Marina Gheorghiu

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.

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.

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.

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.

Immune responses induced by recombinant BCG strains according to level of production of a foreign antigen: malE.

A variety of viral, bacterial and parasitic antigens have been expressed in BCG and the capacity of these recombinant bacteria to induce immune responses has been well documented. However, little is known about the parameters influencing the induction of immune responses by recombinant BCG (rBCG), such as level of production and localization of the recombinant antigen. In the present study, we have constructed several rBCG strains expressing the malE gene from Escherichia coli which is either secreted or targeted to the cytoplasm or plasma membrane. Expression of malE was quantified by ELISA and localization was analyzed by flow cytometry. Even when using the same promoter, levels of cytoplasmic or membrane MalE production were far less than those from secreting strains using either mycobacterial or E. coli secretion signals. Stronger and more rapid immune responses were induced by rBCG strains with the highest levels of secreted MalE compared to cytoplasmic or membrane constructs, including both good humoral and proliferative responses in BALB/c, C57BL6 and even C3H mice, previously shown to be poor MalE responders. These results suggest that the levels of foreign antigen production play an important role in the induction of immune responses by rBCG strains.

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.

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.

Antitumor activity of two BCG vaccine preparations against the lewis lung carcinoma in mice

SummaryTwo BCG vaccine preparations were prepared following different production methods. Immuno-BCG Pasteur F was produced by surface culture on Sauton medium; BCG-RIV was a homogenous stirred deep culture.The antitumor effects of the two BCG vaccines were investigated on the Lewis lung carcinoma (3LL) in C57Bl/6 mice. A direct relationship exists in this tumor model between the log10 dose of single-cell suspension inoculated subcutaneously in the hind footpad of mice and the onset and the degree of local tumor growth and the time of death, which is directly related to the lung metastases. No significant difference from control mice was observed in the two groups of BCG-immunized mice when 3LL tumor cells were injected 2 weeks after BCG immunization. When varying numbers of viable units of the two BCG vaccines were injected together with 105 tumor cells in separate groups of normal mice, a dose-dependent local reaction was observed with Immuno-BCG Pasteur F, which was associated with a delay in the onset and development of tumor growth and an increase in the mean survival time. The local inflammatory reaction produced with BCG-RIV was of lower magnitude, and only the highest concentration (1.8×106 viable units) led to some delay in tumor occurrence and mortality. The antitumor effect of a specific local delayed-type hypersensitivity (DTH) elicited by varying amounts of the two BCG preparations injected together with 105 tumor cells in separate groups of normal or BCG-immunized mice showed that the challenge injection of Immuno-BCG Pasteur F was in all cases more effective than the BCG-RIV, but these two vaccines were more effective in BCG-RIV-immunized mice than in Immuno-BCG F Pasteur-immunized mice.When the same number of viable units within each BCG vaccine was used as a criterion of comparison, Immuno-BCG Pasteur F produced a higher specific and nonspecific local inflammatory reaction (which was associated with a local antitumor effect) than BCG-RIV. But within 2 weeks, the latter was much better able to sensitize the mice to mycobacterial antigens. This was confirmed by the evaluation of local granuloma formation and tuberculin hypersensitivity. BCG vaccines prepared as surface-grown pellets and mechanically dispersed always sensitized mice to a lesser degree and after a much longer period of time than did the well-dispersed deep-cultured vaccine.

Toxicity studies of intravenously administered BCG in baboons

SummaryThe toxicity of fresh preparations of BCG administered intravenously as single or weekly repeated doses of 1 g, 100 mg, 10 mg, and 1 mg, was studied in 9 adult baboons. Clinical, biochemical, histologic, and bacteriologic examinations were performed. Tissues for histopathologic and bacteriologic examinations were obtained by laparotomy and biopsy, or by autopsy. Four and 5 months after the first injection, the surviving animals were killed purposely for examination. With high doses of BCG (2 g, 1 g, 500 mg) the severe toxicity with death of 2 animals was demonstrated. Diffuse cellular infiltrations of macrophages and lymphocytes in the liver, spleen, lungs, and lymph nodes, with formation of early, typical granulomas and great numbers of cultivable units of BCG were found in these animals. With small doses of BCG, general granulomatous reactions occurred in all organs sampled. Four months after BCG injection, granulomas had regressed, but viable organisms persisted in the lymph nodes. Future investigations may prove feasible the possibility of immunotherapy with small doses of BCG administered intravenously, since live bacilli persist for months after injections, presumably creating the chronic BCGitis necessary for effective immunotherapy.

Preservation of fresh BCG scarification vaccine at 4, −25, and −70° C

SummaryBatches of BCG to be used as a scarification vaccine by suspension in a new liquid may be preserved at 4, −25, and −70° C. During storage the colony-forming units have been found to be practically unchanged after 3 months at 4° C, 9 months at −25° C, and 3 years at −70° C. The biological controls in animals agree with those in vitro.