Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer.

Buruli ulcer, caused by Mycobacterium ulcerans, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine M. bovis BCG. To identify potential mechanisms for this cross-protection, we identified and characterized the M. ulcerans homologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in M. ulcerans, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of M. ulcerans- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads of M. ulcerans- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.

Fibronectin-binding antigen 85 and the 10-kilodalton GroES-related heat shock protein are the predominant TH-1 response inducers in leprosy contacts.

Peripheral blood mononuclear cells from 27 healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (interleukin-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa from Mycobacterium leprae and the 30-32-kDa antigen 85 (Ag 85) from Mycobacterium bovis BCG. Cells from 18 and 19 of 19 lepromin-positive contacts proliferated or produced TH-1 cytokines in response to the M. leprae 10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis for two lepromin-positive contacts indicated that about one-third of M. leprae-reactive T cells displayed specificity to the M. leprae 10-kDa protein and Ag 85. The M. leprae 65- and 18-kDa proteins were less potent TH-1 response inducers: gamma interferon and interleukin-2 could be measured in 14 and 19 lepromin-positive contacts, respectively. In contrast, very low or undetectable proliferative and cytokine responses were found for 8 lepromin-negative contacts. Our data demonstrate that the fibronectin-binding Ag 85 and the 10-kDa GroES homolog are powerful mycobacterial TH-1 response inducers in the vast majority of lepromin-positive contacts and suggest that they might be valuable candidates for a future subunit vaccine.

IgG humoral response against the antigen 85 complex homologues in leprosy.

Antigen 85 complex is the major protein component present in M. bovis BCG culture filtrate (CF). It consists of a family of three proteins: 85A, 85B and 85C. Combining isoelectric focusing and Western blot analysis, we have previously identified different antigenically related proteins present in the CF of other mycobacteria (M. tuberculosis, M. kansasii, M. avium, M. gordonae, M. fortuitum and M. phlei) using monoclonal antibodies (MoAbs) directed against the antigen 85 complex of M. bovis BCG. Humoral immune response directed against these cross-reactive homologues was analysed in sera from 20 patients with multibacillary leprosy (BL/LL), from 20 patients with paucibacillary leprosy (BT/TT) and from 15 healthy leprosy contacts. All the antigen 85 homologues identified in the seven CFs by MoAbs were also recognized by IgG present in sera from multibacillary leprosy patients, but not or very faintly in sera from paucibacillary leprosy patients or from healthy subjects. These results suggest that some of the M. leprae epitopes inducing a significant humoral response in multibacillary leprosy are common to the various 85 antigenically related proteins present in all mycobacterial species.

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.

Evolution of cellular and humoral response against Tuberculin and antigen 85 complex during intravesical treatment with BCG of superficial bladder cancer.

Prophylactic treatment with Bacillus Calmette-Guerin (BCG) is an established and effective therapy of bladder cancer. The antitumor effect of BCG seems to be largely related to cellular immunological mechanisms, although its precise mode of action is unknown. Antitumor response of BCG seems to be initiated by the attachment of BCG to bladder wall via Fibronectin (FN). The cellular immune response against Tuberculin PPD and the major secreted BCG antigen (Fibronectin-binding AG 85 complex) has been tested in a control group of 20 untreated bladder tumor patients and before and after 6 weekly intravesical BCG instillations in a group of 20 superficial bladder tumor patients. A major increase in the lymphoproliferative response against PPD and AG 85 was observed in respectively 66% and 57% of the treated patients. In contrast, no detectable antibody response (IgA, IgM, IgG) was observed against AG 85 complex after BCG treatment. On the other hand, antibodies against Tuberculin increased in 13 of 20 patients. This study seems to demonstrate a specific cellular immune activation against AG 85 Fibronectin-binding complex during BCG treatment of superficial bladder tumors. Humoral response against the AG 85 is not activated after BCG treatment. Further studies are needed to elucidate the role of AG 85 in the cellular intravesical penetration of BCG. Presence or absence of cellular response against this antigen could be of clinical value.

T cell reactivity against antigen 85 but not against the 18- and 65-kD heat shock proteins in the early stages of acquired immunity against Mycobacterium leprae.

