Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.     

Bacillus Calmette-Guérin Failures and Beyond: Contemporary Management of Non-Muscle-Invasive Bladder Cancer

In the United States, bacillus Calmette-Guérin (BCG) is the treatment most used for superficial bladder cancer. Patients with carcinoma in situ (CIS) treated with intravesical BCG plus interferon have a 60% to 70% chance of a complete and durable response if they were never treated with BCG or if they failed only 1 prior induction or relapsed more than a year from induction. Intravesical gemcitabine is safe, but its usefulness for BCG-refractory patients is unclear. Valrubicin, approved for intravesical treatment of BCG-refractory CIS of the bladder, has efficacy and acceptable toxicity. Cystectomy should be considered in high-risk, non-muscle-invasive cancer, particularly if intravesical therapy failed.Key words: Bladder cancer, Bacillus Calmette-Guérin, Cystectomy, Gemcitabine, ValrubicinThe management of superficial bladder cancer requires a clear understanding of diagnostic, prognostic, and treatment parameters. Cystoscopy remains the gold standard for detection, but despite good visualization and resection, bladder cancers recur frequently. Because of this, a variety of drugs has been used intravesically. The most commonly used drug in the United States is bacillus Calmette-Guérin (BCG), both with and without interferon and mitomycin.

Cytokine gene polymorphisms can alter the effect of Bacillus Calmette-Guérin (BCG) immunotherapy

Various types of cancer are more frequent in men than women, and bladder cancer is one of the most common of these. Intravesical instillation of Bacillus Calmette-Guérin (BCG) after transurethral resection is the most effective treatment for superficial bladder cancers. The main aim of this study was to investigate for possible links between cytokine gene polymorphisms and different outcomes after BCG immunotherapy. Sixty patients who had been diagnosed with transitional cell cancer were investigated. All genotyping experiments were performed using polymerase chain reaction sequence-specific primers and a commercially available kit. The genes investigated were those that code for interleukin (IL)-1alpha, IL-1beta, IL-1R, IL-1RA, IL-4RA, IL-2, IL-4, IL-6, IL-10, IL-12, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha). Analyses of the data identified TGF-beta codon 25 GG (92.85% vs. 64.44%, p=0.04, OR=7.17), IL-4 -1098 GG (16.6% vs. 0.0%, p=0.05, OR=18.33), IL-10 -1082 GG (28.5% vs. 6.8%, p=0.05, OR=5.47), and IL-10 -1082 GCC/GCC (28.57% vs. 4.5%, p=0.025, OR=8.4) polymorphisms as risk factors for progression of bladder cancer.     

Progression of pulmonary tuberculosis and efficiency of bacillus Calmette-Guérin vaccination are genetically controlled via a common sst1-mediated mechanism of innate immunity

Using a mouse model for genetic analysis of host resistance to virulent Mycobacterium tuberculosis, we have identified a genetic locus sst1 on mouse chromosome 1, which controls progression of pulmonary tuberculosis. In vitro, this locus had an effect on macrophage-mediated control of two intracellular bacterial pathogens, M. tuberculosis and Listeria monocytogenes. In this report, we investigated a specific function of the sst1 locus in antituberculosis immunity in vivo, especially its role in control of pulmonary tuberculosis. We found that the sst1 locus affected neither activation of Th1 cytokine-producing T lymphocytes, nor their migration to the lungs, but rather controlled an inducible NO synthase-independent mechanism of innate immunity. Although the sst1(S) macrophages responded to stimulation with IFN-gamma in vitro, their responsiveness to activation by T cells was impaired. Boosting T cell-mediated immunity by live attenuated vaccine Mycobacterium bovis bacillus Calmette-Guérin or the adoptive transfer of mycobacteria-activated CD4(+) T lymphocytes had positive systemic effect, but failed to improve control of tuberculosis infection specifically in the lungs of the sst1(S) animals. Thus, in the mouse model of tuberculosis, a common genetic mechanism of innate immunity mediated control of tuberculosis progression in the lungs and the efficiency of antituberculosis vaccine. Our data suggest that in immunocompetent humans the development of pulmonary tuberculosis and the failure of the existing vaccine to protect against it, in some cases, may be explained by a similar defect in a conserved inducible NO synthase-independent mechanism of innate immunity, either inherited or acquired.     

Comparative tuberculosis (TB) prevention effectiveness in children of Bacillus Calmette-Guérin (BCG) vaccines from different sources, Kazakhstan

Background

Except during a 1-year period when BCG vaccine was not routinely administered, annual coverage of infants with Bacillus Calmette-Guérin (BCG) in Kazakhstan since 2002 has exceeded 95%. BCG preparations from different sources (Japan, Serbia, and Russia) or none were used exclusively in comparable 7-month time-frames, September through March, in 4 successive years beginning in 2002. Our objective was to assess relative effectiveness of BCG immunization.

