Macrophage Migration Inhibition Studies with Cells from Mice Vaccinated with Cell Walls of Mycobacterium bovis BCG: Relationship Between Inhibitory Activity of Lung Cells and Resistance to Airborne Challenge with Mycobacterium tuberculosis H37Rv

In an effort to evaluate the role of delayed hypersensitivity in acquired resistance of mice to airborne infection with Mycobacterium tuberculosis H37Rv, the ability of lung and peritoneal cells from mice vaccinated in various ways with mycobacterial fractions or with M. bovis BCG to inhibit, in the presence of purified protein derivative, in vitro migration of normal peritoneal cells was determined. The degree of inhibition induced by lung cells was correlated with immunity, but that induced by peritoneal cells could not be associated with enhanced resistance. Live BCG given intravenously to mice stimulated greater resistance to infection and inhibitory activity of lung cells than did live BCG given subcutaneously. Vaccines with a protective index greater than 1 also induced a significant increase in lung weight. Although a correlation between ability of lung cells to inhibit cell migration and acquired resistance of the host to airborne infection with H37Rv was demonstrated, the data do not exclude the possibility that the two phenomena are independent responses to the immunologically complex mycobacterial antigens.     

Listeria Pneumonitis: Influence of Route of Immunization on Resistance to Airborne Infection

Mice that are immunized with an airborne inoculum of BCG are more highly resistant to airborne challenge with Mycobacterium tuberculosis than are mice that are immunized by the subcutaneous or intravenous route. To discover whether this phenomenon is peculiar to tuberculosis, we studied the influence of the route of immunization upon pulmonary resistance in Listeria monocytogenes infection. Mice were immunized by the airborne, intravenous, or footpad route and were subsequently challenged by the same route at 1 to 4 weeks after immunization. Mice were highly and uniformly resistant to intravenous challenge, regardless of the route of immunization. The route of immunization bore no influence upon resistance to footpad infection, but resistance was appreciably better in mice challenged within 2 weeks of immunization than it was at later time points. In mice immunized by the footpad and intravenous routes, the pattern of resistance to airborne and footpad challenges was similar, in that there was substantially less immunity at 4 weeks than at 2 weeks after immunization. However, mice immunized by the airborne route were highly resistant to airborne challenge, regardless of the interval between immunization and reinfection. In this last respect, resistance of the lungs to reinfection was similar after Listeria and tuberculosis pneumonitis. It is suggested that a similar pattern of resistance may prevail in pneumonitis caused by other facultative intracellular parasites.     

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.     

Transfer of Adoptive Immunity to Tuberculosis in Mice

A system is described for studying adoptive immunity to tuberculosis in syngeneic mice. Donor mice were immunized with 10⁴ BCG intravenously, and lymphoid cells were harvested 28 days later. Adoptive immunity was measured in recipient mice in terms of the inhibition of growth of BCG in the liver and spleen following intravenous injection. Adoptive immunity was expressed optimally when recipients were sublethally irradiated (500 R), challenged with 10⁴ to 10⁵ viable organisms, and given sensitized lymphoid cells intravenously. Adoptive immunity was not manifest until 14 days after challenge and was effective against Mycobacterium tuberculosis H37Rv as well as BCG. Immunity could be conferred by spleen, lymph node, peritoneal exudate, and resident peritoneal (washout) cells. The lymphoid cells conferring immunity were shown to be thymus-dependent lymphocytes by virtue of their nonadherence to glass wool and sensitivity to anti-θ serum plus complement. The sensitized cells were relatively susceptible to both in vitro and in vivo X-irradiation.     

