BCG Tokyo Research Articles

Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model.

This study evaluates a short therapy schedule for bladder cancer using BCG Tokyo. BCG Tokyo was evaluated in vitro using bone marrow derived dendritic cells, neutrophils, RAW macrophages and the murine bladder cancer cell line, MB49PSA, and compared to other BCG strains. BCG Tokyo > BCG TICE at inducing cytokine production. In vivo, high dose (1 × 107 colony forming units (cfu)) and low dose (1 × 106 cfu) BCG Tokyo with and without cytokine genes (GMCSF + IFNα) were evaluated in C57BL/6J mice (n = 12–16 per group) with orthotopically implanted MB49PSA cells. Mice were treated with four instillations of cytokine gene therapy and BCG therapy. Both high dose BCG alone and low dose BCG combined with cytokine gene therapy were similarly effective. In the second part the responsive groups, mice (n = 27) were monitored by urinary PSA analysis for a further 7 weeks after therapy cessation. More mice were cured at day 84 than at day 42 confirming activation of the immune system. Cured mice resisted the re-challenge with subcutaneous tumors unlike naïve, age matched mice. Antigen specific T cells recognizing BCG, HY and PSA were identified. Thus, fewer intravesical instillations, with high dose BCG Tokyo or low dose BCG Tokyo with GMCSF + IFNα gene therapy, can induce effective systemic immunity.

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Impact of maintenance therapy using a half dose of the bacillus Calmette\u2013Gu\xe9rin Tokyo strain on recurrence of intermediate and high-risk nonmuscle invasive bladder cancer: a retrospective single-center study

BackgroundData are scarce regarding intravesical maintenance therapy (MT) with the low-dose bacillus Calmette–Guérin (BCG) Tokyo strain. We investigated the efficacy and safety of MT with a half dose of the Tokyo strain for patients following transurethral resection of nonmuscle invasive bladder cancer (NMIBC).MethodsThis study retrospectively reviewed clinical data on 78 patients diagnosed with intermediate or high-risk NMIBC followed by either MT (n = 38) or IT alone (n = 40) between January 2012 and March 2018. Statistical analysis was performed to compare recurrence-free survival (RFS) and adverse effects between the two groups. BCG was instilled once weekly for 6 weeks as IT, then once weekly in 2-week for a total of 20 instillations over 3 years.ResultsKaplan–Meier analyses showed that patients undergoing MT had significantly better RFS than did those undergoing IT alone (hazard ratio (HR):0.32, 95% confidence interval (CI):0.12–0.89, P = 0.02). The 3-year RFS was 65.0% in the IT group and 89.5% in the MT group. Multivariate analysis showed that MT was associated with a reduced risk of recurrence (HR: 0.32, 95% CI:0.11–0.93, P = 0.03). One MT patient (2.6%) exhibited progression.ConclusionsThe BCG Tokyo strain showed acceptable efficacy and safety in patients undergoing MT; thus, it is a potential treatment for preventing bladder cancer recurrence.

Abstract A12: Bladder cancer-derived extracellular vesicles and immune activation

