The BCG Moreau Vaccine Upregulates In Vitro the Expression of TLR4, B7-1, Dectin-1 and EP2 on Human Monocytes.

Background: Tuberculosis (TB) is currently the second greatest killer worldwide and is caused by a single infectious agent. Since Bacillus Calmette−Guérin (BCG) is the only vaccine currently in use against TB, studies addressing the protective role of BCG in the context of inducible surface biomarkers are urgently required for TB control. Methods: In this study, groups of HIV-negative adult healthy donors (HD; n = 22) and neonate samples (UCB; n = 48) were voluntarily enrolled. The BCG Moreau strain was used for the in vitro mononuclear cell infections. Subsequently, phenotyping tools were used for surface biomarker detection. Monocytes were assayed for TLR4, B7-1, Dectin-1, EP2, and TIM-3 expression levels. Results: At 48 h, the BCG Moreau induced the highest TLR4, B7-1, and Dectin-1 levels in the HD group only (p-value < 0.05). TIM-3 expression failed to be modulated after BCG infection. At 72 h, BCG Moreau equally induced the highest EP2 levels in the HD group (p-value < 0.005), and higher levels were also found in HD when compared with the UCB group (p-value < 0.05). Conclusions: This study uncovers critical roles for biomarkers after the instruction of host monocyte activation patterns. Understanding the regulation of human innate immune responses is critical for vaccine development and for treating infectious diseases.

Genome sequence of Mycobacterium bovis BCG Moreau, the Brazilian vaccine strain against tuberculosis.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur.

BACKGROUND: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. RESULTS: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. CONCLUSIONS: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Bacillus Calmette-Guérin Immunotherapy for Cancer.

Bacillus Calmette-Guérin (BCG), an attenuated vaccine from Mycobacterium bovis, was initially developed as an agent for vaccination against tuberculosis. BCG proved to be the first successful immunotherapy against established human bladder cancer and other neoplasms. The use of BCG has been shown to induce a long-lasting antitumor response over all other forms of treatment against intermediate, non-invasive muscle bladder cancer Several types of tumors may now be treated by releasing the immune response through the blockade of checkpoint inhibitory molecules, such as CTLA-4 and PD-1. In addition, Toll-Like Receptor (TLR) agonists and BCG are used to potentiate the immune response against tumors. Studies concerning TLR-ligands combined with BCG to treat melanoma have demonstrated efficacy in treating mice and patients This review addresses several interventions using BCG on neoplasms, such as Leukemia, Bladder Cancer, Lung Cancer, and Melanoma, describing treatments and antitumor responses promoted by this attenuated bacillus. Of essential importance, BCG is described recently to participate in an adequate microbiome, establishing an effective response during cell-target therapy when combined with anti-PD-1 antibody, which stimulates T cell responses against the melanoma. Finally, trained immunity is discussed, and reprogramming events to shape innate immune responses are addressed.

The in vitro Mycobacterium bovis BCG Moreau infection of human monocytes that induces Caspase-1 expression, release and dependent cell death is mostly reliant upon cell integrity.

BACKGROUND: Caused by Mycobacterium tuberculosis, tuberculosis (TB) is an extremely contagious disease predominantly affecting the lungs. TB is found worldwide and has a major impact on public health safety primarily due to its high mortality rate. Applied for over a hundred years as a preventive measure, Mycobacterium bovis BCG remains the only available TB vaccine. Only one seminal study about the apoptotic pathways induced by this vaccine in the monocytic lineage of the host cell has found the effects of BCG on regulation of apoptosis. The aim of this study was to explore beyond that pioneer study the pathway related to the in vitro cell-death pattern and the inflammatory response to the BCG vaccine in human monocytes. METHODS: Cohorts of HIV-negative volunteers were enrolled: adult Healthy Donors (HD) and neonates' Umbilical Cord Blood (UCB) individuals. Host mononuclear cells were infected with the M. bovis Moreau strain of BCG vaccine at 16, 24, 48, and 72 h. The Real-Time RT-PCR for TRADD, Bcl-2, and Caspases-1 and -3 were performed, and supernatants were assayed in parallel for Caspase-1, NLRP3, HO-1, and IL-1ß levels whereas caspases were assessed intracellularly. The effect of a BCG infection in monocytes was characterized via a metabolic activity assay by LDH release profiles. RESULTS: Overall, the BCG vaccine induced significantly higher Caspase-1 and Bcl-2 mRNA levels in both the HD and UCB groups (p-value ≤0.05). In addition, a significant increase solely in Caspase-1 protein levels was also noted in both HD and UCB (p-value ≤0.05) notwithstanding the absence of any damaged cell membranes. CONCLUSIONS: Our data directly corroborate other findings showing that BCG Moreau led to an increased secretion of IL-1ß but not IL-18, two Caspase-1-activated cytokines, and are also in support of the model that the BCG Moreau infection of human mononuclear cells may induce a cell-death pattern involving Caspase-1 activation.

