Biological activity of protein antigens isolated from Mycobacterium tuberculosis culture filtrate.

Mycobacterium tuberculosis culture filtrate (MTCF) protein antigens were isolated from mid-logarithmic-phase cultures grown in liquid medium and examined by high-pressure liquid chromatography and Western blot (immunoblot) analysis. A major protein band with a molecular mass of about 68 kilodaltons (kDa) and several fainter bands in the 38- and 24-kDa range were observed. The MTCF protein produced a significant delayed footpad hypersensitivity response in Mycobacterium bovis BCG-vaccinated C57BL/6 mice, comparable to that observed with standard purified protein derivative (PPD). The same proteins induced a blastogenic response in tuberculin-sensitive human peripheral blood monocytes and in T-cell clones developed from these cells. The proliferative responses to the MTCF antigens were equivalent to those observed following stimulation with PPD or M. tuberculosis sonic extracts. However, the MTCF sensitins were not recognized by five monoclonal antibodies directed against killed M. tuberculosis antigens in an enzyme immunoassay, although some response was seen with a monoclonal antibody (ML34) directed against M. leprae antigens. The ability of the MTCF to stimulate T-cell responses both in vivo and in vitro while not being recognized by antibodies directed against dead mycobacterial antigens suggests that they may be of interest as potential protective immunogens.     

Serodiagnostic potential of culture filtrate antigens of Mycobacterium tuberculosis

Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534-1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49-56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.     

Antibody repertoire against culture filtrate antigens in wild house mice infected with Mycobacterium bovis BCG.

Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 x 10(6) variable units of Mycobacterium bovis BCG and examined by Western blot analysis for IgG secretion against BCG culture filtrate (CF) antigens. Wild mice showed a marked individual variation in antibody pattern when tested 4, 6 and 8 weeks after infection. Some animals reacted to a wide range of antigens and others only to a limited number. Most wild mice recognized preferentially antigens with molecular weight of 24 kD, 32 kD, 37-38-40 kD, 65 kD and 82 kD, i.e. the major CF antigens known to be recognized by sera from BCG-infected inbred laboratory strains, BALB/c, DBA/2, CBA/Ca and C57BL/6. The 32-kD fibronectin-binding protein and the 65-kD heat-shock protein appeared as very immunodominant in wild mice. Furthermore, about 20-25% of the mice reacted strongly with a unique antigen of 35 kD estimated molecular weight, to which the tested inbred laboratory mice did not respond. Monitoring the size of the bacterial population in the spleen indicated that the BCG inoculum did not replicate in wild mice, suggesting that the Bcgr allele is expressed in this population.     

Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein

Tuberculosis continues to ravage humanity, killing 2 million people yearly. Most cases occur in areas of the world to which the disease is endemic, where almost everyone is vaccinated early in life with Mycobacterium bovis BCG, the currently available vaccine against tuberculosis. Thus, while more-potent vaccines are needed to replace BCG, new vaccines are also needed to boost the immune protection of the 4 billion people already vaccinated with BCG. Until now, no booster vaccine has been shown capable of significantly enhancing the level of protective immunity induced by BCG in the stringent guinea pig model of pulmonary tuberculosis, the "gold standard" for testing tuberculosis vaccines. In this paper, we describe a booster vaccine for BCG comprising the purified recombinant Mycobacterium tuberculosis 30-kDa protein, the major secreted protein of this pathogen. In the guinea pig model of pulmonary tuberculosis, boosting BCG-immunized animals once with the 30-kDa protein greatly increased cell-mediated and humoral immune responses to the protein in three consecutive experiments. Most importantly, boosting BCG-immunized animals once with the 30-kDa protein significantly enhanced protective immunity against aerosol challenge with highly virulent M. tuberculosis, as evidenced by a significantly reduced lung and spleen burden of M. tuberculosis compared with those for nonboosted BCG-immunized animals (mean additional reduction in CFU of 0.4 +/- 0.1 log in the lung [P = 0.03] and 0.6 +/- 0.1 log in the spleen [P = 0.002]). This study suggests that administering BCG-immunized people a booster vaccine comprising the 30-kDa protein may enhance their level of immunoprotection against tuberculosis.     

