Bovis Bacillus Calmette-Guérin Strain Research Articles

PD42-02 THE EFFECTS OF RECOMBINANT BACILLUS CALMETTE-GUERIN RESISTANT TO ANTIMICROBIAL PEPTIDES ON ORTHOTOPIC BLADDER CANCER MOUSE MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (PD42)1 Sep 2021PD42-02 THE EFFECTS OF RECOMBINANT BACILLUS CALMETTE-GUERIN RESISTANT TO ANTIMICROBIAL PEPTIDES ON ORTHOTOPIC BLADDER CANCER MOUSE MODEL Jae Hun Shim, Joonhee Gook, Se Young Choi, Byung Hoon Chi, Jin Wook Kim, Tae-Hyoung Kim, Soon Chul Myung, and In Ho Chang Jae Hun ShimJae Hun Shim More articles by this author , Joonhee GookJoonhee Gook More articles by this author , Se Young ChoiSe Young Choi More articles by this author , Byung Hoon ChiByung Hoon Chi More articles by this author , Jin Wook KimJin Wook Kim More articles by this author , Tae-Hyoung KimTae-Hyoung Kim More articles by this author , Soon Chul MyungSoon Chul Myung More articles by this author , and In Ho ChangIn Ho Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002056.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is the most widely used bladder cancer immunotherapy, innate immune responses involving antimicrobial peptides (AMPs) cause BCG failure. Here, we developed genetically modified recombinant BCG (rBCG) strains which escape AMPs and evaluate the efficacy and effects of rBCG. METHODS: We constructed rBCG strains expressing Streptococcal inhibitor of complement (Sic), which confers resistance to human α-defensin-1 and cathelicidin, and d-alanyl carrier protein ligase (dltA), which confers resistance to cationic AMPs. Sic and dltA were separately cloned into the pMV306 plasmid and introduced into BCG via electroporation. The efficacy of the Sic and dltA gene electroporation into BCG was evaluated by quantitative real-time polymerase chane reaction (qRT-PCR). The internalization rates and anti-cancer effects of the rBCG strains containing Sic (rBCG-Sic) and dltA (rBCG-dltA) was evaluated by the orthotopic bladder cancer mouse model. RESULTS: The cycle quantification (Cq) values of rBCG-Sic (y=-4.8823x+13.645, R2=0.9996) and rBCG-dltA (y=-5.438x+11.641, R2=0.9995) were inverse correlations to the amount of Sic and dltA genes dose dependently. The mean introduction proportions of Sic and dltA genes into BCG by electroporation were 22.2%, 27.5% and showed constant efficacy. In the orthotopic bladder cancer mouse model, the relative internalization number of rBCG-Sic, and rBCG-dltA into bladder cell in mouse bladder were higher than that of BCG and the tumor volume at rBCG-Sic were lower than at BCG and rBCG-dltA at 11, 14 m and 18 days. CONCLUSIONS: Our results showed that constructed rBCG-Sic and rBCG-dltA by electroporation and the rBCG-Sic and rBCG-dltA can effectively escape BCG-stimulated AMPs, and significantly improved immunotherapeutic tools to treat bladder cancer in orthotopic bladder cancer mouse model. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e724-e724 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jae Hun Shim More articles by this author Joonhee Gook More articles by this author Se Young Choi More articles by this author Byung Hoon Chi More articles by this author Jin Wook Kim More articles by this author Tae-Hyoung Kim More articles by this author Soon Chul Myung More articles by this author In Ho Chang More articles by this author Expand All Advertisement Loading ...

Phenotypic and Genotypic Drug Susceptibility Assessment of Mycobacterium bovis Bacillus Calmette-Guérin Clinical Strains.

PurposeMycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine licensed against tuberculosis. Despite the protection offered by the vaccine, in some circumstances children and immunocompromised individuals can develop associated infections, known as BCGitis. Drug susceptibility patterns of BCG clinical strains have rarely been described. We aimed to describe the susceptibility pattern of BCG clinical strains isolated in two different countries.MethodsWe performed culture-based drug susceptibility testing of thirty one BCG strains isolated from patients in Brazil (n=5, 16%) and Argentina (n=26, 84%) using the broth micro-dilution method (phenotypic method). Final antibiotic concentrations for susceptibility testing ranged from 0.5 to 16 mg/L for amikacin, 0.3125 to 10 mg/L for ethambutol, 0.05 to 1.6 mg/L for isoniazid and 0.25 to 8 mg/L for rifampicin, streptomycin and ofloxacin. Additionally, we compared the results with genetic data obtained by whole genome sequencing.ResultsBy using the phenotypic method we detected one strain resistant to ethambutol, three strains resistant to rifampicin and one resistant to isoniazid. Additionally, two strains that were phenotypically resistant to both isoniazid and rifampicin carried mutations in the katG and rpoB genes simultaneously.ConclusionThere is evidence of the emergence of BCG-resistant strains isolated from vaccine-related complications. We recommend drug susceptibility testing of the BCG strain causing the infection in order to prevent treatment failure.