可表达结核分枝杆菌融合蛋白Ag85A-ESAT-6重组卡介苗免疫保护作用研究

目的以Balb/c小鼠为动物模型,评价重组卡介苗新型结核病疫苗rBCG-Ag85A-ESAT-6(rBC-AE)的免疫保护效应。方法将重组卡介苗rBCG-AE免疫动物10周后,结核分枝杆菌H37Rv尾静脉注射进行感染攻击,分别于感染攻击后3、6和9周,通过观察肺组织大体病变、脾肺组织细菌载荷量计数、肺组织抗酸染色、HE染色结合肺组织病理变化,综合评价该疫苗诱导的免疫保护作用。结果 rBCG-AE组脾肺组织细菌载荷量在各时间点均显著低于阴性对照组(PBST组,P0.01),但明显高于卡介苗(BCG)组(P0.01)。rBCG-AE组肺组织病变在感染攻击后6～9周逐渐改善,但其病理打分在各时间点均明显高于BCG组(P0.01)。各组肺组织大体病变与组织病理打分变化相似。结论重组卡介苗rBCG-AE仅能诱导产生与BCG疫苗相当甚至较低的免疫保护作用。     

Specific and nonspecific resistance in mice immunized with irradiated Myobacterium leprae.

Following subcutaneous inoculation of irradiated Mycobacterium leprae (I-ML) into the left hind footpad of mice, there was increased resistance to Listeria monocytogenes, indicative of macrophage activation, at the immunization site. In spite of the high level of localized macrophage activation which was proportioned to the immunizing dose of I-ML, no such activity could be demonstrated systemically in these mice, as evidenced by the absence of increased resistance to an intravenous challenge with L. monocytogenes. Under these conditions, I-ML-immunized mice were nonetheless resistant to intravenous infection with either M. tuberculosis or M. bovis BCG, and this immunity was transferred to normal recipients using spleen or lymph node cells. Neonatal thymectomy completely abolished the development of antimycobacterial immunity after vaccination with I-ML, but immunity was restored by an intraperitoneal infusion of syngeneic thymocytes. Systemic nonspecific resistance could be generated in I-ML-immunized mice by an intravenous injection of disrupted I-ML. This study reveals that, after subcutaneous vaccination with I-ML, there is local accumulation of activated macrophages at the inoculation site and a widespread distribution of lymphocytes which are sensitized to mycobacterial antigens. Nonspecific resistance is mediated by the former cells and specific antimycobacterial immunity by the latter.     

Effects of suramin on the in vivo antimicrobial resistance against Listeria monocytogenes and Mycobacterium bovis (BCG) in mice.

Following one intraperitoneal injection of Suramin (400 mg/kg) in mice, biphasic alterations of the resistance to Listeria monocytogenes were observed, depending on the timing of the drug administration in relation to the intravenous challenge. Treatment with suramin, concomitantly or 1 day before, enhanced markedly the bacterial growth in spleen and liver, detected as early as 3 to 6 h after the challenge, the maximum being observed at 48 h in the liver. In contrast, an increased resistance was observed when suramin was given 8 days before the challenge. This late effect was associated with a stimulation of the mononuclear phagocyte system as measured by the increase of the spleen index and accumulation of histiocytic cells in the lymphoid organs, such as peripheral nodes. Intraperitoneal inoculation of suramin (100 mg/kg), before, during and after an intravenous injection of low dose (2 X 10(4)/mouse) of Mycobacterium bovis (BCG) in C57BL/6 and CH3 mice were followed, in both strains, by a similar increase of the mycobacterial growth in spleen and liver during the first 2 weeks. This indicates that suramin was acting on a different non specific mechanism of resistance than the one being expressed by the Bcg gene controlling the natural resistance as described recently. However, when the same schedule of suramin treatment was given to these two strains, during subcutaneous immunization with 1 X 10(7) viable BCG, a significant decrease of 48 h delayed-type hypersensitivity to tuberculin was noted 21 days later only in C57BL/6 but not in C3H mice, in which such a reaction was also of low magnitude in the infected control. The decrease of this cell-mediated immunity parameter in C57BL/6 suramin treated mice was also correlated with a higher number of viable BCG found at the challenge site and in the draining nodes.     

