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PubMed1999

A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice.

The tubercle vaccine, bacille Calmette-Guérin (BCG), is a strong inducer of T-helper type 1 (Th1) responsiveness, and it has been suggested that recombinant BCG (rBCG), which produces and secretes antigens, may be used to prevent allergic diseases. The effects of rBCG vaccination on allergic responses in a murine model were examined in this study. A BCG-Escherichia coli shuttle vector was developed with the promoter and signal sequence of the alpha-antigen of Mycobacterium bovis, and the vector was tested using E. coli beta-galactosidase as the model antigen and allergen. This vector enabled the expression of the E. coli beta-galactosidase gene in BCG, which was detected in its protein extract by immunoblotting analysis. Vaccination of mice with a single dose of 106 recombinant BCG generated a beta-galactosidase-specific antibody response. The splenocytes of vaccinated mice compared with controls produced significantly higher amounts of interferon-gamma (IFN-gamma) (P<0. 01) and interleukin-2 (IL-2) (P<0.05) and lower amounts of IL-5 (P<0. 01). Mice vaccinated with rBCG had significantly less (P<0.01) serum IgE compared with controls. These results together demonstrate that rBCG secreting antigens or allergens may be utilized for the induction of a Th1-like response and the down-regulation of IgE antibody response.

Animals↗

PubMedDate not supplied

Immunotherapy of experimental bladder cancer with recombinant BCG expressing interferon-gamma.

One of the most potent immunotherapies presently used is the application of Bacillus Calmette Guérin (BCG) to prevent recurrences of superficial bladder cancer. Despite its successful use, nonresponders and certain side effects remain a major obstacle. Therefore, current studies aim at developing recombinant BCG (rBCG) strains to further improve the effectiveness of the therapy. In BCG-treated patients a strong local induction of Th1-like cytokines was observed. For this reason rBCG-strains secreting Th1-like cytokines might be potentially useful agents to improve this type of immunotherapy. Because we previously demonstrated the essential role of IFNgamma in BCG-induced antitumor responses, in this study a rBCG strain secreting murine IFNgamma (rBCG-IFNgamma) was generated and tested for its immunostimulatory capacity in several in vitro and in vivo test systems. In vitro rBCG-IFNgamma specifically up-regulated expression of MHC class I molecules on a murine bladder cancer cell line (MB49), compared to the rBCG control strain (transfected with an empty vector). In a murine model of experimental bladder cancer, intravesical instillation of rBCG-IFNgamma resulted in an enhanced recruitment of CD4+ T-cells into the bladder and further induced the local expression of IL-2 and IL-4 cytokines (mRNA) compared to control rBCG. With a low-dose treatment regimen for murine orthotopic bladder cancer, rBCG-IFNgamma significantly prolonged survival, whereas the therapeutic effect of wild-type control BCG did not reach statistical significance. We conclude that this recombinant BCG strain has enhanced immunostimulatory potential and might offer new opportunities in the treatment of bladder cancer.

Animals↗

PubMed1993

Oral immunization with recombinant BCG induces cellular and humoral immune responses against the foreign antigen.

It has been shown recently that BCG can be used as a live recombinant vaccine to stimulate immune responses. Proliferative or cytotoxic T-cell responses against several viral proteins such as HIV Gag, Env or Nef were obtained after parenteral immunization with BCG expressing these proteins. Antibody responses were also obtained after immunization of mice with recombinant BCG strain which expressed lac Z under the control of a promoter sequence isolated from Mycobacterium paratuberculosis. We have used this recombinant vaccine in guinea-pigs to investigate the influence of various routes of immunization on the immunogenicity of a foreign antigen expressed by recombinant BCG. Guinea-pigs were immunized by oral, respiratory or intradermal routes and proliferative responses, delayed-type hypersensitivity and antibody responses specific for beta-galactosidase were followed for 16 weeks. Results demonstrated that humoral and cellular immune responses specific for beta-galactosidase can be produced in all groups of guinea-pigs. However, the respiratory and especially the oral route of administration induced higher local and systemic immune responses than the intradermal route of immunization. Moreover, the oral immunization of mice with this recombinant BCG induced IgA responses which could be detected in both sera and intestinal secretions. Therefore, this study demonstrates for the first time that oral immunization with recombinant BCG can induce strong cellular and humoral immune responses.

