Bovis Bacille Calmette-Guérin Research Articles

Low-dose M.tb infection but not BCG or MTBVAC vaccination enhances heterologous antibody titres in non-human primates

IntroductionMycobacteria are known to exert a range of heterologous effects on the immune system. The mycobacteria-based Freund’s Complete Adjuvant is a potent non-specific stimulator of the immune response used in immunization protocols promoting antibody production, and Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccination has been linked with decreased morbidity and mortality beyond the specific protection it provides against tuberculosis (TB) in some populations and age groups. The role of heterologous antibodies in this phenomenon, if any, remains unclear and under-studied.MethodsWe set out to evaluate antibody responses to a range of unrelated pathogens following infection with Mycobacterium tuberculosis (M.tb) and vaccination with BCG or a candidate TB vaccine, MTBVAC, in non-human primates.ResultsWe demonstrate a significant increase in the titer of antibodies against SARS-CoV-2, cytomegalovirus, Epstein-Barr virus, tetanus toxoid, and respiratory syncytial virus antigens following low-dose aerosol infection with M.tb. The magnitude of some of these responses correlated with TB disease severity. However, vaccination with BCG administered by the intradermal, intravenous or aerosol routes, or intradermal delivery of MTBVAC, did not increase antibody responses against unrelated pathogens.DiscussionOur findings suggest that it is unlikely that heterologous antibodies contribute to the non-specific effects of these vaccines. The apparent dysregulation of B cell responses associated with TB disease warrants further investigation, with potential implications for risk of B cell cancers and novel therapeutic strategies.

Molecular detection of pre-ribosomal RNAs of Mycobacterium bovis bacille Calmette-Guérin and Mycobacterium tuberculosis to enhance pre-clinical tuberculosis drug and vaccine development

Efforts are underway globally to develop effective vaccines and drugs against M. tuberculosis (Mtb) to reduce the morbidity and mortality of tuberculosis. Improving detection of slow-growing mycobacteria could simplify and accelerate efficacy studies of vaccines and drugs in animal models and human clinical trials. Here, a real-time reverse transcription PCR (RT-PCR) assay was developed to detect pre-ribosomal RNA (pre-rRNA) of Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mtb. This pre-rRNA biomarker is indicative of bacterial viability. In two different mouse models, the presence of pre-rRNA from BCG and Mtb in ex vivo tissues showed excellent agreement with slower culture-based colony-forming unit assays. The addition of a brief nutritional stimulation prior to molecular viability testing further differentiated viable but dormant mycobacteria from dead mycobacteria. This research has set the stage to evaluate pre-rRNA as a BCG and/or Mtb infection biomarker in future drug and vaccine clinical studies.

Characterization of mycophage endolysin cell wall binding domains targeting Mycobacterium bovis peptidoglycan

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis, responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics.

Mendelian susceptibility to mycobacterial diseases: State of the puzzle.

The constant progress of genomics and the establishment of new functional tests have paved the way for identifying monogenic defects conferring a selective predisposition to infections by certain microbes as a new type of inborn errors of immunity (IEIs). Mendelian susceptibility to mycobacterial diseases (MSMD) is the most characterized of these IEIs, with 36 different disorders found in 20 distinct genes (IFNGR1, IFNGR2, IFNG, IL12RB1, IL12RB2, IL23R, IL12B, ISG15, USP18, ZNFX1, TBX21, STAT1, TYK2, IRF8, IRF1, CYBB, JAK1, RORC, NEMO, and SPPL2A) over the last 20 years. MSMD confers a selective susceptibility to infections with weakly virulent mycobacteria, including the M. bovis Bacille Calmette-Guerin (BCG) vaccines and various environmental mycobacteria in patients, primarily children, without classical immune defects. These patients may also present severe forms of tuberculosis, and about half of them might develop non-typhoidal salmonellosis. In some cases, patients also suffer from chronic mucocutaneous candidiasis (CMC), while in others, patients also present severe viral, parasitic, fungal, and/or bacterial diseases. Despite this clinical and genetic heterogeneity, almost all genetic etiologies of MSMD alter the interferon-gamma (IFN-γ)- mediated immunity by impairing or abolishing IFN-γ production or the response to this cytokine. It was proven that the human IFN-γ level is a quantitative trait that defines the outcome of mycobacterial infection. The study of these monogenic defects contributes to understanding the molecular mechanism of mycobacterial diseases in humans and to the development of new diagnostic and therapeutic approaches to improve care and prognosis. For example, MSMD patients with impaired production of IFN-γ may benefit from injections of human recombinant IFN-γ, while for patients with abolished response to this cytokine, hematopoietic stem cell transplantation (HSCT) and promising gene therapy are the only current therapeutic options. These discoveries also bridge the gap between simple Mendelian inheritance and complex human genetics.

