Non-specific antimicrobial cellular resistance in sensitized guinea pigs.

Specific delayed-type hypersensitivity was induced in guinea pigs with bovine albumin + complete Freund adjuvant, bovine gamma globulin + complete Freund adjuvant and BCG vaccine. The animals were subsequently tested for nonspecific antimicrobial resistance. Sensitized and control groups were challenged intraperitoneally with Listeria monocytogenes 2 hr after reinjection with the sensitizing antigen. The listeria content of the spleens was determined 1 or 5 days after the infection. The number of organisms recovered from the spleen one day after infection was significantly less in guinea pigs sensitized with bovine gamma globulin and BCG than in the control group; after 5 days no such difference was recorded. There was no difference between the bovine albumin sensitized and the control group 1 day after infection, while on the 5th postinfection day listeria counts were higher in the sensitized than in the control animals.     

Specificity of Acquired Resistance Produced by Immunization with Mycobacterial Cells and Mycobacterial Fractions

Mice were immunized intraperitoneally with 5.0 mg of living and 5.0 mg of heat-killed H37Ra cells of the attenuated strain Mycobacterium tuberculosis and challenged intraperitoneally with Listeria monocytogenes and Klebsiella pneumoniae. The period of protection provided by the living and heat-killed H37Ra cells against both heterologous infections was the same. When mice were immunized intraperitoneally with graded doses of living and heat-killed H37Ra and challenged intraperitoneally with listeria or klebsiella, the lowest immunizing dose providing protection against both klebsiella and listeria challenge was the same for living and heat-killed cells. Living and heat-killed cells also immunized equally effectively when the routes of immunization and challenge were different. Mice also were immunized intraperitoneally with mycobacterial ribosomal fraction, mycobacterial cell walls, and several nonspecific agents (Escherichia coli endotoxin, mineral oil emulsion, and Freund's incomplete adjuvant). The mice were challenged intraperitoneally with listeria or klebsiella at varying times after immunization. The mycobacterial components and all the nonspecific agents provided transitory protection lasting no longer than 4 days after immunization. Only the mycobacterial cell walls and the endotoxin provided protection against listeria challenge. It was concluded that the protection provided by the mycobacterial ribosomal fraction is specific for tuberculosis infection, since this fraction provided no protection against listeria infection and only transitory protection against klebsiella. It was also concluded that the mycobacterial component providing protection against heterologous infections is heat stable and probably is found in the cell wall.     

Specificity of Acquired Resistance Produced by Immunization with Listeria monocytogenes and Listeria Fractions

Mice were immunized with 1.0 mg of an attenuated strain of Listeria monocytogenes to determine the period of protection afforded by this strain when the mice were challenged intravenously with 5 MLD of listeria. Protection appeared 2 days after immunization and was still apparent 4 weeks after immunization. If the challenge dose was decreased to 1 MLD, protection was apparent at 10 weeks. Mice immunized with a comparable dose of mycobacterial cells and challenged intravenously with 1 MLD of listeria showed no protection at 10 weeks. The magnitude of the immune response to listeria challenge was not increased in mice immunized with the same virulent strain as that used for challenge. It was also found that resistance to listeria challenge appeared early after listeria immunization if the immunizing dose was large. As the immunizing dose was decreased and the challenge dose increased, resistance appeared later. Listeria killed by heat or ultraviolet irradiation, living but nonmultiplying streptomycin-dependent listeria, or listeria ribosomal fraction gave no protection against listeria challenge. The magnitude of the immune responses after listeria immunization to listeria challenge and to mycobacteria challenge were compared. It was found that protection after listeria challenge was of longer duration. In addition, a 100-fold larger vaccinating dose was required to give comparable protection against tuberculous infection.     

