Sohkichi Matsumoto

Murine IL-2 secreting recombinant Bacillus Calmette-Guerin augments macrophage-mediated cytotoxicity against murine bladder cancer MBT-2.

PURPOSEnThis study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant (r) Bacillus Calmette-Guérin (BCG) secreting alpha-antigen (alpha-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against murine bladder cancer cell, MBT-2, in vitro.nnnMATERIALS AND METHODSnpSO246 plasmid vector ligated with mIL-2 gene was introduced into BCG by electroporation. Thioglycollate-elicited murine peritoneal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied. Cytokines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay. Cytotoxicity was measured by 51Cr releasing assay.nnnRESULTSnrBCG (alpha-Ag-IL-2) secreted functional IL-2 and augmented more efficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-gamma in PEC than parental BCG did. rBCG (alpha-Ag) had the same activity as BCG. Anti-IL-2 antibody reduced rBCG (alpha-Ag-IL-2)-mediated cytotoxicity and IFN-gamma production. Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required to express the same activity as rBCG (alpha-Ag-IL-2). Anti-IL-12 neutralizing antibody and the depletion of T cells and NK cells reduced IFN-gamma production by PEC stimulated with rBCG (alpha-Ag-IL-2), suggesting that T cells, NK cells and IL-12 participate in the enhancement of IFN-gamma production.nnnCONCLUSIONSnrBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer patient to reduce both clinical dose and side effects of BCG for immunotherapy.

Characterization of the gene encoding the MPB51, one of the major secreted protein antigens of Mycobacterium bovis BCG, and identification of the secreted protein closely related to the fibronectin binding 85 complex

The secreted protein MPB51 is one of the major proteins in the culture filtrate of Mycobacterium bovis BCG (BCG) and is a protein immunologically cross‐reacting with the fibronectin binding 85 complex secreted by this bacterium. The gene encoding MPB51 (mpb51) was cloned, sequenced, and expressed in Escherichia coll. The mpb51 gene was mapped downstream of the gene for 85A component with 179 bp spaces. The mpb51 gene encoded 299 amino acids, including 33 amino acids for the signal peptide, followed by 266 amino acids for the mature protein with a molecular mass of 27807.37 Da. This is the first complete sequence of MPB51. MPB51 showed 37–43% homology to the components of 85 complex. Two‐dimensional electrophoresis of culture fluids of BCG and Western blotting indicated the existence of the other novel protein(s) which strongly cross‐reacted with the α antigen (85B) and MPB51.

Mycobacterium bovis bacillus calmette-guérin induces protective immunity against infection by Plasmodium yoelii at blood-stage depending on shifting immunity toward Th1 type and inducing protective IgG2a after the parasite infection.

Bacillus calmette-guérin (BCG)-vaccination raised dramatically the survival rates of A/J mice from infection by Plasmodium yoelii 17XL at blood-stage. The analysis of the immune response of spleen cells indicated that BCG vaccination biased the immune response toward Th1 type. Neutralization of IFN-gamma and nitric oxide abrogated the protection. The kinetics of Ab production in the course of P. yoelii 17XL infection was monitored. Surprisingly, larger amounts of parasite-specific Abs were produced in BCG-vaccinated mice than in the placebo control. The vast majority of the produced IgG against parasites in BCG-vaccinated mice was IgG2a, which was observed hardly in placebo controls. The peak of IgG2a production coincided with the clearance of infection. The naive mice transferred adoptively with IgG2a from self-cured mice survived the lethal challenge from the parasite. These data indicated that BCG vaccination protected A/J mouse from P. yoelii 17XL infection by biasing immunity toward Th1-type after parasite infection and enhancing production of IgG2a, which ultimately played a major role in protection.

Identification of a Novel DNA-Binding Protein from Mycobacterium bovis Bacillus Calmette-Guérin

A novel DNA‐binding protein expressed (8–10% in total protein) in Mycobacterium bovis bacillus Calmette‐Guérin was observed. This protein was designated mycobacterial DNA‐binding protein 1 (MDP1). MDP1 recognized bases, sugar moieties, phosphate‐backbone on DNA and preferentially bound to DNA guanine and cytosine. In the gel retardation assay, MDP1 preferentially bound to closed circular plasmid DNA than open circular and linear form plasmid DNA and also bound to RNA. MDP1 formed a highly polymerized structure and localized not only in the nucleoid but also at the 50S ribosomal subunits and cell surface. MDP1 was conserved in Mycobacterium thus far examined and the expression was enhanced in stationary growth phases. These results will provide a reasonable basis for further study of the function of MDP1 in living mycobacteria.

Cloning and sequencing of a unique antigen MPT70 from Mycobacterium tuberculosis H37Rv and expression in BCG using E. coli-mycobacteria shuttle vector.

MPB70 is known to be an immunogenic mycobacterial protein secreted in large amounts from Mycobacterium bovis BCG (BCG) Tokyo. The analogous gene for MPT70 was cloned from Mycobacterium tuberculosis H37Rv which produces this protein in only a small amount. The gene encoding 193 amino acid residues inciuding 30 amino acids for the signal peptide. the promoter‐like sequence, and the ribosome‐binding site, was completely identical to that of BCG Tokyo. Computer analysis revealed that the carboxy‐terminal half of MPT70 was homologous to amino acid sequences of fasciclin 1, osteoblast‐specific factor 2 (OSF‐2), and human transforming growth factor‐beta induced gene product (βIG‐H3). Escherichia coli (E. coli) ‐mycobacteria shuttle vectors containing mpt70 or mpb70 genes 0.7kbp upstream of the 5′ end of them were able to be expressed in BCG Pasteur which is a MPB70 low‐producer, but the extent of the expression was not that of a high‐producer.