Bacillus Calmette–Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control

ObjectiveLaboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette–Guérin (BCG)). MethodsSeven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC90. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently. ResultsOur panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance. ConclusionBCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.

Mycobacterium bovis BCG in metastatic melanoma therapy.

Melanoma is the most aggressive form of skin cancer, with a high mortality rate and with 96,480 new cases expected in 2019 in the USS. BRAFV600E, the most common driver mutation, is found in around 50% of melanomas, contributing to tumor growth, angiogenesis, and metastatic progression. Dacarbazine (DTIC), an alkylate agent, was the first chemotherapeutic agent approved by the US Food and Drug Administration (FDA) used as a standard treatment. Since then, immunotherapies have been approved for metastatic melanoma (MM) including ipilimumab and pembrolizumab checkpoint inhibitors that help decrease the risk of progression. Moreover, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) serves as an adjuvant therapy that induces the recruitment of natural killer NK, CD4+, and CD8+ T cells and contributes to antitumor immunity. BCG can be administered in combination with chemotherapeutic and immunotherapeutic agents and can be genetically manipulated to produce recombinant BCG (rBCG) strains that express heterologous proteins or overexpress immunogenic proteins, increasing the immune response and improving patient survival. In this review, we highlight several studies utilizing rBCG immunotherapy for MM in combination with other therapeutic agents.

Recombinant BCG Expressing HTI Prime and Recombinant ChAdOx1 Boost Is Safe and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice.

Despite the availability of anti-retroviral therapy, HIV-1 infection remains a massive burden on healthcare systems. Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, confers protection against meningitis and miliary tuberculosis in infants. Recombinant BCG has been used as a vaccine vehicle to express both HIV-1 and Simian Immunodeficiemcy Virus (SIV) immunogens. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVACAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic Mycobacterium bovis BCG strain (BCGΔLys) to generate the vaccine BCG.HTI2auxo.int. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed, and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for 35 bacterial generations, and that when delivered in combination with chimpanzee adenovirus (ChAd)Ox1.HTI in adult BALB/c mice, it was well tolerated and induced HIV-1-specific T-cell responses. Specifically, priming with BCG.HTI2auxo.int doubled the magnitude of the T-cell response in comparison with ChAdOx1.HTI alone while maintaining its breadth. The use of integrative expression vectors and novel HIV-1 immunogens can aid in improving mycobacterial vaccine stability as well as specific immunogenicity. This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens.

Recombinant BCG strains expressing chimeric proteins derived from Leptospira protect hamsters against leptospirosis

Leptospirosis is a zoonosis that is responsible for one million human cases per year. Fusing multiple immunogenic antigens represents a promising approach to delivering an effective vaccine against leptospirosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a potential vaccine vector due to its adjuvant properties and safety. Two chimeric genes based on genic sequences of ligANI, ligBrep, lipL32, and lemA, were individually cloned into five BioBrick vectors with different promoters (pAN, Hsp60, 18 kDa, Ag85B and Ag85B plus signal sequence) for antigen expression in BCG. Groups of ten hamsters were vaccinated with recombinant BCG (rBCG) strains in two doses of 106 CFU and challenged with 5 × LD50 of L. interrogans serovar Copenhageni. All rBCG vaccines expressing chimera 1, based on antigens LipL32, LigANI, and LemA, under the control of any promoter, protected 80–100% of the hamsters from challenge (P < 0.05) and four of them also protected from renal carrier status; for chimera 2, based on LigANI and LigBrep antigens, the only vaccine that afforded survival rates statistically different from the control was the vaccine that incorporated the pAN promoter (60% of survival). A single vaccine dose was sufficient to induce significant IgG levels by all vaccine compositions evaluated; however, humoral response was not related to protection. These findings suggest that the combination of potential vaccine candidates in chimeric antigens and the use of BCG as a live vector are promising strategies by which it is possible to obtain an effective and sterilizing vaccine against leptospirosis.