The dolabellane diterpene Dolabelladienetriol is a typical noncompetitive inhibitor of HIV-1 reverse transcriptase enzyme.

We recently described that a dollabelane diterpene isolated from the marine algae Dictyota pfaffii (Dolabelladienetriol) inhibits the human immunodeficiency virus type 1 (HIV-1) enzyme reverse transcriptase (RT), and HIV-1 replication in primary cells. Based on these findings, we investigated additional antiretroviral properties of Dolabelladienetriol. Here, we describe that Dolabelladienetriol blocked the synthesis and integration of HIV-1 provirus and completely abrogated viral replication in primary cells. Also, studies of kinetic mode of action revealed that the Dolabelladienetriol is a nonnucleoside RT inhibitor (NNRTI), acting as a noncompetitive inhibitor, with a K(i) value equal to 7.2 microM. To assess whether Dolabelladienetriol could potentiate the anti-HIV-1 effects of other HIV-1 inhibitors, HIV-1-infected cells were treated with Dolabelladienetriol at its EC(50) dose plus sub-optimal concentrations of classical antiretrovirals. Dolabelladienetriol provided an additive effect with the nucleoside RT inhibitor AZT, and a synergistic effect with the protease inhibitor atazanavir sulphate. There was no increment of the anti-HIV-1 effect resulting from the combination between Dolabelladienetriol and the NNRTI nevirapine. Using a large panel of HIV-1 isolates harboring NNRTI resistance mutations, we found no cross-resistance between Dolabelladienetriol and clinical available NNRTIs. Thus, Dolabelladienetriol is an NNRTI, with potent activity against HIV-1 isolates carrying common NNRTI-associated resistance mutations. Dolabelladienetriol may be considered as a potential new agent for anti-HIV-1 therapy.

Modulation of humoral immune response to oral BCG vaccination by Mycobacterium bovis BCG Moreau Rio de Janeiro (RDJ) in healthy adults.

BACKGROUND: Oral administration of BCG was the route initially used by Calmette and Guérin, but was replaced by intradermal administration in virtually all countries after the Lubeck accident. However, Brazil continued to administer oral BCG Moreau RDJ, which was maintained until the mid-1970s when it was substituted by the intradermal route. Although BCG vaccination has been used in humans since 1921, little is known of the induced immune response. The aim of this study was to analyse immunological responses after oral vaccination with M. bovis BCG Moreau RDJ. METHODS: This study in healthy volunteers has measured cellular and humoral aspects of the immunological response to oral M. bovis BCG Moreau RDJ in Rio de Janeiro, Brazil. T-cell trafficking and Th1 and Th2 cytokine responses are described, as well as isotype-specific antibody production using novel techniques. RESULTS: Oral immunisation has no adverse effects. We have shown that there are cellular and humoral immunological responses after oral immunisation. Oral revaccination does not induce a positive skin test in responsive individuals and multiple booster orally was able to induce modulation in humoral immunological responses (switch from IgG to IgA) in previously immunised subjects and incapable of inducing tolerance. In contrast, the cellular immune response does not differ between vaccinated individuals with positive and negative skin test reactions. CONCLUSION: All subjects, including those who did not respond to the skin test at study commencement, were capable of mounting humoral and cellular immune response to the antigens tested.

The role of host soluble inflammatory mediators induced by the BCG vaccine for the initiation of in vitro monocyte apoptosis in healthy Brazilian volunteers.

