BCG Tokyo Research Articles

Activation of Toll‐like receptor 2 by a novel preparation of cell wall skeleton from Mycobacterium bovis BCG Tokyo (SMP‐105) sufficiently enhances immune responses against tumors

The cell wall skeleton of Mycobacterium bovis BCG has been investigated as an immunopotentiating adjuvant for immuno-therapy of malignant tumors via Toll-like receptor (TLR) 2 and TLR4. However, due to its high molecular weight, highly complicated lipoglycan structure, and complicated purification and isolation procedure, its exact structure-activity relationship has not been well established. We have newly isolated the cell wall skeleton from M. bovis BCG Tokyo (SMP-105) and examined the binding of SMP-105 with TLR. It was revealed that highly purified SMP-105 activates the nuclear factor-kB promoter in a TLR2-dependent manner, not a TLR4-dependent manner, using a reporter gene assay system. Peritoneal exudated cells of TLR2 and MyD88 knockout mice severely reduced the induction of tumor necrosis factor-alpha and interleukin-6 in the presence of SMP-105, whereas cells from TLR4 knockout mice produced similar levels of cytokines to wild-type mice. Dendritic cells and macrophages accumulated in the draining lymph nodes of treated mice. When mice were administered both SMP-105 and mitomycin C-inactivated Lewis lung carcinoma cells simultaneously, interferon-gamma-producing cells reacting to the tumor were increased distinctly in draining lymph nodes. When C57BL/6 mice, into which splenocytes from OT-I transgenic mice had been transferred, were administered with both SMP-105 and E.G7-OVA, OVA-specific cytotoxic T lymphocytes (CTL) increased markedly. Mice treated with SMP-105 and inactivated Lewis lung carcinoma cells suppressed the growth of implanted tumors. These results suggest that the activation of TLR2 by SMP-105 sufficiently enhanced immune responses, such as the number of interferon-gamma-producing cells and CTL, and prevented the growth of tumors without the contribution of TLR4.

新しい結核ワクチンの開発

We have developed a novel tuberculosis (TB) vaccine ; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome or-envelope (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CD8 positive CTL activity against TB antigens and improvement of the histopathological tuberculosis lesions, respectively. The Elispot assay showed that HSP65+IL-12 DNA/ HVJ vaccine induced a greater number of IFN-gamma producing T cells than BCG in the mouse model. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses (IFN-gamma, IL-2, IL-6 production , and lymphocyte proliferation of cynomolgus monkey). The combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials.

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105)

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: α-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO 3)-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. α-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (α1), and cis-monocyclopropanoic- cis-monoenoic (α2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic- trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic- cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic- cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/ trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic- trans-monoenoic mycolic acid (m4).

Recombinant BCG Tokyo (Ag85A) protects cynomolgus monkeys ( Macaca fascicularis) infected with H37Rv Mycobacterium tuberculosis

One tuberculosis vaccine candidate that has shown a promising degree of protective efficacy in guinea pigs is recombinant BCG Tokyo (Ag85A)(rBCG-Ag85A[Tokyo]). As a next step, cynomolgus monkeys were utilized because they are susceptible to Mycobacterium tuberculosis and develop a continuous course of infection that resembles that in humans both clinically and pathologically. The recombinant BCG vaccine was administered once intradermally in the back skin to three groups of cynomolgus monkeys, and its protective efficacy was compared for 4 months with that of its parental BCG Tokyo strain. Vaccination of the monkeys with the rBCG-Ag85A[Tokyo] resulted in a reduction of tubercle bacilli CFU (p<0.01) and lung pathology in animals challenged 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 in monkeys that rBCG-Ag85A[Tokyo] induced higher protective efficacy than BCG Tokyo. This warrants further clinical evaluation.

Novel vaccination (HVJ-liposome/HSP65 DNA+ IL-12 DNA against Tuberculosis) (47.15)

Abstract We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in BALB/C mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respetively. This vaccine strongly induced CTL against HSP antigen and M.tuberculosis antigens Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR and immune responses. The combination of HSP65 + IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, the survival of the BCG Tokyo group was 33%. These data indicate that our novel DNA vaccine might be useful against M.tuberculosis for human clinical trials. [H-17-Shinko-5] of Research on Emerging and Re-emerging Infectious Diseases in Health Sciences Research grants from Japan.