Administration, Inhalation↗

PubMed1999

Immunogenicity of recombinant BCG producing the GRA1 antigen from Toxoplasma gondii.

Toxoplasmosis is a major parasitic disease, responsible for foetopathy in humans and domestic animals, especially sheep. Toxoplasma gondii infection generally protects immunocompetent hosts against subsequent reinfection, suggesting that efficacious vaccines can be developed against this disease. Excreted/secreted T. gondii antigens have previously been shown to provide immunoprotection in small rodents, and protective immunity is thought to be cell-mediated. Mycobacterium bovis BCG is known to be a good inducer of cellular immunity. In this study, we have developed a BCG strain which produces and secretes GRA1, one of the major excreted/secreted T. gondii antigens. This strain does not carry antibiotic-resistance determinants and is therefore safe for the environment. The intraperitoneal immunisation of OF1 outbred mice with this BCG strain failed to induce GRA1-specific humoral or cellular immune responses and only conferred a very limited degree of protection against challenge with virulent T. gondii. However, in sheep immunised subcutaneously and boosted intravenously, this recombinant BCG strain induced GRA1-specific cell-mediated responses, as evidenced by the proliferation of peripheral blood mononuclear cells and by the production of IFN-gamma, although it failed to elicit GRA1-specific antibody responses. Following oocyst challenge infection, sheep immunised with recombinant BCG exhibited an abbreviated temperature response compared with controls, suggesting partial protection.

Animals↗

PubMed2004

Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy.

Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses. Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells. We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged. Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination. Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (P<.01) within 28 days. Thus, EPGIT using multicomponent multisubunit BCG is highly effective in the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.

Animals↗

PubMed2005

Novel recombinant BCG and DNA-vaccination against tuberculosis in a cynomolgus monkey model.

We have developed two novel tuberculosis (TB) vaccines: a DNA vaccine combination expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ) and a recombinant BCG harboring the 72f fusion gene (72f rBCG). These vaccines provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current by available BCG vaccine. In the present study, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP65+IL-12/HVJ and 72f rBCG vaccines. Vaccination with HSP65+IL-12/HVJ as well as 72f rBCG vaccines provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings and immune responses than BCG. Most importantly, HSP65+IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination and recombinant BCG vaccination against M. tuberculosis in the monkey model.

Animals↗

PubMed2002

Induction of MUC1-specific cellular immunity by a recombinant BCG expressing human MUC1 and secreting IL2.

MUC1 mucin is aberrantly expressed in many epithelial malignancies and is a promising tumor antigen for target-directed immunotherapy against human breast cancer. Mycobacterium BCG is an effective immunoadjuvant which is known to induce Th1 immune response. Recombinant BCG expressing tumor antigen and secreting cytokine may therefore potentiate the tumor antigen-specific immune responses. In this study, we constructed a recombinant BCG-MUC1-IL2, which expresses a high level of human MUC1 VNTR core protein and secretes functional interleukin 2 (IL2). The immune responses induced by BCG-MUC1-IL2 were examined using a SCID mouse model reconstituted with immunologically competent human lymphocytes, SCID/hu-PBL. The mucin-specific IFN-gamma was secreted only by the lymphocytes derived from animals immunized with BCG-MUC1-IL2, but not with BCG-vector or purified mucin protein for the vaccination. In contrast, in vitro secretion of IL4 by the immunized lymphocytes was only seen in the group of animals which received native MUC1 protein, but not BCG-MUC1-IL2 and BCG-vector. Minimal MUC1-specific IgG and IgM were detected in SCID/hu-PBL mice vaccinated with BCG-MUC1-IL2. These results suggest that BCG-MUC1-IL2 preferentially induces MUC1-specific cellular immune responses and it may serve as a vaccine for breast cancer prevention and treatment.

Animals↗

PubMed2003

Immunogenicity of recombinant BCG vaccine strains overexpressing components of the antigen 85 complex of Mycobacterium tuberculosis.