Biological activities of a murine T-cell clone with reactivity to Mycobacterium leprae

Mice were immunized subcutaneously with killed Mycobacterium leprae in incomplete Freund's adjuvant and draining lymph nodes removed. Lymph node cells were propagated in vitro and cloned at limiting dilution in the presence of syngeneic accessory cells, antigen, and T-cell growth factor. Cloned T cells were restricted by the H-2I-A sublocus. In vitro interaction(s) of cloned T cells with accessory cells presenting M. leprae-derived determinants resulted in T-cell proliferation, interleukin secretion, and macrophage activation. The T cells were stimulated by killed M. leprae and M. bovis (strain BCG), but not Listeria monocytogenes, organisms indicating cross-reactivity between M. leprae and BCG at the clonal level. In vivo, cloned T cells induced protection against the "bystander" bacterium L. monocytogenes. These data suggest that the cloned M. leprae-reactive T cells are involved in acquired antimicrobial resistance.     

Regulation of the immune response by macrophages

Regulation of the immune response by macrophages was studied with cellular resistance to Listeria monocytogenes as parameter. The use of agents which suppress macrophage activity during the induction-phase of immunity enabled the induction of protective immunity with killed listeria. Fractionation of the cell content of listeria yielded an RNA'se sensitive fraction which in a dose of 300 ng and in combination with the cationic surfactant dimethyl dioctadecyl ammonium bromide induced protective immunity against listeria.     

Biochemical characterization of three mycobacterial ribosomal fractions

The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity.     

Formulation of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG in cationic liposomes significantly enhances protection against tuberculosis

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) - D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe, and relatively inexpensive alternative to vaccination with conventional BCG.     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

Specificity of the Anamnestic Response Produced by Listeria monocytogenes or Mycobacterium tuberculosis to Challenge With Listeria monocytogenes

When mice immunized with Listeria monocytogenes were given a second injection of listeria, they showed an anamnestic immune response to intravenous challenge with listeria, as measured by enumeration of the viable infecting organisms in the spleens of the infected animals. This response was independent of the effects of the challenge dose. When mice immunized with living or heat-killed attenuated mycobacterial cells were boosted with living H37Ra, there was also an accelerated response to listeria challenge. The response was greater in the mice given the primary immunization with living cells than in those immunized with heat-killed cells. The response to listeria challenge in mice immunized and boosted with mycobacteria was of less magnitude than that in the mice immunized and boosted with listeria. Growth of listeria in the mice immunized and boosted with mycobacteria was retarded only during the first 2 days of the infection, whereas the infecting listeria in mice immunized and boosted with listeria were permanently inactivated. Mice immunized with mycobacterial ribosomal fraction and restimulated with living mycobacterial cells showed no accelerated response to listeria challenge. It is evident from these results that resistance to these organisms is specifically evoked, but that once evoked it is not completely nonspecific in action. Also, the resistance produced by the mycobacterial ribosomal fraction to challenge with mycobacteria is completely specific in action. Therefore, it has been shown that there are two mechanisms involved in acquired immunity to facultative, intracellular parasites. One is nonspecific and mediated by activated macrophages. The other is specific and mediated by a mechanism as yet unknown.     

Immunizing Capacity of Viable and Killed Attenuated Mycobacterial Cells Against Experimental Tuberculous Infection

The relationship of the dose of vaccine to the immune response was determined in CF-1 mice vaccinated intraperitoneally with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and in mice vaccinated with cells of the same strain killed by autoclaving at 121 C for 15 min. The results showed, in terms of increased resistance to tuberculous infection, that the immune response with both living and killed cells was dependent upon the dose of vaccine, whereas only the living cells were dependent upon the time of challenge after vaccination. The dose response curves show dramatically that viable cells, which do not multiply in vivo, are several hundred times more effective immunizing agents against tuberculous infection than are autoclaved cells. Viable 2-week-old H37Ra cells were far more immunogenic than viable 4-week-old cells. Autoclaved 2-week-old cells, however, were no more immunogenic than autoclaved 4-week-old cells. H37Ra cells killed by boiling (98 C), exposure to 65 C for 30 min, treating with 2% phenol, or by being dried with acetone also lost most of their capacity to immunize mice. The effect of adjuvant on the immune response of mice to tuberculous infection was tested by incorporating both viable and autoclaved cells in Freund's incomplete adjuvant. We found that this vehicle had little or no effect on the immunizing capacity of either viable or heat-killed mycobacterial cells. The relationship of all the findings to the specificity of the immune response to tuberculosis is discussed.     