BCG Vaccination Enhances Resistance to M. Tuberculosis Infection in Guinea Pigs Fed a Low Casein Diet

In order to examine the relationship between malnutrition and tuberculosis development in vivo, a malnourished guinea pig model fed with a low casein (5%) diet was developed. After being fed with the low casein diet, the guinea pigs were infected with Mycobacterium (M.) tuberculosis Kurono strain by aerosol infection, and seven weeks later were subjected to histopathologic examination, colony-forming unit (CFU) assay, fluorescence-activated cell sorter (FACS) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-12 and inducible nitric oxide synthase (iNOS) mRNA. Another group of guinea pigs were vaccinated subcutaneously with 10(6) CFU BCG Tokyo for three weeks and then similarly infected by aerosol. Eighty-eight% (7/8) of the malnourished guinea pigs succumbed to mycobacterial infection within 85 days after infection, while the malnourished guinea pigs vaccinated with BCG Tokyo survived. CFU assay showed that lung and splenic CFUs were higher in the low casein diet-fed groups than in the control diet (20% casein)-fed groups, although both groups had significantly lower CFUs after vaccination with BCG Tokyo (p<0.01). Examination of lung histopathology revealed that pulmonary granulomas were large and disorganized in the groups fed the low casein diet. The number of visible lesions on the surfaces of the fixed lungs in guinea pigs fed control diet+BCG and low casein diet+BCG was low significantly. Pan T-, CD4-, CD8- and Mac antigen-positive cells were also recognized in the infected lung tissues of low casein-fed guinea pigs and Pan T-, CD4- and Mac antigen-positive cells increased after vaccination with BCG Tokyo. Expression of IFN-gamma, TNF-alpha, IL-12 and iNOS mRNA was also recognized in the infected lung tissues of low casein-fed guinea pigs and IFN-gamma and TNF-alpha mRNA expression was enhanced with BCG vaccination. These results indicate that malnutrition exacerbates mycobacterial infection and that malnourished infected hosts may be protected by BCG vaccination.

Actinomadura rudentiformis sp. nov., isolated from soil

As part of an antibiotic-screening programme, the actinomycete strain HMC1(T) was isolated from soil collected from the banks of the Gamka River in the Swartberg Nature Reserve, Western Cape Province, South Africa. The isolate produced branching vegetative mycelia, which entwined to form rope-like structures. It exhibited antibiosis against Enterococcus faecium VanA (a vancomycin-resistant clinical strain) and Mycobacterium aurum A+. Organic solvent extracts of the culture filtrate and mycelial mass of strain HMC1(T) exhibited moderate antibiosis against Mycobacterium aurum A+, Mycobacterium bovis BCG (Tokyo) and Mycobacterium tuberculosis H37Rv(T). The chemotaxonomic characteristics of strain HMC1(T) corresponded with those of members of the genus Actinomadura. Furthermore, phylogenetic analysis based on the 16S rRNA gene showed that the strain was closely related to members of the genus Actinomadura. Phenotypic and 16S rRNA gene sequence analyses indicated that strain HMC1(T) represents a novel species of the genus Actinomadura, for which the name Actinomadura rudentiformis sp. nov. is proposed. The type strain is HMC1(T) (=DSM 44962(T)=NRRL B-24458(T)).

Study on the Cell Wall Skeleton Derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105): Establishment of Preparation and Analytical Methods

Mycobacterial cell walls have diverse adjuvant activities, and in particular, cell wall skeleton (CWS) of Mycobacterium bovis BCG has been expected as a drug for tumor immunotherapy. However, its molecular structure-biological activity relationship has not been fully elucidated despite more than 30 years of intensive research. Since it is important to secure purified CWS for such investigation, we established a preparation method of CWS from M. bovis BCG Tokyo 172 (SMP-105) and developed accurate, precise, and reliable analytical methods, based on previous reports. Furthermore, we confirmed that SMP-105 is composed of mycolic acids; arabinogalactan consisting of arabinose, galactose, and rhamnose; and peptidoglycan consisting of alanine, glutamic acid, diaminopimeric acid, muramic acid, glucosamine, and galactosamine. We also determined the levels of potential impurities that might be contaminated in the original bacterium or arise during the manufacturing process, such as glucose, mannose, non-constituted amino acids, as well as nucleic acid, trehaolse di-mycolate, and bacterial endotoxins. These results demonstrated that the prepared SMP-105 was of sufficient quality for research into the chemistry, bioactivity, and structure-activity relationship of CWS.

