCG restores cell-mediated immunity in the majority of lepromatous patients, and concomitantly reduces the in 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 of TH capable of responding to either specific or cross-reactive mycobacterial antigens. The mode of action of these TS 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.

Demonstration of antibodies reacting with different determinants on Mycobacterium leprae antigen 7.

A purified preparation of M. leprae antigen 7 was used to investigate the specificity of anti-M. Leprae 7 antibodies in leprosy sera in a radioimmunoassay. A solution containing arabinogalactan (AG) and arabinomannan (AM) inhibited the antibodies in some sera to a great extent, whereas the antibody activity was virtually unchanged in other sera under the same conditions. These findings indicate that the antibodies are directed against different determinants on M. leprae antigen 7. Antibodies against determinants other than AG and AM occurred particularly in lepromatous leprosy sera. In 12 out of 14 sera, AG and AM had similar inhibiting capacity. In one serum, AG had markedly higher inhibiting capacity than AM; in the last serum, the reverse was the case, demonstrating variation in anti-polysaccharide specificity in individual sera.