The immunotherapeutic effects of recombinant Bacillus Calmette-Guérin resistant to antimicrobial peptides on bladder cancer cells

PurposeAlthough Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the most widely used bladder cancer immunotherapy, innate immune responses involving antimicrobial peptides (AMPs) cause BCG failure and unwanted side effects. Here, we generated genetically modified BCG strains with improved immunotherapeutic effects by adding genes that confer evasion of AMPs. Materials and methodsWe constructed recombinant BCG (rBCG) strains expressing Streptococcal inhibitor of complement (Sic), which confers resistance to human α-defensin-1 and cathelicidin, and d-alanyl carrier protein ligase (dltA), which confers resistance to cationic AMPs. Sic and dltA were separately cloned into the pMV306 plasmid and introduced into BCG via electroporation. Then, the efficacy of the rBCGs was tested in a growth inhibition assay using two bladder cancer cell lines (5637, T24). ResultsWe confirmed the presence of cDNA segments corresponding to the Sic and dltA genes in total mRNA of the rBCG strains containing Sic (rBCG-Sic) and dltA (rBCG-dltA), and these rBCGs showed higher survival against AMPs. The growth inhibitory effects of rBCGs on bladder cancer cells were significantly enhanced compared to those of the parent BCG, and THP-1 migration also increased. After 8 h of infection, the levels of internalization were higher in rBCG-infected bladder cancer cells than in BCG-infected cells, and cells infected with rBCGs showed increased release of antitumor cytokines, such as IL-6/12, TNF-α, and INF-γ, resulting in inhibition of bacterial killing and immune modulation via antimicrobial peptides.Conclusions: rBCG-Sic and rBCG-dltA can effectively evade BCG-stimulated AMPs, and may be significantly improved immunotherapeutic tools to treat bladder cancer.

The patterns of in vitro cell-death and inflammatory cytokines induced by distinct BCG vaccine strains are differentially induced in human mononuclear cells

ABSTRACTTuberculosis (TB) remains among the world's leading cause of mortality. For its control, studies of TB vaccines are needed. Since live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently in use, studies on the protective role of BCG are required. In this study, we analyzed host cells purified directly from whole blood of human immunodeficiency virus (HIV)-negative volunteers, comprising adult healthy donors (HD) and neonates (umbilical cord bloods, UCB), with the aim to directly compare in vitro immune responses with distinct BCG strains in human mononuclear cells. The Moreau, Pasteur, and Danish BCG strains were used to infect mononuclear cells in vitro for 48 h; bacilli viability and cell-death were subsequently detected by flow cytometry. In addition, cell culture supernatants were used in cytokine detection assays. Overall, the Moreau BCG strain induced higher levels of apoptosis than the Pasteur and Danish BCG strains in both the HD and UCB groups (p-value < 0.05), and a human monocytic cell-line mirrored those cell-death patterns after BCG infection. The Moreau BCG strain, exclusively, induced Th1 cytokines at the highest levels in cells from adults (p-value < 0.05) when compared with both Pasteur and Danish BCG strains, whereas TGF-β1 levels were reduced significantly (p-value < 0.01) in the HD group when cells were infected with the Moreau BCG vaccine. As expected, eight out of 22 pro-inflammatory cytokines were secreted at significant levels (p-value < 0.05) above the baseline rates in all BCG-infected cell cultures, in the HD group only. When analyzing these results, we excluded confounding factors related to storage and viability of the BCG strains used. These findings suggest that Moreau BCG is a more potent immunostimulating agent than the Pasteur and Danish BCG strains. Clinical trials will be needed to confirm these findings.

