Proteome and immunogenicity differences in BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c during planktonic growth in 7H9 and Proskauer Beck media.

Bacillus Calmette-Guèrin (BCG) remains as the only vaccine employed to prevent tuberculosis (TB) during childhood. Among factors likely contributing to the variable efficacy of BCG is the modification in its antigenic repertoire that may arise from in vitro growth conditions. Our vaccine candidate, BCGΔBCG1419c, improved protection against TB in mice and guinea pigs with bacteria grown in either 7H9 OADC Tween 80 or in Proskauer Beck Tween 80 media in independent studies. Here, we compared the proteomes of planktonic cultures of BCG and BCGΔBCG1419c, grown in both media. Further to this, we compared systemic immunogenicity ex vivo elicited by both types of BCG strains and cultures when used to vaccinate BALB/c mice. Both the parental strain BCG Pasteur ATCC 35734, and its isogenic mutant BCGΔBCG1419c, had several medium-dependent changes. Moreover, ex vivo immune responses to a multiantigenic (PPD) or a single antigenic (Ag85A) stimulus were also medium-dependent. Then, not only the presence or absence of the BCG1419c gene in our strains under study affected the proteome produced in vitro but also that this was affected by culture medium, potentially leading to changes in the capacity to induce ex vivo immune responses.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice.

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

Genome sequences of BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c.

BACKGROUND: Bacillus Calmette-Guérin (BCG) remains the only vaccine to prevent tuberculosis (TB) during childhood, with relatively low to no efficacy against pulmonary TB in adolescents and adults. BCG consists of close to 15 different substrains, where genetic variations among them might contribute to the variable protective efficacy afforded against pulmonary TB. We have shown that the vaccine candidate, BCGΔBCG1419c, which is based on BCG Pasteur, improved protection against chronic TB in murine models, as well as against pulmonary and extrapulmonary TB in guinea pigs. Here, to confirm deletion of the BCG1419c gene and to detect possible genetic variations occurring as a consequence of the spontaneous mutations that may arise during in vitro culture of mycobacteria, the genomes of BCG Pasteur ATCC 35734 and its isogenic derivative, BCGΔBCG1419c, were sequenced and subjected to a comparative analysis between them and against BCG Pasteur 1173P2. RESULTS: The complete catalog of variants in genes relative to the reference genome BCG Pasteur 1173P2 (GenBank NC008769) showed that the parental strain BCG Pasteur ATCC 35734, from which the mutant BCGΔBCG1419c originated, showed five synonymous mutations, three missense mutations, and five codon insertions, whereas the BCGΔBCG1419c mutant reported the same changes. When BCG Pasteur ATCC 35734 and BCGΔBCG1419c were compared, we confirmed that the latter was devoid of the BCG1419c gene, with only one unanticipated SNP at position 2, 828, 791  which we consider has no role in vaccine properties reported thus far. CONCLUSION: We provide evidence that the mutagenesis performed to remove BCG1419c from BCG Pasteur ATCC 35734 solely deleted this gene, and that compared with the reference strain BCG Pasteur 1173P2, few changes were present confirming that they are BCG Pasteur strains, and that changes in immunogenicity or efficacy observed thus far in BCGΔBCG1419c are most likely derived solely from the elimination of the BCG1419c gene.

BCGΔBCG1419c increased memory CD8+ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis.

Previously, we reported that a hygromycin resistant version of the BCGΔBCG1419c vaccine candidate reduced tuberculosis (TB) disease in BALB/c, C57BL/6, and B6D2F1 mice infected with Mycobacterium tuberculosis (Mtb) H37Rv. Here, the second-generation version of BCGΔBCG1419c (based on BCG Pasteur ATCC 35734, without antibiotic resistance markers, and a complete deletion of BCG1419c) was compared to its parental BCG for immunogenicity and protective efficacy against the Mtb clinical isolate M2 in C57BL/6 mice. Both BCG and BCGΔBCG1419c induced production of IFN-γ, TNF-α, and/or IL-2 by effector memory (CD44+CD62L-), PPD-specific, CD4+ T cells, and only BCGΔBCG1419c increased effector memory, PPD-specific CD8+ T cell responses in the lungs and spleens compared with unvaccinated mice before challenge. BCGΔBCG1419c increased levels of central memory (CD62L+CD44+) T CD4+ and CD8+ cells compared to those of BCG-vaccinated mice. Both BCG strains elicited Th1-biased antigen-specific polyfunctional effector memory CD4+/CD8+ T cell responses at 10 weeks post-infection, and both vaccines controlled Mtb M2 growth in the lung and spleen. Only BCGΔBCG1419c significantly ameliorated pulmonary inflammation and decreased neutrophil infiltration into the lung compared to BCG-vaccinated and unvaccinated mice. Both BCG strains reduced pulmonary TNF-α, IFN-γ, and IL-10 levels. Taken together, BCGΔBCG1419c increased memory CD8+T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG.

