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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: Strain infectivity, viability, co-localisation with acidified vesicles, macrophage secretion of IL-1 and MCP-1 and lipid droplet biogenesis were evaluated after infection. FINDINGS: 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-1ß and MCP-1 secretion and lipid droplet biogenesis by infected macrophages were more prominent in response to BCG Pasteur. MAIN CONCLUSION: 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.

In the footsteps of Albert Calmette: an ecological study of TB, leprosy and potential exposure to wild-type Mycobacterium bovis.

BACKGROUND: One hundred years ago, Albert Calmette developed an avirulent strain of Mycobacterium bovis, but there is no evidence that his BCG strain was more immunogenic than wild-type M. bovis. Geographic variations in BCG efficacy remain ill-understood. We hypothesized that exposure to M. bovis through unpasteurized milk might protect against Mycobacterium tuberculosis and Mycobacterium leprae. METHODS: After excluding high-income countries (with universal milk pasteurization) and microstates, an ecological study comprising 113 countries was conducted. National data were obtained from United Nations agencies and international organizations about milk production per capita (1980-1999) as a proxy for exposure to wild-type M. bovis, TB (2000-2019) and leprosy (2005-2019) incidence, HIV prevalence (2000-2019), human development index (2010), global hunger index (2010), neonatal BCG coverage (1980-1999), urbanization (2000) and temperature (1990-2020). Multiple linear regression analyses were performed using log-transformed variables. RESULTS: For TB, the association differed by region. An inverse association with milk production was seen in regions outside, but not within, sub-Saharan Africa, after adjustment for confounders. The incidence of leprosy was inversely associated with milk production when combining all countries, but the association was stronger in sub-Saharan Africa. CONCLUSIONS: Exposure to wild-type M. bovis through unpasteurized milk may provide cross-protection against M. tuberculosis and M. leprae and contribute to geographic disparities in BCG efficacy. This needs to be confirmed by individual-level studies.

Tuberculous spondylitis following intravesical Bacillus Calmette-Guérin therapy for bladder cancer surgically treated through the anterior approach.

Intravesical BCG therapy is commonly used to treat superficial bladder cancer. Although various complications associated with this therapy have been reported, tuberculous spondylitis is uncommon. Here, we report a rare case of tuberculous spondylitis that occurred after intravesical BCG therapy for bladder cancer. A man in his 80s received BCG immunotherapy for bladder cancer and developed low back pain after treatment. Remarkably, he presented with neurological symptoms. Spondylitis was suspected on imaging. CT-guided biopsy was performed to confirm the diagnosis. Consequently, Mycobacterium bovis was identified as the causative pathogen by multiplex PCR. Multidrug therapy, administered for several months, was ineffective. Therefore, surgery was performed through an anterior approach. The symptoms, including low back pain, improved and postoperative C reactive protein tests were within the normal range. Tuberculous spondylitis following BCG therapy should be considered in cases with a history of bladder cancer treatment.

Investigating Bacterial Volatilome for the Classification and Identification of Mycobacterial Species by HS-SPME-GC-MS and Machine Learning.

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography-quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.

Bacillus Calmette-Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally.

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Cytological diagnosis in a clinically unsuspected case of disseminated BCGosis: A case report.

Bacille Calmette-Guerin (BCG) vaccine is administered worldwide to neonates and considered safe. Serious complications like disseminated BCGosis are extremely rare occurrences (<1 per million vaccinations). A 6 months male was brought to paediatric outpatient department with fever and swelling over the dorsum of the left hand for 5 days. On examination, he was febrile and had hepatosplenomegaly. X-ray of the hand showed lytic lesions in the first and second metacarpals. Provisional clinical diagnosis included Langerhans cell histiocytosis, congenital syphilis, and haematological malignancy. Fine Needle Aspiration Cytology (FNAC) was done from the swelling and showed diffuse sheets of histiocytes with both intracellular and extracellular rod-shaped unstained structures along with inflammatory cells. These ghost images stained positive with ZN stain. A cytological diagnosis of atypical mycobacteria vs leprosy was made. Child was revisited and found to have an active BCG scar. Further investigations showed low serum IgM and positive AFB culture. These bacilli were confirmed by GenoType MTBDR plus test as Mycobacterium bovis. Despite Antitubercular therapy, the patient succumbed to death. This case highlights the variable clinical presentation of BCGosis. Its occurrence may unmask any underlying immunodeficiency. If unfamiliar with the above cytological features and in absence of routinely performed special stains, the cytopathologist may miss these notorious organisms and treat such cases like suppurative lesions. To conclude, an early and definitive diagnosis of BCGosis can be established on FNAC which would ensure timely management and better outcome in this highly lethal entity.

Rv2617c and P36 are virulence factors of pathogenic mycobacteria involved in resistance to oxidative stress.

In this study, we characterized the role of Rv2617c in the virulence of Mycobacterium tuberculosis. Rv2617c is a protein of unknown function unique to M. tuberculosis complex (MTC) and Mycobacterium leprae. In vitro, this protein interacts with the virulence factor P36 (also named Erp) and KdpF, a protein linked to nitrosative stress. Here, we showed that knockout of the Rv2617c gene in M. tuberculosis CDC1551 reduced the replication of the pathogen in a mouse model of infection and favored the trafficking of mycobacteria to phagolysosomes. We also demonstrated that Rv2617c and P36 are required for resistance to in vitro hydrogen peroxide treatment in M. tuberculosis and Mycobacterium bovis, respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.