The BCGΔBCG1419c strain, which produces more pellicle in vitro, improves control of chronic tuberculosis in vivo

Mycobacterium tuberculosis (Mtb) has been a threat to humans since ancient times, and it is the main causative agent of tuberculosis (TB). Until today, the only licensed vaccine against Mtb is the live attenuated M. bovis Bacillus Calmette-Guérin (BCG), which has variable levels of protection against the pulmonary form of infection. The quest for a new vaccine is a priority given the rise of multidrug-resistant Mtb around the world, as well as the tremendous burden imposed by latent TB. The objective of this study was to evaluate the immunogenicity and capacity of protection of a modified BCG strain (BCGΔBCG1419c) lacking the c-di-GMP phosphodiesterase gene BCG1419c, in diverse mice models. In a previous report, we have shown that BCGΔBCG1419c was capable of increasing biofilm production and after intravenous infection of immunocompetent mice; this strain persisted longer in lungs than parental BCG Pasteur. This led us to hypothesize that BCGΔBCG1419c might therefore possess some advantage as vaccine candidate. Our results in this report indicate that compared to conventional BCG, vaccination with BCGΔBCG1419c induced a better activation of specific T-lymphocytes population, was equally effective in preventing weight loss despite being used at lower dose, reduced tissue damage (pneumonic scores), increased local IFNγ+ T cells, and diminished bacterial burden in lungs of BALB/c mice infected intratracheally with high dose Mtb H37Rv to induce progressive TB. Moreover, vaccination with BCGΔBCG1419c improved resistance to reactivation after immunosuppression induced by corticosterone in a murine model of chronic infection similar to latent TB. Furthermore, despite showing increased persistence in immunocompetent mice, BCGΔBCG1419c was as attenuated as parental BCG in nude mice. To our knowledge, this is the first demonstration that a modified BCG vaccine candidate with increased pellicle/biofilm production has the capacity to protect against Mtb challenge in chronic and reactivation models of infection.

Deletion of nuoG from the Vaccine Candidate Mycobacterium bovis BCG ΔureC::hly Improves Protection against Tuberculosis.

ABSTRACTThe current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG, encoding NADH dehydrogenase 1 subunit G, from BCG ΔureC::hly. In vitro, deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG ΔureC::hly ΔnuoG vaccination was safer than BCG and improved protection over that of parental BCG ΔureC::hly, significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG ΔureC::hly or BCG ΔureC::hly ΔnuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG ΔureC::hly ΔnuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety.

Antitumor activity of recombinant Bacille Calmette-Guérin secreting interleukin-15-Ag85B fusion protein against bladder cancer

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is used for the treatment of bladder cancer. The recruitment of neutrophlis to the bladder after BCG instillation exerts anti-tumor activity against bladder tumor. We have recently demonstrated that interleukin (IL)-17A produced by γδ T cells played a role in the recruitment of neutrophlis to the bladder after BCG instillation. IL-15 is known to play an important role in neutrophil migration during inflammation. We previously constructed a recombinant BCG strain expressing the fusion protein of IL-15 and Ag85B (BCG-IL-15) for prevention of Mycobacterium tuberculosis infection. Here we compared the efficacy of the BCG-IL-15 in protection against bladder cancer with that of rBCG-Ag85B (BCG).Six-week-old female C57BL/6 mice were inoculated with MB49 bladder tumor cells in the bladder and subsequently intravesically inoculated with BCG or BCG-IL-15.BCG-IL-15 treatment significantly prolonged survival of mice inoculated with bladder cancer cells compared with BCG treatment. Infiltration of neutrophils was significantly elevated in BCGB-IL-15 treated mice accompanied by increased chemokines (MIP-2 and MIP-1α) in the bladder. Thus, BCG-IL-15 exerted additive effect on Infiltration of neutrophils in the bladder. BCG-IL-15 may be a promising drug for non-muscle invasive bladder cancer.

Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available for preventing tuberculosis (TB), however, BCG has varying success in preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. Our results indicated that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, and the Ag85A peptide–MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG::Ag85A were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that rBCG::Ag85A can enhance protection against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared to the BCG vaccine alone. Our results demonstrate the potential of rBCG::Ag85A as a candidate vaccine against TB.

Molecular analysis of Mycobacterium isolates from patients with BCG-induced lymphadenitis