Abstract Background: Bladder tumors occur on the luminal surface of the bladder, and cancer cells and cellular components are shed in urine, which permits the isolation of an enriched population of bladder cancer-specific extracellular vesicles (EVs). EVs contain proteins, miRNA, mRNA, and cellular metabolites, which may serve as stimulators of immune cells. The objective of this study was to evaluate the immune potential of tumor-derived EVs (TEVs) produced on exposure to BCG using a murine bladder cancer cell line and immune cells. Methods: TEVs were isolated by differential high-speed centrifugation of cell culture supernatants. They were confirmed to be extracellular vesicles by electron microscopy and size measurement using nanoparticle tracking analysis. Unstimulated murine bladder cancer cells (MB49) were used to prepare TEV1. The cells were then exposed to BCG Tokyo (MOI 4) and TEVs were isolated after 2 hours (TEV2). BCG was then washed off and the cells were allowed to grow in complete media for 2 days before isolation of the third sample, TEV3. EVs from BCG (BEV) were collected to serve as a control. Bone marrow-derived cells were obtained from C57BL/6J mice to generate dendritic cells (DC) and neutrophils. These cells were untreated or treated with 1ug of TEVs or BEV or BCG (MOI 5) for 24 hours. The supernatants were analyzed for cytokine production and the DCs and neutrophils were analyzed for activation markers by flow cytometry. The institutional animal care and use committee approved the use of mice. Results: DCs treated with TEV2 had significantly higher expression of CD86 (at similar levels when DCs were treated with BCG) and MHC2, both of which enhanced the DC's capacity to present antigens to T cells, when compared to TEV1 or TEV3. While neutrophils induced by BCG expressed significantly higher levels of costimulatory molecules CD80, CD83, CD86, and MHC2, TEV2 induced significant expression of CD80 and CD86. BCG caused a massive release of proinflammatory cytokines (IL6, TNFa, and IL12p70) and the anti-inflammatory cytokine IL10. Cytokine production induced by TEV2 was not as high as that induced by direct BCG stimulation, but it was significantly higher than that produced by untreated cells or TEV1 and TEV3. Unlike BCG, TEV2 did not significantly stimulate IL10 production. Conclusion: Our results demonstrate that tumor cells treated with BCG for 2 hours produce EVs that are able to cause significant modulation of DCs and neutrophils. TEVs generated during BCG immunotherapy have immunostimulatory potential. Therefore, TEVs represent a promising source of material to increase immune activation and efficacy of BCG therapy. This could reduce the number of instillations and adverse effects associated with BCG therapy. Citation Format: Sin Mun Tham, Kesavan Esuvaranathan, Ratha Mahendran. Bladder cancer-derived extracellular vesicles and immune activation [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A12.

Evaluating the sensitivity of the bovine BCG challenge model using a prime boost Ad85A vaccine regimen

In the absence of biomarkers of protective immunity, newly developed vaccines against bovine tuberculosis need to be evaluated in virulent Mycobacterium bovis challenge experiments, which require the use of expensive and highly in demand Biological Safety Level 3 (BSL3) animal facilities. The recently developed bovine BCG challenge model offers a cheaper and faster way to test new vaccine candidates and additionally reduces the severity of the challenge compared to virulent M. bovis challenge in line with the remits of the NC3Rs. In this work we sought to establish the sensitivity of the BCG challenge model by testing a prime boost vaccine regimen that previously increased protection over BCG alone against M. bovis challenge. All animals, except the control group, were vaccinated subcutaneously with BCG Danish, and half of those were then boosted with a recombinant adenoviral vector expressing Antigen 85A, Ad85A. All animals were challenged with BCG Tokyo into the prescapular lymph node and the bacterial load within the lymph nodes was established. All vaccinated animals, independent of the vaccination regimen, cleared BCG significantly faster from the lymph node than control animals, suggesting a protective effect. There was however, no difference between the BCG and the BCG-Ad85A regimens. Additionally, we analysed humoral and cellular immune responses taken prior to challenge for possible predictors of protection. Cultured ELISpot identified significantly higher IFN-ɣ responses in protected vaccinated animals, relative to controls, but not in unprotected vaccinated animals. Furthermore, a trend for protected animals to produce more IFN-ɣ by quantitative PCR and intracellular staining was observed. Thus, this model can also be an attractive alternative to M. bovis challenge models for the discovery of protective biomarkers.

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

BackgroundMycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172–1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172–1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains.ResultsBy combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains.ConclusionsWe demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization.

938 - Long term results of BCG Tokyo and GMCSF-IFNa gene therapy in an orthotopic model of bladder cancer

938 - Long term results of BCG Tokyo and GMCSF-IFNa gene therapy in an orthotopic model of bladder cancer

