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M Gheorghiu

Publications and source records attributed to M Gheorghiu.

At least 37 records · Page 2Linked to original sources
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↗
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↗
PubMed1993

BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route.

Since studies on cellular immune responses have demonstrated the role of the mucosal lymphoid system of the respiratory tract, we have studied responses obtained from the local respiratory route, compared to the systemic intradermal route, of BCG immunization. Guinea pigs vaccinated with different doses of BCG via both routes served to follow lymphoid cell proliferation, hilar lymph node and lung BCG clearance, lung granuloma formation and protection induced after virulent challenge. Results demonstrate that the aerogenic route of vaccination with BCG has no harmful side-effects for the host. In comparison with the intradermal route of vaccination, aerogenic vaccination with 10(5) BCG cfu induced higher local cellular immune responses and a substantially improved protective effect.

Administration, Inhalation↗
PubMed1993

Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria.

Increased protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by a previous immunization with living avirulent mycobacteria, usually Mycobacterium bovis BCG. Only a transient and marginal protection is obtained after immunization with bacterial extracts or dead bacteria. Both living and heat-killed bacteria share a number of common antigens. In order to identify mycobacterial molecules which are dominant antigens during immunization with living bacteria, a two-step selection method was used. Two groups of guinea pigs were immunized either with living or with heat-killed BCG. Sera were then collected and used to select and counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of high-pressure liquid chromatography columns (gel filtration, DEAE, and reverse-phase chromatography) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on polyvinylidene difluoride sheets. The molecules present on twin immunoblots were stained with antibodies raised in guinea pigs immunized either with living or with heat-killed BCG. Cross-reactive antigens stained in twin immunoblots were eliminated. Major antigens interacting with antibodies raised after immunization only with living bacteria were further purified. A complex of 45- and 47-kDa major molecules (45/47-kDa complex) was thus identified and further purified. The complex was found to interact only with antibodies present in sera of guinea pigs immunized with living bacteria and not at all with antibodies raised after immunization with dead bacteria. The 45/47-kDa antigen complex molecules were resolved on two-dimensional electrophoresis in three major and seven minor proteins detected with silver staining. All the molecules interacted with the antibodies present in sera of guinea pigs immunized with living BCG. The three major proteins (two at 47 kDa and one at 45 kDa) were amino-terminal sequenced. The sequence A-P-E-P-A-P-P-V-P-P-A-A-A-A-P-P-A, which was not previously reported, was the same for these three molecules. By using a competitive enzyme-linked immunosorbent assay, the concentrations of the 45/47-kDa antigen complex were measured in BCG culture filtrates, freeze-dried BCG, and dried heat-killed BCG; they were, respectively, 2, 0.01, and 0.001% of the total mass. The low or very low values compared with the high antibody concentration emphasized the ability of the 45/47-kDa complex delivered through live BCG to trigger an antibody response.

Amino Acid Sequence↗
PubMed1992

Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains.

Using field inversion gel electrophoresis (FIGE), different Mycobacterium tuberculosis strains, such as phage prototypes, exhibit different DNA restriction patterns which are easy to compare. Virulent and avirulent variants of M. tuberculosis H37, as well as daughter strains of M. bovis BCG, display characteristic DNA profiles. BCG strains isolated from suppurative adenitis following vaccination of French patients showed patterns identical to the BCG Pasteur strain used for vaccination. These results demonstrate that FIGE of DNA restriction fragments generated by DraI represents a suitable technique for the analysis of mycobacteria at a genomic level. The DraI profiles allow the differentiation and precise identification of the BCG Pasteur, Glaxo, Russian and Japanese strains.

DNA, Bacterial↗
PubMed1992

Expression of Escherichia coli beta-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses.

A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase. The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.

Amino Acid Sequence↗
PubMed1991

Expression of heterologous genes in Mycobacterium bovis BCG: induction of a cellular response against HIV-1 Nef protein.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used as a live bacterial vaccine to immunize more than two billion people against tuberculosis. In an attempt to use this vaccinal strain as a vehicle for protective antigens, the human immunodeficiency virus type 1 gene encoding the Nef protein was cloned in a mycobacteria-Escherichia coli shuttle plasmid and transferred into BCG. The nef gene was expressed under the control of an expression cassette carrying the promoter of the groES/groEL1 operon from Streptomyces albus and a synthetic ribosome-binding site. Lymph node cells from mice immunized with BCG-nef proliferated vigorously in response to purified Nef protein. This first report of a proliferative response suggests that recombinant BCG strains may be used to immunize against pathogens for which T-cell-mediated responses are important for protection.

Gene Products, nef↗
PubMed1991

Cellular oxidative responses and mycobacterial growth inhibition in aerosol and intradermal BCG-immunized guinea-pigs.

Although the dissemination of tuberculosis is aerogenic, less than 10% of infected subjects develop the active disease. Local immunity plays a major role in systemic cell-mediated immunity against this disease. BCG immunization may be more effective if administered via aerosol rather than intradermally. In this study, the immune responses seen in guinea-pigs vaccinated with a BCG aerosol were compared with those seen following intradermal vaccination. At regular intervals after each vaccination, the activation of alveolar macrophage was determined by their capacity to produce superoxides, phagosome-lysosome fusion and the inhibition of in vitro BCG growth. Concurrently, BCG multiplication or growth inhibition in the target organs was also determined. This study demonstrates that the alveolar route of BCG administration activated broncho-alveolar macrophage more effectively than the intradermal route. Superoxide production correlated with in vitro and in vivo inhibition of BCG growth. The spread, by the BCG inoculum, to the draining lymph nodes and spleen was similar for both test routes of administration. However, the lung BCG counts were significantly lower following intradermal vaccination. In contrast, the activation of broncho-alveolar macrophage was higher following aerogenic, rather than intradermal, BCG immunization.

