Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene.

The virulence of the mycobacteria that cause tuberculosis depends on their ability to multiply in mammalian hosts. Disruption of the bacterial erp gene, which encodes the exported repetitive protein, impaired multiplication of M. tuberculosis and M. bovis Bacille Calmette-Guérin in cultured macrophages and mice. Reintroduction of erp into the mutants restored their ability to multiply. These results indicate that erp contributes to the virulence of M. tuberculosis.

In vivo efficiency of targeted norfloxacin against persistent, isoniazid-insensitive, Mycobacterium bovis BCG present in the physiologically hypoxic mouse liver.

Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.

[Preliminary study on isoniazid-epiroprim combination in a tuberculosis murine model]. / Etude préliminaire de l'association isoniazide-épiroprim dans un modèle murin de tuberculose.

The purpose of this study regarding isoniazid-epiroprim's association applied to antituberculosis chemotherapy, carried through murine model, initiated into Institut Pasteur of Côte d'Ivoire and worked out at Institut Pasteur of Paris was to evaluate the epiroprim's effect alone and associated with isoniazid on Mycobacterium tuberculosis. Sixteen mouses (lineage C57Bl/6) were inoculated by venous way with 10(5) viable bacillus (strain H37Rv) suspended in 500 microliters sterile physiological aqueous solution and were shared out into 4 sets. Fifteen days later the sets have been submitted or not to a daily treatment by gavage during three weeks (epiroprim, isoniazid, isoniazid plus epiroprim). The mouses were euthanasied, spleen and lung were removed from each animal. The titres of determined bacillus into those organs prove that isoniazid and epiroprim associated seem more efficacious than the isoniazid monotherapy for mouses pulmonary tuberculosis. Bacillus obtained are sensitive to isoniazid.

Immunogenicity and protective capacity of Mycobacterium bovis BCG after oral or intragastric administration in mice.

After oral or intragastric administration of BCG to mice, comparable numbers of IFN gamma and TNF gamma producing cells were detected in both local (Peyer's patches) and central (spleen) lymphoid organs. Similar levels of precursors of CD8+ cytotoxic T lymphocytes specific for mycobacterial antigens were also found in the spleen and the mesenteric lymph nodes. These immune responses remained high over the course of 3 months, the duration of observation. Oral administration of BCG led to an enlargement of the cervical lymph nodes, which contained high levels of viable bacteria. In contrast, no adverse effects were observed in mice given the BCG via the intragastric route. These two routes of immunization induced similar levels of protective immunity to those observed in mice immunized via the subcutaneous route against a challenge with a virulent Mycobacterium tuberculosis strain (H37Rv).

Mycobacterium bovis BCG induces similar immune responses and protection by rectal and parenteral immunization routes.

We compared cellular immune responses to rectal, subcutaneous, and intradermal administration of Mycobacterium bovis BCG for 5 to 20 weeks in mice, guinea pigs, and macaques. Strong lymphoproliferative responses were induced in spleen cells after in vitro stimulation with purified protein derivative in guinea pigs and macaques, whatever the route of immunization. Comparable high numbers of gamma interferon- and tumor necrosis factor alpha-producing cells were found in the spleen after rectal, subcutaneous, and intradermal immunization of mice and macaques. Similar levels of precursors of cytotoxic T lymphocytes specific for mycobacterial antigens were observed in mice for all immunization routes. In macaques, cytotoxic activity, determined only at the end of the experiment (20 weeks), was similar after rectal and intradermal immunization. Six months after immunization, rectal and subcutaneous routes induced in mice similar levels of protective immunity against challenge with a virulent Mycobacterium tuberculosis strain (H37Rv). Rectal immunization gave immune responses and protective capacity similar to those for parenteral immunization and seemed to be a promising new route of vaccination against tuberculosis; in our study, immunization via the rectal route never induced side effects associated with parenteral routes (axillary adenitis) and could also effectively reduce the risks of viral transmission associated with unsafe injections in the developing world.

Development of mixed Th1/Th2 type immune response and protection against Mycobacterium tuberculosis after rectal or subcutaneous immunization of newborn and adult mice with Mycobacterium bovis BCG.

Cell-mediated immunity plays a key role in containing the growth of Mycobacterium tuberculosis in the host. The induction of an antibody response or a mixed cell-mediated and humoral response is frequently associated with tuberculosis disease or a decrease in the ability to control M. tuberculosis load. We recently reported the induction of similar immune responses and protection by rectal, subcutaneous (SC) or intradermal administration of Mycobacterium bovis BCG in adult mice, guinea pigs and macaques. The rectal immunization, which did not induce the side-effects associated with parenteral routes (axillary adenitis) and which could be used to reduce the risks of viral transmission associated with unsafe injections in the developing world, was analysed and compared in newborn and adult BALB/c mice. The rectal and SC immunization induced, in mice immunized as newborns or as adults, a mixed T helper 1/T helper 2 (Th1/Th2) immune response; however, particularly in adult mice, after SC administration of BCG, the level of Th2 immune response is significantly higher than it is by the rectal route. Six months after immunization with BCG, rectal and SC delivery induced similar levels of protective immunity against a virulent challenge with M. tuberculosis strain (H37Rv) in mice immunized as adults, but the rectal BCG delivery triggered stronger protection than the SC delivery if mice were immunized as newborns.

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.

Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine.

New vaccines against tuberculosis are urgently required because of the impressive incidence of this disease worldwide and the highly variable protective efficacy of the current vaccine. The possibility of creating new live vaccines by the rational attenuation of strains from the Mycobacterium tuberculosis complex was investigated. Two auxotrophic mutants of M. tuberculosis and M. bovis BCG were constructed by disruption of one of their purine biosynthetic genes. These mutants appeared unable to multiply in vitro within mouse bone-marrow derived macrophages. They were also attenuated in vivo in the mouse and guinea pig animal models. In guinea pigs, the two mutants induced strong delayed-type hypersensitivity response to purified protein derivative. In a preliminary experiment, the two mutants were compared to the BCG vaccine for their protective efficacy in a challenge against aerosolized virulent M. tuberculosis in the guinea pig model. Both mutants conferred some level of protection. These experiments demonstrate that the rational attenuation of M. tuberculosis could lead to the design of new candidate live vaccines against tuberculosis.