Mycobacterium bovis BCG vaccine fails to protect protein-deficient guinea pigs against respiratory challenge with virulent Mycobacterium tuberculosis.

Specific-pathogen-free Hartley guinea pigs were maintained on isocaloric-purified diets either adequate (30%) or moderately deficient (10%) in protein. Half of each diet group was vaccinated with viable Mycobacterium bovis BCG. Six weeks later, all animals were challenged by the respiratory route with virulent Mycobacterium tuberculosis H37Rv. At intervals of 1, 2, and 3 weeks postchallenge, guinea pigs from each diet and vaccination group were skin tested with tuberculin and sacrificed. Protein deficiency resulted in loss of tuberculin hypersensitivity. Vaccination with M. bovis BCG protected control animals, as determined by significant reductions in the number of M. tuberculosis H37Rv organisms recovered from lungs, spleen, and bronchotracheal lymph nodes 2 and 3 weeks postchallenge. Based upon the same criteria, the degree of protection afforded protein-deficient animals by M. bovis BCG vaccine ranged from partial (spleen and lymph nodes) to none at all (lungs). Approximately the same numbers of tubercle bacilli were recovered from nonvaccinated guinea pigs in both diet groups. Protein deficiency appears to impair M. bovis BCG-induced immunity while not affecting primary pulmonary infection with virulent M. tuberculosis.     

Identification of a Mycobacterium bovis BCG auxotrophic mutant that protects guinea pigs against M. bovis and hematogenous spread of Mycobacterium tuberculosis without sensitization to tuberculin

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.     

MTSA-10, the product of the Rv3874 gene of Mycobacterium tuberculosis, elicits tuberculosis-specific, delayed-type hypersensitivity in guinea pigs

In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of Mycobacterium tuberculosis elicited delayed-type hypersensitivity in M. tuberculosis-infected guinea pigs but not in control animals immunized with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium avium. The antigen, which was named MTSA-10 (for M. tuberculosis-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent M. tuberculosis infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria.     

Influence of Mycobacterium bovis BCG vaccination on cellular immune response of guinea pigs challenged with Mycobacterium tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) currently remains the only licensed vaccine for the prevention of tuberculosis. In this study, we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs and lymph nodes of na?ve and vaccinated guinea pigs challenged by low-dose aerosol infection with virulent Mycobacterium tuberculosis. As anticipated, vaccinated guinea pigs controlled the growth of the challenge infection more efficiently than controls did. This early phase of bacterial control in immune animals was associated with increased accumulation of CD4 and CD8 T cells, including cells expressing the activation marker CD45, as well as macrophages expressing class II major histocompatibility complex molecules. As the infection continued, the numbers of T cells in the lungs of vaccinated animals waned, whereas the numbers of these cells expressing CD45 increased. Whereas BCG vaccination reduced the influx of heterophils (neutrophils) into the lungs, an early B-cell influx was observed in these vaccinated animals. Overall, vaccine protection was associated with reduced pathology and lung damage in the vaccinated animals. These data provide the first direct evidence that BCG vaccination accelerates the influx of protective T-cell and macrophage populations into the infected lungs, diminishes the accumulation of nonprotective cell populations, and reduces the severity of lung pathology.     

Association of tuberculin-boosted antibody responses with pathology and cell-mediated immunity in cattle vaccinated with Mycobacterium bovis BCG and infected with M. bovis

Vaccine development and our understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by definition of the immunological correlates of protection and/or pathology. In this study we analyzed humoral immune responses in Mycobacterium bovis BCG-vaccinated and control cattle (in particular, the relationship between the intradermal comparative tuberculin skin test and serum immunoglobulin G [IgG] responses) against a range of mycobacterial antigens (MPB59, MPB64, MPB70, MPB83, ESAT-6, CFP-10, Acr1, and PstS-1) by multiantigen print immunoassay and conventional enzyme-linked immunosorbent assay. Following M. bovis infection, the comparative tuberculin skin test strongly boosted IgG, IgG1, and IgG2 antibody responses, particularly against MPB83 and MPB70, in unvaccinated cattle but failed to boost these responses, or did so only weakly, in BCG-vaccinated calves. In addition, the skin test-induced increases in MPB83-specific IgG responses correlated positively with bacterial loads and ESAT-6-induced in vitro gamma interferon responses. In conclusion, both the negative correlation of skin test-enhanced MPB83-specific antibody responses with BCG-induced protection and their positive correlation with bacterial loads can serve as useful markers for vaccine efficacy after challenge.     

Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51

We report here the induction of specific protective cellular immunity against Mycobacterium tuberculosis by the employment of vaccination with recombinant attenuated Listeria monocytogenes strains. We constructed self-destructing attenuated L. monocytogenes Delta 2 strains carrying eukaryotic expression plasmids for the antigen 85 complex (Ag85A and Ag85B) and for MPB/MPT51 (mycobacterial protein secreted by M. bovis BCG/mycobacterial protein secreted by M. tuberculosis) molecules. Infection of these recombinant bacteria allowed expression of the genes in the J774A.1 murine macrophage cell line. Intraperitoneal vaccination of C57BL/6 mice with these recombinant bacteria was capable of inducing purified protein derivative-specific cellular immune responses, such as foot pad reactions, proliferative responses of splenocytes, and gamma interferon production from splenocytes, suggesting the efficacy of vaccination against mycobacterial infection by use of these recombinant L. monocytogenes strains. Furthermore, intravenous vaccination with recombinant bacteria carrying expression plasmids for Ag85A, Ag85B, or MPB/MPT51 in BALB/c mice elicited significant protective responses, comparable to those evoked by a live Mycobacterium bovis BCG vaccine. Notably, this is the first report to show that MPB/MPT51 is a major protective antigen in addition to Ag85A and Ag85B, which have been reported to be major mycobacterial protective antigens.     

Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs

Tuberculosis is rising in the developing world due to poor health care, human immunodeficiency virus type 1 infection, and the low protective efficacy of the Mycobacterium bovis BCG vaccine. A new vaccination strategy that could protect adults in the developing world from tuberculosis could have a huge impact on public health. We show that BCG boosted by poxviruses expressing antigen 85A induced unprecedented 100% protection of guinea pigs from high-dose aerosol challenge with Mycobacterium tuberculosis, suggesting a strategy for enhancing and prolonging the efficacy of BCG.     

Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge

Infection with Mycobacterium tuberculosis remains a major cause of morbidity and mortality all over the world. Since the effectiveness of the only available tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is suboptimal, there is a strong demand to develop new tuberculosis vaccines. As tuberculosis is an airborne disease, the intranasal route of vaccination might be preferable. Live influenza virus vaccines might be considered as potential vectors for mucosal immunization against various viral or bacterial pathogens, including M. tuberculosis. We generated several subtypes of attenuated recombinant influenza A viruses expressing the 6-kDa early secretory antigenic target protein (ESAT-6) of M. tuberculosis from the NS1 reading frame. We were able to demonstrate the potency of influenza virus NS vectors to induce an M. tuberculosis-specific Th1 immune response in mice. Moreover, intranasal immunization of mice and guinea pigs with such vectors induced protection against mycobacterial challenge, similar to that induced by BCG vaccination.     

Biological Activity in Sensitized Guinea Pigs of MPB 70, a Protein Specific for Some Strains of Mycobacterium bovis BCG

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.     

Purification of MPB70 and production of specific monoclonal antibodies.

MPB70 is a protein secreted into the culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172), which is able to induce a delayed-type hypersensitivity (DTH) skin reaction in guinea pigs immunized with BCG-Tokyo. By high-pressure chromatofocusing and size-exclusion high performance liquid chromatography, a further purified MPB70 protein was obtained, which was visualized as a single band with a molecular mass of 22 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A series of hybridoma cell lines that produced monoclonal antibodies (MAbs) against the purified MPB70 protein was prepared, and three MAbs, Bov-1, Bov-2, and Bov-3, with strong antigen-binding capacities were established. Bov-1 was the most potent MAb among them and binds to only a 22 kDa protein band in culture filtrates of M. bovis, but not to bands in those of M. tuberculosis by Western immunoblotting analysis, suggesting that Bov-1 recognize different epitope of MPB70 from MAbs that have been shown previously to recognize several species of molecules in culture filtrates of M. bovis. The purified MPB70 protein elicited a strong DTH skin reaction in guinea pigs sensitized with BCG-Tokyo vaccine. Bov-1 had no inhibitory effect on generation of the DTH skin reaction, showing that MAb bound to an epitope distinct from that inducing the skin reaction. All of the three MAbs were specific to MPB70 and each recognized a different epitope on MPB70. MPB70 was not detected in the culture filtrate of M. tuberculosis H37Rv. Thus, these MAbs may be useful for detecting MPB70 in studies on discriminating infection with M. bovis in domestic animals or in distinguishing vaccination with BCG-Tokyo from other mycobacterial infections in humans.     

Protection against Mycobacterium avium by DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.     

T-cell determinants and antibody binding sites on the major mycobacterial secretory protein MPB59 of Mycobacterium bovis.