BACKGROUND: Tuberculosis (TB) is the second greatest killer worldwide that is caused by a single infectious agent. For its control, studies of TB vaccines are needed. Since Bacillus Calmette-Guerin (BCG) is the only vaccine against TB currently in use, studies addressing the protective role of BCG in the context of inducible inflammatory mediators are urgently required. METHODS: In this study, groups of HIV-negative adult healthy donors (HD; n = 42) and neonates (UV; n = 18) have been voluntarily enrolled, and BCG Moreau strain was used for the in vitro mononuclear cell infections for an initial period of 48 h. Subsequently, harvested conditioned medium (CM) was added to autologous resting cells for an additional 24, 48, and 120 h, and Annexin V, in conjunction with a vital dye, was then used for apoptosis detection. CM was also assayed for nitric oxide (NO), prostaglandin E2 (PGE2), leukotriene B4 (LTB4), interferon (IFN)-ß, and transforming growth factor (TGF)-ß1 levels. The p values were set up for any differences between two groups of individuals using Student's t-test and considered significant when ≤ 0.05. RESULTS: At 120 h, CM induced the highest apoptosis levels in both group studied, but necrosis was high in UV group only (p-value < 0.05). NO was released equally during BCG infection in both groups, but higher levels were found in HD when compared with UV group (p-value < 0.05). Overall, BCG Moreau triggered high PGE2, LTB4 and IFN-ß productions in macrophages from the UV group (p-value ≤ 0.05), whereas the prostanoid PGE2 and TGF-ß1 had an opposite pattern in the HD group. CONCLUSIONS: This study uncovers critical roles for endogenous compounds in the instruction of host macrophage cell death patterns. Understanding the regulation of human immune responses is critical for vaccine development and the treatment of infectious diseases. These findings shed new light on the potential condition for a booster immunization in individuals already vaccinated with BCG for TB protection, and further studies are warranted.

A Single Dose of Oral BCG Moreau Fails to Boost Systemic IFN-γ Responses to Tuberculin in Children in the Rural Tropics: Evidence for a Barrier to Mucosal Immunization.

Immune responses to oral vaccines are impaired in populations living in conditions of poverty in developing countries, and there is evidence that concurrent geohelminth infections may contribute to this effect. We vaccinated 48 children living in rural communities in Ecuador with a single oral dose of 100 mg of BCG Moreau RDJ and measured the frequencies of tuberculin-stimulated peripheral blood mononuclear cells expressing IFN-γ before and after vaccination. Vaccinated children had active ascariasis (n = 20) or had been infected but received short- (n = 13) or long-term (n = 15) repeated treatments with albendazole prior to vaccination to treat ascariasis. All children had a BCG scar from neonatal vaccination. There was no evidence of a boosting of postvaccination IFN-γ responses in any of the 3 study groups. Our data provide support for the presence of a barrier to oral vaccination among children from the rural tropics that appeared to be independent of concurrent ascariasis.

Oral delivery of BCG Moreau Rio de Janeiro gives equivalent protection against tuberculosis but with reduced pathology compared to parenteral BCG Danish vaccination.

There is a need for an improved vaccine to better control human tuberculosis (TB), as the only currently available TB vaccine, bacillus Calmette-Guerin (BCG) delivered parenterally, offers variable levels of efficacy. Therefore, recombinant strains expressing additional antigens are being developed alongside alternative routes to parenteral delivery. There is strong evidence that BCG Moreau (RdJ) is a safe and effective vaccine in humans when given by the oral route. This study compared the efficacy of a single oral dose of wild type BCG Moreau Rio de Janeiro (RdJ), or a recombinant RdJ strain expressing Ag85B-ESAT6 fusion protein, formulated with and without lipid to enhance oral delivery, with subcutaneous BCG Danish 1331 and saline control groups in a guinea pig aerosol infection model of pulmonary tuberculosis. Protection was measured as survival at 30 weeks post-challenge and reduced bacterial load and histopathology in lungs and spleen. Results showed that a single oral dose of BCG Moreau (RdJ) or recombinant BCG Moreau (RdJ)-Ag85B-ESAT6, formulated with or without lipid, gave protection equivalent to subcutaneously delivered BCG Danish in the 30 weeks post-challenge survival study. The orally delivered vaccines gave reduced pathology scores in the lungs (three of the four formulations) and spleens (all four formulations) compared to subcutaneously delivered BCG Danish. The oral wild type BCG Moreau (RdJ) in lipid and the unformulated oral wild type BCG Moreau (RdJ) vaccine also gave statistically lower bacterial loads in the lungs and spleens, respectively, compared to subcutaneously delivered BCG Danish. This study provides further evidence to show that lipid formulation does not impair vaccine efficacy and may enhance the delivery and stability of oral vaccines intended for use in countries with poor health infrastructure. Oral delivery also avoids needles (and associated cross-infection risks) and immunisation without the need for specially trained medical professional staff.

