T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy.

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.

IgG response to purified 65- and 70-kDa mycobacterial heat shock proteins and to antigen 85 in leprosy.

IgG antibody response to mycobacterial heat-shock proteins (hsp) (the 70-kDa antigen from Mycobacterium tuberculosis and M. bovis BCG; the 65-kDa antigen from M. leprae and M. bovis BCG) and to the fibronectin-binding antigen 85 from M. bovis BCG was analyzed in a dot-blot assay in plasma from leprosy patients and their contacts. Most plasma--whatever the status of the subjects--reacted to the hsp 70; 8 of 9 (89%) of paucibacillary patients recognized the 65 mycobacterial hsp but only 2 of 9 (22%) recognized the antigen 85. In contrast, 12 of 12 (100%) of multibacillary patients reacted with the antigen 85 and only 4 of 12 (33%) reacted to the hsp 65 from M. leprae. On the one hand, 7 of 25 (28%) of the lepromin-positive contacts and 2 of 9 (22%) of the lepromin-negative contacts recognized the antigen 85. On the other hand, 11 of 25 (44%) of the lepromin-positive contacts but only 1 of 9 (11%) of the lepromin-negative contacts reacted to the hsp65 from M. leprae. Finally, very few (10%) of the lepromin-positive controls showed a positive reaction to the M. leprae 65-kDa antigen, the BCG 65-kDa antigen, and the 85-kDa antigen of BCG. Thus, differences in binding to the hsp65 from M. leprae and to antigen 85 could be helpful in distinguishing different forms of the disease.

T-cell stimulation with purified mycobacterial antigens in patients and healthy subjects infected with Mycobacterium leprae: secreted antigen 85 is another immunodominant antigen.

Peripheral blood leucocytes from 9 paucibacillary and 12 multibacillary leprosy patients, from 18 healthy controls and from 34 healthy leprosy contacts were stimulated with three mycobacterial heat shock proteins with respective molecular weights of 70, 65 and 18 kDa and with the secreted 30-32 kDa protein, also called antigen 85. Antigen 85 was found to be the most powerful T-cell antigen (as measured by lymphoproliferation and IFN-gamma secretion), eliciting a positive response in all (100%) paucibacillary patients and in all lepromin-positive controls and contacts. The three heat shock proteins (hsp) were less active T-cell stimuli. Reactivity to the 70 kDa hsp was found in only 44% of the paucibacillary patients, in 80% of the lepromin-positive controls and in 60% of the lepromin-positive leprosy contacts. The 65 kDa hsp stimulated T cells in 89% of the paucibacillary patients and in 80% of the lepromin-positive controls and contacts. Responsiveness to the 18 kDa hsp, finally, was clearly more frequent in tuberculoid leprosy patients (78%) than in lepromin-positive controls (40%) or lepromin-positive leprosy contacts (4%). T-cell reactivity of 8 lepromin-negative controls, of 9 lepromin-negative contacts and of 12 multibacillary leprosy patients was low to all the antigens tested. Although proliferative and IFN-gamma responses were generally closely related, some subjects demonstrated a dissociation of these two immune parameters. Our data confirm previous findings on the powerful T-cell stimulatory properties of antigen 85 during M. leprae infection and suggest that this antigen is indeed a potentially protective T-cell immunogen.

The major secreted antigen complex (Ag 85) from Mycobacterium bovis bacille Calmette-Guérin is associated with protective T cells in leprosy: a follow-up study of 45 household contacts.

T cell proliferation and interferon (IFN)-gamma secretion were analyzed in 45 leprosy contacts stimulated with antigen 85 (Ag85), the major culture filtrate antigen from Mycobacterium bovis bacille Calmette-Guérin. All 14 Mitsuda reaction-positive contacts reacted to Mycobacterium leprae and Ag85. Three Mitsuda reaction-negative contacts reacted weakly to M. leprae and Ag85. The other 28 Mitsuda reaction-negative contacts did not react to M. leprae, but 9 reacted to Ag85. Thirty-four contacts were retested 16 months later. Eleven contacts initially positive by the Mitsuda test remained lepromin positive and reactive to M. leprae and Ag85. Fourteen contacts initially negative by the Mitsuda test converted, and all reacted in vitro to M. leprae and Ag85. Finally, 9 contacts remained Mitsuda test-negative, and 7 were unreactive to Ag85. In vitro reactivity to Ag85 at baseline in Mitsuda test-negative contacts was associated with subsequent conversion to lepromin reactivity in 7 of 9 subjects. These data suggest that reactive T cells against Ag85 develop very early during M. leprae infection and that Ag85 is a potentially protective T cell immunogen.

Human phagocyte respiratory burst by Mycobacterium bovis BCG and M. leprae: functional activation by BCG is mediated by complement and its receptors on monocytes.

We have measured the role of serum components on two parameters of the phagocytosis reaction: a) the chemiluminescence (CL) response associated with the oxidative respiratory burst in response to Mycobacterium bovis BCG and M. leprae, and b) the uptake of these two mycobacteria by healthy human monocytes. Pre-incubations of fresh or heat-inactivated serum or serum containing EGTA or EDTA indicate that these two mycobacteria activate the alternative complement pathway. Monoclonal antibodies against CR1 and CR3 inhibit the responses of M. bovis BCG and M. leprae, demonstrating that complement receptors mediate the phagocytosis of these two mycobacteria. Thus, complement and its receptors on the surface of the monocytes (CR1 and CR3) are important in the functional activation of phagocytosis of M. bovis BCG and M. leprae.

TNF-alpha failed to reverse the M. leprae-induced defective chemiluminescence response of human mononuclear cells.

The effect of phagocyte activation by TNF-alpha on the ability to trigger a chemiluminescence (CL) response, associated with the release of oxidizing species was evaluated in healthy human mononuclear cells in the presence of Mycobacterium leprae. Recombinant TNF-alpha (r-TNF-alpha) increased the CL response of unstimulated M. bovis BCG- and PMA-stimulated cells but did not reverse the M. leprae defective activation of the human phagocyte oxidative burst. M. leprae was less well phagocytosed than M. bovis BCG but phagocytosis of mycobacteria was not altered by addition of r-TNF-alpha. The failure of activation of oxygen-free radical production might have some relevance to the pathogenesis of leprosy.

[The value of electromyographic studies in leprosy]. / Intérêt de l'examen électromyographique dans la lèpre.

In Dakar, during a definite period of time, all the new leprosy cases have been subjected to an electromyographic examination before treatment: a total of 37 patients and 518 examined nerves including all clinical forms: NCV: 33% of the examined nerves are found to be affected. The sensory nerves are frequently and early involved. In frequency order: sural (54%), posterior tibial (50%), sensory ulnar (35%), sensory median (29%), motor ulnar (28%), lateral popliteal (17%) and motor median (12%). The study of the SCV seems relatively more reliable than the sensory testing in the case of the ulnar and the median (75 comparisons): concordance in 69% of the cases; SCV only abnormal in 19%; sensory testing only abnormal in 12%. The EMG detection is superior to the motor testing and to the motor nerve conduction for the lateral popliteal (32 comparisons): 41% of concordant examination; 59% of differences among which 44% of anomalies revealed only by detection.