Mycobacterium bovis BCG killed by extended freeze-drying induces an immunoregulatory profile and protects against atherosclerosis.

OBJECTIVES: Atherosclerosis is an inflammatory disease of the arterial wall that leads to myocardial infarction and stroke. Regulatory T cells (Tregs) and IL-10 exert significant anti-atherogenic effects in experimental models of atherosclerosis by modulating vascular inflammation. We have previously shown that Mycobacterium bovis BCG killed by extended freeze-drying (EFD BCG) decreases lung and colon inflammation by recruiting IL-10-producing Tregs. Therefore, the aim of this study was to investigate the effect of EFD BCG on the development of atherosclerosis. DESIGN: We used two strains of atherosclerosis-prone mice: Ldlr(-/-) (four or six EFD BCG injections) and Apoe(-/-) (six injections). RESULTS: In both models, EFD BCG significantly reduced the size of atherosclerotic lesions, increased IL-10 production and reduced the serum levels of pro-inflammatory cytokines (IL-6, IL-13, KC and tumour necrosis factor-α). Shortly after treatment with EFD BCG, the number of plasmacytoid dendritic cells (pDCs) and Foxp3(+) Tregs in the draining lymph nodes increased. EFD BCG also led to accumulation of Tregs, but not of pDCs in the spleen, and reduced activity of NF-κB and increased activity of PPAR-γ in both the spleen and vascular tissue of treated mice. CONCLUSION: EFD BCG has atheroprotective effects through IL-10 production and Treg expansion. These findings support a novel approach to the prevention and treatment of atherosclerosis.

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.

Extended freeze-dried Mycobacterium bovis Bacillus Calmette-Guérin induces the release of interleukin-12 but not tumour necrosis factor-alpha by alveolar macrophages, both in vitro and in vivo.

BACKGROUND: Airway hyper-responsiveness (AHR), chronic airway inflammation and predominance of the T helper type-2 (Th2; IL-4, IL-5, IL-13) over the Th1 (IL-2, IFN-gamma) immune response are hallmarks of asthma. Alveolar macrophages (AM) are the most numerous cells in the airway lumen, where they represent the first immune cell population encountered by inhaled antigens. AM act as antigen-presenting cells (APC) and they release various soluble mediators and enzymes. AM thus play a prominent role in the modulation of the local immunity in airways. In allergic airways, AM have been implicated in the pathogenesis of inflammation by promoting the Th2 versus the Th1 cytokine patterns. OBJECTIVES: Infections with attenuated bacteria or challenges with bacterial products may involve AM. Such stimuli have been shown to potentially restore the Th1/Th2 balance in asthmatic airways, but they also induce the release of inflammatory mediators. We investigated the response of AM when stimulated by two preparations of non-proliferating Bacillus Calmette-Guérin (BCG). METHODS: We evaluated the cytokine production by AM from BP2 and C57BL/6 mice when cultured with heat-killed (HK) and extended freeze-dried (EFD) BCG. We then investigated in vivo the release of soluble factors in the airway lumen of mice after instillation of these BCG preparations. Finally, we studied the profile of cytokine transcripts in the lung of mice pre-treated with BCG and then challenged with ovalbumin (OVA). RESULTS: HK BCG induced the production of both TNF-alpha and IL-12, and did not prevent high levels of Th2 cytokine transcripts. In contrast, EFD BCG induced a response dominated by the production of IL-12, with no later over-expression of Th2 cytokine transcripts. CONCLUSION: Our results show that EFD BCG induce the release of the Th1-promoting cytokine IL-12 by AM, without the deleterious effects of HK BCG. These data suggest that EFD BCG may be considered as a potential novel treatment to restore the Th1/Th2 imbalance in asthma.

Mixed immune response induced in rodents by two naked DNA genes coding for mycobacterial glycosylated proteins.

