Immunogenicity of Mycobacterium bovis BCG expressing Anaplasma marginale MSP1a antigen

Humoral and cellular immune responses of mice inoculated with recombinant Mycobacterium bovis BCG expressing the MSP1a antigen of Anaplasma marginale were evaluated. The msp1a gene was amplified by PCR and cloned into the mycobacterial expression vectors pUS2000 and pMIP12. Immunization of isogenic BALB/c mice with the rBCG/pUS2000-msp1a construct induced significant seroconversion to MSP1a (p<0.001), which was 26 times above pre-immunization levels at day 63 post-initial immunization and which remained stable for the duration of the experiment (6 months). In contrast, rBCG/pMIP12-msp1a induced seroconversion at a level of 6 times above pre-immunization values, which peaked at day 63. Western blot analysis showed that sera derived from mice vaccinated with either rBCG construct recognized both native and recombinant forms of A. marginale MSP1a. In contrast to the humoral response data, immunization with rBCG/pMIP12-msp1a was found to induce a markedly stronger cellular response than that recorded for BCG/pUS2000-msp1a. These observations clearly demonstrated the immunogenicity of recombinant BCG expressing the MSP1a antigen and suggested that the immune responses were influenced by the level of antigen expression. The results of this research warrant studies of recombinant M. bovis BCG expressing MSP1a in cattle to test for protective antibody production for control of bovine anaplasmosis.     

Absorption of Mycobacterium bovis BCG administered by the oral route

The action of gastric and duodenal juices on BCG as well as on its absorption and its distribution in the organs after intragastric administration in mice were studied. A significant decrease in BCG oxygen uptake and a moderate loss of viability were found after 2 h treatment with gastric juice. Using duodenal juice, a marked decrease of respiration and a notable fall in viability were observed. Labelling of BCG with carbon-14 was accomplished using [14C]glycerol as a precursor of mycobacterial lipids. Similar levels of radioactivity were recovered in organs of mice 24 h after intragastric administration of 14C-BCG, sonicated 14C-BCG and [14C]glycerol. The level of 14C-BCG remained stable from 6 to 24 days, while sonicated 14C-BCG and [14C]glycerol defined a biological decay process. Studies of biological decay from the small intestine and liver indicated that the absorptive process started rapidly and reached its highest level at 24 h, declining thereafter according to the complexity of the material given to mice. However, living bacilli were not cultured from organs of mice given single doses of unlabelled BCG. Therefore, judging from the above data it may be concluded that the majority of BCG bacilli absorbed intact were not alive.     

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Effect of reducing or eliminating BCG load on cell-mediated immunity

Oral delivery of lipid-encapsulated BCG represents an effective method for vaccination against tuberculosis (Tb). This method establishes live, replicating BCG in the lymphatic tissues of the alimentary tract, and promotes systemic-level cell-mediated immunity (CMI) and consequent protection against virulent mycobacterial challenge. Here, we investigated the effects of reducing or eliminating the BCG load on CMI responses in mice. Mice receiving a standard immunising dose of approximately 10(7) BCG (range, 1-5 x 10(7)) developed mycobacterial antigen-specific lymphocyte transformation (LT) responses, as well as interleukin-2 (IL-2) and gamma-interferon (IFN-gamma) secretion, at 8 and 18 weeks post-oral vaccination. These responses were concurrent with establishment of viable, replicating BCG in the alimentary tract lymphatics in over 90% of cases. Reducing the immunising dose by 10-fold reduced the magnitude of CMI, concurrent with abridged establishment of BCG in the lymphatics; reducing the dose 100-fold ablated BCG establishment, and diminished the production of IFN-gamma by antigen-stimulated lymphocytes of these mice. In mice immunised using the standard dose, replicating BCG were eliminated from the alimentary tract lymphatics using selective antibiotics. Interestingly, while lymphocyte transformation and interleukin-2 responses remained largely unaltered in these mice, levels of IFN-gamma produced by antigen-stimulated lymphocytes were shown to be reduced significantly. This study identifies a dosage threshold for effective oral vaccination using lipid-encapsulated BCG, and furthermore highlights the requirement of on-going intra-lymphatic BCG replication for the maintenance of strong IFN-gamma production, above other indicator CMI responses.     