Protective immunity to experimental tuberculosis by mannophosphoinositides of mycobacteria

Mannophosphoinositides isolated from mycobacterial cells were found to induce both humoral and cell-mediated immune responses in mice when injected as mannoside-methylated bovine serum albumin (MBSA) complexes. Immunization of mice with mannoside-MBSA complexes elicited significant protection against challenge with LD₅₀ dose of M. tuberculosis H₃₇Rv as revealed by high survival rate, low values of root-specific lung weight, lung densities and colony forming units recovered from lung, liver and spleen, compared to the nonimmunized group. These observations were further substantiated by histopathological studies. The protective immunity elicited by mannoside-MBSA complexes against challenge with M. tuberculosis H₃₇Rv was mediated by the cooperation of T-B cells, as shown by the passive transfer of immune cells/sera into syngeneic sublethally irradiated recipient mice.     

Efficacy of Mycobacterium bovis BCG vaccination in mice undergoing prior pulmonary infection with atypical mycobacteria

The efficacy of Mycobacterium bovis BCG immunization in mice with established pulmonary infections caused by atypical mycobacteria was studied. In all four strains of Mycobacterium tested (M. kansasii, M. simiae, M. avium, and M. scrofulaceum), intravenous inoculation with 10(6) BCG had no discernible effect upon the course of atypical mycobacterial infection within the lungs; despite this, however, all BCG-vaccinated groups of mice were fully resistant to a subsequent acute aerogenic challenge with M. tuberculosis H37Rv, regardless of the presence of the pulmonary atypical mycobacterial infections. Furthermore, animals infected with M. kansasii, M. simiae, or M. avium but not vaccinated with BCG expressed considerable antituberculous resistance within the lungs, resulting in significant prolonged survival of these animals. The relevance of these findings to the expression of antituberculous resistance in human populations in areas in which atypical mycobacteria are endemic and the failure of these findings to support the hypothesis that prior contact with atypical mycobacteria might in some way jeopardize or interfere with the efficacy of subsequent BCG vaccination are discussed.     

Immunosuppressive effect of cyclosporin A on Mycobacterium bovis BCG infections in mice.

The effect of increasing doses of cyclosporin A (CsA) given to mice infected intravenously with Mycobacterium bovis BCG was investigated. Development of both tuberculin hypersensitivity and acquired antituberculous resistance was suppressed in a dose-responsive manner. Daily dosages at 100 mg/kg of body weight prevented any reduction in the BCG counts within the lungs, liver, or spleen. This effect was associated with lowered nonspecific resistance to a Listeria monocytogenes challenge and a decline in specific protective immunity adoptively transferred to naive recipients. CsA treatment had no effect on antilisterial activity by activated macrophages or on the antituberculous immunity expressed by specific memory T cells. CsA treatment inhibited the ability of BCG-vaccinated mice to produce gamma interferon (IFN-gamma) after a secondary stimulation with live BCG or with lipopolysaccharide. Spleen cells from BCG-infected mice which were exposed to daily treatment with CsA showed reduced IFN-gamma production in response to purified protein derivative or concanavalin A stimulation, suggesting that the immunosuppressive effect of CsA on BCG-infected mice was expressed by inhibiting the development of effector T cells responsible for the production of IFN-gamma.     

Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis.

In this study highly purified culture filtrate proteins obtained from Mycobacterium tuberculosis strains Erdman and H37Rv were tested for their capacity to stimulate immune T cells in vitro, and to immunize mice in vivo. Analysis of the culture filtrate antigen pool revealed a complex mixture of proteins; after separation of this pool into fractions of defined molecular size using an electrophoretic method, it was found that multiple fractions strongly stimulated interferon-gamma (IFN-gamma) secretion by immune CD4 T cells in vitro. In a further series of experiments mice were given multiple immunizations with the culture filtrate protein pool suspended in emulsions of incomplete Freund's adjuvant. Such mice were as resistant as mice given live bacillus Calmette-Guérin (BCG) vaccine to a low dose aerosol challenge infection with M. tuberculosis, but this resistance waned to low levels by 5 months post-vaccination. Furthermore, experiments using the filtrate antigens to boost or augment immunity induced by the BCG vaccination itself were unsuccessful. These data therefore support the hypothesis that the culture filtrate proteins of M. tuberculosis contain multiple antigens that are strongly recognized by T cells acquired during the initial expression of protective immunity to tuberculosis. Conventional immunization with these purified protein antigens can engender a strong degree of protective immunity, but this immunity is apparently not sustained at the same level as that induced by the live vaccine, perhaps suggesting a lack of suitable stimulation of memory immunity.     

