Bacillus Calmette-Guérin Substrains Research Articles

Procedure for preparation, certification, and storage of a seed lot of the <i>Mycobacterium bovis</i> BCG-I (Russia) vaccine strain

To date, the Bacillus Calmette–Guérin (BCG) vaccine has been the only medicinal product for active mass childhood immunisation against tuberculosis in the Russian Federation. Industrial-scale batches of the BCG vaccine are manufactured using a seed-lot system, which provides for producing the vaccine for civil circulation from a single batch of seed material, a lyophilisate of Mycobacterium bovis BCG. National and international documents touch upon the evaluation of BCG vaccine seed material in terms of its quality attributes in small separate sections containing brief descriptions and/or lists of attributes and control methods. It is relevant to bring together the information on receipt, certification, and storage of the inoculum (the seed lot) for production of the Russian BCG vaccine. The aim of the study was a comparative assessment of the main characteristics of and control methods for the inoculum of the Russian vaccine strain, M. bovis BCG-I, set out in the national and international requirements for BCG vaccines. The article summarises literature data on the history of BCG substrains and the variability of their characteristics and presents a brief account of the origin of the Russian BCG-I substrain. It considers the control methods specified in the national and international requirements for the inoculum for the BCG vaccine. The study demonstrated the practical possibility of identifying BCG down to the substrain level with subsequent determination of genetic properties that characterise genomic stability of the substrain. The article presents the results of the comparative analysis of data on stability of lyophilisates of M. bovis BCG-I seed lots (Russia). Particular attention is paid to biological methods for controlling the seed lot (determination of residual virulence, including BCG survival) and the immunobiological method for controlling BCG for the absence of the genes responsible for virulence antigen expression (animal skin tests with Diaskintest®). The authors concluded that the control of stability of genetic and biological properties throughout the entire period of seed lot production and storage makes it possible to obtain BCG vaccines that meet all the regulatory requirements.

BCG Moreau Polish Substrain Infections in Patients With Inborn Errors of Immunity: 40 Years of Experience in the Department of Immunology, Children's Memorial Health Institute, Warsaw.

ObjectiveWe aimed to assess BCG (Bacillus Calmette-Guérin) complications in patients with Inborn Errors of Immunity (IEI), according to the inherited disorders and associated immunological defects, as well as the different BCG substrains.MaterialWe studied adverse reactions to the locally-produced BCG Moreau vaccine, analyzed in patients with IEI diagnosed between 1980 and 2020 in the Department of Immunology, Children's Memorial Health Institute (CMHI), Warsaw. These results were compared with previously published studies.ResultsSignificantly fewer disseminated BCG infections (BCGosis) were found in 11 of 72 (15%) SCID (Severe Combined Immunodeficiency) NK (Natural Killer)-phenotype patients, when compared with the 119 out of 349 (34%) (p = 0.0012) patients with SCID with BCG in other countries. Significantly fewer deaths caused by BCGosis were observed (p = 0.0402). A significantly higher number of hematopoietic stem cell transplantations (HSCTs) were performed in the CMHI study (p = 0.00001). BCGosis was found in six patients with Mendelian susceptibility to mycobacterial diseases (MSMD). Other patients with IEI prone to BCG complications, such as CGD (Chronic Granulomatous Disease), showed no case of BCGosis.ConclusionThe BCG Moreau substrain vaccine, produced in Poland since 1955, showed genetic differences with its parental Brazilian substrain together with a superior clinical safety profile in comparison with the other BCG substrains, with no BCGosis in patients with IEI other than SCID and MSMD. Our data also confirmed significantly fewer cases of BCGosis and deaths caused by BCG infection in patients with SCID with this vaccine substrain. Finally, they confirmed the protecting role of NK cells, probably via their production of IFN-γ.

Treatment options and results of adjuvant treatment in nonmuscle-invasive bladder cancer (NMIBC) during the Bacillus Calmette-Guérin shortage.