The components of antigen 85 complex of Mycobacterium tuberculosis (Ag 85A, Ag 85B and Ag 85C), due to their immunodominant and secretory nature, represent promising protective antigen candidates and have been used in numerous vaccine preparations. We have used recombinant Bacille Calmette Guerin (BCG) strains overexpressing Ag 85A and Ag 85C to immunize BALB/c mice to investigate the immunogenicity of these strains. Mice immunized with recombinant BCG strains exhibited an increased humoral immune response when compared to mice immunized with wild-type BCG. The recombinant BCG strain overexpressing Ag 85A also induced an increased Th1-like response, characterized by elevated levels of IFN-gamma in antigen stimulated splenocyte cultures and a strong IgG2a antibody response, when compared to wild-type BCG. Immunization with recombinant BCG strain overexpressing Ag 85C, on the other hand did not elicit increased IFN-gamma secretion on restimulation of splenocytes in vitro.

Acyltransferases↗

PubMed1995

Recombinant BCG therapy suppresses melanoma tumor growth.

BACKGROUND: Melanoma is the fastest rising cancer in the United States. Bacillus Calmette-Guerin (BCG) has been genetically engineered to actively express and secrete the cytokine interleukin-2 (IL-2). Both BCG and IL-2 have known potent antitumor and immunomodulatory properties. METHODS: This recombinant BCG (rBCG 3A) has been tested as an intratumoral injection and a vaccine therapy in conjunction with irradiated tumor cells against melanoma in the murine B16 melanoma model. RESULTS: The transfection process did not adversely alter the function of the wild-type (WT) BCG. rBCG 3A and WT BCG are equally effective intratumoral and vaccine therapies against melanoma when compared with normal saline control groups. Tumor burdens were significantly smaller (p < or = 0.01 and 0.05) for the treatment groups for both intratumoral and vaccine administration of therapy. Immunization with rBCG 3A and WT BCG 14 days before a B16 challenge resulted in an approximately 45% smaller tumor burden when compared with controls. CONCLUSIONS: Novel therapies based on the immunogenic properties of melanoma combined with molecular technologies may offer promise for an effective and safe treatment of melanoma.

Animals↗

PubMed1992

Standardized viability test required for preventive, immunomodulator and recombinant BCG vaccine.

The protective and immunostimulating effect of the living attenuated BCG vaccine cannot be expressed by bacterial mass (mg/dose). Colony forming units (CFU 10(6)/ml) and 95% confidence limit estimates as precise in vitro potency tests are required for the exact evaluation of the immunogenicity and reactogenicity of the BCG vaccine. The laboratory technique and computerized statistical analysis presented here ensure the precision and validity of the BCG viability test, permit the standardized BCG CFU 10(6)/ml unbiased point and confidence interval estimation for preventive and immunomodulator administration of the BCG vaccines and the evaluation of the genetic transformation frequency in recombinant BCG experiments.

BCG Vaccine↗

PubMed2004

Construction, expression and identification of a recombinant BCG vaccine encoding human Mycobacterium tuberculosis heat shock protein 65.

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

BCG Vaccine↗

PubMedDate not supplied

Recombinant BCG as a candidate oral vaccine vector.

Bacille Calmette-Guerin (BCG), currently the most widely used vaccine in the world, was originally administered for many years as an oral vaccine. The low frequency of serious complications, inexpensive production, and adjuvanticity make BCG an ideal candidate for a recombinant vaccine vehicle. Although mycobacteria are slow growing and not yet well characterized genetically, we have recently developed technology for the genetic manipulation of BCG and other mycobacteria. Phage and plasmid systems based on a shuttle strategy to manipulate DNA in Escherichia coli and transfer it to mycobacteria have been developed. We have established that the aminoglycoside phosphotransferase gene can be used as an effective selectable marker in the mycobacteria and that a foreign antigen from Mycobacterium leprae can be expressed in BCG. Furthermore, a thorough analysis of mycobacterial expression sequences has been undertaken to optimize the expression of foreign antigens in BCG. We constructed an expression probe shuttle plasmid with beta-galactosidase as reporter gene, and have used it successfully to identify multiple mycobacteriophage DNA sequences with varying levels of constitutive or regulable promoter activity. Further genetic advances required for development of recombinant BCG into an effective recombinant vaccine vehicle, including possibilities for oral administration, are adumbrated.

Administration, Oral↗

PubMed2005

Extraordinarily few organisms of a live recombinant BCG vaccine against tuberculosis induce maximal cell-mediated and protective immunity.