结核杆菌PEPCK表达及保护性免疫反应效果

目的研究结核杆菌磷酸烯醇型丙酮酸羧激酶(PEPCK)的表达,并观察此融合蛋白使机体产生的保护性免疫反应。方法将含有结核杆菌磷酸烯醇型丙酮酸羧激酶(pckA)基因的重组质粒转化大肠埃希菌HB₁₀₁,表达出融合蛋白PEPCK,并进行蛋白质印迹(Western blot)分析;选BALB/c小鼠60只,随机分为实验组和对照组。实验组每只小鼠用表达的PEPCK 10μg加弗氏不完全佐剂进行腹腔免疫注射,对照组小鼠仅用弗氏不完全佐剂注射。每隔2周免疫1次,共免疫3次。末次免疫2周后,分别取小鼠脾脏、血清,检测CD4⁺T细胞和CD8⁺、T细胞及各细胞因子。结果成功表达出融合蛋白PEPCK,并能够与小鼠抗卡介苗(BCG)血清反应;实验组小鼠的脾脏明显增大,粘连严重;CD4⁺T细胞增殖明显(73.5±3.69),CD4⁺/CD8⁺比值显著升高(5.1±0.98)(P<0.01);血清中γ干扰素(IFN-γ)、白介素12(IL-12)和α肿瘤坏死因子(TNF-α)均有不同程度的升高。结论PEPCK能够诱发小鼠产生细胞免疫和体液免疫反应,是很好的抗结核疫苗候选分子之一。     

Overexpression of Rv3097c in Mycobacterium bovis BCG abolished the efficacy of BCG vaccine to protect against Mycobacterium tuberculosis infection in mice

Rv3097c of Mycobacterium tuberculosis encoding lipase (LipY) was overexpressed in Mycobacterium bovis BCG. Efficacy of recombinant BCG to protect against infection of M. tuberculosis was evaluated in mice. Whereas the parent BCG vaccine protected the mice against infection, recombinant BCG overexpressing LipY offered no protection as judged by viable counts of tubercule bacilli in lungs, weight of infected mice, pathology of lungs and survival of challenged mice. Downregulation of overexpression of LipY by antisense approach considerably restored protection of infected mice as observed with parent BCG vaccine. Overexpression of lipase in BCG caused extensive hydrolysis of triacylglycerol (TG) as identified by TLC, HPLC and NMR spectroscopy. A good correlation could be inferred between hydrolysis of TG and decrease in Th1 secreted IFNγ and IL-2, proinflammatory cytokines and survival of infected mice. Mice immunized with purified LipY antigen were protected and both proinflammatory and Th1 specific cytokines were augmented. TG was found to be a poor vaccine providing no protection, which appears to be due to attenuation of Th1 and proinflammatory immune responses. In conclusion this is the first experimental report to show that immunogenicity of BCG vaccine was impaired by LipY-induced hydrolysis of specific lipids leading to suppression of host immune responses.     

In vitro culture medium influences the vaccine efficacy of Mycobacterium bovis BCG

The varied rates of protection induced by Mycobacterium bovis BCG vaccine against tuberculosis has been attributed to many factors such as genetic variability among BCG strains, rapid clearance of BCG in some populations, and different levels of previous exposure of vaccinated populations to environmental mycobacteria. However, the methods and conditions employed to prepare this vaccine for human usage by various manufacturers have not been investigated as potential factors contributing to the variation in vaccine efficacy. A review of the literature indicates discrepancies between the approach for growing BCG vaccine in the laboratory to assess immune responses and protective ability in animal models, and that employed for production of the vaccine for administration to humans. One of the major differences is in the growth medium used for routine propagation in the laboratory and the one used for bulk vaccine production by manufacturers. Here we compared the immunogenicity of the BCG vaccine grown in Middlebrook 7H9 medium, the most commonly used medium in laboratory studies, against that grown in Sauton medium, which is used for growing BCG by most manufacturers. Our results showed clear differences in the behavior of BCG grown in these different culture media. Compared to BCG grown in Middlebrook 7H9 medium, BCG grown in Sauton media was more persistent inside macrophages, more effective at inhibiting apoptosis of infected cells, induced stronger inflammatory responses and stimulated less effective immunity against aerosol challenge with a virulent Mtb strain. These findings suggested that the growth medium used for producing BCG vaccine is an important factor that deserves increased scrutiny in ongoing efforts to produce more consistently effective vaccines against Mtb.     