The Bacillus Calmette-Guérin (BCG) vaccine is administered to all newborns within the first days of life for the prevention of tuberculosis (TB) in many of the developing countries, including Iran. BCG vaccine is prepared from a special strain of Mycobacterium bovis. This vaccine is safe, but local and systemic side effects are associated with BCG vaccine. Severe adverse effects of BCG vaccination are extremely rare in immunocompetent children, but lymphadenitis, which is the most common side effect of BCG vaccination in children, is seen with high frequency in Iran. Different studies have shown that the vaccine strain and its genetic variations are important in inducing these side effects. Therefore, in this study, the aim is to analyze the genetic characterizations of vaccine strains used in Iran. MethodsThe samples were collected from infants showing lymphadenitis induced by BCG vaccination. After acid-fast staining and culture of samples on Lowenstein–Jensen medium, DNA was extracted from samples using Phenol–chloroform method. Using primer for 16sRNA gene, the genus of the isolates was characterized. Then using RD1, RD14 and DU1 primers and followed by the next step using RD9, RD4 Deleted, RD4 Present and RD1 Deleted primers, the species and strains of the isolates were characterized. ResultsAll 30 samples of acid-fast bacilli that were isolated from infants with BCG complications confirmed as Mycobacterium genus using 16sRNA PCR and using RD1, RD14, DU1, RD9, RD4 Deleted, RD4 Present and RD1 Deleted primers confirmed that all isolates were M. bovis BCG strain Pasteur. ConclusionAs all of the strains isolated from the patients were confirmed as M. bovis BCG strain Pasteur, the other possible factors causing BCG complications, including BCG overdose, faulty intradermal technique, and disturbance of cellular immunity, must be considered.

Gene deletions in Mycobacterium bovis BCG stimulate increased CD8+ T cell responses.

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.

Impact of in vitro evolution on antigenic diversity of Mycobacterium bovis bacillus Calmette-Guerin (BCG)

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.

Development of a recombinant BCG based vaccine that prevents the infection with the human metapneumovirus stimulating Th1 immunity (VAC7P.975)

Abstract The human metapneumovirus (hMPV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, due to an inefficient immunological memory, hMPV infection provides limited immune protection. The inadequate immune response causes severe lung inflammation leading to airway obstruction. It is likely that an effective clearance of hMPV require a balanced Th1 type immune response, involving the participation of activated, IFN-γ secreting, T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guerin (BCG), which has been used for decades to protect newborns from tuberculosis. Here, we show that a re-infection worsens the viral illness in Balb/c mice, questioning the safety of vaccines based on attenuated virus. We also observed a Th1 pattern of cytokines animals infected as normal infection curse. The immunization with our recombinant BCG strains expressing the Phosphoprotein (P) from hMPV induces a protective Th1 immunity. The mice were protected from clinical disease such us weight loss, inflammatory cells recruitment in airway and viral replication in the lungs. Our recombinant vaccine also induced the activation of hMPV-specific T cells producing IFN- γ and IL-2, which protect from hMPV infection when transferred to naïve mice. These data strongly support the notion that recombinant BCG induces protective Th1 immunity and could be considered as an efficient vaccine against hMPV.

A TetR Family Transcriptional Factor Directly Regulates the Expression of a 3-Methyladenine DNA Glycosylase and Physically Interacts with the Enzyme to Stimulate Its Base Excision Activity in Mycobacterium bovis BCG

3-Methyladenine DNA glycosylase recognizes and excises a wide range of damaged bases and thus plays a critical role in base excision repair. However, knowledge on the regulation of DNA glycosylase in prokaryotes and eukaryotes is limited. In this study, we successfully characterized a TetR family transcriptional factor from Mycobacterium bovis bacillus Calmette-Guerin (BCG), namely BCG0878c, which directly regulates the expression of 3-methyladenine DNA glycosylase (designated as MbAAG) and influences the base excision activity of this glycosylase at the post-translational level. Using electrophoretic mobility shift assay and DNase I footprinting experiments, we identified two conserved motifs within the upstream region of mbaag specifically recognized by BCG0878c. Significant down-regulation of mbaag was observed in BCG0878c-overexpressed M. bovis BCG strains. By contrast, about 12-fold up-regulation of mbaag expression was found in bcg0878c-deleted mutant M. bovis BCG strains. β-Galactosidase activity assays also confirmed these results. Thus, BCG0878c can function as a negative regulator of mbaag expression. In addition, the regulator was shown to physically interact with MbAAG to enhance the ability of the glycosylase to bind damaged DNA. Interaction between the two proteins was further found to facilitate AAG-catalyzed removal of hypoxanthine from DNA. These results indicate that a TetR family protein can dually regulate the function of 3-methyladenine DNA glycosylase in M. bovis BCG both at the transcriptional and post-translational levels. These findings enhance our understanding of the expression and regulation of AAG in mycobacteria.