Induction of protective immunity against Mycobacterium tuberculosis by delivery of ESX antigens into airway dendritic cells

As the Bacillus Calmette–Guérin (BCG) vaccine does not confer long-lasting protection against lung Mycobacterium tuberculosis infection, the development of more efficient vaccines is greatly needed. Here, we used mycobacterial low-molecular weight proteins of the 6-kDa Early Secreted Antigenic Target (ESAT-6) protein family (ESX) antigens for the evaluation of a novel vaccine delivery strategy that enables versatile in vivo targeting of antigens into specialized dendritic cell (DC) subsets. ESX antigens were genetically fused to the tetramerizing core of streptavidin (SA) to form high-affinity complexes with biotin (biot)-conjugated antibodies recognizing DC surface receptors. When directed through the CD11b or CD11c β₂-integrins or diverse C-type lectins, the ESX-SA:biot-antibody complexes were efficiently captured and presented on major histocompatibility complex molecules of DCs to specific T-cell receptors. Robust ESX-specific T-cell responses were induced by immunization with as little as several picomoles of ESX-SA targeted to DC subsets. Moreover, directing of TB10.4-SA to airway CD205⁺ cells enabled the induction of mucosal T-cell responses and provided significant protection against virulent M. tuberculosis.     

Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis (phoP) SO2 strain

The Mycobacterium tuberculosis phoP mutant strain SO2 has been shown previously to be more attenuated than Mycobacterium bovis bacillus Calmette-Guérin (BCG) and confers protective immunity against tuberculosis in mice and guinea pig models. In this study we have investigated the survival and immunological responses of Balb/c mice infected with the M. tuberculosis SO2 strain. All Balb/C mice survived intratracheal infection with M. tuberculosis SO2 strain under conditions where all the mice infected with the parental M. tuberculosis MT103 had died after 9 weeks. Infection of Balb/c mice with M. tuberculosis SO2 was associated with comparatively lower levels of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha and higher levels of inducible nitric oxide synthase (iNOS) during the late stage of infection, when compared with M. tuberculosis MT103 infection. The delayed-type hypersensitivity (DTH) response against M. tuberculosis culture filtrates was similar in mice infected with either the M. tuberculosis phoP SO2 strain or M. tuberculosis MT103. The protective efficacy of M. tuberculosis SO2 was compared with M. bovis BCG when delivered subcutaneously to groups of Balb/C mice. Following intratracheal challenge with M. tuberculosis H37Rv, protection was generated by 60 days post-challenge in mice vaccinated with either vaccine. At day 120 post-challenge the levels of protection were still significantly greater when compared with the non-vaccinated control group. The levels of protection conferred by vaccination with M. tuberculosis SO2 or with M. bovis BCG were similar, as measured by granuloma coalescence and pneumonia in addition to growth reduction of M. tuberculosis H37Rv.     

Induction of high levels of protective immunity in mice after vaccination using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis

Adoptively transferred dendritic cells presenting antigens derived from different pathogens have been shown to elicit specific T cell responses and to induce protective antibacterial immunity. We describe here the induction of high levels of protective immunity in mice using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis. We provide evidence that protection is superior to BCG and that it is associated with increased priming of CD4+ and CD8+ T cells specific for mycobacterial antigens. This method for generating high levels of anti-bacterial protective immunity could be helpful in the design of novel vaccines against tuberculosis and other intracellular pathogens.     