Oral delivery of lipid-encapsulated Mycobacterium bovis BCG extends survival of the bacillus in vivo and induces a long-term protective immune response against tuberculosis

The success of oral-route vaccination using Mycobacterium bovis bacille Calmette-Guérin (BCG) relies on delivery of live, actively metabolising bacilli to confer protection. Here, we describe that lipid-microencapsulation can extend the in vivo survival of bacilli when fed to mice, and can induce a long-lasting protective immune response. Feeding mice with lipid-encapsulated BCG (L-BCG) resulted in greater recovery of viable BCG bacilli from the mesenteric lymph nodes (MLN) compared to mice fed non-encapsulated BCG. A time-course study indicated persistence of viable BCG bacilli in MLN up to 30 weeks post-vaccination, similar to the duration of viable BCG recovery from the spleen following subcutaneous vaccination. The persistence of viable bacilli in the MLN of L-BCG mice invoked long-lasting systemic cell-mediated immune reactivity, with responses similar to those observed in subcutaneously-vaccinated mice. Further, L-BCG-vaccinated mice showed a high degree of protection against aerogenic challenge with virulent M. bovis at 30 weeks post-vaccination, with significant reductions in lung and spleen pathogen burdens. This study identifies that lipid-encapsulation of live BCG bacilli can facilitate increased in vivo survival and immunogenicity of the vaccine in orally-vaccinated mice, and highlights protection via this route for up to 7 months post-immunisation.     

Mycobacterium bovis DeltaleuD auxotroph-induced protective immunity against tissue colonization, burden and distribution in cattle intranasally challenged with Mycobacterium bovis Ravenel S

Bovine tuberculosis is a chronic granulomatous disease caused by Mycobacterium bovis. Lack of definitive diagnostics and effective vaccines for domestic animals are major obstacles to the control and eradication of bovine tuberculosis. Auxotrophic mutants of Mycobacterium tuberculosis have shown promise as vaccine candidates for preventing human tuberculosis. Similarly, we constructed a leucine auxotroph of M. bovis, by using allelic exchange to delete leuD (encoding isopropyl malate isomerase), creating a strain requiring exogenous leucine for growth in vitro. We vaccinated 10 cattle subcutaneously with 10(9)CFU of M. bovis DeltaleuD and 10 age-matched, gender-matched controls were injected with phosphate-buffered saline. Vaccinated cattle had significantly increased in vitro antigen-specific T-cell-mediated responses. All cattle were challenged intranasally on day 160 post-immunization with 10(6)CFU of virulent M. bovis Ravenel S. On day 160 post-challenge vaccinated cattle had significantly reduced tissue mycobacterial burdens and 6 of 10 had complete clearance of the challenge strain and histopathological lesions were dramatically less severe in the vaccinated group. Thus, a single subcutaneous immunization of the M. bovis DeltaleuD mutant produced highly significantly protective immunity as measured by a reduction in tissue colonization, burden, bacilli dissemination, and histopathology caused by virulent M. bovis Ravenel S challenge.     

Evidence for enhanced central memory priming by live Mycobacterium bovis BCG vaccine in comparison with killed BCG formulations

Development of cattle vaccines against bovine tuberculosis is a GB research priority. Recently, it has been shown that formalin-killed Bacille Calmette-Guérin (BCG) delivered with the liposomal adjuvant NAX687 imparted significant protection against Mycobacterium bovis infection in the guinea pig aerosol infection model. Extending these studies, we inoculated calves with live BCG, formalin-killed BCG and formalin-killed BCG formulated in NAX687. Live and killed BCG vaccine formulations induced primary effector T-cell populations comparably, both killed BCG formulations also induced potent humoral immune responses. In contrast, live BCG generated enhanced central memory responses against the protective antigen Ag85A whilst killed BCG-induced such responses only poorly. However, the poor capacity of killed BCG to generate central memory could be partially overcome by formulation with NAX687. Measurement of central memory responses induced by TB vaccine candidates in cattle may provide a useful correlate of protection and warrants further investigation in challenge experiments.     

A comparison of ethanol absorption and narcosis in long- and short-sleep mice following intraperitoneal or intragastric ethanol administration

Blood ethanol concentrations (BEC) were determined in Long-Sleep (LS) and Short-Sleep (SS) mice during a 30 min period following ethanol (ETOH) administration. Absorption of ETOH was rapid and followed a similar time course in the two lines after intraperitoneal (IP) administration of 3.8 or 4.5 g/kg. Following intragastric (IG) administration, slower absorption and lower peak BECs were noted in both lines, but in LS mice this effect was more pronounced. The two routes of administration were not effective in altering duration of loss of the righting reflex (LRR), or waking BECs following 4.5 g/kg ETOH. LS mice had the expected longer LRR durations and lower BECs at waking than did SS mice. Differences in absorption rate and peak BEC are concluded to be unrelated to ETOH neurosensitivity in these mice.     