Immunization with a Recombinant Bacillus Calmette–Guérin Strain Confers Protective Th1 Immunity against the Human Metapneumovirus

Along with the human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, owing to an inefficient immunological memory, hMPV infection provides limited immune protection against reinfection. Furthermore, hMPV can induce an inadequate Th2 type immune response that causes severe lung inflammation, leading to airway obstruction. Similar to hRSV, it is likely that an effective clearance of hMPV would require a balanced Th1 type immunity by the host, involving the activation of IFN-γ-secreting T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which has been used in newborns for many decades and in several countries as a tuberculosis vaccine. We have previously shown that immunization with BCG strains expressing hRSV Ags can induce an efficient immune response that protects against this virus. In this study, we show that immunization with rBCG strains expressing the phosphoprotein from hMPV also can induce protective Th1 immunity. Mice immunized with rBCG were protected against weight loss, airway inflammation, and viral replication in the lungs after hMPV infection. Our rBCG vaccine also induced the activation of hMPV-specific T cells producing IFN-γ and IL-2, which could protect from hMPV infection when transferred to recipient mice. These data strongly support the notion that rBCG induces protective Th1 immunity and could be considered as an efficient vaccine against hMPV.

Immunogenicity and Protective Efficacy Conferred by a Novel Recombinant Mycobacterium bovis Bacillus Calmette‐Guérin Strain Expressing Interleukin‐12p70 of Human Cytokine and Ag85A of Mycobacterium tuberculosis Fusion Protein

Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunization provides protection against tuberculosis (TB) in infants, but the antituberculosis protective immunity wanes gradually after initial immunization and lasts less than 15 years. Therefore, more efficacious vaccines are urgently needed. In this study, we constructed a new tuberculosis vaccine of recombinant BCG strain (rBCG-IA), which could express IL-12p70 of human cytokine and Ag85A of M. tuberculosis fusion protein, and investigated its immunogenicity in BALB/c mice by measuring antibody titres, proliferation rate of splenocytes, ratios of CD4(+) T and CD8(+) T cells stimulated by specific antigens and levels of IFN-γ production in antigen-stimulated splenocyte cultures. Meanwhile, we evaluated its protective efficacy against M. tuberculosis H37Rv infection through detecting lung histopathology, organ bacterial loads and lung acid-fast stain. Immunogenicity experiments illustrated that from 2nd to 8th week after immunization, the rBCG-IA vaccine was able to induce the highest level of antibody titres, proliferation rate of splenocytes and IFN-γ production among groups and gained improved ratio of CD4(+) T and CD8(+) T cells from 6th to 8th week after vaccination. And from 2nd to 8th week after M. tuberculosis H37Rv infection, the score of pathology and bacterial loads in the rBCG-IA group were obviously lower than that in rBCG-I group, rBCG-A group or control group (PBST group), but similar to that in BCG group. This study suggested that rBCG-IA was able to elicit stronger humoral and cellular immune responses, but could only confer similar protective efficacy compared with its parental BCG vaccine.

Mycobacteria-Infected Dendritic Cells Attract Neutrophils That Produce IL-10 and Specifically Shut Down Th17 CD4 T Cells through Their IL-10 Receptor

Neutrophils participate in the control of mycobacterial infection both by directly eliminating bacilli and by interacting with macrophages and dendritic cells (DCs). Despite host defenses, slow-growing mycobacteria can persist in the host for decades, mostly inside macrophages and DCs, and eventually destroy tissues after exacerbated inflammation. IL-17A-driven neutrophil recruitment may participate in this process. We report that mouse bone marrow-derived DCs infected with live Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produced large amounts of CXCL1 and CXCL2, and attracted neutrophils. After physical contact with DCs infected with live BCG, the neutrophils produced large quantities of the immunosuppressive cytokine IL-10 via the MyD88 and spleen tyrosine kinase pathways. The CD11b integrin was involved in this neutrophil-DC interaction and allowed IL-10 production. TCR OVA transgenic mice immunized with a BCG strain producing OVA mounted an OVA-specific Th17 and Th1 CD4 response. Interestingly, IL-10-producing neutrophils specifically shut down IL-17A production by Th17 CD4 cells, but not IFN-γ production by Th1 cells. This was due to Th17 CD4 cell-restricted expression of the receptor for IL-10. After neutrophil depletion, total mouse lung cells produced less IL-10 but more IL-17A; IFN-γ production was not affected. Therefore, we suggest that during mycobacterial infection, regulatory neutrophils are instructed by infected reservoir DCs to produce IL-10 that specifically targets IL-10Rα-expressing Th17 CD4 T cells. This could be important to control the otherwise exuberant Th17 response.