Mechanisms of resistance to delamanid, a drug for Mycobacterium tuberculosis

Delamanid, a bicyclic nitroimidazooxazole, is effective against M. tuberculosis. Previous studies have shown that resistance to a bicyclic nitroimidazooxazine, PA-824, is caused by mutations in an F420-dependent bio-activation pathway. We investigated whether the same mechanisms are responsible for resistance to delamanid. Spontaneous resistance frequencies were determined using M. bovis BCG Tokyo (BCG) and M. tuberculosis H37Rv. F420 high-performance liquid chromatography (HPLC) elution patterns of homogenates of delamanid-resistant BCG colonies and two previously identified delamanid-resistant M. tuberculosis clinical isolates were examined, followed by sequencing of genes in the F420-dependent bio-activation pathway. Spontaneous resistance frequencies to delamanid were similar to those of isoniazid and PA-824. Four distinct F420 HPLC elution patterns were observed, corresponding to colonies with mutations on fgd1, fbiA, fbiB, and fbiC with no change in the ddn mutants from the wildtype. Complementation with the wildtype sequence of the mutated gene restored susceptibility. The two delamanid-resistant clinical isolates had ddn mutations and the wildtype F420 HPLC elution pattern. In conclusion, delamanid-resistant bacilli have mutations in one of the 5 genes in the F420-dependent bio-activation pathway with distinct F420 HPLC elution patterns. Both genetic and phenotypic changes may be considered in the development of a rapid susceptibility test for delamanid.

Evaluation of Recombinant <i>Mycobacterium tuberculosis</i> Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs

The tuberculin Purified Protein Derivative (PPD) skin test is widely used; however, the results are often inaccurate. Positive results can be observed in patients with active tuberculosis (TB) as well as in BCG-vaccinated persons and individuals who are infected with mycobacteria but have not developed the disease. MPT64, an antigen secreted from actively growing Mycobacterium tuberculosis and some strains of M. bovis BCG such as BCG Tokyo and BCG Russia, is immunogenic and elicits delayed-type hypersensitivity (DTH) in guinea pigs and humans. This antigen has been used to develop a new skin test for the diagnosis of active TB infection. Two of the antigens encoded by the M. tuberculosis-specific region of difference 1 (RD1, deleted in M. bovis BCG strains), CFP10 (culture filtrate protein 10) and ESAT6 (early secreted antigenic target-6), also induce M. tuberculosis-specific DTH responses. The aim of this study was to investigate the DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo elicited by three purified recombinant proteins (rMPT64, rCFP10 and rESAT6) compared to those elicited by PPD. In this study genes encoding MPT64, CFP10, and ESAT6 were cloned and expressed as recombinant proteins with the addition of a C-terminal His6 tag for ease of purification by Immobilized Metal ion Affinity Chromatography (IMAC). The recombinant proteins (rMPT64, rCFP10, and rESAT6) were purified to homogeneity and were used to elicit DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo. The results showed that rMPT64 elicits a DTH response comparable to that of PPD in M. bovis BCG Tokyo-vaccinated animals. However, M. tuberculosis-infected animals show less reactivity to rMPT64 than they do to PPD. Although single rCFP10 or rESAT6 did not readily elicit a DTH response in M. tuberculosis-infected animals, combining these antigens with rMPT64 led to an increased DTH response, thus enabling the detection of TB infection.

MycoProtease-DB: Useful resource for Mycobacterium tuberculosis complex and nontuberculous mycobacterial proteases.

MycoProtease-DB is an online MS SQL and CGI-PERL driven relational database that domiciles protease information of Mycobacterium tuberculosis (MTB) complex and Nontuberculous Mycobacteria (NTM), whose complete genome sequence is available. Our effort is to provide comprehensive information on proteases of 5 strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, F11 and KZN 1435), 3 strains of Mycobacterium bovis (AF2122/97, BCG Pasteur 1173P2 and BCG Tokyo 172) and 4 strains of NTM (Mycobacterium avium 104, Mycobacterium smegmatis MC2 155, Mycobacterium avium paratuberculosis K-10 and Nocardia farcinica IFM 10152) at gene, protein and structural level. MycoProtease-DB currently hosts 1324 proteases, which include 906 proteases from MTB complex with 237distinct proteases & 418 from NTM with 404 distinct proteases. Flexible database design and easy expandability & retrieval of information are the main features of MycoProtease-DB. All the data were validated with various online resources and published literatures for reliable serving as comprehensive resources of various Mycobacterial proteases.AvailabilityThe Database is publicly available at http://www.bicjbtdrc-mgims.in/MycoProtease-DB/

Earlier protective response and milder clinical course of Mycobacterium bovis BCG infection in mice lacking an adapter molecule CARD9 (55.6)