Nanoscale clustering of mycobacterial ligands and DC-SIGN host receptors are key determinants for pathogen recognition.

The bacterial pathogen Mycobacterium tuberculosis binds to the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on dendritic cells to evade the immune system. While DC-SIGN glycoconjugate ligands are ubiquitous among mycobacterial species, the receptor selectively binds pathogenic species from the M. tuberculosis complex (MTBC). Here, we unravel the molecular mechanism behind this intriguing selective recognition by means of a multidisciplinary approach combining single-molecule atomic force microscopy with Förster resonance energy transfer and bioassays. Molecular recognition imaging of mycobacteria demonstrates that the distribution of DC-SIGN ligands markedly differs between Mycobacterium bovis Bacille Calmette-Guérin (BCG) (model MTBC species) and Mycobacterium smegmatis (non-MTBC species), the ligands being concentrated into dense nanodomains on M. bovis BCG. Upon bacteria-host cell adhesion, ligand nanodomains induce the recruitment and clustering of DC-SIGN. Our study highlights the key role of clustering of both ligands on MTBC species and DC-SIGN host receptors in pathogen recognition, a mechanism that might be widespread in host-pathogen interactions.

Maintenance and recall of memory T cell populations against tuberculosis: Implications for vaccine design.

Despite the widespread use of standardised drug regimens, advanced diagnostics, and Mycobacterium bovis Bacille-Calmette-Guérin (BCG) vaccines, the global tuberculosis (TB) epidemic remains uncontrollable. To address this challenge, improved vaccines are urgently required that can elicit persistent immunologic memory, the hallmark of successful vaccines. Nonetheless, the processes underlying the induction and maintenance of immunologic memory are not entirely understood. Clarifying how memory T cells (Tm cells) are created and survive long term may be a crucial step towards the development of effective T cell-targeted vaccines. Here, we review research findings on the memory T cell response, which involves mobilization of several distinct Tm cell subsets that are required for efficient host suppression of M. tuberculosis (Mtb) activity. We also summaries current knowledge related to the T cell response-based host barrier against Mtb infection and discuss advantages and disadvantages of novel TB vaccine candidates.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity.

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

Selection of IS6110 conserved regions for the detection of Mycobacterium tuberculosis using qPCR and LAMP.

IS6110 insertion sequence is a frequently used target for Mycobacterium tuberculosis detection. However, its sequence variability is studied insufficiently. We aimed to identify the most conservative and variable regions in IS6110 sequences and develop qPCR and LAMP oligonucleotide sets for the conservative regions. Using in-house Python scripts, 3609M. tuberculosis genome sequences from the NCBI database were aligned; conservative regions were identified to design oligonucleotide sets. IS6110 fragments located within the 31-231bp region were the most conservative and represented in genomes and were used to design qPCR and LAMP oligonucleotides. The in silico sensitivity of the qPCR oligonucleotides on the whole genome set was 99.1% and 98.4%. For the LAMP primers developed, the sensitivity was 96.9%. For qPCR, the limit of detection with 95% confidence (LoD95%) was four IS6110 copies per reaction, with LoD90% being 200 BCG cells per ml of artificial sputum. For LAMP, LoD95% was 16 copies per reaction, with LoD90% being 400 Mycobacterium bovis Bacille Calmette-Guerin (BCG) cells per ml of artificial sputum. We have demonstrated the IS6110 sequence variability and designed highly sensitive qPCR and LAMP oligonucleotides to detect M. tuberculosis.

Differences in responses to the intracellular macrophage environment between Mycobacterium bovis BCG vaccine strains Moreau and Pasteur.