Comparative analysis of B- and T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture filtrate protein 10

Culture filtrate protein 10 (CFP-10) from Mycobacterium tuberculosis is a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells from M. tuberculosis-infected mice and humans. The sequence of the Mycobacterium leprae homologue of CFP-10 shows only 40% identity (60% homology) at the protein level with M. tuberculosis CFP-10 and thus has the potential for development as a T- or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinant M. leprae and M. tuberculosis CFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for either M. leprae or M. tuberculosis CFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against either M. leprae or M. tuberculosis CFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections of M. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting that M. leprae CFP-10, like its homologue in M. tuberculosis, is a secreted protein.     

Mycobacterium bovis BCG immunization induces protective immunity against nine different Mycobacterium tuberculosis strains in mice

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.     

Specific skin-reactive protein from culture filtrate of Mycobacterium bovis BCG.

A highly purified protein, named MPB70, was isolated from the culture filtrate of Mycobacterium bovis BCG. This protein accounted for more than 10% of the proteins secreted into the culture medium. MPB70 was purified by precipitation with ammonium sulfate, followed by treatment with diethylaminoethyl ion exchanger, with or without 3 M urea, and by gel filtration. The final MPB70 preparation was homogenous as judged by several analyses. The molecular weight was estimated to be 18,000 by electrophoresis or molecular sieve and 15,100 by sedimentation equilibrium. The preparation did not contain sugars. The amino acid composition did not include cysteine or tryptophan. In skin reaction, MPB70 was a strictly BCG-specific antigen and, among the guinea pigs sensitized with the heat-killed cells of the various species of mycobacteria--Mycobacterium tuberculosis strains H37Rv and Aoyama B, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium phlei, and BCG, it elicited a delayed cutaneous reaction only in the guinea pigs sensitized with BCG. The potency of MPB70 in the skin reaction was about one-twentieth of the standard purified protein derivative.     

Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins

Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC-C18 chromatography column followed by separation based on their molecular mass by SDS-PAGE and subsequent immunoblotting or (ii) proteins were separated by two-dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using I mage master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody-to-protein-ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv3881c is located in the region of difference 1 (RD1) which is deleted from Mycobacterium bovis BCG, and is therefore a particularly promising candidate for development of serodiagnostic assays to detect active tuberculosis. The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.     

Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate

Ten major antigens from Mycobacterium bovis culture filtrate of 39, 32, 30, 25, 24, 22 (a and b forms), 19, 15, and 12 kDa have been purified and characterized by classical physicochemical methods. With monoclonal antibodies and/or N-terminal amino acid sequencing data, it was found that the antigens of 32, 30, 24, 22 (a), 19, and 12 kDa are related to M. bovis or M. tuberculosis antigens P32, MPB59, MPB64, MPB70, 19 kDa, and 12 kDa, respectively. The 39-, 25-, 22 (b)-, and 19-kDa antigens showed concanavalin A-binding properties and were positive in a glycan detection test, suggesting that they are glycoproteins. The 25- and 22 (b)-kDa proteins were found to be glycosylated forms of MPB70.     

Repertoires of antibodies to culture filtrate antigens in different mouse strains infected with Mycobacterium bovis BCG.

Two susceptible (Bcgs) mouse strains, BALB/c and C57BL/6, were compared by Western blot (immunoblot) analysis for their immunoglobulin G response to 14-day-old BCG culture filtrate (CF) following intravenous infection with live Mycobacterium bovis BCG. The two strains demonstrated a completely different antibody repertoire. BALB/c antibodies were directed against a wide range of CF antigens between 20 and about 100 kilodaltons (kDa), with a preferential recognition of the 65-kDa heat shock protein and the 32-kDa fibronectin-binding protein. C57BL/6 sera, on the other hand, showed a much more restricted antibody pattern, almost exclusively directed against three antigens with estimated molecular sizes of 37, 38, and 40 kDa. Whereas the 37- and 38-kDa antigens were also recognized by BALB/c mice, the 40-kDa antigen was very intensely stained by C57BL/6 sera only. F1 mice had the restricted antibody pattern of C57BL/6 after one injection of BCG and had a hybrid BALB/c-C57BL/6 phenotype following a boost injection of BCG 2 months after the initial infection. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments suggests that a gene in the K-IA region of the H-2 locus is associated with the preferential recognition of certain CF antigens. Inoculation with the same dose of killed BCG failed to elicit an antibody response to these filtrate antigens.     