Evaluation of early innate and adaptive immune responses to the TB vaccine Mycobacterium bovis BCG and vaccine candidate BCGΔBCG1419c.

The vaccine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) elicits an immune response that is protective against certain forms of tuberculosis (TB); however, because BCG efficacy is limited it is important to identify alternative TB vaccine candidates. Recently, the BCG deletion mutant and vaccine candidate BCGΔBCG1419c was demonstrated to survive longer in intravenously infected BALB/c mice due to enhanced biofilm formation, and better protected both BALB/c and C57BL/6 mice against TB-induced lung pathology during chronic stages of infection, relative to BCG controls. BCGΔBCG1419c-elicited protection also associated with lower levels of proinflammatory cytokines (i.e. IL6, TNFα) at the site of infection in C57BL/6 mice. Given the distinct immune profiles of BCG- and BCGΔBCG1419c-immunized mice during chronic TB, we set out to determine if there are early immunological events which distinguish these two groups, using multi-dimensional flow cytometric analysis of the lungs and other tissues soon after immunization. Our results demonstrate a number of innate and adaptive response differences between BCG- and BCGΔBCG1419c-immunized mice which are consistent with the latter being longer lasting and potentially less inflammatory, including lower frequencies of exhausted CD4+ T helper (TH) cells and higher frequencies of IL10-producing T cells, respectively. These studies suggest the use of BCGΔBCG1419c may be advantageous as an alternative TB vaccine candidate.

BCG and BCGΔBCG1419c transiently protect hypersusceptible I/St mice and induce different influx of macrophages and neutrophils during pulmonary tuberculosis.

Background. Host genetic factors influence both susceptibility to Mycobacterium tuberculosis infection and immune responses generated by vaccination. Genetically susceptible mice help to study mechanisms of immune protection which may differ from those operating in more resistant models.Methods. In this work, we compared the efficacy of protection conferred by subcutaneous vaccination of hypersusceptible I/St mice with BCG and the first-generation, hygromycin resistant version of the vaccine candidate BCGΔBCG1419c, against tuberculosis (TB), measured as survival, weight loss and replication in lungs. We further characterized the relative presence of immune cells in lungs.Results. We found that in I/St mice, vaccination with BCG or BCGΔBCG1419c provided similar level of protection against TB-driven weight loss and M. tuberculosis replication in lungs, while prolonging median survival time compared with unvaccinated controls. Despite affording similar protection to parental BCG, BCGΔBCG1419c led to a reduced presence of macrophages in lungs during early TB and to an increased neutrophil recruitment to the lungs during chronic TB.Conclusions. BCGΔBCG1419c protects I/St mice in a different manner than wild-type BCG against pulmonary TB by promoting different influx of macrophages and neutrophils at distinct times post-infection. These findings prompt us to suggest that preclinical evaluation of novel TB vaccine candidates should include evaluation of efficacy not only in commonly used resistant inbred mice, but also in susceptible hosts, to further determine their potential application to populations varying in their genetic. This would likely impact their intended use depending on host resistance or susceptibility to TB.

Construction of Novel Live Genetically Modified BCG Vaccine Candidates Using Recombineering Tools.

One of the strategies for the construction of live vaccine candidates is through the generation of genetically defined isogenic strains, containing single or multiple mutations in target-specific genes generated by allelic exchange. This approach allows to produce rational attenuation of or, alternatively, sequence-specific modifications to produce variants of antigenic molecules or change their expression levels. Genetic tools amenable for their use in mycobacterial strains have allowed the identification and validation of potential targets for the diagnosis, prevention, and treatment of tuberculosis. However, the genetic manipulation of Mycobacterium tuberculosis and other slow-growing strains such as Mycobacterium bovis BCG has been delayed by various factors related to their physiology and cell wall characteristics. Notwithstanding the foregoing, the high frequency of illegitimate recombination and the availability of few antibiotic selection markers limit the feasibility of genetic manipulation of mycobacterial strains. This chapter describes a protocol for the generation of defined mutants using recombination tools in an inducible recombination system driven by mycobacterial Che9c phage RecET proteins, originally developed in Dr. Graham Hatfull's group, combined with linearized recombination substrates containing flanking sequences of a locus of interest and an antibiotic resistance gene. These recombination substrates contain sites for removal of antibiotics selection markers. This system allows to make marked and unmarked mutations by homologous recombination in a single step as a result of a double crossover between the homologous regions on the genome and the allelic exchange substrate. In addition, this genetic tool used for engineering mycobacterial genomes performs with lower rates of illegitimate recombination and take on average less time to create knock-out (KO) mutant compared with other techniques.