The Bacille Calmette-Guérin (BCG) vaccine comprises a family of strains with variable protective efficacy against pulmonary tuberculosis (TB) and leprosy, partly due to genetic differences between strains. Previous data highlighting differences between the genomes and proteomic profiles of BCG strains Moreau and Pasteur led us to evaluate their behaviour in the macrophage microenvironment, capable of stimulating molecular responses that can impact the protective effect of the vaccine. Strain infectivity, viability, co-localisation with acidified vesicles, macrophage secretion of IL-1 and MCP-1 and lipid droplet biogenesis were evaluated after infection. We found that BCG Moreau is internalised more efficiently, with significantly better intracellular survival up to 96 h p.i., whereas more BCG Pasteur bacilli were found co-localised in acidified vesicles up to 6 h p.i. IL-1β and MCP-1 secretion and lipid droplet biogenesis by infected macrophages were more prominent in response to BCG Pasteur. Overall, our results show that, compared to Pasteur, BCG Moreau has increased fitness and better endurance in the harsh intracellular environment, also regulating anti-microbial responses (lower IL-1b and MCP-1). These findings contribute to the understanding of the physiology of BCG Moreau and Pasteur in response to the intraphagosomal environment in a THP-1 macrophage model.

2351. Systemic Influenza Infection Impairs Whole Blood Control of Mycobacteria in a Human Challenge Study

Abstract Background Of the millions of people infected with Mycobacterium tuberculosis (Mtb) each year, only a small proportion will develop TB disease, with most immunologically containing or clearing Mtb. Factors influencing TB progression risk are incompletely understood. Co-infections, including influenza, have been proposed as a risk factor for TB progression via disruption of anti-Mtb immune responses. We employed a human influenza challenge study to investigate the effect of systemic influenza infection on host mycobacterial control. Methods A whole blood (WB) mycobacterial growth inhibition assay was utilised to compare mycobacterial growth and anti-mycobacterial immune responses before and after influenza infection. Thirty adults were per-nasally inoculated with H3N2 influenza virus (Day [D] 0). Influenza PCR assay of D4 nasal swab confirmed infection. WB, collected pre- (D0, “pre-influenza”) and post-inoculation (D6, “post-influenza”), was infected with Mycobacterium bovis Bacille Calmette Guerin (BCG)-lux, incubated for 72 hours (h) and WB mycobacterial growth (growth ratio [GR]) measured. In parallel, BCG-lux-infected and uninfected blood aliquots were incubated for 0, 6, 24 and 72 h and measurements of cytokines (Meso Scale) and gene expression (RNA-Sequencing) undertaken. Comparisons between pre- and post-influenza infection blood samples were made (Fig. 1). Blood was aliquoted into triplicate for each timepoint (0 hours [h], 72 h), diluted 1:1 with Gibco Roswell Park Memorial Institute 1640 Medium (RPMI), inoculated with a fixed quantity of BCG-lux and incubated at 37°C in a rocker-incubator. Previous experiments had confirmed and quantified the correlation between BCG-lux luminescence (measured in Relative Light Units [RLU] by a luminometer) and number of mycobacterial colony forming units. At 0 h and 72 h, the luminescence of each triplicate sample was measured in duplicate. The growth ratio (GR) was calculated by division of the median 72 h luminescence value by the median 0 h luminescence value. In parallel, BCG-lux inoculum or sterile PBS were added to additional whole blood aliquots (to give infected and uninfected aliquots), which were incubated at 37°C in a rocker-incubator. At timepoints 0 h, 6 h, 24 h, and 72 h, aliquots were removed from the incubator and centrifuged. Supernatants were frozen and stored for future cytokine analyses (quantification using Meso Scale Discovery [MSD] U-plex). Cell pellets were stabilised and stored for future RNA-sequencing and transcriptomic analysis. Figure created with BioRender.com. Results In 22 influenza PCR-positive (+) subjects, median GR was significantly higher in post- (1.69) vs pre-influenza samples (1.03, p=0.0016) with no significant difference in the PCR-negative subjects (Fig. 2). Significant differences in BCG-lux-stimulated cytokine production were observed between post- and pre-influenza samples in PCR+ subjects (Fig. 3). Transcriptomic analysis identified significantly differentially expressed genes in the post- vs pre-influenza samples and differences between the groups over time, mapping to pathways related to TB susceptibility. Complete BCG-lux growth ratio (GR) data were available for 28/30 participants. Comparisons of median GRs pre- and post-influenza infection for (A) 22 participants who were influenza PCR-positive on day 4 and (B) 6 participants who were influenza PCR-negative on day 4. Significance (paired analysis): ** p≤0.01; ns p>0.05. (C) Change in GR from pre-influenza baseline was calculated for each individual (GR-Post divided by GR-Pre, expressed as a percentage) and compared between the influenza PCR-positive and PCR-negative participants. Significance: ** p≤0.01. Cytokine concentrations were measured in duplicate in supernatants from blood incubated with BCG-lux for 0 hours (h), 6 h, 24 h and 72 h, using Meso Scale Discovery U-plex. Results are available for a subset of 15 participants who had influenza infection confirmed by influenza PCR-positive assay on day 4 nasal swab. Comparisons of median concentrations between pre- and post-influenza samples and individual participants’ paired samples are shown for (A, B) Interleukin-1β (IL-1β) at 24 h; (C, D) Tumour necrosis factor-α (TNF-α) at 6 h; (E, F) Interleukin-10 (IL-10) at 0 h; (G, H) IL-10 at 24 h. Significance (paired analysis): **** p≤0.0001; ** p≤0.01. Conclusion Systemic influenza infection reduces whole blood control of mycobacteria through modulation of anti-mycobacterial immune responses. Cytokine differences may contribute. These novel findings suggest that influenza may be a risk factor for TB disease and influenza vaccine could have a non-specific effect on TB immunity. Disclosures Christopher W. Woods, MD MPH, Predigen, Inc: Co-founder Christopher Chiu, BMBCh PhD, GlaxoSmithKline: Grant/Research Support|Merck: Grant/Research Support|Reithera: Advisor/Consultant.