Combined intrarectal/intradermal inoculation of recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces enhanced immune responses against the inserted HIV-1 V3 antigen.

The development of a successful recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vector-based vaccine for human immunodeficiency virus type 1 (HIV-1) requires the induction of high levels of HIV-1-specific immunity while at the same time maintaining immunity to tuberculosis. To examine a combined vaccination strategy for enhancement of immune responses specific for HIV-1, guinea pigs were inoculated with either a single or combination intradermal (i.d.), intrarectal (i.r.) and intranasal (i.n.) administration of rBCG-pSOV3J1 which secretes a chimeric protein of HIV-1 V3J1 peptide and alpha-antigen. Significant level of delayed-type hypersensitivity to both V3J1 peptide and tuberculin was induced in guinea pigs inoculated with human doses of rBCG-pSOV3J1 by a combination of intrarectal and intradermal routes. Guinea pigs inoculated by combined routes also had significantly higher titers of HIV-1-specific serum IgG and IgA compared with those animals immunized only intrarectally, which led to the enhanced neutralization activity against HIV-1(MN). In addition, the induction of high levels of IFNgamma and interleukin-2 (IL-2) mRNA in PBMC, splenocytes, and intraepithelial lymphocytes from the immunized animals was detected until at least 110 weeks post-inoculation. These results suggest that enhanced immune responses specific for HIV-1 are efficiently induced by combined intrarectal and intradermal immunization with rBCG-HIV, and antigen-specific Th1-type memory cells are maintained for more than 2 years in the immunized animals. Thus, inoculation with rBCG-HIV by combined routes represents an effective vaccination strategy to elicit high levels of HIV-1-specific immune responses.

Prostaglandin E2 down-regulates viable Bacille Calmette–Guérin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro

The regulatory effect of prostaglandin (PG) E2 and a cyclooxygenase (COX) inhibitor on Bacille Calmette–Guérin (BCG)‐induced macrophage cytotoxicity in a bladder cancer cell, MBT‐2, was studied in vitro. BCG stimulated thioglycollate‐elicited murine peritoneal exudate cells (PEC) to induce cytotoxic activity and to produce cytokines such as interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and PGE2. NS398, a specific COX‐2 inhibitor, and indomethacin (IM), a COX‐1 and COX‐2 inhibitor, enhanced viable BCG‐induced cytotoxic activity and IFN‐γ and TNF‐α production of PEC. However, NS398 and IM did not enhance these activities induced by killed BCG. Enhanced cytotoxicity was mediated by increased amounts of IFN‐γ and TNF‐α. Exogenous PGE2 reduced cytotoxic activity and IFN‐γ and TNF‐α production of PEC. These results suggest that PGE2 produced by BCG‐activated macrophages has a negative regulatory effect on the cytotoxic activity of macrophages. Accordingly, a PG synthesis inhibitor may be a useful agent to enhance BCG‐induced antitumour activity of macrophages.

Cloning and Sequencing of an MPB70 Homologue Corresponding to MPB83 from Mycobacterium bovis BCG

MPB70 is secreted in high concentrations by Mycobacterium bovis BCG substrain Tokyo (BCG Tokyo), but little by substrains Pasteur (BCG Pasteur) and M. tuberculosis. The gene encoding a MPB70 homologue secreted by BCG Tokyo was found at the upstream region of the gene encoding MPB70, with approximately 2.3 kilobase pairs (kbp) spacing: the same gene was also found in BCG Pasteur. This gene was cloned and sequenced from BCG Tokyo. The DNA sequence which contained a 663 base pair (bp) open reading frame beginning at position 1 and ending with a TAA codon at position 661 was found. Its theoretical molecular mass was calculated to be 22.068u2003kDa. This gene was highly homologous to the coding region of mpb70 and the deduced amino acid sequence was very similar to MPB83 reported by Harboe et al. It was speculated that the gene the authors characterized probably corresponded to the mpb83 gene.

Long-lasting protective immunity against rodent malaria parasite infection at the blood stage by recombinant BCG secreting merozoite surface protein-1.

Previously, we constructed a recombinant live BCG (rBCG) secreting a 15 kDa C-terminal region of MSP-1 from Plasmodium yoelii (MSP-1(15)) and succeeded in the induction of more efficient protective immunity against parasite infection than observed with artificial adjuvants (Matsumoto S, Yukitake H, Kanbara H, Yamada T. Recombinant Mycobacterium bovis bacillus Calmette-Guerin secreting merozoite surface protein 1 (MSP-1) induces protection against rodent malaria parasite infection depending on MSP-1-stimulated interferon gamma and parasite-specific antibodies. J Exp Med 1998;188:845-54 [1]). In this study, we examined the endurance of the protective effects. The protective effect generated by rBCGMSP-1(15) was observed even 9 months after final immunization, whereas the effects of immunization by MSP-1(15) together with incomplete Freund adjuvant (IFA) were found to last only 4 months.

Long‐Lasting Immune Response Induced by Recombinant Bacillus Calmette–Guérin (BCG) Secretion System

The recombinant bacillus Calmette–Guérin (rBCG) secretion system utilizing an extracellular α antigen of Mycobacterium kansasii (α‐K) was characterized biochemically and immunologically. The human immunodeficiency virus type1 (HIV‐1) p17gag B cell epitope fused to α‐K was secreted in extremely large amounts. At least three mice out of seven inoculated with rBCG generated high titres of antibody to the epitope. The long‐lasting antibody production persisted more than 14 months.