Abstract CARD9 plays a crucial role as an adaptor molecule in signal transduction triggered through C-type lectin receptors that are known to recognize polysaccharides from various fungal microorganisms. In this study, we examined the role of CARD9 in the host defense to Mycobacterium bovis BCG that has polysaccharide cell-wall components such as lipoarabinomannan. C57BL/6 (WT) and CARD9 knockout (KO) mice were infected intratracheally with M. bovis BCG (Tokyo strain) per mouse. The number of viable bacterium, cytokine synthesis and histopathological findings in lungs were evaluated. CARD9KO mice cleared more bacteria in lungs than WT mice on day 28 after infection. In a histopathological analysis, the lung tissues from CARD9KO mice showed poorer granuloma formation and fewer bacilli in lungs than WT mice on days 14 and 28 after infection. The levels of IFN-γ, a key cytokine for the host defense to this infection, in the bronchoalveolar lavage fluids and its expression in T, NK and γδ T cells on day 7 were increased in CARD9KO mice compared to those in WT mice, although the production of this cytokine was significantly reduced in Card9KO mice on day 28. By contrast, the expression of IL-17A in lungs was not much different between WT and CARD9KO mice. These results indicate that CARD9 is dispensable for elimination of M. bovis BCG and granulomatous response, which suggests a complicated mechanism involved in the role of this molecule in the earlier host response to M. bovis BCG.

Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection

BackgroundStudies of Mycobacterium bovis BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico.ResultsBCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests.ConclusionsBCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (fadD23) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time.

Evaluation of cell-mediated immune responses to two BCG vaccination regimes in young children in South Korea

Children in South Korea are vaccinated with either BCG Pasteur vaccine intradermally (ID), or with BCG Tokyo vaccine given by multipuncture device (MP). Data from a recent national survey indicated that in children under 6 years old, 31.1% had received the ID vaccine and 64.5% the MP vaccine. To compare the T cell responses induced by the two vaccines, children aged 3–7 were recruited and tested for tuberculin skin test reactivity and for in vitro IFN-γ responses to mycobacterial antigens. DTH responses were not significantly different in children vaccinated by either the ID or MP vaccines. PPD-induced IFN-γ was measured in supernatants of 6-day diluted whole blood cultures. IFN-γ production to PPD was not significantly different in the two vaccine groups, although there is a trend that the MP group gives a higher proportion of IFN-γ positivity than the ID group. In addition, when IFN-γ responses to the antigens ESAT-6 and CFP-10 were assessed in the 6–7 year old group, there was no significant difference between the two vaccine groups. Thus, there was no evidence that the increasing use of MP vaccination has reduced protection against M. tuberculosis in young children in South Korea, based on immunogenicity as assessed by DTH and IFN-γ responses to PPD, and also equivalent frequency of responses to ESAT-6 and CFP-10.

Development of slide-method to distinguish alive and dead mycobacteria by fluorescent staining--a trial for solving the biohazard problem in TB laboratories

An acid-fast staining can detect mycobacteria in clinical specimens rapidly and specifically. It equally stains living and dead bacteria. It would be of more clinical use if the viability of mycobacteria in a sample was determined by the staining. In the present paper, the problems of FDA/EB staining, which detects live or dead bacteria, were solved by establishing a new technique, a slide-method. An air-dried smear of Mycobacterium bovis BCG (Tokyo 172) on a glass slide was covered by a filter paper fully soaked in the staining solution (500 micrograms FDA and 40 micrograms EB per ml PBS). This was kept in an incubator at 37 degrees C for 20 min. The filter paper was removed after incubation and the slide was examined using a fluorescent microscope with a blue filter. Live bacteria were stained greenish yellow taking the FDA stain in while dead bacteria were stained red with EB. This new slide technique eliminated the problems associated with FDA/EB staining. Moreover, stained smears appeared to be more stable compared with the conventional tube method. To overcome the biohazard problems in smear examination of tubercle bacilli, heating of the slides on a heat block at 100 degrees C for 20 min or passing air dried smears in a flame 5 to 30 times was tried to kill the bacteria. The heat-treated slides were stained with FDA/EB and the number of green and red bacteria were counted. Samples of the smeared bacteria were taken after heating and cultured on a solid medium to determine the presence of any colony-forming unit. It was found that no CFU was observed after heating and the morphology of the stained sample was the same to that before heating. These facts suggest that the above mentioned method is a simple, safe yet inexpensive diagnostic tool for mycobacterial clinical specimens.