Given the worldwide shortage of Bacillus Calmette-Guérin (BCG), we review the efficacy of alternative BCG application schedules, doses or strains and intravesical chemotherapy in patients with nonmuscle-invasive bladder cancer (NMIBC). Modifying BCG schedules by reducing the dose is preferable to reducing the frequency of BCG that increases recurrence rates and should be avoided if possible. Changing the BCG substrain represents a reasonable option, as current evidence does not suggest different oncological outcomes with specific BCG substrains. Mitomycin C (MMC) alone is inferior to BCG with maintenance, but promising results have been demonstrated when used with chemohyperthermia and electromotive drug administration. Several other intravesical chemotherapies including Gemcitabine and Epirubicin should be used when both BCG and MMC are in short supply. In case of BCG shortage, much will depend on the severity and length of the BCG shortage, but our review supports several solutions: First, we recommend contacting the local pharmacist or BCG supplier to consider alternative BCG strains or sterile splitting of BCG doses. In the complete absence of BCG, consideration should be given to MMC with chemohyperthermia or electromotive drug administration where available or other intravesical chemotherapy. High-risk patients should be considered for cystectomy.

Clinical and molecular insights into BCG immunotherapy for melanoma.

The incidence of cutaneous melanoma and the mortality rate of advanced melanoma patients continue to rise globally. Despite the recent success of immunotherapy including ipilimumab and pembrolizumab checkpoint inhibitors, a large proportion of patients are refractory to such treatment modalities. The application of mycobacteria such as Bacillus Calmette-Guérin (BCG) in the treatment of various malignancies, including cutaneous melanoma, has been clearly demonstrated after almost a century of observations and experimentation. Intralesional BCG (IL-BCG) immunotherapy is a highly efficient and cost-effective treatment option for inoperable stage III in-transit melanoma, as recommended in the National Comprehensive Cancer Network Guidelines. IL-BCG has shown great efficacy in the regression of directly injected metastatic melanoma lesions, as well as distal noninjected nodules in immunocompetent patients. Clinical and preclinical studies have shown that BCG serves as a strong immune modulator, inducing the recruitment of various immune cells that contribute to antitumour immunity. However, the specific mechanism of BCG-mediated tumour immunity remains poorly understood. Comparative genome analyses have revealed that different BCG strains exhibit distinct immunological activity and virulence, which might impact the therapeutic response and clinical outcome of patients. In this review, we discuss the immunostimulatory potential of different BCG substrains and highlight clinical studies utilizing BCG immunotherapy for the treatment of cutaneous melanoma. Furthermore, the review focuses on the cellular and molecular mechanisms of the BCG-induced immune responses of both the innate and adaptive arms of the immune system. Furthermore, the review discussed the administration of BCG as a monotherapy or in combination with other immunotherapeutic or chemotherapeutic agents.

Ruptured aortic aneurysm due to Mycobacterium bovis BCG with a delayed bacteriological diagnosis due to false negative result of the MPB 64 immunochromatographic assay

BackgroundAdjuvant therapy with bacillus Calmette–Guerin (BCG), a live attenuated strain of Mycobacterium bovis, has become the treatment of choice for low-risk superficial bladder carcinoma following transurethral resection of the bladder. Complications following vesical BCG instillations are uncommon but, in some cases, severe side-effects can occur such as sepsis or mycotic aneurysm. Besides usual laboratory techniques used for the diagnosis of Mycobacterium tuberculosis complex (MTBC) infections (smear microscopy and cultures), commercial immunochromatographic assays detecting MBP64, a 24 kDa M. tuberculosis complex-specific secretory protein, can rapidly distinguish MTBC and non-tuberculosis mycobacteria (NTM). MPB64 is found in M. tuberculosis, M. bovis and some but not all substrains of M.bovis BCG. Therefore, these immunochromatographic tests can lead to false negative results and delayed bacteriological diagnosis depending on the presence or absence of MPB64 protein in BCG substrains used for intravesical therapy.Case presentationWe report the case of a 78-year-old male patient who was admitted to the hospital because of a 1-month history of unexplained fever, thrill, weight-loss and general malaise. His past medical history was marked by a non-muscle-invasive bladder carcinoma treated by transurethral resection followed by BCG instillations (Oncotice, Merck, USA). The patient was initially treated for a urinary tract infection but as fever persists after 72 h of antibiotherapy, urinary tract ultrasound was performed and revealed a large abdominal aortic aneurysm confirmed by computed tomography. Surgery was performed after multidisciplinary discussion. Direct smear of perioperative samples revealed acid-fast bacilli and both solid and liquid cultures were massively positive. Rapid identification of the positive mycobacterial culture was performed using an immunochromatographic assay based on the detection of the Mycobacterium tuberculosis MPB 64 antigen. The result was negative for Mycobacterium tuberculosis complex. After review of the medical record, a polymerase chain reaction (PCR) was performed and gave a positive result for M. tuberculosis complex. Anti-tuberculosis therapy was started immediately and the patient evolved favorably.ConclusionsThrough this case, we showed how the utilisation of MPB64 immunochromatographic assays can provide misleading information due to the variable presence of this protein among the different BCG strains. This case further illustrates the utility of rapid TB complex-specific PCR assays which provide a more reliable identification of all MTBC species.

