Live Tuberculosis Vaccine Research Articles

Correlation between systemic allergen desensitisation and long-term asthma protection in mice following intravenous administration of the live tuberculosis vaccine MTBVAC

MTBVAC is a live attenuated tuberculosis vaccine, currently undergoing phase III evaluation for tuberculosis prevention. In previous preclinical studies, we found that local pulmonary administration of MTBVAC via the intranasal route had a strong therapeutic effect against asthma. This effect correlated with the abrogation of allergen-specific Th2 response in the lungs. Using different mouse models of asthma, we investigated the effect of MTBVAC administered by intravenous (IV) route and its potential as immunotherapeutic agent to induce desensitisation of allergen-specific responses at a systemic level. We explored the effects of this process in the efficacy against airway hyperresponsiveness (AHR) induced by exposure to different allergens. IV MTBVAC was highly efficient at reducing AHR induced by different allergens. Additionally, IV MTBVAC was found to be well-tolerated, being progressively eliminated from the different organs analysed. From a mechanistic standpoint, we observed that MTBVAC intravenous, but not intranasal, impaired allergen-specific Th2 response in both lungs and spleen. This reduction at a systemic level correlated with long-term therapeutic protection against allergen exposure. Our results also revealed differential immunological mechanisms governing systemic and local pulmonary allergen desensitisation processes. Notably, in a cohort of patients with asthma sensitive to house dust mite (HDM), invitro incubation of peripheral blood mononuclear cells (PBMCs) with MTBVAC prevented allergen-specific production of Th2 cytokines IL-4 and IL-5. Altogether, our results suggest that intravenous MTBVAC could be a plausible allergen desensitising approach for treatment of asthma, and could provide long-term protection against allergen exposure. MCIN/AEI/10.13039/501100011033 [grants number RTI2018-097625-B-I00 and PID2022-138624OB-I00]; Consorcio Centro de Investigación Biomédica en Red- (Groups CB06/06/0020 and CB06/06/0013), Instituto de Salud Carlos III.

Immunogenicity and vaccine potential of clinical isolate Mycobacterium kansasii strain against Mycobacterium tuberculosis infection.

Mycobacterium kansasii is a bacterium included in non-tuberculous mycobacteria (NTM) that can cause lung disease. It shares a significant number of antigens with Mycobacterium tuberculosis (Mtb), suggesting that it has the potential to be used as a tuberculosis (TB) vaccine. Therefore, we subcutaneously vaccinated mice with reference strain, M. kansasii-ATCC12478 [M. kansasii-American Type Culture Collection (ATCC)], and clinically isolated strain, M. kansasii-SM-1 to evaluate potential as a TB vaccine by comparing with bacille Calmette-Guerin (BCG) vaccine. Ten weeks after vaccination, we evaluated immunogenicity of M. kansasii-ATCC and M. kansasii-SM-1, and M. kansasii-SM-1 immunization induces potent Mtb antigen-specific IFN-γ-producing CD4+ T cells than M. kansasii-ATCC. Upon Mtb infection, M. kansasii-SM-1 provided better protection than M. kansasii-ATCC, which was comparable to the efficacy of BCG. These results showed that the clinical strain M. kansasii-SM-1, which exhibits an enhanced Mtb antigen-specific Th1 response, shows greater vaccine efficacy compared to M. kansasii-ATCC. In this study, we demonstrated that vaccine efficacy can vary depending on the strain of M. kansasii and that its efficacy can be comparable to BCG. This suggests that M. kansasii has the potential to be a live TB vaccine candidate.IMPORTANCEMycobacterium kansasii, a non-tuberculous mycobacteria (NTM) species causing lung disease, shares key antigens with Mycobacterium tuberculosis (Mtb), indicating its potential for TB vaccine development. Subcutaneous vaccination of mice with M. kansasii strains reference strain M. kansasii-ATCC12478 [(M. kansasii-American Type Culture Collection (ATCC)] and clinically isolated strain M. kansasii-SM-1 revealed differences in immunogenicity. M. kansasii-SM-1 induced a robust Mtb antigen-specific IFN-γ-producing CD4+ T cell response compared to M. kansasii-ATCC. Additionally, M. kansasii-SM-1 conferred better protection against Mtb infection than M. kansasii-ATCC, which is comparable to bacille Calmette-Guerin (BCG). These findings underscore the variable vaccine efficacy among M. kansasii strains, with M. kansasii-SM-1 exhibiting promising potential as a live TB vaccine candidate, suggesting its comparative effectiveness to BCG.

