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At least 19 recordsLinked to original sources
PubMed1995

[Induction of protective immune responses by a chimeric soluble protein from a recombinant BCG vector candidate vaccine to HIV-1].

We have been isolated HIV strains from blood specimens of HIV infected individuals in Japan for these 6 years. The number of specimens tested reached approximately 1,700 that ninety percent of them were from hemophiliacs repeatedly injected blood products from the United States. More than 300 of field HIV were successfully isolated from the samples. The isolation rates has decreased to 30 percent in 1993 from 40 percent in 1992, suggesting that treatment with anti-HIV drugs such as AZT and/or ddI may be effective to HIV-infected individuals. Further, both of the viral and genomic sequences of HIV were classified to be clade B virus. The clinical isolates that expressed IHIGPGRAFY sequence at the center of the HIV-V3 domain were found to be neutralized by an anti-clade B-V3 monoclonal antibody, mu 5.5. By individual levels, when asymptomatic seropositives have progressed to disease-states, neutralization core motif of GPGR in approximately 6% of the viruses has changed to GPGG and hydrophilic amino acid changed to hydrophobic amino acid, correlating the loss of binding activity to PND-peptide of Japanese Consensus virus. Further, rapid progressors to HIV-induced diseases showed decreased activity of the binding antibody. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein, and established the PND-peptide secretion system in BCG. The recombinant BCG inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide followed by passive transfer of the DTH by the systemic route.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Vaccines↗
PubMed1994

Mycobacterium bovis BCG priming induces a strong potentiation of the antibody response induced by recombinant BCG expressing a foreign antigen.

Several recent studies have demonstrated that strong cellular or humoral immune responses can be induced against foreign antigens expressed by recombinant Mycobacterium bovis BCG. It has therefore been suggested that BCG could represent one of the best candidate vectors for live recombinant vaccines. However, a large percentage of the human population has been immunized by BCG, and this priming could modify the immune response to future recombinant BCG vaccines. In the present study, we have therefore compared the immune responses induced in naive and BCG-primed mice by two recombinant BCG vaccines expressing either beta-galactosidase or human immunodeficiency virus type 1 Nef antigens. Our results demonstrated that BCG priming limits the growth of recombinant BCG in mouse spleen or lymph nodes. This reduction in BCG growth was associated with decreased proliferative responses against Nef or beta-galactosidase antigens. This suppression, however, never exceeded 50%. Interestingly, in contrast to these reduced T-cell responses, BCG-primed mice developed high levels of anti-beta-galactosidase antibodies after immunization with recombinant BCG expressing this antigen. This stimulation of antibody responses was not due to polyclonal stimulation or to a nonspecific adjuvant effect of BCG. The isotypic patterns of anti-beta-galactosidase antibody responses induced by the recombinant BCG were similar in naive and BCG-primed mice. These results indicate that priming with BCG will not be a limitation for the use of recombinant BCG vaccines in humans.

Animals↗
PubMed1995

Progress on development of the live BCG recombinant vaccine vehicle for combined vaccine delivery.

BCG, the current vaccine for tuberculosis, has been administered to approximately three billion people. This live vaccine has a low incidence of serious side effects and can be given at birth. Within the past six years, systems for the manipulation and expression of foreign genes in mycobacteria have been developed, allowing the evaluation of rBCG as a vaccine delivery vehicle for heterologous antigens. Recent studies from our group have shown that rBCG expressing outer surface protein A of Borrelia burgdorferi can completely protect mice from an intradermal challenge with this organism. Immune responses protective against Streptococcus pneumoniae challenge have also been achieved by immunization of mice with rBCG expressing PspA. The simplest means of administering multiple vaccine antigens in a rBCG vehicle would be to coexpress these simultaneously in the same BCG recombinant. Currently two general classes of vectors exist for the expression of foreign proteins in BCG: shuttle plasmid vectors, which replicate extrachromosomally in mycobacteria, and shuttle "phasmid" vectors, which integrate as a single copy into the mycobacterial chromosome by means of vector-encoded integration functions of the lysogenic mycobacteriophage L5. The genetic capacity of the multicopy plasmid vectors may be 20 kb or more, while the potential exists for stable integration of much larger amounts of DNA into the mycobacterial genome (L5 itself is 52 kb). Additionally, these two expression systems can have the compatibility to coexist in a single BCG cell. Otitis media is caused by infections of the middle ear chiefly with either S. pneumoniae or H. influenzae. Thus, an effective vaccine would necessarily include antigens from both these pathogens. Our initial attempt at construction of a BCG multivaccine vehicle was to express proteins from each of these pathogens from the same multicopy plasmid. We have recently succeeded in coexpressing the S. pneumoniae PspA and H. influenzae PAL proteins in BCG. Future work will address how the biochemical characterization of and immune responses to the recombinant antigens of the "bivalent" rBCG:PspA/PAL vaccine compare to those of the respective "monovalent" rBCG vaccines.