Monoclonal antibodies produced in BALB.B10 mice define new antigenic determinants in culture filtrate preparations of Mycobacterium tuberculosis.

A panel of monoclonal antibodies were derived from BALB.B10 mice immunized with a culture filtrate from Mycobacterium tuberculosis H37Rv. Of these antibodies, 10 were examined more closely for antigen specificity and interspecies reactivity. Six antibodies were used as immunosorbents for affinity purification of their corresponding antigens. Two monoclonal antibodies (HBT 2 and HBT 11) reacted with a 17-kilodalton antigen, and a competition assay showed that these antibodies are directed against the same epitope or against epitopes that are sterically very close to each other. Monoclonal antibody HBT 12 reacted with the same molecule with which a previously described 38-kilodalton reactive antibody reacted but was directed against a different epitope. Antibody HBT 10 reacted with a culture filtrate of M. tuberculosis but not of Mycobacterium bovis BCG. This latter finding was further studied by testing different preparations of M. tuberculosis H37Rv antigens and, additionally, culture filtrates of four M. tuberculosis and two BCG strains. Interspecies reactivity was assayed by immunoblotting and revealed that the majority of the monoclonal antibodies were specific to M. tuberculosis complex.     

Immunogenicity and safety of Mycobacterium tuberculosis culture filtrate proteins in non-human primates

The development of improved vaccines is considered a high priority in the effort to control tuberculosis (TB) world wide. Results from several studies performed in relevant animal models have demonstrated that Mycobacterium tuberculosis secreted antigens may represent major components of improved TB vaccines. To characterize further the M. tuberculosis secreted antigens as they relate to specific features important for vaccine development, rhesus macaques were immunized with either one of two different preparations containing M. tuberculosis culture filtrate (CF) proteins. These preparations differed in relative protein content and in the presence or absence of lipoarabinomannan. Animals received a total of three monthly intramuscular injections consisting of CF proteins resuspended in RIBI adjuvant and were tested for development of specific antibody and cellular proliferative responses. In addition, all animals were constantly monitored for local and systemic reactions as well as for the development of DTH reactions to intradermal tuberculin injection. Results from this study show that the two CF preparations are relatively safe and immunogenic in non-human primates. These two CF preparations differed in their ability to induce specific antibody responses, but were comparable in their ability to induce specific cellular proliferative responses. Induction of both humoral and cellular responses occurred even in presence of pre-existing antibodies directed against M. tuberculosis antigens. However, these responses appeared to be short-lived. Only one of the four animals produced interferon-gamma (IFN-gamma) in response to immunization with CF proteins. No DTH reaction to intradermal tuberculin injection was observed in any immunized animal. Although it is clear that additional studies are required to design strategies for the improvement of the immunogenicity of CF proteins, our observations support the currently accepted view that secreted protein-based preparations may represent promising vaccine candidates for TB.     

Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis.

Five actively secreted proteins (MPT32, MPT45, MPT51, MPT53, and MPT63) and the MPT46 protein were purified to homogeneity from Mycobacterium tuberculosis culture fluid and compared with proteins previously purified by ourselves and other investigators. Antisera were obtained by immunization of rabbits with all of the newly isolated proteins identified to be immunogenic. Two-dimensional electrophoresis of culture fluids obtained each week for 2 to 10 weeks of culturing of M. tuberculosis revealed characteristic changes, permitting identification of two distinct groups of proteins being actively secreted from the mycobacterial cells or appearing later in the culture fluids as a result of the release of soluble proteins from the cytosol after lysis of bacteria. The N-terminal amino acid sequences of five MPTs were shown to be identical to those of proteins previously isolated by other investigators and given different designations, and five new sequences are given. These sequences and the use of the antisera may serve to identify these proteins with mycobacterial constituents isolated by other investigators. The previously identified but not isolated MPT45 protein was shown to correspond to the C component of the antigen 85 complex. The 27-kDa MPT51 protein was demonstrated to cross-react with the three components of the antigen 85 complex, and the N-terminal amino acid sequences of MPT51 and MPT59 showed 60% homology. This finding and the extensive cross-reactivity between the components of the antigen 85 complex may indicate that there is a family of closely related secreted proteins in mycobacteria.     