The Mycobacterium tuberculosis PhoPR virulence system regulates expression of the universal second messenger c-di-AMP and impacts vaccine safety and efficacy

Cyclic (di)nucleotides act as universal second messengers endogenously produced by several pathogens. Specifically, the roles of c-di-AMP in Mycobacterium tuberculosis immunity and virulence have been largely explored, although its contribution to the safety and efficacy of live tuberculosis vaccines is less understood. In this study, we demonstrate that the synthesis of c-di-AMP is negatively regulated by the M. tuberculosis PhoPR virulence system. Accordingly, the live attenuated tuberculosis vaccine candidate M. tuberculosis vaccine (MTBVAC), based on double phoP and fadD26 deletions, produces more than 25- and 45-fold c-di-AMP levels relative to wild-type M. tuberculosis or the current vaccine bacille Calmette-Guérin (BCG), respectively. Secretion of this second messenger was exclusively detected in MTBVAC but not in M. tuberculosis or in BCG. We also demonstrate that c-di-AMP synthesis during in vitro cultivation of M. tuberculosis is a growth-phase- and medium-dependent phenotype. To uncover the role of this metabolite in the vaccine properties of MTBVAC, we constructed and validated knockout and overproducing/oversecreting derivatives by inactivating the disA or cnpB gene, respectively. All MTBVAC derivatives elicited superior interleukin-1β (IL-1β) responses compared with BCG during an in vitro infection of human macrophages. However, both vaccines failed to elicit interferon β (IFNβ) activation in this cellular model. We found that increasing c-di-AMP levels remarkably correlated with a safer profile of tuberculosis vaccines in the immunodeficient mouse model. Finally, we demonstrate that overproduction of c-di-AMP due to cnpB inactivation resulted in lower protection of MTBVAC, while the absence of c-di-AMP in the MTBVAC disA derivative maintains the protective efficacy of this vaccine in mice.

Therapeutic efficacy of pulmonary live tuberculosis vaccines against established asthma by subverting local immune environment.

BackgroundSubstantial recent advances in the comprehension of the molecular and cellular mechanisms behind asthma have evidenced the importance of the lung immune environment for disease outcome, making modulation of local immune responses an attractive therapeutic target against this pathology. Live attenuated mycobacteria, such as the tuberculosis vaccine BCG, have been classically linked with a type 1 response, and proposed as possible modulators of the type 2 response usually associated with asthma.MethodsIn this study we used different acute and chronic murine models of asthma to investigate the therapeutic efficacy of intranasal delivery of the live tuberculosis vaccines BCG and MTBVAC by regulating the lung immune environment associated with airway hyperresponsiveness (AHR).FindingsIntranasal administration of BCG, or the novel tuberculosis vaccine candidate MTBVAC, abrogated AHR-associated hallmarks, including eosinophilia and lung remodeling. This correlated with the re-polarization of allergen-induced M2 macrophages towards an M1 phenotype, as well as with the induction of a strong allergen-specific Th1 response. Importantly, vaccine treatment was effective in a scenario of established chronic asthma where a strong eosinophil infiltration was already present prior to immunization. We finally compared the nebulization efficiency of clinical formulations of MTBVAC and BCG using a standard commercial nebulizer for potential aerosol application.InterpretationOur results demonstrate that pulmonary live tuberculosis vaccines efficiently revert established asthma in mice. These data support the further exploration of this approach as potential therapy against asthma.FundingSpanish Ministry of Science [grant numbers: BIO2014-5258P, RTI2018-097625-B-I00], Instituto de Salud Carlos III, Gobierno de Aragón/Fondo Social Europeo, University of Zaragoza [grant number: JIUZ-2018-BIO-01].

Potential of recombinant Mycobacterium paragordonae expressing HIV-1 Gag as a prime vaccine for HIV-1 infection

Recombinant Mycobacterium strains such as recombinant BCG (rBCG) have received considerable attention for the HIV-1 vaccine development. Recently, we described a temperature-sensitive Mycobacterium paragordonae (Mpg) strain as a novel live tuberculosis vaccine that is safer and showed an enhanced protective effect against mycobacterial infection compared to BCG. We studied the possibility of developing a vaccine against HIV-1 infection using rMpg strain expressing the p24 antigen (rMpg-p24). We observed that rMpg-p24 can induce an increased p24 expression in infected antigen presenting cells (APCs) compared to rBCG-p24. We also observed that rMpg-p24 can induce enhanced p24 specific immune responses in vaccinated mice as evidenced by increased p24-specific T lymphocyte proliferation, gamma interferon induction, antibody production and cytotoxic T lymphocyte (CTL) responses. Furthermore, an rMpg-p24 prime and plasmid DNA boost showed an increased CTL response and antibody production compared to rBCG or rMpg alone. In summary, our study indicates that a live rMpg-p24 strain induced enhanced immune responses against HIV-1 Gag in vaccinated mice. Thus, rMpg-p24 may have potential as a preventive prime vaccine in a heterologous prime-boost regimen for HIV-1 infection.