Protective efficacy of recombinant (Ag85A) BCG Tokyo with Ag85A peptide boosting against Mycobacterium tuberculosis-infected guinea pigs in comparison with that of DNA vaccine encoding Ag85A

A recombinant form of BCG Tokyo with an Ag85A gene insert was administered once subcutaneously to guinea pigs and its protective efficacy was compared with that of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis administered twice to guinea pigs by epidermal gene gun bombardment. Vaccination with either the recombinant BCG Tokyo or Ag85A DNA significantly reduced the severity of pulmonary pathology and the number of pulmonary and splenic colony-forming units (cfu) (p<0.001). The recombinant BCG Tokyo was better than Ag85A DNA in terms of protective efficacy against M. tuberculosis. When immunogenic synthetic Ag85A peptide was further used as a booster together with recombinant BCG Tokyo (Ag85A) or Ag85A DNA, lung pathology was improved significantly and the number of pulmonary CFU was reduced significantly. Neither recombinant BCG Tokyo, Ag85A DNA, nor the parental BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. The spleen tissues from guinea pigs vaccinated with recombinant BCG Tokyo or Ag85A DNA expressed IFN-gamma and IL-2 mRNA at significantly high levels (p<0.001) as evaluated by reverse transcription polymerase chain reaction. It is concluded that peptide boosting is important for the induction of higher protective efficacy by recombinant BCG Tokyo or a tuberculosis DNA vaccine and both recombinant BCG Tokyo (Ag85A) and Ag85A DNA vaccine induce Th2 cytokine mRNA expression significantly.

Intact molecular characterization of cord factor (trehalose 6,6′-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry

Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium-intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

Clinical evaluation of serodiagnosis of active tuberculosis by multiple-antigen ELISA using lipids from Mycobacterium bovis BCG Tokyo 172

The humoral immune responses of 69 active tuberculosis (TB) patients against three major mycobacterial lipid antigens, monoacyl phosphatidylinositol dimannoside (Ac-PIM2), trehalose 6,6'-dimycolate (TDM-T) and trehalose 6-monomycolate (TMM-T) from Mycobacterium bovis BCG Tokyo 172, were examined by ELISA. IgG antibodies from active TB patients were reactive against each of the three lipid antigens (Ac-PIM2, TDM-T and TMM-T), giving positive results of 42.0-59.4%. If tests were combined and an overall positive was scored when any of the three tests was positive, sensitivity was 71.0%, showing better discrimination between patients and normal subjects. Although this value is not satisfactory for the clinical diagnosis of active TB, it is still higher than values for Determinar TBGL (56.5%) and MycoDot (31.9%) test results, both of which are commercially available. IgG antibody responses to particular lipid antigens in an individual patient differed diversely between patients. Positive IgG antibody rates and IgG antibody levels to lipid antigens were mostly paralleled by the amount of mycobacteria excreted and by the severity of pathological lesions (size and cavity formation). Serologically positive responsiveness was shown in 60.0% of tuberculin skin test (TST)-negative TB patients. Furthermore, seropositivity for multiple-antigen ELISA in active TB patients was demonstrated in other possible immune-suppressed cases, such as senile, diabetes mellitus and fulminant TB patients. Therefore, in contrast to tests based on cellular immune responses such as the TST, the humoral immune responses of TB patients against mycobacterial lipid antigens were disease-specific and were shown to be useful for the early diagnosis of active TB disease in conjunction with smear and cultivation tests, even if cellular immune responses are decreased.

Nucleotide sequence of MPB63 gene in Mycobacterium bovis BCG Tokyo.