Mycobacterium lepraemurium infection of nude athymic (nu/nu) mice.

Nude athymic (nu/nu) mice on a BALB/c background and their heterozygous euthymic litter mates (nu/+) were infected with either 10(8) or 10(6) Mycobacterium lepraemurium organisms intravenously or in the left hind footpad (LHF). After LHF infection with 10(8) M. lepraemurium organisms, nu/+ mice slowly developed a response that consisted of LHF swelling and local resistance to Listeria monocytogenes. The lower inoculum induced a proportionately lower response in nu/+ mice, but the nu/nu mice developed neither LHF swelling nor resistance to L. monocytogenes in response to either dose of M. lepraemurium. Counts of M. lepraemurium in the LHF revealed no difference between the nu/+ mice and nu/nu mice. After intravenous infection the nu/+ mice developed splenomegaly, but did not otherwise differ from nu/nu mice with respect to resistance to intravenous challenge with L. monocytogenes or growth of M. lepraemurium in the spleen. In light of the poor responsiveness of nu/+ mice in this experiment, they were then compared with CB6 and B6D2 mice, which are genetically susceptible and resistant to M. lepraemurium, respectively. These mice were infected with either 10(8) or 10(6) M. lepraemurium cells or 10(6) Mycobacterium bovis BCG cells in the LHF. Once again the nu/+ mice responded poorly to M. lepraemurium, the CB6 mice responded very strongly, and the B6D2 mice gave an intermediate response with respect to LHF swelling and resistance to L. monocytogenes. However, M. lepraemurium grew to higher numbers in the LHF of nu/+ and CB6 mice than in B6D2 mice, revealing, in CB6 mice, a dissociation between resistance to L. monocytogenes and M. lepraemurium. All three mouse strains responded strongly to M. bovis BCG, but there was a suggestion that nu/+ mice might be more susceptible to this agent than the other two strains. I concluded that the failure of nu/+ mice to restrict the growth of M. lepraemurium more than nu/nu mice was due to the intrinsic genetic susceptibility of both types of mice. In effect, the nu/+ mice behaved like nu/nu mice, as if they too were deficient in T lymphocytes that were responsive to M. lepraemurium.     

Interleukin-15 as an immune adjuvant to increase the efficacy of Mycobacterium bovis bacillus Calmette-Guérin vaccination

Interleukin-15 (IL-15) transgenic mice which had been inoculated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) 24 weeks previously showed resistance against airborne infection with Mycobacterium tuberculosis H37Rv accompanied by an increased CD8(+)-Tc1-cell response. IL-15 may be used as an immune adjuvant given with BCG vaccination to enhance its biologic efficacy.     

Distinct H-2 complex control of mortality, and immune responses to tuberculosis infection in virgin and BCG-vaccinated mice.