Prospects and challenges of a new live tuberculosis vaccine

Prospects and challenges of a new live tuberculosis vaccine

Recombinant BCG Overexpressing phoP-phoR Confers Enhanced Protection against Tuberculosis

The live tuberculosis vaccine Mycobacterium bovis BCG (Bacille Calmette-Guérin) comprises a number of genetically distinct substrains. In BCG-Prague, phoP of the PhoP-PhoR two-component system is a pseudogene due to a single insertion mutation. We hypothesized that this mutation partially accounts for the low immunogenicity of BCG-Prague observed in the 1970s. In this study, we showed that complementation with the M. bovis allele of phoP restored BCG-Prague’s immunogenicity. Furthermore, we showed that overexpression of the M. bovis allele of phoP-phoR in BCG-Japan, a strain already containing a copy of phoP-phoR, further enhanced immunogenicity and protective efficacy. Vaccination of C57BL/6 mice with the recombinant strain rBCG-Japan/PhoPR induced higher levels of interferon-γ (IFN-γ) production by CD4+ T cells than that with the parental BCG. Guinea pigs vaccinated with rBCG-Japan/PhoPR were better protected against challenge with Mycobacterium tuberculosis than those immunized with the parental BCG, showing significantly longer survival time, reduced bacterial burdens, and less severe pathology. Taken together, our study has identified a genetic modification that could be generally applied to generate new recombinant BCG vaccines.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial

Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Central memory CD4+ T cells are responsible for the recombinant Bacillus Calmette-Guérin ΔureC::hly vaccine's superior protection against tuberculosis.

Bacillus Calmette-Guérin (BCG) has been used for vaccination against tuberculosis for nearly a century. Here, we analyze immunity induced by a live tuberculosis vaccine candidate, recombinant BCG ΔureC::hly vaccine (rBCG), with proven preclinical and clinical safety and immunogenicity. We pursue in-depth analysis of the endogenous mycobacteria-specific CD4+ T-cell population, comparing the more efficacious rBCG with canonical BCG to determine which T-cell memory responses are prerequisites for superior protection against tuberculosis. rBCG induced higher numbers and proportions of antigen-specific memory CD4+ T cells than BCG, with a CXCR5+CCR7+ phenotype and low expression of the effector transcription factors T-bet and Bcl-6. We found that the superior protection of rBCG, compared with BCG, correlated with higher proportions and numbers of these central memory T cells and of T follicular helper cells associated with specific antibody responses. Adoptive transfer of mycobacteria-specific central memory T cells validated their critical role in protection against pulmonary tuberculosis.

Newly attenuated Mycobacterium bovis mutants as vaccines against bovine tuberculosis, particularly for possums

Bovine tuberculosis costs New Zealand more than $80 million per year, mostly because extensive areas of the country are occupied by brushtail possums infected with Mycobacterium bovis. AgResearch has a major programme to produce new live tuberculosis vaccines that can be delivered to possums. Primary work involved development of molecular biological methods to enable genetic manipulation of M. bovis, including the production of random and specific mutants. Many avirulent mutants of M. bovis have been produced and their vaccine efficacy has been compared to BCG in guinea pigs. Selected mutants that perform at least as well as BCG are retested in guinea pigs using an extended vaccination protocol in which animals are pre-sensitized to environmental mycobacteria to mimic natural exposure. Ten candidate vaccines that have induced good protection in guinea pigs have been subsequently tested as vaccines in possums. While the protective efficacy of an M. bovis mutant inoculated into guinea pigs reliably indicated that some protection would be induced in possums, the most protective mutant in guinea pigs was different from that in possums. This illustrates the importance of testing in the target species as part of new vaccine development. An important outcome of this work was the identification of an operon in M. bovis whose inactivation produced an avirulent M. bovis vaccine candidate that was better than BCG in protecting possums from experimental tuberculosis. Allelic exchange methods are now being used to produce vaccine strains with multiple specific mutations to improve safety and immunological characteristics.