New Insights into the Methylation of Mycobacterium tuberculosis Heparin Binding Hemagglutinin Adhesin Expressed in Rhodococcus erythropolis.

In recent years, knowledge of the role that protein methylation is playing on the physiopathogenesis of bacteria has grown. In Mycobacterium tuberculosis, methylation of the heparin binding hemagglutinin adhesin modulates the immune response, making this protein a subunit vaccine candidate. Through its C-terminal lysine-rich domain, this surface antigen interacts with heparan sulfate proteoglycans present in non-phagocytic cells, leading to extrapulmonary dissemination of the pathogen. In this study, the adhesin was expressed as a recombinant methylated protein in Rhodococcus erythropolis L88 and it was found associated to lipid droplets when bacteria were grown under nitrogen limitation. In order to delve into the role methylation could have in host-pathogen interactions, a comparative analysis was carried out between methylated and unmethylated protein produced in Escherichia coli. We found that methylation had an impact on lowering protein isoelectric point, but no differences between the proteins were found in their capacity to interact with heparin and A549 epithelial cells. An important finding was that HbhA is a Fatty Acid Binding Protein and differences in the conformational stability of the protein in complex with the fatty acid were observed between methylated and unmethylated protein. Together, these results suggest that the described role for this mycobacteria protein in lipid bodies formation could be related to its capacity to transport fatty acids. Obtained results also provide new clues about the role HbhA methylation could have in tuberculosis and point out the importance of having heterologous expression systems to obtain modified proteins.

Vaccination with BCGΔBCG1419c protects against pulmonary and extrapulmonary TB and is safer than BCG.

A single intradermal vaccination with an antibiotic-less version of BCGΔBCG1419c given to guinea pigs conferred a significant improvement in outcome following a low dose aerosol exposure to M. tuberculosis compared to that provided by a single dose of BCG Pasteur. BCGΔBCG1419c was more attenuated than BCG in murine macrophages, athymic, BALB/c, and C57BL/6 mice. In guinea pigs, BCGΔBCG1419c was at least as attenuated as BCG and induced similar dermal reactivity to that of BCG. Vaccination of guinea pigs with BCGΔBCG1419c resulted in increased anti-PPD IgG compared with those receiving BCG. Guinea pigs vaccinated with BCGΔBCG1419c showed a significant reduction of M. tuberculosis replication in lungs and spleens compared with BCG, as well as a significant reduction of pulmonary and extrapulmonary tuberculosis (TB) pathology measured using pathology scores recorded at necropsy. Evaluation of cytokines produced in lungs of infected guinea pigs showed that BCGΔBCG1419c significantly reduced TNF-α and IL-17 compared with BCG-vaccinated animals, with no changes in IL-10. This work demonstrates a significantly improved protection against pulmonary and extrapulmonary TB provided by BCGΔBCG1419c in susceptible guinea pigs together with an increased safety compared with BCG in several models. These results support the continued development of BCGΔBCG1419c as an effective vaccine for TB.

Proteomic characterization of a second-generation version of the BCGΔBCG1419c vaccine candidate by means of electrospray-ionization quadrupole time-of-flight mass spectrometry.

Tuberculosis (TB) is the most important infectious disease worldwide, based on the number of new cases and deaths reported by the World Health Organization. Several vaccine candidates against TB have been characterized at preclinical and clinical levels. The BCGΔBCG1419c vaccine candidate, which lacks the BCG1419c gene that encodes for a c-di-GMP phosphodiesterase, provides improved efficacy against chronic TB, reactivation from latent-like infection and against chronic TB in the presence of type 2 diabetes in murine models. We previously reported that compared with wild type BCG, BCGΔBCG1419c changed levels of several proteins. Here, using a label-free proteomic approach, we confirmed that a novel, second-generation version of BCGΔBCG1419c maintains changes in antigenic proteins already reported, and here we further found differences in secreted proteins, as well as that this new BCGΔBCG1419c version modifies its production of proteins involved in redox and nitrogen/protein metabolism compared with wild type BCG. This work contributes to the proteomic characterization of a novel vaccine candidate that is more effective against TB than parental BCG in diverse murine models.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication.