Antigens, Bacterial↗
PubMed1995

Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice.

We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum beta-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the beta-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice.

Animals↗
PubMed1994

Recombinant BCG as an antigen delivery system.

BCG, an attenuated vaccine strain of Mycobacterium bovis, has been engineered for use as an antigen delivery vehicle. Various genetic and molecular biological techniques were recently developed for use in the manipulation of mycobacteria. Vectors for the expression of foreign antigens in BCG have been constructed and used to express genes from bacteria, protozoa and viruses. Here, an overview of recombinant BCG is given, with emphasis on experiments with three different foreign antigens. Antibody, T-helper and cytotoxic T cell responses can be induced in mice following immunization with recombinant BCG (rBCG). In addition, vectors allowing expression of foreign antigens on the surface of rBCG have been constructed; use of these systems can lead to enhanced antibody response against some antigens. Finally, expression in BCG of a protein from the parasite Leishmania can provide protection against a virulent Leishmania challenge in mice.

Animals↗
PubMed1995

Efficacy and safety of live recombinant BCG vaccines.

BCG has a long history of safe use in humans and is one of the best adjuvants known. The use of newer production methods may further reduce the risk of adverse side-effects. Early results with experimental animals have shown BCG to be an effective live recombinant delivery vehicle for several foreign vaccine antigens. Additional refinements to the safety and efficacy of the recombinant BCG vaccine vehicle are under development.

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↗
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↗
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↗
PubMed1990

Analysis of variables of plasmid transformation of a bacterial vaccine: studies on recombinant BCG.

Stepwise regression analysis was applied to evaluate the strength order of the effect of five explanatory variables on the transformation of BCG substrains by electroporation with hybrid shuttle plasmid [pAL5000:pIJ666] and derived as YUB plasmids containing a kanamycin resistance selection marker. From 66 successful transformations, data of 42 transformations of the Pasteur 1172P2 BCG substrain are analysed. The estimated parameters in the multiple linear regression model show that the association of the explanatory variables with the explained variable, i.e. the efficiency of the transformation of BCG expressed in c.f.u./microgram DNA, in decreasing strength order is: concentration of the plasmid DNA; viability of the non-electroporated concentrated BCG suspensions; viability of the BCG cultures; age of the BCG cultures; and time constant of the electroporation. More extended analysis of other factors of the transformation will improve objective statistical inference regarding the biophysical and molecular biological interpretation of the transformation mechanism by electroporation of the different bacterial species. More precise understanding of optimal conditions of genetic transformation will be particularly important for developing recombinant BCG vaccines.

BCG Vaccine↗
PubMed1994

Cytotoxic T lymphocyte response in mice induced by a recombinant BCG vaccination which produces an extracellular alpha antigen that fused with the human immunodeficiency virus type 1 envelope immunodominant domain in the V3 loop.