Histopathologic differences in granulomas of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccinated and non-vaccinated cattle with bovine tuberculosis.

Mycobacterium bovis (M. bovis) is the zoonotic bacterium responsible for bovine tuberculosis. An attenuated form of M. bovis, Bacillus Calmette-Guerin (BCG), is a modified live vaccine known to provide variable protection in cattle and other species. Protection for this vaccine is defined as a reduction in disease severity rather than prevention of infection and is determined by evaluation of the characteristic lesion of tuberculosis: the granuloma. Despite its recognized ability to decrease disease severity, the mechanism by which BCG imparts protection remains poorly understood. Understanding the histopathologic differences between granulomas which form in BCG vaccinates compared to non-vaccinates may help identify how BCG imparts protection and lead to an improved vaccine. Utilizing special stains and image analysis software, we examined 88 lymph nodes obtained from BGC-vaccinated and non-vaccinated animals experimentally infected with M. bovis. We evaluated the number of granulomas, their size, severity (grade), density of multinucleated giant cells (MNGC), and the amounts of necrosis, mineralization, and fibrosis. BCG vaccinates had fewer granulomas overall and smaller high-grade granulomas with less necrosis than non-vaccinates. The relative numbers of high- and low- grade lesions were similar as were the amounts of mineralization and the density of MNGC. The amount of fibrosis was higher in low-grade granulomas from vaccinates compared to non-vaccinates. Collectively, these findings suggest that BCG vaccination reduces bacterial establishment, resulting in the formation of fewer granulomas. In granulomas that form, BCG has a protective effect by containing their size, reducing the relative amount of necrosis, and increasing fibrosis in low-grade lesions. Vaccination did not affect the amount of mineralization or density of MNGC.

What's Old and New in Tuberculosis Vaccines for Children.

Tuberculosis (TB) is a leading cause of global child mortality. Until the turn of the 21st century, Mycobacterium bovis bacille Calmette-Guerin (BCG) was the only vaccine to prevent TB. The pediatric TB vaccine pipeline has advanced in the past decade to include the evaluation of novel whole cell vaccines to replace infant BCG and investigation of subunit and whole cell vaccines to boost TB immunity during adolescence. We describe the history of BCG, current TB vaccine candidates in clinical trials, and the challenges and opportunities for future TB vaccine research in children. Children are a critical target population for TB vaccines, and expansion of the pediatric TB vaccine pipeline is urgently needed to end the TB pandemic.

Progressive Host-Directed Strategies to Potentiate BCG Vaccination Against Tuberculosis.

The pursuit to improve the TB control program comprising one approved vaccine, M. bovis Bacille Calmette-Guerin (BCG) has directed researchers to explore progressive approaches to halt the eternal TB pandemic. Mycobacterium tuberculosis (M.tb) was first identified as the causative agent of TB in 1882 by Dr. Robert Koch. However, TB has plagued living beings since ancient times and continues to endure as an eternal scourge ravaging even with existing chemoprophylaxis and preventive therapy. We have scientifically come a long way since then, but despite accessibility to the standard antimycobacterial antibiotics and prophylactic vaccine, almost one-fourth of humankind is infected latently with M.tb. Existing therapeutics fail to control TB, due to the upsurge of drug-resistant strains and increasing incidents of co-infections in immune-compromised individuals. Unresponsiveness to established antibiotics leaves patients with no therapeutic possibilities. Hence the search for an efficacious TB immunization strategy is a global health priority. Researchers are paving the course for efficient vaccination strategies with the radically advanced operation of core principles of protective immune responses against M.tb. In this review; we have reassessed the progression of the TB vaccination program comprising BCG immunization in children and potential stratagems to reinforce BCG-induced protection in adults.