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

Transcriptional portrait of M. bovis BCG during biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment.

Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BCG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. Our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria.

Transcriptional and Mycolic Acid Profiling in Mycobacterium bovis BCG In Vitro Show an Effect for c-di-GMP and Overlap between Dormancy and Biofilms.

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

BCG and BCGΔBCG1419c protect type 2 diabetic mice against tuberculosis via different participation of T and B lymphocytes, dendritic cells and pro-inflammatory cytokines.

Comorbidity between Tuberculosis (TB) and type 2 diabetes (T2D) is one of the greatest contributors to the spread of Mycobacterium tuberculosis (M. tuberculosis) in low- and middle-income countries. T2D compromises key steps of immune responses against M. tuberculosis and it might affect the protection afforded by vaccine candidates against TB. We compared the protection and immune response afforded by the BCGΔBCG1419c vaccine candidate versus that of wild-type BCG in mice with T2D. Vaccination with both BCGΔBCG1419c, BCG or infection with M. tuberculosis reduced weight loss, hyperglycemia, and insulin resistance during T2D progression, suggesting that metabolic changes affecting these parameters were affected by mycobacteria. For control of acute TB, and compared with non-vaccinated controls, BCG showed a dominant T CD4+ response whereas BCGΔBCG1419c showed a dominant T CD8+/B lymphocyte response. Moreover, BCG maintained an increased response in lung cells via IFN-γ, TNF-α, and IL-4, while BCGΔBCG1419c increased IFN-γ but reduced IL-4 production. As for chronic TB, and compared with non-vaccinated controls, both BCG strains had a predominant presence of T CD4+ lymphocytes. In counterpart, BCGΔBCG1419c led to increased presence of dendritic cells and an increased production of IL-1 ß. Overall, while BCG effectively reduced pneumonia in acute infection, it failed to reduce it in chronic infection, whereas we hypothesize that increased production of IL-1 ß induced by BCGΔBCG1419c contributed to reduced pneumonia and alveolitis in chronic TB. Our results show that BCG and BCGΔBCG1419c protect T2D mice against TB via different participation of T and B lymphocytes, dendritic cells, and pro-inflammatory cytokines.

BCG Vaccination Prevents Reactivation of Latent Lymphatic Murine Tuberculosis Independently of CD4+ T Cells.

Tuberculosis (TB) is a major global public health problem causing significant mortality and morbidity. In addition to ~10.4 million cases of active TB annually, it is estimated that about two billion people are latently infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB. Reactivation of latent Mtb infection is the leading cause of death in patients with immunodeficiency virus (HIV) infection. The low efficiency of the only licensed anti-TB vaccine, Bacille Calmette-Guérin (BCG) to reduce pulmonary TB in adults contributes to this problem. Here we investigated if vaccination with conventional BCG or the genetically modified experimental BCGΔBCG1419c strain can prevent reactivation of latent lymphatic TB in a mouse model of induced reactivation, following the depletion of CD4+ T cells, as it occurs in HIV+ individuals. Vaccination with conventional BCG or BCGΔBCG1419c prevented reactivation of Mtb from the infected lymph node and the systemic spread of Mtb to spleen and lung. Prevention of reactivation was independent of vaccination route and was accompanied by reduced levels of circulating inflammatory cytokines and the absence of lung pathology. Our results demonstrate that vaccine-induced CD4+ T cells are not essential to prevent reactivation of latent lymphatic murine TB, and highlight the need to better understand how non-CD4+ immune cell populations participate in protective immune responses to control latent TB.

Macrophage infection with combinations of BCG mutants reduces induction of TNF-α, IL-6, IL-1ß and increases IL-4.

Tuberculosis (TB) is the most prevalent infectious disease worldwide, with no fully effective vaccine yet available. Considering that BCG strains devoid of the BCG1416c or BCG1419c genes afforded protection in mice versus highly virulent M. tuberculosis challenge, or in chronic infection models compared to BCG, respectively, we hypothesized that a synergistic effect of these strains might occur and provide enhanced protection against TB. Herein, we evaluated this hypothesis throughout an experimental design approach, where different combinations of these strains were tested for their capacity to induce cytokines in vitro, compared to individual strains. Our results show that mixed-infection of murine macrophages using these strains significantly decreases induction of TNF-α, IL-1ß, IL-6 but increases IL-4 induction compared with individual strains. These results suggest the existence of interaction effects during infection, which reduce induction of pro-inflammatory cytokines, even though individual intracellular replication is not altered when strains are combined. This is the first report of the evaluation of a potential whole-live combined vaccine against tuberculosis, which paradoxically seems to reduce production of pro-inflammatory cytokines while induces IL-4, leading us to further hypothesize that this combination might contribute as a therapeutic vaccine to reduce inflammation in severe TB cases.