Two genes of Mycobacterium tuberculosis, apa (Rv1860) and pro (Rv1796), coding for two glycosylated excreted proteins have been injected to mice and guinea pigs. They produce an extended immunological response of Th1 and Th2 types. Despite the fact that mycobacterial glycosylation is necessary for a high level of delayed-type hypersensitivity (DTH) reaction, plasmids bearing each of the two genes induced an elevated level of DTH sensitization. An inverse relation between the CpG-N hexamer cluster frequency and the protective effect of injected genes is described. A comparison of the strength of several eukaryotic promoters based on the diameter of the DTH reaction shows that CMVIE followed by the ubiquitin promoter are the most efficient among those tested. A significant protective effect (0.7 log unit CFU) in mice was found for the apa gene while the pro gene had no effect.

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.

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).

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.

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.

Oral immunization with recombinant Mycobacterium bovis BCG simian immunodeficiency virus nef induces local and systemic cytotoxic T-lymphocyte responses in mice.

Recombinant live Mycobacterium bovis BCG vectors (rBCG) induce strong cellular and humoral immune responses against various antigens after either systemic or oral immunization of mice. Cytotoxic T-lymphocyte (CTL) responses may contribute to the control of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infections whose portal of entry is the gastrointestinal or genital mucosa. In this study, we immunized BALB/c mice with a recombinant BCG SIV nef and observed its behavior in oropharyngeal and target organ lymphoid tissues. The cellular immune responses, particularly the intestinal intraepithelial and systemic CTL responses, were investigated. The results showed that rBCG SIV nef translocated the oropharyngeal mucosa and intestinal epithelium. It diffused to and persisted in target lymphoid organs. Specific SIV Nef peptide proliferative responses and cytokine production were observed. Strong systemic and mucosal CTL responses were induced. In particular, we demonstrated direct specific anti-Nef CTL in intestinal intraepithelial CD8beta+ T cells. These findings provide evidence that orally administered rBCG SIV nef may contribute to local defenses against viral invasion. Therefore, rBCG SIV nef could be a candidate vaccine to protect against SIV infection and may be used to develop an oral rBCG HIV nef vaccine.

Recombinant Mycobacterium bovis BCG producing the N-terminal half of SIVmac251 Env antigen induces neutralizing antibodies and cytotoxic T lymphocyte responses in mice and guinea pigs.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) represents a good candidate for the development of vaccines against AIDS. Several HIV or SIV genes including nef, gag, and env have already been expressed by rBCG strains and shown to induce strong humoral and cellular immune responses in experimental animals. Because a broad immune response directed to multiple HIV/SIV antigens is highly desirable in order to develop effective vaccines, we have also investigated the immune response induced by an rBCG strain expressing a large N-terminal portion of the SIVmac251 Env gp110-encoding gene. The rBCG(SIVmac251Env) strain obtained was able to induce strong CTL responses in mice as well as humoral immune responses in mice and guinea pigs immunized by parenteral routes. The anti-gp110 IgGs produced were able to neutralize in vitro growth of virulent SIVmac251 field isolates. Moreover, guinea pigs immunized by the oral route produced significant levels of anti-gp110 IgAs in the feces, demonstrating that rBCG is able to induce local humoral immunity in the intestinal mucosa. These data provide further evidence of the utility of BCG as a candidate vaccine vector against AIDS.

Comparison of immune responses of mice immunized with five different Mycobacterium bovis BCG vaccine strains.