Aerosols↗
PubMed1990

Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector.

Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker. Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.

Chromosome Deletion↗
PubMed1989

Relapses after stopping chemotherapy for experimental tuberculosis in genetically resistant and susceptible strains of mice.

Three strains of mice (Swiss albinos, C57BL/6,C3H/OuJ) were injected intravenously with 3.7 x 10(6) colony forming units (CFU) of M. tuberculosis, H37RV, sensitive and resistant to antibiotics (90% of bacilli sensitive, 9% resistant to Streptomycin and 0.9% resistant to Kanamycin). Two weeks later, chemotherapy was started 6 days a week for a 6-month period with isoniazid (INH) and rifampicin (RMP). Twenty mice of each strain were killed at the end of the chemotherapy and the others were kept without antibiotics for a second 6-month follow-up period before being killed. The early multiplication of bacilli during the first 2 weeks following infection and before chemotherapy, was similar in the three strains of mice. Chemotherapy had the same apparent efficacy in the three strains of mice, nearly all the mice being cured as assessed by a negative spleen culture on Loewenstein-Jensen medium at the end of chemotherapy. But after the 6-month follow-up period, the C3H strain presented a statistically significantly higher level of positive spleen culture ('relapse') than seen in the C57BL/6 strain, and an increased number of mycobacteria per relapsing mouse spleen. It has been estimated with the help of resistant and sensitive bacilli that the relapses were due in most of the cases to the regrowth of one or very few bacilli, giving a clone. It seems that the C3H strain of mice, known to carry the Bcg-r allele of the Bcg gene, might be less able to develop a specific acquired resistance capable of stopping the delayed development of a highly virulent strain of mycobacteria.

Animals↗
PubMed1989

Structure of pAL5000, a plasmid from M. fortuitum and its utilization in transformation of mycobacteria.

We have developed a gene cloning system for mycobacteria. Based on the nucleotide sequence determined for the M. fortuitum plasmid pAL5000, we have constructed an E. coli/mycobacteria shuttle vector. This vector, pAL8, is composed of pAL5000, pTZ19R (an E. coli plasmid) and a kanamycin resistance gene (from Tn903). We were unable to obtain viable kanamycin resistant pAL8 transformants of M. smegmatis using a PEG-mediated DNA uptake system, in spite of the fact that we could show efficient DNA uptake by transfection using the mycobacterial lytic phage D29. However, kanamycin resistant transformants of M. smegmatis or BCG could be obtained by electroporation. This plasmid cloning system provides a tool for studies of the expression of cloned genes (e.g. virulence) or epitopes in mycobacteria and allows the rational construction of recombinant BCG polyvalent vaccines.

Chromosome Mapping↗
PubMed1988

The stability and immunogenicity of a dispersed-grown freeze-dried Pasteur BCG vaccine.

The level of antituberculous immunity seems to be related to the number of memory T cells induced. This may vary as a function of the multiplication and persistence of BCG in host tissues. The most important requirements for a BCG vaccine are, therefore, the immunogenicity of the strain, the high proportion of live to dead bacilli, and adequate dispersion and low levels of soluble antigens. The surface-grown Pasteur BCG vaccine contains a very high proportion of bacilli killed by ball-milling and freeze-drying. It also contains clumps and soluble antigens, all factors influencing cell-mediated immune processes and viability control. Therefore, several batches of vaccine were prepared on an industrial scale using one of the most immunogenic strains (French 1173 P2) and grown as dispersed bacilli by a modified cell type culture method. This method provided fully viable, well-dispersed vaccines which have a viability and heat stability superior to that of the classical surface-grown BCG. The immunogenicity was checked by multiplication and persistence in mouse organs and the skin reactivity and tuberculin hypersensitivity in guinea-pigs showed results comparable to those obtained with classical vaccine. Small-scale tests in children showed superior immunogenicity of the dispersed as opposed to the classical vaccine and there was no suppurative adenitis.

Animals↗
PubMed1985

Resistance of high and low antibody responder lines of mice to the growth of avirulent (BCG) and virulent (H37Rv) strains of mycobacteria.

The resistance to Mycobacterium bovis (BCG) of lines of mice selected for high (H) or low (L) antibody responsiveness was estimated from the rate of BCG multiplication in the organs. During the first 2 weeks after i.v. infection with 5 X 10(6) CFU, BCG multiplied faster in the spleens of H than of L mice. Afterwards there was a more drastic reduction of viable BCG counts in H mice than in L mice so that the residual BCG counts were significantly lower in H mice than in L mice, not only in the spleen but also in the liver and lungs. On the 14th day of infection, the spleen and liver enlargement and the increase of phagocytic activity were similar in the two lines, suggesting an identical T lymphokine release. In contrast with BCG, during the first 2 weeks after infection with 7 X 10(5) CFU, M. tuberculosis (H37Rv) multiplied in the spleens of L mice at a similar or a slightly faster rate than in the spleens of H mice. On the 4th week, the viable H37Rv counts were reduced in H mice whereas L mice did not survive the infection. In mice vaccinated with BCG 5 months before virulent challenge, the multiplication of H37Rv was inhibited in the H and L lines. The protective effect of BCG is therefore stronger in L mice taking into account their higher innate susceptibility to H37Rv. This might be due to the higher level of living BCG found in L mice at the time of challenge.

Animals↗