Significance of immune response to Mycobacterium tuberculosis culture filtrate protein antigens in cerebrospinal fluid of tuberculous meningitis patients: A search for diagnostic marker

Mycobacterium tuberculosis (H37Ra) culture filtrate proteins (CFP) are explored as a diagnostic marker for tuberculous meningitis (TBM). Cerebrospinal fluid (CSF) samples from patients were categorized as confirmed (n = 47), suspected (n = 20), and non-TBM (n = 25) cases. Immune response by Western blot revealed TBM CSF samples are having heterogeneous response to CFP. CFP ELISA was 92% sensitive and 38.30% specific. ODs of confirmed TBM and non-TBM cases were significantly different (P < 0.0001) and also the suspected TBM and non-TBM cases (P = 0.0001). No significant difference noticed in TBM and suspected TBM (P = 0.90). Thus, CFP can be a better biomarker for the diagnosis of TBM.     

Immunization of mice with mycobacterial culture filtrate proteins.

Culture filtrate proteins were obtained from Mycobacterium tuberculosis cultures after 7 days growth in Proskauer and Beck medium. The protein yield increased substantially to peak about the time the number of viable organisms reached its maximum level (day 8). Examination of the protein concentrate by SDS-PAGE revealed the presence of at least 12 separate protein bands varying from 10 to 90 kD. Mice were injected subcutaneously with 20 micrograms of M. tuberculosis culture filtrate (MTCF) protein suspended in saline or Freund's complete or incomplete adjuvant. The vaccinated mice were subjected to an aerogenic challenge with 10(3) colony-forming unit (CFU) M. tuberculosis Erdman and a significant reduction in the number of viable organisms was observed in the spleens and lungs determined over a 21-day period compared with age-matched normal controls. Mice immunized with the same culture filtrate proteins bound to nitrocellulose particles also showed some resistance to the virulent challenge, suggesting that individual antigens present in the culture filtrate were able to induce a protective T cell-mediated immune response in appropriately immunized mice.     

Enhancement of the human T cell response to culture filtrate fractions of Mycobacterium tuberculosis by microspheres

An improved method of assessment of the human immune response against Mycobacterium tuberculosis antigens will assist the development of new vaccines or diagnostic reagents. In this study, we have analyzed human T cell responses to culture filtrate fractions (CFF) of actively replicating M. tuberculosis strain H37Rv using peripheral blood mononuclear cells from healthy PPD skin test positive and negative individuals. Adsorption of CFF onto polystyrene microspheres, that were approximately the size of the M. tuberculosis (bead-adsorbed antigens, BAA) significantly enhanced IFN-gamma production compared to soluble antigens (SA) in PPD skin test positive individuals in an antigen-specific manner. Further, BAA induced activation of both CD4(+) and CD8(+) T cell subsets. However, CD4(+) responses in general were higher and their antigenic repertoire was wider than the CD8(+) responses. By contrast, CD8(+) responses were strongest to the lower molecular weight BAA. When CFF were chemically coupled to carboxyl modified microspheres (bead-coupled antigens, BCA), induction of IFN-gamma was similar to BAA. Enhancement of T cell responses to particulate M. tuberculosis antigens may prove useful in vaccine design strategies.     

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.