S&T-47 A NETWORK META-ANALYSIS OF INTRAVESICAL BCG SUBSTRAIN EFFICACY IN THE TREATMENT OF NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyScience & Technology Posters1 Apr 2016S&T-47 A NETWORK META-ANALYSIS OF INTRAVESICAL BCG SUBSTRAIN EFFICACY IN THE TREATMENT OF NON-MUSCLE INVASIVE BLADDER CANCER Brock Boehm, John Cornell, Michael Liss, Dharam Kaushik, and Robert Svatek Brock BoehmBrock Boehm More articles by this author , John CornellJohn Cornell More articles by this author , Michael LissMichael Liss More articles by this author , Dharam KaushikDharam Kaushik More articles by this author , and Robert SvatekRobert Svatek More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2876AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bacillus Calmette-Guerin (BCG) is not a defined strain, but a family of regional substrains with marked genetic differences that influence phenotype, immunogenicity, and reactogenicity. The influence of BCG substrains on efficacy in non-muscle invasive bladder cancer (NMIBC) is not defined. Although a number of BCG substrains exist, recent manufacturing shortages have adversely affected patients in the U.S. due to nationwide dependence on the TICE substrain. We sought to explore the efficacy of BCG on tumor recurrence rates across BCG substrains using network meta-analyses. METHODS MEDLINE served as the primary data source, with search from inception to April 2014 for clinical trials involving patients with NMIBC receiving BCG. Additionally, bibliographies were manually searched for related references. Network meta-analyses were performed based on all possible comparators and using intravesical chemotherapy as the common comparator (Network in Figure). Probabilistic rank ordering was derived using surface under the cumulative ranking curve (SUCRA). RESULTS In analyses using all possible comparators (n=65 trials, 12,246 patients, 9 substrains), TICE (OR = 0.57, 95% CI 0.38-0.86) and Connaught (OR = 0.60, 95%CI 0.40-0.89) substrains demonstrated significant decrease in recurrence compared to chemotherapy. In analyses with chemotherapy as common comparator (n=29 trials, 5,837 patients, 5 substrains), 3 substrains demonstrated significant decrease in recurrence: Tokyo 127 (OR 0.39, 95% CI 0.16-0.93), Pasteur (OR 0.49, 95% CI 0.28-0.86), and TICE (OR 0.61, 95% CI 0.40-0.93). No significant differences were observed in recurrence rates when comparing Connaught and TICE substrains. Probabilities of most effective treatment based on SUCRA criteria were 50.7% (Tokyo 127), 24.5% (Pasteur), 15% (TICE), and 6.2% (Connaught). CONCLUSIONS When compared against chemotherapy, BCG substrain selection influences bladder cancer recurrence rates. Tokyo 127 substrain was identified as having the highest probability for superiority. However, no definitive conclusions are reached regarding superiority and head-to-head trials are greatly needed to further understand the importance of substrains in determining BCG efficacy. © 2016FiguresReferencesRelatedDetailsCited byMeeks J, Lerner S and Svatek R (2016) Bacillus Calmette-Guérin Manufacturing and SWOG S1602 Intergroup Clinical TrialJournal of Urology, VOL. 197, NO. 3 Part 1, (538-540), Online publication date: 1-Mar-2017. Volume 195Issue 4SApril 2016Page: e327 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Brock Boehm More articles by this author John Cornell More articles by this author Michael Liss More articles by this author Dharam Kaushik More articles by this author Robert Svatek More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Moreau Strain of Bacillus Calmette-Guerin (BCG) for High-Risk Non-Muscle Invasive Bladder Cancer: An Alternative during Worldwide BCG Shortage?