Novel vaccines against M. tuberculosis

Novel vaccines against M. tuberculosis

Editorial Comment to Maintenance intravesical bacillus Calmette‐Guérin instillation for Ta, T1 cancer and carcinoma in situ of the bladder: Randomized controlled trial by the BCG Tokyo Strain Study Group

Editorial Comment to Maintenance intravesical bacillus Calmette‐Guérin instillation for Ta, T1 cancer and carcinoma <i>in situ</i> of the bladder: Randomized controlled trial by the BCG Tokyo Strain Study Group

Isolation and identification of arabinose mycolates of Cell Wall Skeleton (CWS) derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105)

A unique hydrolysis method using a two-layer solution, consisting of diluted hydrochloric acid and toluene was developed to isolate whole arabinose mycolates from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) in order to reveal its pivotal role in enhancing immune responses against tumors.

Separation and molecular characterization of mycolic acid from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) and BCG substrains by normal-phase high performance liquid chromatography and liquid chromatography/mass spectrometry

Since mycolic acids, the most characteristic major lipid component in mycobacterial cell envelopes, play pivotal roles in the cell surface-based host immune responses, normal-phase HPLC has been developed to quantify and identify mycolic acids of the cell wall skeleton from Mycobacterium bovis BCG Tokyo 172 (SMP-105).

Morphological study on Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105)

Mycobacterial cell wall consists of rigid cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidiglycan complex, in which mycoloyl structure varies by the mycobacterial species diversely, whereas the arabinogalactan peptidoglycan structure is consistent comparatively. The CWS of Mycobacterium bovis BCG has long been expected as a potent adjuvant for immunotherapy of malignant tumor. Although the chemical structure of CWS has been established in the last few decades, the physicochemical properties of CWS having highly amphipathic micelle structure with very long mycoloyl and carbohydrate chains are not unveiled. In this study, the ultrastructure of CWS of M. bovis BCG Tokyo 172 (SMP-105), suspended in several solvents with different polarity, was investigated with a particle size analyzer, a transmission electron microscope (TEM) and other techniques. As a result, the particle size was about 4.7 to 67.8 μm in physiological saline, but it became smaller and more compact when suspended in hydrophobic solvents. TEM images showed two different morphological forms distinctively: double folded sheet structure in hydrophilic conditions and multilayered rolled sheet structure in hydrophobic conditions. These studies have revealed characteristic surface features of SMP-105, the hydrophobic moiety occupying dominant space and the hydrophilic moiety smaller space, respectively, which may lead to the acceleration of immunological studies on this product.

Protective efficacy of recombinant BCG Tokyo (Ag85A) in rhesus monkeys ( Macaca mulatta) infected intratracheally with H37Rv Mycobacterium tuberculosis

We have reported previously that recombinant BCG Tokyo (Ag85A) (rBCG-Ag85A[Tokyo]) shows promise as a tuberculosis vaccine, demonstrating protective efficacy in cynomolgus monkeys. As a next step, rhesus monkeys were utilized because they are also susceptible to Mycobacterium tuberculosis and show a continuous course of infection resembling human tuberculosis. The recombinant BCG vaccine (5x10(5) CFU per monkey) was administered once intradermally into the back skin to three groups of rhesus monkeys, and its protective efficacy was compared for 4months with that of its parental BCG Tokyo strain. Eight week vaccination of the monkeys with rBCG-Ag85A[Tokyo] resulted in a reduction of tubercle bacilli CFU (p<0.01) and lung pathology in animals infected intratracheally with 3000 CFU H37Rv M. tuberculosis. Vaccination prevented an increase in the old tuberculin test after challenge with M. tuberculosis and reaction of M. tuberculosis-derived antigen. Thus, it was shown that even in rhesus monkeys rBCG-Ag85A[Tokyo] induced higher protective efficacy than BCG Tokyo.