BCG constitutively expressing the adenylyl cyclase encoded by Rv2212 increases its immunogenicity and reduces replication of M. tuberculosis in lungs of BALB/c mice.

Mycobacterium tuberculosis remains as a threat to public health around the world with 1.7 million cases of TB-associated deaths during 2016. Despite the use of Bacillus Calmette-Guerin (BCG) vaccine, control of the infection has not been successful. Because of this, several efforts have been made in order to develop new vaccines capable of boosting previous immunization or attempted for replacing current BCG. We previously showed that over expression of the M. tuberculosis adenylyl cyclase encoding gene Rv2212 in BCG bacilli (BCG-Rv2212), induced an attenuated phenotype when administered in BALB/c mice. Moreover, two-dimensional proteomic analysis showed that heat shock proteins such as GroEL2 and DnaK were overexpressed in this BCG-Rv2212. In this report, we show that immunization of mice with BCG-Rv2212 significantly increments IFN-γ+ CD4+ and CD8+ T-lymphocytes after PPD stimulation in comparison with BCG vaccinated mice. Mice vaccinated with BCG-Rv2212 significantly reduced the bacterial load in lungs after four-month post infection with M. tuberculosis H37Rv but was similar to BCG after 6 month-post-challenge. Survival experiment showed that both vaccines administered separately in mice induce similar levels of protection after 20-week post-challenge with M. tuberculosis H37Rv. Virulence experiments developed in nude mice, showed that BCG-Rv2212 and BCG bacilli were equally safe. Our results suggest that BCG-Rv2212 is capable of stimulating cellular immune response effectively and reduce bacterial burden in lungs of mice after challenge. Particularly, it seems to be more effective in controlling bacterial burden during the first steps of infection.

Vaccination of guinea pigs with BCGΔBCG1419c transiently reduces hematogenous spread of M. tuberculosis to the spleen.

The guinea pig has proven to be a reliable model for testing vaccine candidates against tuberculosis (TB) because of its capacity to produce human-like disease associated to primary TB, thus providing a more stringent test of the ability of a vaccine to prevent disease and deaths. Here, the BCGΔBCG1419c vaccine candidate, which previously has been shown to provide protection in mice, was tested in a guinea pig model. We found that this vaccine candidate was as effective as parental BCG in reducing M. tuberculosis H37Rv replication in lungs, and significantly reducing hematogenous spread to spleen at 60 days post-infection in comparison with BCG. Moreover, lung histopathological examination revealed comparable protection between the parental and mutant BCG strains, with some differences in prevention of primary lesions or necrosis at a single time point post-infection in a strain-dependent manner. Our results show that the BCGΔBCG1419c vaccine candidate is as effective as BCG in reducing M. tuberculosis H37Rv replication in lungs and reducing lung pathology, as well as significantly improving control of its dissemination to spleens up to 60 days post-infection.

The BCGΔBCG1419c Vaccine Candidate Reduces Lung Pathology, IL-6, TNF-α, and IL-10 During Chronic TB Infection.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of human tuberculosis (TB), is estimated to be harbored by up to 2 billion people in a latent TB infection (LTBI) state. The only TB vaccine approved for use in humans, BCG, does not confer protection against establishment of or reactivation from LTBI, so new vaccine candidates are needed to specifically address this need. Following the hypothesis that mycobacterial biofilms resemble aspects of LTBI, we modified BCG by deleting the BCG1419c gene to create the BCGΔBCG1419c vaccine strain. In this study, we compared cytokine profiles, bacterial burden, and lung lesions after immunization with BCG or BCGΔBCG1419c before and after 6 months of aerosol infection with M. tuberculosis H37Rv in the resistant C57BL/6 mouse model. Our results show that in infected mice, BCGΔBCG1419c significantly reduced lung lesions and IL-6 in comparison to the unmodified BCG strain, and was the only vaccine that decreased production of TNF-α and IL-10 compared to non-vaccinated mice, while vaccination with BCG or BCGΔBCG1419c significantly reduced IFN-γ production. Moreover, transcriptome profiling of BCGΔBCG1419c suggests that compared to BCG, it has decreased expression of genes involved in mycolic acids (MAs) metabolism, and antigenic chaperones, which might be involved in reduced pathology compared to BCG-vaccinated mice.