We have studied the impact of distinct haplotypes and of different alleles at specific H-2 loci on: (i) the susceptibility to lethal form of experimental tuberculosis; (ii) the level of DTH to mycobacterial antigens; (iii) the efficacy of vaccination with bacille Calmette-Guérin (BCG); and (iv) the IgG production and T cell proliferative response to H37Rv antigens. On the basis of median survival time (MST) following primary inoculation with lethal dose of Mycobacterium tuberculosis, susceptibility to infection associated with I-Ab and Db alleles, host resistance associated with I-Ak and Dd alleles. Mice bearing a disease-resistant phenotype also developed a vigorous DTH response. Vaccination with BCG before H37Rv infection significantly prolonged the survival time of both resistant and susceptible animals, except in B10.M (H-2f) mice. The latter exhibited intermediate resistance to infection before but slight decrease in the MST following a high-dose BCG vaccination. Distinct H-2 regulation of susceptibility to lethal infection and of BCG vaccination efficacy was confirmed in another relatively resistant H-2f-bearing strain A.CA, in which mortality occurred more rapidly in vaccinated compared with primarily infected animals. The expression of the H-2f haplotype was associated with a low DTH response to tuberculin following vaccination and subsequent lethal infection. The lack of BCG protection against Myco, tuberculosis challenge in B10.M mice associated with the high titre of specific IgG. In addition, these mice exhibited a unique ability to respond to 65-kD antigen by both IgG synthesis and T cell proliferation.     

Influence of the mouse Bcg, Tbc-1 and xid genes on resistance and immune responses to tuberculosis infection and efficacy of bacille Calmette–Guérin (BCG) vaccination

We have studied the role of three mouse distinct non-H-2 genes (BcgTbc-1xid) in several phenomena of antituberculosis immunity and resistance. On the basis of median survival time (MST) of mice following infection with virulent Mycobacterium tuberculosis H37Rv, Bcg gene did not control resistance to the lethal dose of H37Rv infection in non-vaccinated and Myco. bovis (BCG)-vaccinated mice. However, Bcg^r allele, in comparison with Bcg^s allele, determined more effective suppression of an early multiplication in spleens of H37Rv mycobacteria after a low dose (5 × 10⁴ colony-forming units (CFU)) injection. CBA/N mice, which are not protected efficiently against tuberculous challenge by BCG vaccination, were characterized by a decreased in vitro proliferation of immune lymph node cells, both spontaneous and stimulated with mycobacterial antigens. The decreased proliferation was due to immunosuppression caused by interactions between responding T cells and CBA/N antigen-presenting cells (APC). We have confirmed that the defective response to BCG-vaccination in CBA/N mice is linked with the X-chromosome and thus is presumably determined by the xid gene itself. I/St mice (Tbc-1^s), supersusceptible to H37Rv infection, were not able to restrict the growth of H37Rv mycobacteria in spleens, even following infection with a low dose (5 × 10⁴), but restricted the growth of Mycobovis BCG more effectively than Bcg^s mice.     

Enhancement activity of anti-mycobacterial sera in experimental Mycobacterium bovis (BCG) infection in mice.

The passive transfer of rabbit anti-mycobacterial immunoglobulins was directed against either living or soluble extracts of Mycobacterium tuberculosis strain H37Rv, which promotes the multiplication of the BCG strain of M. tuberculosis in the spleen of mice infected with low doses of this latter strain. This enhancing effect was reduced significantly when antisera were absorbed with living BCG. Moreover, such treatment led to the removal of all hemagglutinating antibodies when antisera were tested against either BCG or H37Rv soluble extracts. Therefore, it is most probable that the enhancing effect is related to the presence of mycobacterial antibodies.     

结核分枝杆菌DNA疫苗pVS85B的构建及免疫保护实验研究

评价构建的结核DNA疫苗pVS85B免疫小鼠后脾细胞体外产生细胞因子和抵抗结核分枝杆菌H37Rv攻击的能力。利用基因操作技术将结核菌ag85b插入pVAX1载体,构建表达结核菌Ag85B分泌型蛋白的DNA疫苗pVS85B。将雌性C57BL/6小鼠分成5组,每组20只,分别用pVS85B、pVAX1、pIL2S和PBS分别免疫3次,均间隔2周,以相同剂量加强免疫2次。另一组用BCG(105CFU)免疫1次。每组10只鼠在最后一次加强免疫后,无菌取脾培养,检测上清细胞因子。另10只鼠用结核菌H37Rv经静脉攻击,2周后取脾、肝和肺培养结核菌并进行菌落计数。结果是pVS85B免疫组鼠脾淋巴细胞培养上清的mIL-2和mIFN-γ平均含量分别为(311.3±46.2)和(273.6±55.4)pg/ml,显著高于3个阴性对照组(P<0.001),与BCG免疫组无显著性差异(P>0.05)。5个组的平均mIL-6和mIL-10无显著性差异。pVS85B免疫组小鼠的脾、肝和肺的平均结核菌载量分别为(47 716.6±5 689.0)、(50 113.4±6 532.2)和(51 095.3±2 788.9)CFU/g,分别低于pVAX1、pIL2S和PBS三个阴性对照组的相应器官的结核菌载量(P<0.001)。pVS85B免疫组脾、肝和肺的结核菌载量显著高于BCG免疫对照组。提示,表达结核菌分泌型Ag85B的DNA疫苗pVS85B能够刺激机体产生抗结核菌所需的Th1型免疫应答,免疫鼠获得抵抗H37Rv攻击的能力。     