In previous studies, we have described a live recombinant BCG vaccine (rBCG30) overexpressing the 30 kDa major secretory protein of Mycobacterium tuberculosis that induces greater protective immunity against tuberculosis than the current vaccine in the demanding guinea pig model of pulmonary tuberculosis. In this study, we have investigated the impact of vaccine dose on the development of cell-mediated and protective immunity in the guinea pig model. We found that the protective efficacy against M. tuberculosis aerosol challenge of both BCG and rBCG30 was essentially dose-independent over a dose range of 10(1)-10(6) live organisms. As previously observed, rBCG30 was more potent, reducing colony-forming units (CFU) below the level observed in animals immunized with the parental BCG vaccine by 0.7 logs in the lungs and 1.0 logs in the spleen (P<0.0001). To gain a better understanding of the influence of dose on bacterial clearance and immunity, we assessed animals immunized with 10(1), 10(3), or 10(6)CFU of rBCG30. The higher the dose, the higher the peak CFU level achieved in animal organs. However, whereas humoral immune responses to the 30 kDa protein reflected the disparate CFU levels, cell-mediated immune responses did not; high and low doses of rBCG30 ultimately induced comparable peak lymphocyte proliferative responses and cutaneous delayed-type hypersensitivity responses to the 30 kDa protein. We estimate that the amount of the 30 kDa protein required to induce a strong cell-mediated immune response when delivered via 10 rBCG30 organisms is about 9 orders of magnitude less than that required when the protein is delivered in a conventional protein/adjuvant vaccine. This study demonstrates that a very low inoculum of rBCG30 organisms has the capacity to induce strong protective immunity against tuberculosis and that rBCG30 is an extremely potent delivery system for mycobacterial antigens.

Animals↗

PubMed2005

Improved protection by recombinant BCG.

Mycobacterium bovis bacille Calmette Guérin (BCG) is one of the most widely used live vaccines. Technologic advancement in genome manipulation enables the construction of recombinant BCG (rBCG) strains, which can be employed as highly immunogenic vaccines against tuberculosis with improved safety profile.

BCG Vaccine↗

PubMed2005

Specific immune response and pathological findings in BALB/c mice inoculated with recombinant BCG expressing HIV-1 antigen.

Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.

Animals↗

PubMed2001

Immune response induced by recombinant BCG expressing merozoite surface antigen 2 from Plasmodium falciparum.

Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used as a live bacterial vaccine to immunize >3 billion people against tuberculosis. In an attempt to use this vaccinal strain as a vehicle for protective antigens, the recombinant BCG (rBCG), expressing merozoite surface antigen 2 (MSA2) from Plasmodium falciparum under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from M. tuberculosis, was constructed and used to immunize BABL/c mice. The administration of rBCG producing MSA2 (BCG-MSA2) resulted in the induction of a strong humoral and cellular response directed against MSA2. These results encourage the further protection testing of BCG-MSA2 vaccines in primate models.

Animals↗

PubMed2003

Skewing of the Th1/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system.

In spite of rapid developments in the study of mycobacteria during the last two decades, tuberculosis (TB) has maintained its status as the leading killer among all infectious diseases. Extensive evidence exists to support a central role for a T-helper type 1 (Th1) immune response for protection against TB in mice and humans. Bacille Calmette-Guerin (BCG), the only vaccine against TB, although not perfect in its ability to protect against the adult form of TB, is a strong inducer of Th1 responses and is being increasingly used as a delivery vehicle for the presentation of foreign antigens to the immune system. It has been proposed that expression of immunodominant antigens or cytokine genes in BCG can enhance the ability of BCG to induce a Th1 immune response. Since dose of the antigen is considered as one of the parameters that influence the Th cell responses, the level of expression of the candidate antigen should influence the final Th response against the recombinant BCG (rBCG). In the present study, the effect of over-expression of a candidate antigen Antigen 85B (Ag 85B) in a rBCG system, on the Th-priming ability of BCG has been investigated in the murine model. BALB/c mice were immunized with three different rBCG constructs expressing Ag 85B to various levels. Induction of Th1/Th2 responses was analyzed by measuring levels of interferon-gamma (Th1) and interleukin-10 (Th2) in antigen-stimulated splenocyte cultures and by quantifying the antigen-specific IgG2a (Th1) and IgG1 (Th2) antibody responses. By varying the level of expression of Ag 85B, specific immune responses against Ag 85B were observed to range from mixed Th1/Th2 to Th1. However, the BCG-specific immune responses in case of all rBCG-immunized animals remained predominantly Th1.

Animals↗

PubMed1999

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.

Animals↗