Pathogenesis of tuberculosis: the 1930 Lübeck disaster revisited.

During the 1930 Lübeck Mycobacterium bovis bacille Calmette-Guérin (BCG) disaster, 251 neonates received three oral BCG doses accidentally contaminated by virulent Mycobacterium tuberculosis; 67 (26.7%) infants died of tuberculosis. BCG reversion to pathogenicity did not occur. Detailed post mortem examinations clarified contested aspects of tuberculosis pathogenesis. Gastrointestinal infection was seldom "silent" and did not cause typical primary pulmonary lesions. In 15 infants, primary pulmonary foci were found but these resulted from vaccine ingestion and aspiration and were not caused by gastrointestinal infection spreading to the lungs without trace of its journey, as claimed by prominent researchers such as Calmette and von Behring. Further, among 60 infants in whom post mortem evaluation was completed, a "silent" gastrointestinal infection without an intestinal primary focus was found in only one. Lymphohaematogenous-disseminated tuberculosis caused death in 24/67 (35.8%) infants and tuberculous meningitis in a further 17/67 (25.4%). Gastrointestinal tuberculosis complications caused death in 26/67 (38.8%) infants. Half of the tuberculosis-attributed deaths had occurred by 3 months, 93% by 6 months and 100% by 12 months; remarkably no further deaths or tuberculosis recurrences occurred within 5 years post-vaccination/infection. These findings provide graphic confirmation that the early introduction of chemoprophylaxis in recently M. tuberculosis-infected young children is critical and urgent.

A next generation BCG vaccine moves forward

The Mycobacterium Bovis bacille Calmette–Guerin (BCG) vaccine is now over 100 years old. During this century, we have learnt a lot about what the BCG vaccination does and does not do, including that it does not provide consistent or optimal protection against tuberculosis (TB), and particularly, the most common pulmonary form of the disease, that is responsible for Mycobacterium tuberculosis transmission.1,2 Many new candidate TB vaccines are in the development pipeline, including individual antigens delivered by viral vectors or with adjuvant and live mycobacterial vaccines such as genetically modified BCG or M tuberculosis itself.

Cyclooxygenase-Derived Prostaglandin E2 Drives IL-1-Independent Mycobacterium bovis Bacille Calmette-Guérin-Triggered Skin Dendritic Cell Migration to Draining Lymph Node.

Inoculation of Mycobacterium bovis Bacille Calmette-Guérin (BCG) in the skin mobilizes local dendritic cells (DC) to the draining lymph node (dLN) in a process that remains incompletely understood. In this study, a mouse model of BCG skin infection was used to investigate mechanisms of skin DC migration to dLNs. We found enhanced transcription of cyclooxygenase (COX)-2 and production of COX-derived PGE2 early after BCG infection in skin. Animals treated with antagonists for COX or the PGE2 receptors EP2 and EP4 displayed a marked reduction in the entry of skin DCs and BCG to dLNs, uncovering an important contribution of COX-derived PGE2 in this migration process. In addition, live BCG bacilli were needed to invoke DC migration through this COX-PGE2 pathway. Having previously shown that IL-1R partially regulates BCG-induced relocation of skin DCs to dLNs, we investigated whether PGE2 release was under control of IL-1. Interestingly, IL-1R ligands IL-1α/β were not required for early transcription of COX-2 or production of PGE2 in BCG-infected skin, suggesting that the DC migration-promoting role of PGE2 is independent of IL-1α/β in our model. In DC adoptive transfer experiments, EP2/EP4, but not IL-1R, was needed on the moving DCs for full-fledged migration, supporting different modes of action for PGE2 and IL-1α/β. In summary, our data highlight an important role for PGE2 in guiding DCs to dLNs in an IL-1–independent manner.