An investigation of clinical and immunological events following repeated aerodigestive tract challenge infections with live Mycobacterium bovis Bacille Calmette Guérin.

Bacille Calmette Guérin substrain Moreau Rio de Janeiro is an attenuated strain of Mycobacterium bovis that has been used extensively as an oral tuberculosis vaccine. We assessed its potential as a challenge model to study clinical and immunological events following repeated mycobacterial gut infection. Seven individuals received three oral challenges with approximately 10(7) viable bacilli. Clinical symptoms, T-cell responses and gene expression patterns in peripheral blood were monitored. Clinical symptoms were relatively mild and declined following each oral challenge. Delayed T-cell responses were observed, and limited differential gene expression detected by microarrays. Oral challenge with BCG Moreau Rio de Janeiro vaccine was immunogenic in healthy volunteers, limiting its potential to explore clinical innate immune responses, but with low reactogenicity.

Report of an international collaborative study to evaluate the suitability of multiplex PCR as an identity assay for different sub-strains of BCG vaccine.

Current methods for the identification of BCG vaccine in quality control settings involve acid-fast staining with microscopic examination. However, this method is unable to distinguish the many different sub-strains of BCG, or to differentiate BCG strains from virulent members of the Mycobacterium tuberculosis complex. A multiplex PCR (mPCR) which uses six target regions in mycobacteria has been developed to identify specific sub-strains of BCG. This study reports the findings from an international collaborative study to assess the accuracy, robustness and reproducibility of this mPCR method to differentiate BCG sub-strains. The method was found to fulfil these criteria successfully and was able to distinguish BCG sub-strains in vaccine preparations. The majority of the participants in the study generated the expected PCR product profiles indicating the method is also robust.

Protection against tuberculosis induced by oral prime with Mycobacterium bovis BCG and intranasal subunit boost based on the vaccine candidate Ag85B-ESAT-6 does not correlate with circulating IFN-gamma producing T-cells.

The potent IFN-gamma inducing fusion antigen Ag85B-ESAT-6 (85B6) is a lead subunit candidate to improve current vaccination against Mycobacterium tuberculosis (Mtb). The recombinant M. bovis BCG strain Myc3504 was constructed to secrete 85B6. It was based on commercial BCG strain Moreau Rio de Janeiro (BCG(MoWT)) which remains available for human oral administration. Myc 3504 induced higher levels of 85B6-specific IFN-gamma circulating T-cells as compared to BCG(MoWT). A novel needle-free mucosal immunization regimen combining oral prime with Myc3504 or BCG(MoWT) with intranasal boost with LTK-63-adjuvanted 85B6 was compared to subcutaneous prime-boost immunization. Strikingly whereas parenteral immunization induced sustained levels of 85B6-specific IFN-gamma secretion by circulating T-cells, mucosal regimens induced barely detectable IFN-gamma. Despite this, mice and guinea pigs immunized with the mucosal regimens were as efficiently protected against aerosol Mtb challenge as parenterally immunized animals. After Mtb challenge, anti-ESAT-6 IFN-gamma responses sharply increased in non-vaccinated mice as a hallmark of infection. Parenterally immunized mice that controlled Mtb infection, displayed anti-ESAT-6 IFN-gamma responses as high as non-immunized infected mice, compromising the possible use of ESAT-6 as a diagnostic tool. Interestingly, in mucosally immunized mice that were equally protected, post-challenge ESAT-6-specific IFN-gamma T-cell response remained low.

Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine.