Methods/Findings

We compared outcomes of birth cohorts from the 4 time-frames retrospectively. Three cohorts received vaccine from one of three manufacturers exclusively, and one cohort was not vaccinated. Cohorts were followed for 3 years for notifications of clinical TB and of culture-confirmed TB, and for 21 months for TB meningitis notifications. Prevention effectiveness based on relative risk of TB incidence was calculated for each vaccinated cohort compared to the non-vaccinated cohort.Although there were differences in prevention effectiveness observed among the three BCG vaccines, all were protective. The Japanese vaccine (currently used in Kazakhstan), the Serbian vaccine, and the Russian vaccine respectively were 69%, 43%, and 22% effective with respect to clinical TB notifications, and 92%, 82%, and 51% effective with respect to culture confirmed TB. All three vaccines were >70% effective with respect to TB meningitis.

Limitations

Potential limitations included considerations that 1) the methodology used was retrospective, 2) multiple risk factors could have varied between cohorts and affected prevention effectiveness measures, 3) most cases were clinically diagnosed, and TB culture-positive case numbers and TB meningitis case numbers were sparse, and 4) small variations in reported population TB burden could have affected relative risk of exposure for cohorts.

Conclusions/Significance

All three BCG vaccines evaluated were protective against TB, and prevention effectiveness varied by manufacturer. When setting national immunization policy, consideration should be given to prevention effectiveness of BCG preparations.     

Ion-exchange chromatography method for the purification of genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin

The goal of this study was to provide practical strategies for purifying genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin (BCG-DNA) by ion-exchange chromatography. A multistep process was developed to purify BCG-DNA. The process consisted of sonication, heating, trypsin digestion, ion-exchange chromatography, gel-filter chromatography, and lyophilization. After ion-exchange chromatography, BCG-DNA was highly purified and possessed potent biological effects. The methods described were efficient and had good reproducibility. Further, this was the first reported chromatography method to purify BCG-DNA.     

Effect of Bacillus Calmette-Guérin infection on delayed footpad reaction to Listeria monocytogenes

The role of peritoneal macrophages induced by Bacillus Calmette-Guérin (BCG) in the induction of immune responses to Listeria monocytogenes was studied in mice. The peritoneal macrophages from mice treated with BCG 14 days previously contained a high proportion of Ia-bearing macrophages (approximately 56%) and the cells showed not only a high level of listericidal activity but also a strong ability for presentation of listerial antigen to Listeria-immune T cells. An intraperitoneal inoculation with a low dose of Listeria, which can induce the maximal level of delayed footpad reaction (DFR) and positive migration inhibitory activity of macrophages in untreated mice, did not induce a detectable level of such responses in BCG-treated mice. The bacterial growth at an early stage of infection was suppressed by scavenger macrophages in these mice. On the other hand, BCG-treated mice showed the early development of DFR and macrophage migration inhibitory activity after an inoculation with a high dose of Listeria. It is revealed in transfer experiments that Listeria-pulsed peritoneal exudate cells induced by BCG elicited the highest level of DFR and positive migration inhibition of macrophages in normal mice at the earlier period of injection compared with Listeria-pulsed resident peritoneal cells. These results suggested that the increased activities of macrophages acting as scavenger cells and as antigen-presenting cells play important roles in the modification of immune responses to Listeria in BCG-treated mice.     

Influence of Mycobacterium bovis bacillus Calmette-Guérin on antibody and cytokine responses to human neonatal vaccination.

The immaturity of the immune system increases the susceptibility of young infants to infectious diseases and prevents the induction of protective immune responses by vaccines. We previously reported that Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination induces a potent Th1 response to mycobacterial Ags in newborns. In this study, we evaluated the influence of BCG on the response to unrelated vaccines given in early life. Newborns were randomly allocated to one of three study groups receiving BCG at birth, when infants received their first dose of hepatitis B and oral polio vaccines; at 2 mo of age, when infants received their first dose of diphtheria and tetanus vaccines; or at 4.5 mo of age, when immune responses to vaccines were measured. Administration of BCG at the time of priming markedly increased the cellular and Ab responses to the hepatitis B vaccine, but had only a limited influence on the cytokine response to tetanus toxoid and no effect on the Ab responses to tetanus and diphtheria toxoids. Although BCG induced a potent Th1-type response to mycobacterial Ags, it promoted the production of both Th1- and Th2-type cytokines in response to unrelated vaccines. The effect of BCG was apparent at the systemic level, as it increased the Ab response to oral polio vaccine. These results demonstrate that BCG influences the immune response to unrelated Ags in early life, likely through its influence on the maturation of dendritic cells.     