Mycobacterium bovis BCG has been used for the prevention of tuberculosis and as therapy for bladder tumor. MPB63 in M. bovis BCG is one of the immunogenic proteins and is secreted in large quantities. Therefore, it is of interest that the MPB63 gene be examined for the determination of its nucleotide sequence. A fragment of 820 base pairs (bp) including the MPB63 gene was prepared by amplification using polymerase chain reaction (PCR) employing M. bovis BCG Tokyo chromosomal DNA as a template and its nucleotide sequence was determined. The nucleotide sequence of mpb63 in M. bovis BCG was then compared with that of mpt63 in M. tuberculosis. The result indicated that the nucleotide sequences between two protein genes were quite agreeable in the genes' structural and upstream regions, except that one base change from C in mpt63 to G in mpb63 was detected in the downstream trailer sequence. This suggests that the genetic information of M. bovis BCG is not entirely identical to that of M. tuberculosis, although the characteristics of both microorganism are very similar to each other.

Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment

A DNA vaccine encoding Ag85A from Mycobacterium tuberculosis was administered to guinea pigs by epidermal gene gun bombardment and its protective efficacy was determined. Vaccination with Ag85A DNA twice significantly reduced the severity of pulmonary pathology and number of pulmonary colony-forming units (CFU) ( p<0.01). When immunogenic synthetic Ag85A peptide was used as a booster, lung pathology was improved significantly and pulmonary CFU were reduced dramatically. Neither Ag85A DNA nor BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. When the vaccinated guinea pigs were followed up for 7 months, the pulmonary lesions became fibrotic in guinea pigs vaccinated with Ag85A DNA plus Ag85A peptide, or BCG Tokyo, and no tubercle bacilli were detected. The protective efficacy of the tuberculosis Ag85A DNA vaccine was improved significantly by peptide boosting. It is concluded that dosage and peptide boosting are important in the induction of higher protective efficacy by a tuberculosis DNA vaccine.

Isolation of MPB63 in the culture fluid of Mycobacterium bovis BCG Tokyo: genetical and immunological characterization

MPT63 is a secretory protein first isolated from a culture fluid of M. tuberculosis H37Rv by Nagai et al. In this study, this protein was isolated from an 8-day-culture fluid (Sauton synthetic medium) of M. bovis BCG Tokyo 172 according to Nagai's method. It was shown that M. bovis BCG Tokyo 172 secreted this protein in the medium. The mpt63 gene was detected only in the species of M. tuberculosis complex by polymerase chain reaction (PCR) among 40 different mycobacterial species. Therefore, it is appropriate to designate this protein as MPB63 or MPB/T63 from M. bovis BCG, in similar way as other major secretory proteins of Mycobacteria, such as MPB59 and MPB64. Comparison of the nucleotide sequences of the genes encoding MPB63 protein of M. bovis BCG and MPT63 protein of M. tuberculosis showed only single nucleotide difference at the position 474 where thymine (T) in the former was replaced by adenine (A) in the latter. Amino acid sequences of both proteins were completely identical. MPB63 didn't show delayed-type hypersensitivity (DTH) skin reaction in the sensitized guinea pigs with live or heat-killed M. bovis BCG or heat-killed M. tuberculosis. However, the measurements of serum IgG antibody titers of active tuberculosis patients by enzyme-linked immunosorbent assay (ELISA) showed 74% sensitivity and 96% specificity compared to healthy subjects. Therefore, MPB63 seems to be a promising candidate as an antigen for serodiagnosis of active tuberculosis.

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

Protective efficacy of BCG Tokyo 172 in the guinea pig model of pulmonary tuberculosis