Background: Bacillus Calmette-Guerin (BCG) is the standard of care for adjuvant intravesical instillation therapy for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC) after complete transurethral resection. Increasing evidence suggests that there are marked differences in outcomes according to BCG substrains. BCG-Moreau was recently introduced to the European market to cover the issue of BCG shortage, but there are little data regarding the oncologic efficacy. Methods: We retrospectively analyzed 295 consecutive patients, who received adjuvant intravesical instillation therapy with BCG-Moreau for intermediate- and high-risk NMIBC between October 2007 and April 2013 at a single institution. The end points of this study were time to first recurrence and progression to muscle-invasive disease. Results: Median age was 66 years (interquartile range 59-74, mean 65.9 years). According to the EAU risk group, 76 patients presented with intermediate-risk and 219 patients with high-risk NMIBC. The 5-year recurrence-free survival and progression-free survival rate was 64.8% (95% CI 52.8-74.4) and 81.4% (95% CI 65.2-90.2), respectively. Conclusions: BCG-Moreau is an effective substrain for adjuvant instillation therapies of NMIBC, and outcomes appear to be comparable to series using other substrains. During worldwide shortage of BCG-TICE, Connaught and RIVM, BCG-Moreau may serve as an equally effective alternative.

A DNA vaccine expressing CFP21 and MPT64 fusion protein enhances BCG-induced protective immunity against Mycobacterium tuberculosis infection in mice

The efficacy of Bacillus Calmette-Guérin (BCG) vaccine in preventing adult tuberculosis (TB) is highly variable. Genetic differences between BCG vaccine substrains, which can be divided into early strains and late strains based on the loss of region of difference two (RD2), may result in the variability and BCG substrains. The effect of lack of RD2 on the protective efficacy of BCG substrains against TB remains unknown. In this study, we demonstrated that CFP21 and MPT64(rCM) fusion protein, encoded by RD2 of Mycobacterium tuberculosis, could stimulate higher level of interferon (IFN)-γ in tuberculin skin test (TST)-positive healthy population than in TST-negative healthy population. Compared with naive mice challenged with virulent M. tuberculosis H37Rv, C57BL/6 mice vaccinated with pcD2164 DNA expressing rCM protein resulted in a greater decrease in the bacterial load of lung. Moreover, pcD2164 could boost the protective immunity in mice primed by BCG than BCG alone or DNA vaccination alone, as evidenced by lower bacterial load in the lung tissue and reduced lung pathology. The protection induced by BCG prime-DNA vaccine boost strategy was associated with significant increases in rCM protein-specific IFN-γ. Therefore, our results clearly indicate that the loss of RD2 has an important influence on the protective efficacy of different BCG substrains. These findings will benefit the optimal choice of BCG substrain for neonatal immunization and rational design of new vaccines for the prevention of TB.