Intranasal bacille Calmette-Guerin (BCG) vaccine dosage needs balancing between protection and lung pathology

Intranasal vaccination may offer practical benefits and better protection against respiratory infections, including tuberculosis. In this paper, we investigated the persistence of the Mycobacterium bovis-strain bacille Calmette-Guerin (BCG) Pasteur, lung granuloma formation and protection against pathogenic tuberculous challenge in mice. A pronounced BCG dose-dependent granulomatous infiltration of the lungs was observed following intranasal, but not after subcutaneous, vaccination. Corresponding doses of BCG, over a 100-fold range, imparted similar protection against H37Rv challenge when comparing the intranasal and subcutaneous vaccination routes. Interestingly, a BCG dose-dependent reduction of the H37Rv challenge infection was observed in the lungs, but not in the spleens, following both intranasal and subcutaneous vaccination. In the light of the observed concurrence between the extent of granuloma formation and the level of protection of the lungs, we conclude that intranasal vaccination leading to best protective efficacy needs to be balanced with an acceptable safety margin avoiding undue pathology in the lungs.     

Influence of BCG-induced immunity on the bactericidal activity of isoniazid and rifampicin in experimental tuberculosis of the mouse and guinea-pig.

The influence of previous BCG vaccination on the bactericidal activity of isoniazid and rifampicin has been studied using serial counts of viable tubercle bacilli in the spleens and lungs of mice and guinea-pigs infected intravenously with M. tuberculosis, strain H37Rv. In mice, BCG vaccination decreased the bactericidal activity of isoniazid in the spleen, but did not affect its activity in the lungs, where immunity is less strongly expressed. BCG did not influence the bactericidal activity of rifampicin in either organ. In contrast, previous BCG vaccination in the guinea-pig increased the bactericidal activity of isoniazid and rifampicin in the spleen and lungs. The differences between the animal species might result from the immune response being mainly bacteriostatic in the mouse but bactericidal in the guinea-pig.     

Aerosol-Induced Tuberculosis in Subhuman Primates and the Course of the Disease After Intravenous BCG Vaccination

Suitability of the rhesus monkey (Macaca mulatta) as an experimental host for evaluation of vaccines against airborne infection with Mycobacterium tuberculosis strain H37Rv was investigated. Nonvaccinated monkeys were exposed to estimated doses of 12, 25, or 49 units of H37Rv in a modified Henderson apparatus, and the course of the disease was followed by chest X rays, skin testing with purified protein derivative, body-weight determinations, and autopsy 8 weeks postinfection. These animals developed progressive and extensive tuberculosis with pathological changes proportional to the infecting dose. Four of seven monkeys vaccinated intravenously with 1 mg of live BCG 8 weeks prior to challenge with 40 units of H37Rv had no gross evidence of disease at autopsy 13 weeks postinfection; the other three monkeys had minimal disease. These data demonstrated that (i) reproducible and progressive infection could be induced in rhesus monkeys infected in a manner which simulated natural infection of man and (ii) a high level of resistance to infection could be induced by BCG vaccine in the rhesus monkey, which in nature is highly susceptible to tuberculous infection.