The influence of adjuvant on induction of protective immunity by a non-living vaccine against schistosomiasis

Mice were protected against subsequent infection with Schistosoma mansoni by intradermal or s.c. vaccination with killed schistosomula or soluble parasite extracts and bacillus Calmette-Guérin (BCG). Treatment with i.p. immunization was somewhat less effective, whereas i.m. vaccination failed to elicit protective immunity. The level of resistance induced by intradermal immunization was influenced by the strain of BCG used, and isolated BCG cell walls did not reliably substitute for whole BCG organisms as adjuvant. Bordetella pertussis vaccine and saponin were also able to function as adjuvants for protective immunity in this model, whereas other immunopotentiators including Corynebacterium parvum and aluminum hydroxide were ineffective. No correlation between resistance to challenge infection and antibody levels was detected. Animals immunized intradermally using either protective or non-protective adjuvants all showed minimal humoral reactivity against schistosomulum surface Ag but strong IgG response to soluble parasite components including paramyosin, which is the major serologically recognized Ag in mice vaccinated intradermally with schistosome Ag plus BCG and is protective in this model. In contrast, a strong correlation was observed between resistance and Ag-specific cell-mediated reactivity, including IFN production by T lymphocytes in vitro and macrophage activation in vivo. These results further substantiate the hypothesis that protection in this model is based on cell-mediated immune effector mechanisms. Moreover, they may be of general relevance in the design of vaccination protocols using other Ag or against other infectious agents.     

In vivo persistence and protective efficacy of the bacille Calmette Guerin vaccine overexpressing the HspX latency antigen

New strategies to control infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently required, particularly in areas where acquired immunodeficiencies are prevalent. In this report we have determined if modification of the current tuberculosis vaccine, Mycobacterium bovis BCG, to constitutively express the mycobacterial HspX latency antigen altered its protective effect against challenge with virulent M. tuberculosis. Overexpression of M. tuberculosis HspX in BCG caused reduced growth in aerated cultures compared to control BCG, but growth under limited oxygen availability was not markedly altered. Upon infection of mice, BCG:HspX displayed tissue-specific attenuation compared to control BCG, with reduced growth within the lung and liver but not the spleen. Both BCG:HspX and control BCG protected mice against aerosol M. tuberculosis challenge to a similar extent, however, immunodeficient mice infected with BCG:HspX survived significantly longer than mice infected with the control BCG strain. Therefore, altering the in vivo persistence of BCG by overexpression of HspX may be one important step towards developing a new tuberculosis vaccine with an improved safety profile and suitable protective efficacy against M. tuberculosis infection.     

Strain-specific differences in the genetic control of two closely related mycobacteria

The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans.     

Adjuvants in tuberculosis vaccine development

Tuberculosis remains a major public health problem around the world. Because the Mycobacterium bovis Bacilli–Calmette–Guerin (BCG) vaccine fails to protect adults from pulmonary tuberculosis, there is an urgent need for improved vaccine formulations. Unlike BCG, recombinant vaccines purified from bacterial expression vectors, as well as naked DNA, require an additional adjuvant. Recent improvements in our understanding of disease immunopathology, together with advances in biochemical and molecular techniques, have permitted the successful development of promising tuberculosis vaccine delivery and adjuvant combinations for human use. Here, we summarize the current state of adjuvant development and its impact on tuberculosis vaccine progress.     