Among the various parameters which may contribute to Mycobacterium bovis BCG vaccination efficiency, the choice of the vaccine strain may play an important role. In the present study, we therefore compared the immunogenicity of five different BCG strains that are commonly used for BCG vaccine production (Glaxo 1077, Japanese 172, Pasteur 1173P2, Prague, and Russian strains). The comparison of the growth capacity of these BCG strains in BALB/c and C3H mice demonstrated that a great difference exists between the capacity of various BCG strains to multiply and persist in target organs. A much lower recovery of BCG could be shown in mice immunized with Prague and Japanese BCG strains. T-cell responses of BCG-immunized mice were also examined by analyzing T-cell proliferative responses, cytokine production, delayed-type hypersensitivity responses, and cytotoxic activity. All these assays demonstrated that BCG immunization induced strong CD4+ T-cell responses, mostly of the Th1 type, as demonstrated by interleukin-2 and gamma interferon production. These studies also demonstrated that there are differences between BCG strains in stimulating these T-cell responses. A lack of induction of cytotoxic activity was observed following immunization with the Japanese strain. Lower anti-purified protein derivative antibody responses were also observed after intravenous or oral immunization with this BCG strain. Finally, the protective activity of these BCG strains was tested by measuring the capacity of immunized mice to eliminate recombinant Pasteur and Japanese BCG strains which expressed beta-galactosidase. The results of these experiments clearly demonstrated that the Prague and Japanese strains were unable to protect mice against a second mycobacterial challenge whereas mice immunized with the Glaxo, Pasteur, or Russian strain eliminated the recombinant BCG very efficiently. Altogether, the results of the present study strongly support the view that there are considerable differences in the immunogenicity of various BCG vaccine strains and that these differences may play a major role in BCG vaccination efficiency.

Stabilisation of BCG vaccines.

Aquired resistance against tuberculosis depends on the survival, multiplication and persistence of BCG in the host organs. Therefore, the viability and stabilisation during storage of BCG vaccine constitute a major attribute for good immunogenicity. Different factors which influence BCG viability have been studied. Among the more important ones it was found that a very large number of viable bacilli are killed when vaccine is manufactured in the conventional way by ball milling the surface-grown bacillary mass. The dispersed deep-grow BCG offers the advantage that all bacilli are live in the fresh suspension before stabilisation procedures are applied. Two procedures of stabilisation have been used, freezing and freeze-drying. Preservation by resuspending BCG in different cryoprotective solutions has been followed by storage at low temperatures. Complete survival is obtained when this vaccine was frozen in glycerol solutions and stored for many years at -70 degrees C. Freeze-drying killed more than 50% of the live bacilli in the fresh suspension; the remaining freeze-dried live bacilli were preserved at -30 degrees C for 20 years or at 4 degrees C for at least one year. They also resist exposure to 37 degrees C for one month. Nevertheless, most of the BCG bacilli in freeze-dried vaccines are dead. In conclusion the best BCG viability is obtained with young dispersed-grown bacilli by both freezing and freeze-drying. Freezing is a good method of stabilisation for research purposes or cancer immunotherapy, and freeze-drying for BCG vaccination campaings in tuberculosis prevention. However, BCG stabilisation still needs improvement.

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.

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.

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.

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.

[Stability and immunogenicity of the fresh BCG vaccine applied with a new multiple puncture device (author's transl)]. / Stabilité et immunogénicité du vaccin BCG frais appliqué par un nouveau multipuncteur.

A new multiple puncture instrument with interchangeable pieces has been devised. It permits the application of 12 or 28 cutaneous punctures, depending on the age of the patient (child). Fresh concentrated (50 mg/ml) BCG vaccine was distributed either into neutral glass ampoules or into neutral polypropylene tubes. No difference in viability was observed after storage at 4 degrees C and in a series of 10 batches of vaccine, a good survival (about 50%) was found after three months. Also, survival was identical among 9 batches of vaccine, whether it was resuspended in a protective solution containing human albumin plus 5% glycerol or in 6% glycerol only. The immunogenicity of the fresh concentrated vaccine, when applied with the new multiple puncture device, was studied using different tests: --the growth of BCG in organs of mice, --the survival time after virulent challenge of vaccinated versus unvaccinated mice, --tuberculin-delayed hypersensitivity of vaccinated guinea-pigs. The immunogenic potency has been found similar to that observed using other methods of BCG vaccination. These experimental results confirm the merit of the new method of vaccination using the multiple puncture device.