Effects of Mycobacterium bovis BCG on the development of allergic inflammation and bronchial hyperresponsiveness in hyper-IgE BP2 mice vaccinated as newborns

Asthma may result from excessive Th-2 response in children not previously exposed to Th-1-inducing infections. We tested the hypothesis that BCG vaccination in Th-2-susceptible newborn BP2 mice blocks allergic inflammation and bronchial hyperreactivity (BHR). Ten day-old BP2 mice received 10(5) CFU of BCG 1173P2 intranasally (IN), and 6, 10 or 14 weeks thereafter were sensitized with 100 microg ovalbumin (OVA) in aluminium hydroxide twice subcutaneously (SC) at 1 week interval, and challenged 1 week after the second sensitization with 10 microg OVA IN. Compared to OVA-challenged unvaccinated mice, those that received BCG 8 weeks before challenge developed intense bronchial inflammation, BHR, and high IgE titers. Inflammation involved T cells, macrophages, dendritic cells and was accompanied by increased levels of Interleukin-5 (IL-5) in the bronchoalveolar lavages (BAL). However, animals challenged 16 weeks after BCG vaccination did not develop BHR nor bronchial hypereosinophilia, and showed reduced IgE levels. Bronchial infiltration by immunocompetent cells was also significantly reduced. Increased levels of gamma-interferon (IFN-gamma) after in vitro stimulation of tracheo-bronchial lymph node cells accompanied this blockage, but levels of IL-5 remained high. We demonstrate that 16 weeks after vaccination with BCG in newborn BP2 mice which have a high Th-2 background, allergic inflammation and BHR were blocked, even though a clear Th-1 shift was not achieved.     

Role of IgA in the defense against respiratory infections IgA deficient mice exhibited increased susceptibility to intranasal infection with Mycobacterium bovis BCG

IgA is the predominant Ig isotype in mucosal tissue and is believed to be involved in defense against viral and bacterial infections at these sites. Here, we examined the role of IgA in the protection against intranasal (i.n.) infection with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). IgA deficient (IgA-/-) mice and wild type non-targeted littermate (IgA+/+) mice were immunized by i.n. route with the mycobacterium surface antigen PstS-1 formulated with cholera toxin (CT). Our data showed that IgA-/- mice were more susceptible to BCG infection compared to IgA+/+ mice, as revealed by the higher bacterial loads in the lungs and bronchoalveolar lavage (BAL). Analysis of the Ig levels and the antibody responses to PstS-1 showed that IgA-/- mice had no detectable IgA either in the saliva or in the BAL. However, these mice displayed higher levels of total and specific IgM than IgA+/+ mice in both mucosal fluids. More importantly, analysis of the cytokine responses revealed a reduction in the IFN-gamma and TNF-alpha production in the lungs of IgA-/- compared to IgA+/+ mice. Altogether, our results suggest that IgA may play a role in protection against mycobacterial infections in the respiratory tract by blocking the pathogen entrance and/or by modulating the pro-inflammatory responses.     

Pharmacokinetics of oral contraceptive steroids after morning or evening administration

An oral contraceptive containing ethynyloestradiol and norethisterone was administered to six women in the morning and in the evening using a cross-over design. Serum levels of ethynyloestradiol and norethisterone were measured at various times' after administration. There was no significant difference in a number of pharmacokinetic parameters between the two times of administration, suggesting that morning or evening administration of the contraceptive are equally effective.     

Assessment of safety and interferon gamma responses of Mycobacterium bovis BCG vaccine in goat kids and milking goats

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed.BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination.BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.     

Protective efficacy of recombinant BCG over-expressing protective,stage-specific antigens of Mycobacterium tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. CysVac2, a fusion protein comprising stage-specific Mycobacterium tuberculosis antigens, provided superior protective efficacy against chronic M. tuberculosis infection in mice, compared to BCG. To determine if the delivery of CysVac2 in the context of BCG could improve BCG-induced immunity and protection, we generated a recombinant strain of BCG overexpressing CysVac2 (rBCG:CysVac2). Expression of CysVac2 in BCG was facilitated by the M. tuberculosis hspX promoter, which is highly induced inside phagocytic cells and induces strong cellular immune responses to antigens expressed under its regulation. Intradermal vaccination with rBCG:CysVac2 resulted in increased monocyte/macrophage recruitment and enhanced antigen-specific CD4⁺ T cell priming compared to parental BCG, indicating CysVac2 overexpression had a marked effect on rBCG induced-immunity. Further, rBCG:CysVac2 was a more potent inducer of antigen-specific multifunctional CD4⁺ T cells (CD4⁺IFN-γ⁺TNF⁺IL-2⁺) than BCG after vaccination of mice. This improved immunogenicity however did not influence protective efficacy, with both BCG and rBCG:CysVac2 affording comparable level of protection aerosol infection with M. tuberculosis. Boosting either BCG or rBCG:CysVac2 with the CysVac2 fusion protein resulted in a similar improvement in protective efficacy. These results demonstrate that the expression of protective antigens in BCG can augment antigen-specific immunity after vaccination but does not alter protection against infection, further highlighting the challenge of developing effective vaccines to control TB.     