Hsp65与IL-2融合蛋白诱导小鼠细胞免疫应答及保护力

目的研究Hsp65与hIL-2的融合蛋白在小鼠体内诱导的免疫应答及保护力。方法在大肠杆菌中诱导表达Hsp65与hIL-2的融合蛋白,通过Ni-NTA亲合柱纯化后的蛋白经鉴定后,与佐剂DDA和MPL联合免疫小鼠,连续免疫3次,每次间隔2周,最后一次免疫结束后两周,分离5只小鼠脾淋巴细胞,测定淋巴细胞增殖指数,IFN-γ和IL-2水平,以及特异性淋巴细胞杀伤功能,其余5只免疫小鼠用于MTB毒株攻击实验。结果获得融合蛋白可分别与抗Hsp65和抗hIL-2的单抗发生特异性反应。融合蛋白免疫小鼠后,小鼠脾淋巴细胞被有效活化,诱导产生的-γIFN和IL-2的水平以及CTL杀伤功能均显著高于BCG和单纯Hsp65免疫组（P〈0.05）。融合蛋白免疫组可有效抵抗MTB毒株攻击,脾脏细菌数显著减少（4.36±0.48）,提供的保护力与BCG相当（4.30±0.53）。结论Hsp65与hIL-2的融合蛋白是一种有效的亚单位疫苗,可用于TB的预防。     

Fasciola hepatica: attempts to induce protection against infection in rats and mice by injection of excretory/secretory products of immature worms

In vitro excretory/secretory products of 4-week (immature) and 8-week-old (mature) Fasciola hepatica parasites, derived from rats, were injected together with adjuvant into naive rats and mice. Resistance to infection was assessed in rats by counting adults in the bile ducts at 9 weeks, or in mice by recording deaths after oral challenge with a high dose of viable metacercariae. Exposure of rats to excretory/secretory products of immature F. hepatica conferred a significant degree of resistance which was comparable to the level of resistance induced following oral administration of a low number of metacercariae. No protection against infection was seen in rats injected with excretory/secretory products from mature, bile duct-derived worms. In mice, no obvious mouse strain variation in susceptibility to first infection existed and hypothymic nude mice were as susceptible to infection as intact mice. As determined by protection against death, vaccination with excretory/secretory products derived from immature F. hepatica was without effect in mice. It is concluded that "host protective antigens", at least for rats, were present in the excretory/secretory products of immature F. hepatica larvae.     

Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages

The current vaccine for tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is an attenuated strain of Mycobacterium bovis bacillus Calmette-Guerin (BCG). BCG has proven to be effective in children, however, efficacy wanes in adulthood. Lactoferrin, a natural protein with immunomodulatory properties, is a potential adjuvant candidate to enhance efficacy of BCG. These studies define bovine lactoferrin as an enhancer of the BCG vaccine, functioning in part by modulating macrophage ability to present antigen and stimulate T-cells. BCG-infected bone marrow derived macrophages (BMMs) cultured with bovine lactoferrin increased the number of MHC II(+) expressing cells. Addition of IFN-gamma and lactoferrin to BCG-infected BMMs enhanced MHC II expressiona dna increased the ratio of CD86/CD80. Lactoferrin treated BCG-infected BMMs were able to stimulate an increase in IFN-gamma production from presensitized CD3(+) splenocytes. Together, these results demonstrate that bovine lactoferrin is capable of modulating BCG-infected macrophages to enhance T-cell stimulation through increased surface expression of antigen presentation and co-stimulatory molecules, which potentially explains the observed in vivo bovine lactoferrin enhancement of BCG vaccine efficacy to protect against virulent MTB infection.     

The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.     

Granuloma Formation Induced in Mice by Chemically Defined Mycobacterial Fractions

Amounts of trehalose-6,6-dimycolate as small as 1 to 5 mug can, after intravenous injection, induce in the lungs of mice formation of tubercles in which the cellular composition is indistinguishable from that in tubercles formed after an infection with living BCG bacilli. The strongest cellular response in mice was induced by cord factor from Mycobacterium kansasii; the weakest was induced by cord factor from the BCG strain of M. bovis. It was found that three intravenous injections of cord factor induced a more extensive cellular response than did one injection of the same total amount of cord factor. Mice treated intravenously with cord factor were protected against an intravenous challenge with the virulent H37Rv strain of M. tuberculosis. The cellular response in the lungs of mice to intraperitoneal injections of living BCG and cord factor was very weak compared with that after intravenous injections. Intraperitoneal vaccination of mice with cord factor did not protect the mice against a challenge with virulent tubercle bacilli. Mice vaccinated intraperitoneally with BCG were immunized although no granulomas, or very few, were present in the lungs at the time of the challenge. The significance of the cellular response induced by cord factor is discussed.