Cross-Reactive Immune Responses Against Mycobacterium bovis BCG in Mice Infected with Non-Tuberculous Mycobacteria Belonging to the MAIS-Group

Two bacillus Calmette–Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare , M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum . Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-γ production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum -infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis .     

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.     

Assessment of an Oral Mycobacterium bovis BCG Vaccine and an Inactivated M. bovis Preparation for Wild Boar in Terms of Adverse Reactions,Vaccine Strain Survival,and Uptake by Nontarget Species

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.     

Induction of alpha and beta interferons during the hyporeactive state of gamma interferon by Mycobacterium bovis BCG cell wall fraction in Mycobacterium bovis BCG-sensitized mice.

Characterization of interferon (IFN) induced by a second challenge with specific antigen was investigated during the development of hyporeactivity established after challenge of Mycobacterium bovis BCG-sensitized mice with BCG cell wall fraction (BCG-CW). When BCG-sensitized mice were challenged with BCG-CW, IFN-gamma was detected in the circulation 4 h later and rapidly disappeared. After IFN-gamma disappeared from their blood, mice became completely hyporeactive to the second challenge with BCG-CW for 1 day, and thereafter they recovered from this hyporeactive state. However, we always observed that IFN induced at first by the second challenge with BCG-CW during the hyporeactive state was type I IFN-alpha/beta, but thereafter was entirely IFN-gamma. Induction of IFN-alpha/beta by the specific antigen during restoration from the hyporeactive state in BCG-sensitized mice is discussed.     

The immune response to the cell wall of Mycobacterium bovis BCG.

Mice were immunized with the cell wall of BCG suspended in an oil-in-saline emulsion, and examined against time for the emergence of T cell-mediated acquired immunity. Evidence is presented that shows that levels of acquired resistance expressed in these animals over the first month following inoculation, and which enabled them to substantially resist an intravenous challenge infection with Mycobacterium tuberculosis, were completely nonspecific in nature, in that they were equally well expressed in normal and T cell-deficient mice, and were present at a time when no protective T cell activity could be passively transferred from the inoculated host. Paradoxically, in contrast, weak but statistically significant protective immunity could be detected in the spleens of CW-immunized mice approximately 3 months after inoculation, at a time when the donor animals were devoid of resistance to rechallenge. Finally, evidence is presented that shows that the CW material, if given subcutaneously, is highly immunogenic for the generation of delayed-type hypersensitivity effector T cells; however, these cells do not themselves contribute to protective immunity.     

Specific Differentiation between Mycobacterium bovis BCG and Virulent Strains of the Mycobacterium tuberculosis Complex

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. The senX3-regX3 IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the senX3-regX3 IR, whereas all non-BCG M. tuberculosis complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.     

Exposure to Mycobacterium avium primes the immune system of calves for vaccination with Mycobacterium bovis BCG

The objective of the investigation was to provide data on how a prior exposure of cattle to Mycobacterium avium, used here as a model of exposure to an environmental mycobacterium, affected the cellular immune response that follows vaccination with Mycobacterium bovis BCG. The assessment of cellular immune responses included lymphocyte proliferation assays, the delayed hypersensitivity skin test and IFN-gamma synthesis in whole blood cultures. One group of calves was inoculated subcutaneously with M. avium followed 12 weeks later by M. bovis-BCG. The other group was vaccinated subcutaneously with BCG alone. Calves previously exposed to M. avium responded more rapidly, as assessed in the in vitro assays, to purified protein derivative (PPD) from M. avium (PPD-A) or M. bovis (PPD-B) than did calves inoculated with BCG only, indicating that the exposure to M. avium had primed the immune response in these calves. Following inoculation of BCG the intensity of the in vitro responses and the delayed hypersensitivity skin test to PPD-A was higher for the M. avium-primed animals while the responses to PPD-B were similar in the M. avium-primed and BCG-only groups. The results are consistent with a model in which prior exposure to environmental mycobacteria does not necessarily inhibit the immune response to the vaccine strain, BCG. They suggest that M. avium infection primes the immune system of calves and that the detection of an immune response specific for M. bovis BCG is masked by reactivity to antigens also present in M. avium.     

Characterization of exochelins of the Mycobacterium bovis type strain and BCG substrains

Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of Mycobacterium bovis, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of M. bovis, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The M. bovis type strain, five substrains of M. bovis BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent Mycobacterium tuberculosis all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in M. tuberculosis, intermediate in M. bovis, and smallest in M. bovis BCG.     

Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG.

ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on a synthetic Sauton medium. ESAT-6 has recently been demonstrated to induce strong T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis. In Western blotting (immunoblotting), the monoclonal antibody HYB76-8, reacting with ESAT-6, gave a 6-kDa region was observed in filtrates from four of eight substrains of M. bovis BCG that produced high levels of MPB64, while no band occurred in the 6-kDa region with any of these BCG substrains. Southern blotting and PCR experiments with genomic mycobacterial DNA showed the presence of the esat-6 gene in reference strains and clinical isolates of M. tuberculosis as well as in virulent M. bovis. The esat-6 gene could not be demonstrated in any of the eight substrains of M. bovis BCG tested by these techniques. Two gene deletions that distinguish M. bovis BCG from virulent M. bovis have thus now been demonstrated. Deletion of mpb64 affects four of the eight substrains tested; deletion of esat-6 affects all of them. The reaction of HYB76-8 AT 26 kDa with four of the BCG substrains was demonstrated to result from cross-reactivity with MPB64. HYB76-8 was also shown to cross-react with the A, B, and C components of the antigen 85 complex and MPT51.     

Investigating the Induction of Vaccine-Induced Th17 and Regulatory T Cells in Healthy,Mycobacterium bovis BCG-Immunized Adults Vaccinated with a New Tuberculosis Vaccine,MVA85A

Tuberculosis (TB) remains a threat to global health. While advances in diagnostics and treatment are crucial to the containment of the epidemic, it is likely that elimination of the disease can only be achieved through vaccination. Vaccine-induced protection from Mycobacterium tuberculosis is dependent, at least in part, on a robust Th1 response, yet little is known of the ability of TB vaccines to induce other T-cell subsets which may influence vaccine efficacy. Interleukin-17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells which has been associated with both immune pathology and protection against infectious disease. Following vaccination with MVA85A, a viral vector vaccine aimed at enhancing immune responses to M. tuberculosis, antigen-specific IL-17A-producing T cells were induced in the peripheral blood of healthy volunteers. These T cells are detected later than gamma interferon (IFN-γ)-secreting T cells and are of a low magnitude. Preexisting immune responses to mycobacterial antigens were associated with higher CD4⁺ CD25^hi CD39⁺ T-cell levels in the periphery and a reduced capacity to produce IL-17A following immunization. These data highlight the intricate balance of effector and regulatory immune responses induced by vaccination and that preexisting immunity to mycobacterial antigens may affect the composition of vaccine-induced T-cell subsets.Tuberculosis (TB) remains a global health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, HIV-TB coinfection, and failure of the BCG vaccine to control adult pulmonary TB (9, 13). There is evidence that protection from M. tuberculosis is, at least in part, dependent on a robust Th1 response and the secretion of gamma interferon (IFN-γ) by antigen-specific T cells (1, 15, 22). Although IFN-γ alone is not sufficient for protection, in the absence of a better biomarker, early clinical trials of new TB vaccines use antigen-specific IFN-γ production as the primary gauge of vaccine-induced immune responses (16). In early clinical trials, modified vaccinia virus Ankara expressing antigen 85A from M. tuberculosis (MVA85A), a subunit vaccine designed to increase immune protection conferred by BCG, has been found to induce high levels of antigen-specific IFN-γ-secreting CD4⁺ T cells in individuals previously vaccinated with BCG (28, 29, 31).CD4⁺ T cells can differentiate into diverse effector cell subsets upon antigenic stimulation and the classical Th1/Th2 paradigm has now been expanded to include Th17 and T-regulatory (Treg) cells. Th17 cells are potent inflammatory cells which produce interleukin-17A (IL-17A) as their hallmark cytokine (17, 30). Th17 cells are mainly known for their role in mediating autoimmune pathology (19, 35) but are also thought to be involved in mediating protection against certain extracellular pathogens and fungi which are not effectively cleared by Th1- and Th2-type responses (12, 20). In contrast to Th17 cells, Treg cells comprise a regulatory cell subset of CD4⁺ T cells which act to suppress T-cell responses and are thereby thought to prevent pathology from chronic or excessive immune responses (21, 32, 33).Although a role has been defined for Th17 cells and Treg cells in other diseases, their role in TB remains unclear and even less is known about the effect of vaccination on these T-cell subsets.Clinical trials evaluating the safety and immunogenicity of MVA85A in BCG-vaccinated adults provide an opportunity to further investigate the induction and dynamics of vaccine-induced Th17 cells and Treg cells. Determining the effect of vaccination with MVA85A on these T-cell subsets is important, as protection from TB is likely to be dependent not only upon a Th1 response but also upon the balance between this effector response and the Th17 and Treg responses.     

Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice.

BALB/c mice were infected with Mycobacterium lepraemurium in the footpad or with Mycobacterium bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 U), as a single course of five injections (400 U each), or as a 6-month course starting 3 days after the M. lepraemurium infection. BCG-infected mice received a single dose (1,000 U) or five daily injections of 100 or 1,000 U each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph nodes, and liver of M. lepraemurium-infected mice (50 to 85%) by 6 months and viable counts in the spleen (30 to 50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than those started at 3 days after M. lepraemurium infection (P less than 0.05 to 0.001), and for BCG, 100 U of IL-2 was better than 1,000 U (P less than 0.05 to 0.01). These results indicate that IL-2 limits mycobacterial infections in mice and raise the question of its possible use in humans.     

Homology Between the MPB70 and MPB83 Proteins of Mycobacterium bovis BCG

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS-PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross-reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.     

Purification and characterization of two protein antigens from the heterogeneous BCG85 complex in Mycobacterium bovis BCG

The heterogeneous BCG85 complex is a major component of BCG culture fluid. BCG85A and BCG85B were purified by combining ammonium sulphate precipitation with chromatography on hydroxyapatite, DEAE-Sephacel and phenyl-Sepharose columns. Twenty percent of BCG85B was recovered. The chromatographic separation procedures were monitored by fused rocket immunoelectrophoresis. The BCG85 complex was found to consist of three antigens, which were heterogenous with regard to electrophoretic mobility, molecular weight (MW), hydrophobic and immunological properties. They were designated A, B and C in increasing order, according to their electrophoretic mobilities. Thus BCG85A had the lowest electrophoretic mobility, BCG85C the highest. The MW of BCG85A was found to be 31,000, while BCG85B had a slightly lower MW, 29,000, as determined by SDS-PAGE. The antigenic relationship between the components was evaluated by crossed immunoelectrophoresis and double diffusion, and reactions of partial identity between the antigens were found. The BCG85 complex occurs in far lower concentration in sonicates of BCG than in culture fluid.     

Filterable forms and L-forms of Mycobacterium bovis BCG: impact for live vaccine features

Bacterial L-form conversion, or existence without cell walls, is assumed a universal phenomenon in nature. An interesting aspect of this phenomenon is occurrence of L-forms in vaccine strains. Since BCG is currently a widely used and extensively studied live vaccine for tuberculosis, understanding L-form conversion of M. bovis BCG bacilli can provide new insight into behavior of BCG vaccine. In this respect, specific features, concerning the ability of BCG vaccine to produce viable filterable forms and L-forms, were studied by filtration and starvation stress experiments in vitro. The filterable forms obtained after filtration of BCG suspension, grew on Middlebrook 7H9 semisolid agar and formed typical "fried eggs" L-form colonies. Electron microscopy clearly demonstrated presence of L-form elements with size smaller than the size of bacterial filter pores of 0.2 μm in M. bovis BCG strains. Development of L-form subpopulation with typical morphological appearance of self-replicating cell wall-defective forms was observed after filtration, as well as after starvation stress. Specific DNA detection of pncA gene in derived L-form cultures from filterable and stressed BCG strains verified their identity as M. bovis BCG. In conclusion, the results confirm existence of filterable forms in commercial BCG vaccine, which are able to develop L-form population under appropriate conditions. L-form transformation of BCG bacilli displays a new intriguing aspect concerning exhibition of unusual features and atypical behavior of live BCG vaccine. Further research is requested to explore the influence of L-form phenomenon on BCG vaccine effects in vivo.     

A peptide permease mutant of Mycobacterium bovis BCG resistant to the toxic peptides glutathione and S-nitrosoglutathione

Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database for Mycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele of oppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of the opp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (gamma-glutamyl-L-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [(3)H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that the opp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [(3)H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.