BCG and Adverse Events in the Context of Leprosy.

Background: Notwithstanding its beneficial immunoprophylactic outcomes regarding leprosy and childhood TB, BCG vaccination may cause adverse events, particularly of the skin. However, this local hyper-immune reactivity cannot be predicted before vaccination, nor is its association with protection against leprosy known. In this study we investigated the occurrence of adverse events after BCG (re)vaccination in contacts of leprosy patients and analyzed whether the concomitant systemic anti-mycobacterial immunity was associated with these skin manifestations. Methods: Within a randomized controlled BCG vaccination trial in Bangladesh, 14,828 contacts of newly diagnosed leprosy patients received BCG vaccination between 2012 and 2017 and were examined for adverse events 8 to 12 weeks post-vaccination. From a selection of vaccinated contacts, venous blood was obtained at follow-up examination and stimulated with Mycobacterium leprae (M. leprae) antigens in overnight whole-blood assays (WBA). M. leprae phenolic glycolipid-I-specific antibodies and 32 cytokines were determined in WBAs of 13 individuals with and 13 individuals without adverse events after vaccination. Results: Out of the 14,828 contacts who received BCG vaccination, 50 (0.34%) presented with adverse events, mainly (80%) consisting of skin ulcers. Based on the presence of BCG scars, 30 of these contacts (60%) had received BCG in this study as a booster vaccination. Similar to the pathological T-cell immunity observed for tuberculoid leprosy patients, contacts with adverse events at the site of BCG vaccination showed elevated IFN-γ levels in response to M. leprae-specific proteins in WBA. However, decreased levels of sCD40L in serum and GRO (CXCL1) in response to M. leprae simultaneously indicated less T-cell regulation in these individuals, potentially causing uncontrolled T-cell immunity damaging the skin. Conclusion: Skin complications after BCG vaccination present surrogate markers for protective immunity against leprosy, but also indicate a higher risk of developing tuberculoid leprosy. Clinical Trial Registration: Netherlands Trial Register: NTR3087.

Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae.

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.

F420H2 Is Required for Phthiocerol Dimycocerosate Synthesis in Mycobacteria.

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.

STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen.

M. tuberculosis DNA detection in nasopharyngeal mucosa can precede tuberculosis development in contacts.

BACKGROUND: The nasopharynx is a known gateway for some mycobacterial species such as Mycobacterium bovis and M. leprae. M. tuberculosis can cross lymphoepithelial barriers in vitro, but its ability to colonise the nasopharyngeal mucosa in vivo has not been established. OBJECTIVE: To determine if M. tuberculosis can be transiently detected in nasopharyngeal mucosa of tuberculosis (TB) contacts as a preliminary step in the development of tuberculous infection. DESIGN: Exploratory study conducted among asymptomatic household contacts of pulmonary TB cases. A chest X-ray, QuantiFERON(®) TB-Gold or tuberculin skin test and a bilateral nasopharyngeal swab for Xpert(®) MTB/RIF and mycobacterial culture were performed at baseline and repeated 8-12 weeks later. RESULTS: Eighty-nine contacts were enrolled a median of 9 days after the diagnosis of the index case. At baseline, 29.9% were positive for latent tuberculous infection and one subject (1.1%) had a positive Xpert in the nasopharyngeal swab with a normal chest X-ray, negative QuantiFERON and negative induced sputum. After 12 weeks' follow-up, this subject developed a new cough and upper lobe infiltrates and M. tuberculosis grew in sputum. No other cases of active TB were detected at follow-up. CONCLUSION: The detection of M. tuberculosis DNA in the nasopharyngeal mucosa of contacts is an infrequent event that in this instance preceded the development of pulmonary TB. Its pathogenic role requires further investigation.

Mycobacterial r32-kDa antigen-specific T-cell responses correlate with successful treatment and a heightened anti-microbial response in human leprosy patients.

BACKGROUND: Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. METHODS: Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). RESULTS: Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. CONCLUSIONS: These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients.

STING-Dependent 2'-5' oligoadenylate synthetase-like production is required for intracellular Mycobacterium leprae survival

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen.

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis.

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0(+)CD69(+)CD4(+) memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.

Disseminated cutaneous BCG infection following BCG immunotherapy in patients with lepromatous leprosy.

Cutaneous complications of Bacillus Calmette-Guerin (BCG) vaccine, especially in the form of generalised disease, are uncommon and mostly occur in immunocompromised individuals. There is a paucity of data on the cutaneous adverse reactions secondary to BCG immunotherapy in leprosy. We report two unique cases of disseminated cutaneous BCG infection following immunotherapy in patients with lepromatous leprosy. To our knowledge, cutaneous BCG infection presenting as widespread lesions after immunotherapy and confirmed by isolation of Mycobacterium bovis by polymerase chain reaction (PCR) has not been described. A high index of suspicion is required when leprosy patients who receive BCG immunotherapy develop new lesions that cannot be classified as either reaction or relapse, and diagnosis may be confirmed on histopathology and PCR.

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.

rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B.

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

ProTides of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide as potential anti-tubercular and anti-viral agents.

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.