Self-adjuvanting nanovaccines boost lung-resident CD4+ T cell immune responses in BCG-primed mice

Heterologous vaccine regimens could extend waning protection in the global population immunized with Mycobacterium bovis Bacille Calmette-Guerin (BCG). We demonstrate that pulmonary delivery of peptide nanofibers (PNFs) bearing an Ag85B CD4+ T cell epitope increased the frequency of antigen-specific T cells in BCG-primed mice, including heterogenous populations with tissue resident memory (Trm) and effector memory (Tem) phenotype, and functional cytokine recall. Adoptive transfer of dendritic cells pulsed with Ag85B-bearing PNFs further expanded the frequency and functional repertoire of memory CD4+ T cells. Transcriptomic analysis suggested that the adjuvanticity of peptide nanofibers is, in part, due to the release of damage-associated molecular patterns. A single boost with monovalent Ag85B PNF in BCG-primed mice did not reduce lung bacterial burden compared to BCG alone following aerosol Mtb challenge. These findings support the need for novel BCG booster strategies that activate pools of Trm cells with potentially diverse localization, trafficking, and immune function.

RNA Microarray-Based Comparison of Innate Immune Phenotypes between Human THP-1 Macrophages Stimulated with Two BCG Strains.

Currently, the only available vaccine against tuberculosis is Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary tuberculosis protection provided by the vaccine varies depending on the strain, the patient’s age and the evaluated population. Although the adaptive immune responses induced by different BCG strains have been widely studied, little conclusive data is available regarding innate immune responses, especially in macrophages. Here, we aimed to characterize the innate immune responses of human THP-1-derived macrophages at the transcriptional level following a challenge with either the BCG Mexico (M.BCG) or Phipps (P.BCG) strains. After a brief in vitro characterization of the bacterial strains and the innate immune responses, including nitric oxide production and cytokine profiles, we analyzed the mRNA expression patterns and performed pathway enrichment analysis using RNA microarrays. Our results showed that multiple biological processes were enriched, especially those associated with innate inflammatory and antimicrobial responses, including tumor necrosis factor (TNF)-α, type I interferon (IFN-I) and IFN-γ. However, four DEGs were identified in macrophages infected with M.BCG compared to P. BCG. These findings indicated the proinflammatory stimulation of macrophages induced by both BCG strains, at the cytokine level and in terms of gene expression, suggesting a differential expression pattern of innate immune transcripts depending on the mycobacterial strain.

Mycobacterium bovis bacille Calmette-Guerin-derived extracellular vesicles as an alternative to live BCG immunotherapy.

For over 40years, the gold standard treatment for non-muscular invasive bladder cancer (NMIBC) has been repeated administration of Mycobacterium bovis bacille Calmette-Guerin (BCG). Upon administration, BCG initiates a cascade of immunological events that lead to the recruitment of immune cells to the bladder that eliminates NMIBC cells in a multi-mechanistic, yet incompletely defined manner. Despite its effectiveness, live BCG immunotherapy is often impacted by limited supply and availability and can cause rare but serious side effects. Bacterial extracellular vesicles (EV) are nanoparticles secreted by live bacteria. EVs are composed of multiple surface proteins, sugars, and lipid that can elicit cellular responses and host recognition similar to live bacteria. In this study, we sought to evaluate the cellular responses of epithelial bladder cancer cells (BCC) to BCG EVs and live BCG. We compared the effect of each treatment on BCC cytokine production, cellular viability and apoptosis. Our data suggest that BCG EVs are as effective as live BCG in eliciting cytokine responses and halting cancer cell growth by, in part, inducing apoptosis. These results indicate that BCG EVs warrant investigation as an alternative to live BCG for NMIBC immunotherapy.

After 100 Years of BCG Immunization against Tuberculosis, What Is New and Still Outstanding for This Vaccine?

In 2021, most of the world was reasonably still concerned about the COVID-19 pandemic, how cases were up and down in different countries, how the vaccination campaigns were ongoing, and most people were familiar with the speed with which vaccines against SARS-Co-V2 were developed, analyzed, and started to be applied in an attempt to curb the pandemic. Because of this, it may have somehow passed relatively inadvertently for people outside of the field that the vaccine used to control tuberculosis (TB), Mycobacterium bovis Bacille Calmette-Guérin (BCG), was first applied to humans a century ago. Over these years, BCG has been the vaccine applied to most human beings in the world, despite its known lack of efficacy to fully prevent respiratory TB. Several strategies have been employed in the last 20 years to produce a novel vaccine that would replace, or boost, immunity and protection elicited by BCG. In this work, to avoid potential redundancies with recently published reviews, I only aim to present my current thoughts about some of the latest findings and outstanding questions that I consider worth investigating to help develop a replacement or modified BCG in order to successfully fight TB, based on BCG itself.