The effect of BCG vaccination on macrophage phenotype in a mouse model of intranasal Mycobacterium bovis challenge

In order to develop improved vaccinations against tuberculosis, it is essential to understand the effect of vaccination on the immune response, and to overcome the mechanisms by which mycobacteria regulate this immune response. In this study, we examine the effect of intradermal vaccination with Mycobacterium bovis bacille Calmette-Guèrin on macrophage phenotype following intranasal challenge with virulent Mycobacterium bovis. Preserved lung tissues used in the present study were obtained from a previous vaccination trial in BALB/c mice. Vaccinated mice showed less extensive pulmonary lesions along with a significant decrease in bacterial lung burden when compared to control mice. Immunohistochemical markers of classically activated macrophages (iNOS) and alternatively activated macrophages (Arg1, FIZZ1) were applied to lung sections. Vaccination led to a statistically significant decrease in the number of Arg1+ macrophages. The presence of macrophages that expressed Arginase 1 in pulmonary lesions was much smaller than the presence of macrophages expressing iNOS. The low presence of Arg1+ macrophages induced by vaccination may be caused by Th1 polarization and may reduce alternative activation of macrophages, with an overall more effective intracellular killing of bacteria.     

A recA deletion mutant of Mycobacterium bovis BCG confers protection equivalent to that of wild-type BCG but shows increased genetic stability

The widely used vaccine against tuberculosis, BCG, shows evidence of genetic instability. It has undergone major genetic rearrangements resulting in deletion and duplication of segments of its chromosome. In order to produce a BCG strain with more favourable genetic properties, we inactivated the recA gene. Targeted deletion of the recA gene of BCG resulted in a complete loss of recombination between homologous, chromosomally-located sequences, as well as between plasmid- and chromosomally-located sequences. The deltarecA mutant BCG was as effective as the wild-type in conferring protection in mice against an intravenous challenge with virulent Mycobacterium tuberculosis, indicating that the loss of an SOS response-mediated DNA repair mechanism did not compromise the immunological properties of BCG. The availability of a genetically stable, fully immunogenic BCG is important for the future development of BCG as a live vaccine.     

Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C

In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.     

The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs

The Mycobacterium tuberculosis phoP mutant strain SO2 has previously been shown to have reduced multiplication in mouse macrophages and in vivo using the mouse intravenous-infection model. In this study we demonstrate that the M. tuberculosis SO2 is highly attenuated when compared with the parental M. tuberculosis MT103 strain and also more attenuated than BCG in severe combined immunodeficiency disease (SCID) mice. Complementation of the M. tuberculosis SO2 with the wild-type phoP gene restored the virulence of the strain in the SCID mice, confirming that the attenuated phenotype is due to the phoP mutation. In Balb/c mice subcutaneously vaccinated with either M. tuberculosis SO2 or BCG, the proportions of CD4+ and CD8+ populations measured in the spleen were significantly higher in the M. tuberculosis SO2 vaccinated group. In addition, the proportion of antigen-stimulated CD4+/CD8+ cells expressing IFN-gamma was significantly higher in the M. tuberculosis SO2 vaccinated group when compared with the BCG group. Balb/c mice subcutaneously vaccinated with the M. tuberculosis SO2 strain were also protected against intra-venous challenge with M. tuberculosis H37Rv at levels comparable to mice vaccinated with BCG, as measured by reduced bacterial counts in lung and spleens. Guinea pigs subcutaneously vaccinated with the M. tuberculosis SO2 strain were protected against aerosol challenge with M. tuberculosis H37Rv delivered at different doses. A high dose aerosol challenge of M. tuberculosis SO2 vaccinated guinea pigs resulted in superior levels of protection when compared with BCG vaccination, as measured by guinea pig survival and reduction in disease severity in the lung.     

Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge

The bacillus Calmette--Guerin (BCG), a live attenuated Mycobacterium bovis strain is considered a promising candidate as a vector system for delivery of foreign antigens to the immune system. The gene coding for the Leptospira interrogans external membrane protein LipL32, a highly immunogenic antigen found in all pathogenic leptospira, was cloned into several mycobacterial vectors for expression in BCG. Hamsters immunized with recombinant BCG (rBCG) expressing LipL32 were protected against mortality (P相似文献