As part of the World Health Organisation (WHO) initiative to update the current requirements for BCG vaccine a collaborative study was carried out to establish the robustness, reproducibility and the suitability of the modified ATP assay. This assay was developed by Statens Serum Institut, Denmark, as a potential replacement of the method for detection of viable counts of BCG vaccine which is routinely used as a quality control test for lot release. Two BCG preparations, of same strain but different production methods, were tested. For each preparation, two different storage conditions of -20 or 37 degrees C were used in order to establish the suitability of this assay for testing heat-treated BCG vaccine as in the temperature stability test. The lyophilised BCG samples were tested using the ATP reagents from the same source and same principle of testing but some procedural modifications were allowed to accommodate different equipment and resource availability in different laboratories. Data from four laboratories showed that the heat-treated BCG samples contained significantly lower ATP content per sample than the untreated control stored at -20 degrees C. Three laboratories gave consistent mean ATP contents, especially for control samples, even with variations in testing protocol. The present study showed that this modified ATP assay is very robust and can be reproducible. Once the correlation of cultural viable count and ATP content of a BCG vaccine product has been established, this rapid alternative assay may be used to monitor BCG viable count. Due to the fact that this study was small, further investigation is planned. A collaborative study will be carried out using this modified ATP assay in parallel with the cultural viable count method in the establishment of the replacement of the WHO International Reference Preparation of BCG vaccine.

Inhibition of HIV-1 replication in human primary cells by a dolabellane diterpene isolated from the marine algae Dictyota pfaffii.

We describe in this paper that the dolabellane diterpene 8,10,18-trihydroxy-2,6-dolabelladiene (3), isolated from the marine algae Dictyota pfaffii, inhibits the HIV-1 infection in human primary cells and tumor cell lines. We initially observed that compound 3 inhibited the activity of a purified HIV-1 enzyme reverse transcriptase (RT) in a dose-dependent manner, with an IC (50) value of 16.5 +/- 4.3 microM. Next, we found that compound 3 inhibited HIV-1 infection by an R5-tropic isolate in peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner with an EC (50) value of 8.4 +/- 2.8 microM. The replication of HIV-1 isolates presenting distinct tropism for chemokine receptors was also inhibited, as analyzed in PBMCs or U87 cells infected with R5-, X4- or R5X4-tropic isolates. Likewise, compound 3 blocked HIV-1 infection in macrophages by R5 and R5X4 viruses in a dose-dependent manner with EC (50) values of 1.7 +/- 0.6 microM and 1.85 +/- 0.75 microM, respectively. Compound 3 sustained antiretroviral activity even when added to HIV-1-infected Sup-T1 cells at 12 h after infection, suggesting that, as well as inhibiting HIV-1 RT, it also blocks HIV-1 replication at a post transcriptional step. Our results support further investigations on compound 3 pharmacokinetics and we propose that this diterpene could be considered as a potential compound for HIV-1 therapy.

Boosting of cellular immunity against Mycobacterium tuberculosis and modulation of skin cytokine responses in healthy human volunteers by Mycobacterium bovis BCG substrain Moreau Rio de Janeiro oral vaccine.

Oral immunization of healthy adults with 10(7) CFU BCG Moreau Rio de Janeiro was well tolerated and significantly boosted gamma interferon responses to purified protein derivative, Ag85, and MPB70 from previous childhood intradermal BCG immunization. Oral BCG offers the possibility of a needle-free tuberculosis vaccine and of boosting the protective immunity from intradermal tuberculosis vaccines.

BCG Moreau Rio de Janeiro: an oral vaccine against tuberculosis--review.

The vaccine Bacillus of Calmette Guérin (BCG) was originally developed in France as an oral vaccine against tuberculosis. The oral use of this vaccine was replaced by the parenteral route in almost all countries after the Lubeck disaster. In contrast, Brazil retained the oral delivery of the vaccine until the mid-seventies when it was replaced by the intradermal route. This change in route of delivery was mainly secondary to pressure by medical practitioners based on the poor responses of oral immunized subjects to purified protein derivative (PPD) skin tests. Even after the change of route of delivery, Ataulpho de Paiva Foundation continued making the oral vaccine. Currently, BCG Moreau has been described as one of the most immunogenic and with fewer side effects than other BCGs. The genomics, proteomics and vaccine trials for oral BCG Moreau Rio de Janeiro are currently under investigation. In this review, we intend to describe the history of BCG Moreau Rio de Janeiro in Brazil.