BCG Tokyo 172 strain was examined for its protective efficacy against pulmonary tuberculosis in a guinea pig model. Guinea pigs were vaccinated with an intradermal injection of 10(3) CFU of BCG Tokyo 172 strain. BCG Copenhagen 1331 was employed as a control strain. Eight weeks after the vaccination, the animals were infected with about 10 CFU of M. tuberculosis H37Rv by a respiratory route in an aerosol chamber. Five weeks after infection, the animals were euthanized and their spleens, lungs and livers were obtained for enumeration of M. tuberculosis H37Rv and histopathological examinations. The mean log10 CFU of M. tuberculosis H37Rv recovered from right lower lung lobes of guinea pigs vaccinated with BCG Tokyo 172 (frozen), BCG Tokyo 172 (freeze-dried), BCG Copenhagen 1331 (freeze-dried) and placebo were 4.72, 4.23, 4.35 and 5.76, respectively. The mean log10 CFU of the bacteria recovered from spleens were 2.11, 1.51, 1.37 and 5.90, respectively. There was a significant difference in bacterial recovery from both lung and spleen between the vaccinated and the non-vaccinated groups. No significant difference was seen among the groups vaccinated with different strains of BCG in any organ. The lungs exhibited just small granulomatous nodules and the spleens showed no granulomatous nodules in the BCG-vaccinated guinea pigs. On the other hand, the lungs and spleens from non-vaccinated guinea pigs showed much larger granulomatous nodules with central necrosis. These histopathological difference between the vaccinated and the non-vaccinated guinea pigs was consistent with the difference of bacterial growth between them. The results of this study have clearly indicated that BCG Tokyo 172 strain possesses a significant protective efficacy against M. tuberculosis as well as BCG Copenhagen 1331 strain. These results have also shown that the respiratory infection model in guinea pigs is very useful to evaluate efficacy of vaccines against pulmonary tuberculosis.

Intravesical bacillus Calmette-Guerin instillation therapy for carcinoma in situ of the urinary bladder and prediction of effects by urinary cytologic examination

We investigated the effects of intravesical BCG instillations for carcinoma in situ (CIS) of the urinary bladder. And we have retrospectively analyzed the prediction of effects by fresh urinary cytologic examinations before instillation. 33 patients were treated for bladder CIS (1991-1997) with a median follow-up of 30 months (range from 9 to 90 months). The patients (27 males and 6 females) ranged in age from 46 to 91 (average 71 years) and received 6 to 12 weekly BCG Tokyo 172 strain 80 mg instillations. They were divided into 3 groups based on tumor history: primary (9), secondary (15), concurrent (9). The prediction of effects were analyzed by scoring fresh urinary cytologic examinations before instillation. 22 cases (67%) were responded and they have remained free of disease for follow-up period. The statistic evaluation proved to show the significance between the effects of treatment and the sum of scoring (cellular appearance and existence of large nuclear cells). We confirmed the effects of this treatment. The prediction of effect of this treatment seemed to be indicative by fresh urinary cytologic examinations before instillation, especially cellular appearance and existence of large nuclear cells.

B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

MPB70 and MPB83 as indicators of protein localization in mycobacterial cells.

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

Detection of Pyrazinamide-Resistant Mycobacteriumtuberculosis by PCR-SSCP of pncA Gene

Background : Recently the incidence of tuberculosis is increasing in many countries and control of the disease is further threatened by the emergence of multi-drug resistant tuberculosis. So rapid detection of drug resistance is very important. Pyrazinamide (PZA) is a first-line chemotherapeutic agent for tuberculosis. Now in Korea, we perform PZase activity test instead of actual pyrazinamide susceptibility test for the detection of PZA resistant M. tuberculosis. Recently the pncA gene, encoding the PZase of M. tuberculosis, was completely sequenced. And it was reported that the mutation of pncA gene would be associated with PZA resistance of M. tuberculosis. Therefore we performed this study to evaluate the possibility for the rapid detection of PZA resistant M. tuberculosis using PCR-SSCP of pncA gene. Method : 44 cultured clinical isolates of M. tuberculosis, BCG Tokyo strain. BCG French strain, and one M. bovis isolate were studied. We used H37Rv as the reference strain, The PZase activity test was done at the reference laboratory of Korean Tuberculosis Institute. DNA was extracted by bead-beater method and 561 bp fragment including pncA gene was amplified by PCR. The PCR product were digested by BstB I enzyme. SSCP was done using MDE gel. Of the 44 strains of M. tuberculosis, 22 strains were PZase-positive and other 22 strains were PZase negative. Results : Of the 22 PZase positive strains, 18 strains(82%) showed the same mobility compared with that of H37Rv and 4(18%) showed different mobility. Of the 22 PZase-negative strains, 19(86%) strains showed the same mobility pattern compared with that of H37Rv and 3(14%) showed different mobility. Naturally PZA-resistant BeG-French strain, BCG-Tokyo strain, and one M. bovis isolate showed the same band pattern each other, but their mobility were different from that of H37Rv. The results of PZase activity test and PCR-SSCP of pncA of M. tuberculosis were statistically significantly correlated each other (p