Bacillus Calmette-Guérin and TLR4 agonist prevent cardiovascular hypertrophy and fibrosis by regulating immune microenvironment

Hypertension-induced cardiovascular hypertrophy and fibrosis are critical in the development of heart failure. The activity of TLRs has been found to be involved in the development of pressure overload-induced myocardial hypertrophy and cardiac fibrosis. We wondered whether vaccine bacillus Calmette-Guérin (BCG), which activated TLR4 to elicit immune responses, modulated the pressure overload-stimulated cardiovascular hypertrophy and cardiac fibrosis in the murine models of abdominal aortic constriction (AAC)-induced hypertension. Before or after AAC, animals received BCG, TLR4 agonist, IFN-gamma, or TLR4 antagonist i.p. BCG and TLR4 agonist significantly prevented AAC-induced cardiovascular hypertrophy and reactive cardiac fibrosis with no changes in hemodynamics. Moreover, TLR4 antagonist reversed the BCG- and TLR4 agonist-induced actions of anti-cardiovascular hypertrophy and cardiac fibrosis. BCG decreased the expression of TLR2 or TLR4 on the heart tissue but TLR4 agonist increased the expression of TLR2 or TLR4 on the immune cells that infiltrate into the heart tissue. This led to an increased expression ratio of IFN-gamma/TGF-beta in the heart. The cardiac protective effects of BCG and TLR4 agonist are related to their regulation of ERK-Akt and p38-NF-kappaB signal pathways in the heart. In conclusion, the activity of TLR4 plays a critical role in the mediation of pressure overload-induced myocardial hypertrophy and fibrosis. The regulation of immune responses by BCG and TLR4 agonist has a great potential for the prevention and treatment of hypertension-induced myocardial hypertrophy and cardiac fibrosis.     

Association of polymorphisms in oxidative stress genes with clinical outcomes for bladder cancer treated with Bacillus Calmette-Guérin

Genetic polymorphisms in oxidative stress pathway genes may contribute to carcinogenesis, disease recurrence, treatment response, and clinical outcomes. We applied a pathway-based approach to determine the effects of multiple single nucleotide polymorphisms (SNPs) within this pathway on clinical outcomes in non-muscle-invasive bladder cancer (NMIBC) patients treated with Bacillus Calmette-Guérin (BCG). We genotyped 276 SNPs in 38 genes and evaluated their associations with clinical outcomes in 421 NMIBC patients. Twenty-eight SNPs were associated with recurrence in the BCG-treated group (P<0.05). Six SNPs, including five in NEIL2 gene from the overall and BCG group remained significantly associated with recurrence after multiple comparison adjustments (q<0.1). Cumulative unfavorable genotype analysis showed that the risk of recurrence increased with increasing number of unfavorable genotypes. In the analysis of risk factors associated with progression to disease, rs3890995 in UNG, remained significant after adjustment for multiple comparison (q<0.1). These results support the hypothesis that genetic variations in host oxidative stress genes in NMIBC patients may affect response to therapy with BCG.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Cytokine-mediated antitumor effect of bacillus Calmette-Guérin on tumor cells in vitro

Intravesical instillation therapy of bacillus Calmette-Guérin (BCG) is a useful modality for recurrent superficial transitional-cell carcinoma (TCC) of the urinary bladder. The mechanism of BCG effect has not yet been well characterized. BCG was tested in vitro for cytokine-mediated antiproliferative activity against T24 and KK47 cells (cell lines established from human TCC of the urinary bladder), and ACHN cells (cell line established from human renal cell carcinoma) using a modified human tumor clonogenic assay. Continuous exposure of cells to BCG at concentrations of more than 5 g/ml in the presence of peripheral blood mononuclear cells (PBMC) consisting of a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors, significantly inhibited colony formation of T24 and ACHN cells in comparison with growth inhibition in the absence of PBMC (P<0.05). Slightly inhibited colony formation was observed with KK47 cells under the same conditions. At the same time various cytokines were measured in supernatants when BCG and the same conditioned PBMC were co-cultured. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) were detected at markedly high levels at 24 h, and interferon (IFN) was detected at 120 h. IL-2 and macrophage-colony-stimulating factor were not detected. Neutralizing anti-TNF monoclonal antibody significantly reduced the anti-proliferative activity of ACHN cells, and anti-IFN antibody reduced that of T24 cells. The results obtained suggest that cytokines mediated by BCG play an important role in the antitumor activity of BCG and that the sensitivity of bladder cancer cells to the cytokines induced by BCG may differ considerably.     

Growth inhibition of HeLa cell by internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Tokyo

Background  

Intravesical BCG immunotherapy is effective for preventing recurrence and progression in none muscle-invasive bladder cancer but the dosing schedule and duration of treatment remain empirical. The mechanisms by which intravesical BCG treatment mediates antitumor activity are currently poorly understood.     

Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.