T cell proliferation and interferon-gamma (IFN-gamma) production of peripheral blood mononuclear cells (PBMC) from 20 household contacts were tested against the 18- and 65-kD heat shock proteins from Mycobacterium leprae (ML18 and ML65 respectively) and antigen 85 from Myco. bovis bacille Calmette-Guérin (BCG) (Ag 85) during a 12-months follow-up study. Among the eight contacts that became positive, eight showed positive reactivity against Ag 85, 5/8 against ML65 and 4/8 against ML18 at the end of the study. Of the 16 contacts who were lepromin-positive either at first or second testing, all responded to Ag 85, 11 to ML 65, but only eight reacted to ML18 antigen. Contacts who were lepromin-positive at first testing developed responses to ML18 only at second testing. In contrast, among the four contacts that remained lepromin-negative during the follow up, three proliferated to Ag 85 either at first or second testing, but only one produced IFN-gamma against Ag 85 at the end of the study. These results demonstrated that T cell reactivity and particularly IFN-gamma secretion against Ag 85, but not against ML18 and ML65, might be a predominant mechanism in the early stages of acquired protective immunity against Myco. leprae.

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.

IL-6 production in response to purified mycobacterial heat-shock proteins and to antigen 85 in leprosy.

IL-6 production was examined in PBMC cultures from healthy leprosy contacts and from leprosy patients stimulated with the purified mycobacterial 18-, 65-, and 70-kDa heat-shock proteins (hsp) and the secreted fibronectin-binding antigen 85 (Ag85). In lepromin-negative contacts, the 70-kDa hsp was the only antigen capable of eliciting significant IL-6 production. In lepromin-positive contacts, Ag85, the 65- and the 70-kDa hsp induced substantial IL-6 titers. IL-6 levels induced with the 70-kDa antigen were about fourfold higher than with the 65-kDa hsp or with Ag85. The 18-kDa antigen did not induce any IL-6 in these healthy contacts. PBMC from tuberculoid leprosy patients produced even more elevated levels of IL-6, and PBMC from lepromatous leprosy patients produced extremely high levels of IL-6. All antigens were capable of inducing IL-6 in leprosy patients. Highest levels were found in cultures stimulated with the 65-kDa hsp, and lowest levels were in cultures stimulated with the 18-kDa hsp.

Effects of chemotherapy on antibody levels directed against PGL-I and 85A and 85B protein antigens in lepromatous patients.

IgG antibodies against antigens 85A and 85B from Mycobacterium bovis BCG, IgM antibodies against phenolic glycolipid-I (PGL-I) and circulating PGL-I antigen were measured in the serum of 11 patients with lepromatous leprosy receiving multidrug therapy (MDT). Before treatment, 6 patients were reactive to antigen 85A, 10 patients to antigen 85B, and 11 patients to PGL-I; circulating PGL-I was detected in the sera of all of them. After 2 years of MDT PGL-I antigen could no longer be detected in all of the patients, except for two who were not compliant with treatment. IgG antibodies directed against the 85A and 85B antigens and IgM antibodies against the PGL-I antigen also decreased significantly during treatment but more slowly. The determination of circulating PGL-I antigen remains the most appropriate tool for monitoring lepromatous leprosy under MDT.

Humoral responses against the 85A and 85B antigens of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.

Immunoglobulin G antibodies against the 85A and 85B components of the Mycobacterium bovis BCG antigen 85 complex separated by isoelectric focusing were investigated in serum samples from 129 patients representing the major forms of leprosy, 111 tuberculous patients, and 153 healthy subjects. For both of the antigens, a higher degree of staining was observed for lepromatous leprosy patients and patients with active tuberculosis than for the other groups. Because sera from some healthy subjects recognized the 85A antigen, we suggest that antigen 85B is the most useful component of the antigen 85 complex for the serodiagnosis of the multibacillary forms of leprosy or of the active forms of tuberculosis.

An isoelectric focusing method for the study of the humoral response against the antigen 85 complex of Mycobacterium bovis BCG in the different forms of leprosy.

Isoelectric focusing was used to separate the three components of the antigen 85 complex of Mycobacterium bovis BCG. Antibody responses of leprosy patients against each Mycobacterium bovis BCG. Antibody responses of leprosy patients against each component were quantitated by densitometric analysis of immunoblot assays. The 85A component was recognized by 40% (8/20) of the lepromin positive and negative healthy subjects, by 76% (19/25) of the tuberculoid and by 96% (24/25) of the lepromatous leprosy sera. In contrast, the 85B component was not stained by the control sera, nor by the tuberculoid leprosy sera but by 64% (16/25) of the lepromatous leprosy sera. The results suggest that antigen 85B contains one or several epitopes that are specifically recognized by sera of lepromatous leprosy patients only.