The host immune response of cell-mediated immunity, particularly that of cytotoxic T lymphocytes (CTLs), is a major immune defence mechanism which may provide resistance to a human immunodeficiency virus type 1 (HIV-1) spread leading to acquired immune deficiency syndrome (AIDS). To prevent the accompanying activity of HIV-1 proteins responsible for the loss of helper T-lymphocyte function, it is crucial to develop a live attenuated recombinant vaccine expressing only T- or both T- and B-cell epitopes. Here, we examined the expression of the HIV-1 Env protein V3 region (15 amino acids from Arg315 to Lys329) in Mycobacterium bovis BCG as a fused form with an extracellular alpha antigen of Mycobacterium kansasii. Balb/c mice inoculated with this recombinant BCG (rBCG), rapidly induced V3 peptide-specific CTLs. Target cell lysis was restricted to the murine class I major histocompatibility complex, H-2d. A similar CTL response was also elicited after Balb/c mice were immunized with the same rBCG even when pre-inoculated with non-recombinant BCG. Thus, the rapid induction of HIV-1-specific CTLs indicates that this vaccine may be a therapeutic approach to preventing progression to AIDS.

AIDS Vaccines↗
PubMed1995

Recombinant BCG strains expressing the SIVmac251nef gene induce proliferative and CTL responses against nef synthetic peptides in mice.

CTL responses are known to be important for the control of HIV and SIV infections. Such responses are targeted against various components of these viruses including regulatory proteins like Nef. The SIVmac251nef gene was cloned in Mycobacterium bovis BCG under the control of P(AN), a promoter from Mycobacterium paratuberculosis. Nef was expressed as a fused polypeptide with ORF2, an open reading frame adjacent to P(AN). Mice inoculated with rBCG(SIVmac251nef) exhibited proliferative and CD8+ cytotoxic T-cell (CTL) responses against several Nef synthetic peptides. A mapping of the epitopes recognized by CTLs revealed that the central region of Nef is mainly involved in responses. This region had already been demonstrated to induce CTLs in experimentally SIV-infected macaques as well as in HIV-infected individuals. These results demonstrate the feasibility of constructing BCG vaccine strains expressing nef for eliciting cytotoxic responses.

Animals↗
PubMed1991

[Study on recombinant BCG].

For the purpose to establish the system to express foreign antigen from Mycobacterium bovis BCG. We have cloned, sequenced and expressed genes for secreting proteins, alpha antigen, MPB64, MPB57 and MPB70 from M. bovis BCG. The upstreams and structural genes were characterized. The gene for alpha antigen of Mycobacterium kansasii was also characterized. The gene for alpha antigen of M. kansasii (k-alpha) was chosen for the further study at first. This gene was fused with shuttle plasmid PIJ666-PAL5000 obtained from T. Kisser and transfected to M. bovis BCG (Tokyo). Transformant was obtained by a selection with kanamycin. It was able to secrete k-alpha antigen. DNA-containing a B-cell epitope (Glu-12-Leu-Asp-Arg-Trp-Glu-Lys-Ile-19) of human immunodeficiency virus type 1 P17 gag was fused to this vector at C terminal of k-alpha. Using this vector, we have succeeded to express foreign antigen in M. bovis BCG. The products were analyzed in one or two dimensional electro-phoresis. The results thus obtained will be reported elsewhere.

Amino Acid Sequence↗
PubMed1991

New use of BCG for recombinant vaccines.

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.

Animals↗
PubMedDate not supplied

BCG-induced mucosal immune responses.

The induction of mucosal immune responses is particularly important for protection against diseases for which entry and pathogenesis are clearly related to the mucosal system, such as salmonellosis, AIDS or tuberculosis. We investigated the immune responses in guinea-pigs vaccinated by BCG via the respiratory compared to the intradermal route. The results demonstrate that the aerogenic BCG induced a better activation of broncho-alveolar macrophages and a substantially improved protective effect against a virulent challenge with Mycobacterium tuberculosis. We also used a DNA recombinant BCG expressing LacZ gene to investigate the influence of various routes of administration on the immunogenicity of the beta-galactosidase, a foreign antigen expressed by the LacZ-BCG recombinant. Thus, lymph-node proliferative responses, delayed type hypersensitivity and antibody responses specific for beta-galactosidase can be produced in guinea-pigs immunized orally, respiratorily and intradermally. The respiratory and especially the oral route of administration produced higher mucosal and systemic immune responses compared with the intradermal route of immunization. Moreover, the oral immunization of mice with recombinant BCG induced IgA responses which can be detected both in sera and in intestinal secretions. In conclusion, BCG recombinants may be of potential use as an adjuvant vaccine.

Animals↗