Regions of Differences Encoded Antigens as Targets for Immunodiagnosis of Tuberculosis in Humans

Tuberculosis is one of the major global health problems causing nearly 2 million deaths every year. It continues to be a leading cause of morbidity and mortality in developing countries. Accurate early diagnosis and proper treatment can control the spread of tuberculosis in the community. Currently used diagnostic tests have certain limitations such as low sensitivity and suboptimal turn-around times. Hence, introduction of diagnostic methods that are comparatively more sensitive and specific can increase the efficiency of strategies to control tuberculosis. In recent years, there has been a remarkable progress in identifying new and potentially useful antigens for diagnosis of both latent and active tuberculosis. Regions of differences (RD) encoded proteins are among such promising candidate antigens (RD antigens). Some of these antigens are encoded by regions of differences located in the genome of Mycobacterium tuberculosis, M. africanum, M. bovis but are absent in all the Bacillus Calmette Guerin substrains and many of the environmental mycobacteria. Over the past few years, RD antigens, particularly RD-based diagnostic methods such as improved tuberculin skin testing, interferon-gamma release assays, and RD1-based serological assays are being tested and have shown promising results. This article provides an overview of the use of RD antigens in the immunodiagnosis of tuberculosis infection and disease.

Identification of two subpopulations of Bacillus Calmette-Guérin (BCG) Tokyo172 substrain with different RD16 regions

Two types of colonies with different morphologies (smooth: S and rough: R) formed when Bacillus Calmette-Guérin (BCG) Tokyo172 substrain was cultured on Middlebrook 7H10 agar medium, and their genotypes were analyzed by multiplex PCR on five RD regions and SenX3-RegX3. In most cases these two colony types had different genotypes, i.e., S colonies showed a characteristic 22bp deletion in Rv3405c of the RD16 region (type I), and R colonies did not have this deletion (type II) similar to many other BCG substrains. Thus, there was a strong relationship between colony morphology and genotype. Both genotypes were found in every Tokyo172 preparation tested, including the seed lot for production, the origin of seed lot from the 1960s and ATCC BCG Japan. Type I was always in the majority. It was suggested that types I and II constituted independent subpopulations within the Tokyo172 substrain. Type I was shown to have a growth advantage over type II both on culture media and in mice organs.

Modified multiplex PCR for identification of Bacillus Calmette–Guérin substrain Tokyo among clinical isolates

When an adverse reaction occurs and a mycobacterial species is isolated from a person vaccinated with Bacillus Calmette–Guérin (BCG) or a patient receiving BCG immunotherapy, it is essential to identify whether the isolate is BCG or another mycobacterial species. However, differentiation of BCG from other members of Mycobacterium tuberculosis complex has been very difficult. Using several specific primer-pairs, Bedwell et al. [Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine 2001; 19: 2146–51] recently reported that they could distinguish BCG substrains. We modified their method to improve differentiation of Tokyo 172 from other members of the M. tuberculosis complex, and examined whether this modified method could be applied to clinical isolates. Our method clearly identified BCG substrain (BCG Tokyo 172) among clinical isolates and easily distinguished between M. tuberculosis and wild-type Mycobacterium bovis.

The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue.

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

Characterization of exochelins of the Mycobacterium bovis type strain and BCG substrains.

Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of Mycobacterium bovis, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of M. bovis, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The M. bovis type strain, five substrains of M. bovis BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent Mycobacterium tuberculosis all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in M. tuberculosis, intermediate in M. bovis, and smallest in M. bovis BCG.

Interaction between fibronectin-bearing surfaces and Bacillus Calmette-Guérin (BCG) or gelatin microparticles.

Gelatin, prepared commercially by degradation of animal collagen, was studied to see whether it had an affinity for fibronectin, which has a known affinity for collagen, and whether gelatin-based drugs could be used to target fibronectin-excreting tumours. Bacillus Calmette-Guérin (BCG) vaccine, an attenuated strain of Mycobacterium bovis, is currently the most effective treatment for superficial transitional cell carcinoma of the bladder. The living cells of the BCG vaccine associate with the fibronectin-bearing surfaces of the tumour. Using a multi-well culture plate technique, gelatin microparticles were shown to be adsorbed onto murine S180 sarcoma cells and this reaction was substantially inhibited by the addition of human plasma fibronectin. The avidities of various BCG substrains and gelatin microparticles for glass-bound fibronectin were measured and the association constants determined. The gelatin microparticles associated with the fibronectin with equal avidity as the BCG cells. The results suggest that this model system may allow the investigation of gelatin-based drug delivery devices capable of targeting fibronectin-bearing surfaces associated with some tumours.