Among the first proteins encountered by the host immune system upon infection or vaccination with mycobacteria are those secreted by the bacillus during growth. The antigen 85 complex of Mycobacterium bovis bacillus Calmette-Gúerin (BCG) is composed of three closely related members. The mature 85B protein of M. bovis (MPB59) has a high degree of amino acid identity with the M. bovis 85A protein (76%) and the Mycobacterium tuberculosis 85B (99%) and 85A (76%) proteins. We have examined the regions of MPB59 which stimulate human T- and B-cell responses by use of a set of 28 synthetic peptides, 20 amino acids (aa) in length and overlapping by 10 aa. Initial proliferative assays with recombinant MPB59 demonstrated that peripheral blood mononuclear cells from 95% of BCG vaccinees and 52% of tuberculosis patients responded to the whole mature protein. Peripheral blood mononuclear cells from MPB59 responders, but not nonresponders, were stimulated by peptides in a dose-dependent fashion. Five peptides were reactive in more than half of the MPB59 responders. The T-cell-reactive regions were essentially identical in the M. bovis and M. tuberculosis 85B proteins. Subjects with a variety of HLA-DR phenotypes responded to a number of these peptides. The dominant T-cell-reactive regions were distinct from the peptides recognized by sera from tuberculosis patients (aa 71 to 100) and the murine monoclonal antibody HYT27 (aa 61 to 90). The region reactive with antibodies overlapped part of the MPB59 sequence recently shown to participate in the binding of MPB59 to fibronectin.     

The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.     

Modulation of expression of delayed hypersensitivity by mycobacterial antigen 85 fibronectin-binding proteins.

Although demonstration of delayed hypersensitivity to purified protein derivative of tuberculin (PPD) is an important element in the diagnosis of infection with Mycobacterium tuberculosis, many patients with tuberculosis are anergic. Several possible mechanisms for this specific lack of response have been described. We have now uncovered an additional one. T-cell fibronectin (FN), a lymphokine secreted by activated T cells, is closely associated with the initiation of delayed hypersensitivity reactions. Mycobacterial antigen 85 (Ag85) proteins have been shown to bind to plasma FN. The ability of Ag85 to bind to T-cell FN and modulate expression of delayed hypersensitivity was therefore studied. Purified Ag85 proteins from M. tuberculosis, Mycobacterium bovis BCG, or Mycobacterium kansasii bound to T-cell FN, fibroblast FN, and plasma FN in vitro. Purified 65-kDa heat shock protein (hsp65) from M. bovis BCG did not bind to any FN. Ag85, but not hsp65, inhibited the ability of T-cell FN to agglutinate monocytes in vitro in a dose-dependent manner. In vivo, mixtures of PPD or dinitrophenyl-ovalbumin and purified M. tuberculosis or M. bovis BCG Ag85 proteins elicited significantly smaller delayed hypersensitivity inflammatory reactions in sensitized guinea pigs than did PPD or dinitrophenyl-ovalbumin alone. Purified hsp65 did not inhibit expression of delayed hypersensitivity to PPD or dinitrophenyl-ovalbumin. We suggest that Ag85 proteins could inhibit in vivo expression of delayed hypersensitivity during mycobacterial infections because of their interaction with T-cell FN.     

Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis

The development of more-effective antituberculosis vaccines would assist in the control of the global problem of infection with Mycobacterium tuberculosis. One recently devised vaccination strategy is immunization with DNA plasmids encoding individual microbial genes. Using the genes for the M. tuberculosis secreted proteins MPT64 (23 kDa), Ag85B (30 kDa), and ESAT-6 (6 kDa) as candidate antigens, DNA vaccines were prepared and tested for immunogenicity and protective efficacy in a murine model of aerosolized tuberculosis (TB). Intramuscular immunization with DNA-64 or DNA-85B resulted in the activation of CD4(+) T cells, which produce gamma interferon (IFN-gamma), and high titers of specific immunoglobulin G antibodies. Further, DNA-64 induced major histocompatibility complex class I-restricted CD8(+) cytotoxic T cells. The addition of a eukaryotic leader sequence to mpt64 did not significantly increase the T-cell or antibody response. Each of the three DNA vectors stimulated a significant reduction in the level of M. tuberculosis infection in the lungs of mice challenged 4 weeks after immunization, but not to the levels resulting after immunization with Mycobacterium bovis BCG. The vaccines showed a consistent hierarchy of protection, with the most effective being Ag85B, followed by ESAT-6 and then MPT64. Coimmunization with the three vectors resulted in a greater degree of protection than that induced by any single vector. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells earlier than in infected animals immunized with a control vector. The efficacy of these DNA vaccines suggests that multisubunit vaccination may contribute to future vaccine strategies against TB.     

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.     

Generation of attenuated Mycobacterium bovis strains by signature-tagged mutagenesis for discovery of novel vaccine candidates

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.     

Vaccination of guinea pigs with nutritionally impaired avirulent mutants of Mycobacterium bovis protects against tuberculosis

Four nutritionally impaired strains of Mycobacterium bovis produced by illegitimate recombination were tested for their ability to protect guinea pigs against intratracheal challenge with virulent M. bovis. All four strains and M. bovis BCG induced significant levels of protection as measured by the reduced spread of infection to the spleen and liver. In animals vaccinated with BCG or two of the other strains, the bacterial counts from the lungs were significantly lower than those of the nonvaccinated animals.     

Duration of Immunity against Mycobacterium bovis following Neonatal Vaccination with Bacillus Calmette-Guerin Danish: Significant Protection against Infection at 12, but Not 24, Months

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulent M. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route with M. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinated M. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time of M. bovis challenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.