Reducing the activity and secretion of microbial antioxidants enhances the immunogenicity of BCG.

BackgroundIn early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production.Methodology/Principal FindingsTo determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli.Conclusions/SignificanceWe conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG's ability to protect against pulmonary TB.

Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant

Safety is one of the main concerns for attenuated live vaccine candidates. Here we extend the stability and attenuation studies of the promising tuberculosis vaccine candidate based on Mycobacterium tuberculosis phoP mutant strain, SO2. Stability of the phoP mutation was tested after sub-culturing SO2 strain for 6 months in laboratory media and also after 3 months of infection in SCID mice. Results showed no reversion of the phoP mutation either in vitro or in vivo. In addition, SO2 was fully sensitive to four major first-line antituberculous drugs against tuberculosis. Safety and toxicity studies were performed in guinea pigs. Animals were infected with a quantity of SO2 equivalent to 50 vaccination doses (2.5 × 10 6 CFUs) and weight was monitored for 6 months. All animals survived and no histological lesions were found, showing full attenuation of SO2. Studies in a post-exposure model of guinea pigs and mice, previously infected with M. tuberculosis, were performed and no toxicity effects were found after inoculation of SO2. All these results together confirm that SO2 has a secure safety profile that encourages its use in clinical trials.

Live tuberculosis vaccines based on phoP mutants: a step towards clinical trials

Bacillus Calmette–Guérin (BCG) is the only preventive treatment for tuberculosis in humans, but this live vaccine confers variable protection against pulmonary tuberculosis in adults. Advances in the understanding of Mycobacterium tuberculosis immunopathogenesis have renewed hopes of developing new prophylactic vaccines conferring better protection than BCG. The authors describe here state-of-the-art attenuated live vaccines based on inactivation of the phoP gene, a transcriptional regulator of key virulence networks in M. tuberculosis. Recent preclinical testing of live vaccines based on phoP inactivation has demonstrated proof of concept, with a high degree of attenuation and protection against disease observed in various animal models. These results demonstrate that phoP mutants are promising new live vaccines for tuberculosis prevention. The steps that now need to be followed, to take these live vaccines towards clinical trials, are also reviewed, together with the potential of these vaccines to replace BCG.

The efficacy of live tuberculosis vaccines after presensitization with Mycobacterium avium

The variable efficacy of BCG in humans has been extensively documented but its cause is still not well understood. One possible reason for this variation is the effect of presensitization with environmental mycobacteria. To investigate in guinea pigs the effects of presensitization with well characterized Mycobacterium avium strains on the vaccine efficacy of BCG and of two recently developed, avirulent strains of Mycobacterium bovis. Two strains of M. avium containing the DNA insertion element IS901 (M. avium+) and two strains not containing this element (M. avium-) were inoculated subcutaneously or by oral administration into guinea pigs to assess their virulence in these animals and their ability to induce delayed type hypersensitivity to tuberculins. Subsequently, groups of guinea pigs presensitized with orally administered M. avium+ and M. avium- and a control group were vaccinated with BCG, or one of two newly attenuated strains of M. bovis. All groups were then challenged by the aerosol route with virulent M. bovis. Vaccine efficacy was assessed 5 weeks later by the presence of macroscopic lesions and bacterial counts of spleen and lung. No macroscopic lesions were observed in any of the guinea pigs inoculated with strains of M. avium+ or M. avium- and all animals gave delayed-type hypersensitivity skin-test reactions to avian PPD. In the vaccine experiment, presensitization with orally administered M. avium+ alone produced a low level of protection against subsequent challenge with virulent M. bovis. In the absence of presensitization with M. avium or after presensitization with an M. avium- strain, BCG and two attenuated strains of M. bovis produced significant levels of protection. No additional protection was observed in lungs of guinea pigs presensitized with M. avium+ and subsequently vaccinated with BCG. In contrast, both newly attenuated strains of M. bovis induced significant protection in lungs after such presensitization. Presensitization of guinea pigs by the oral administration of M. avium+ provides a model for testing vaccines under conditions where the efficacy of BCG has been compromised by prior sensitization with environmental mycobacteria.

Dendritic cells derived from BCG-infected precursors induce Th2-like immune response.

Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor alpha, IL-1beta, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.

Effect of Mycobacterium tuberculosis chaperonins on bronchial eosinophilia and hyper-responsiveness in a murine model of allergic inflammation.

Epidemiological evidence suggests that infection with Mycobacterium tuberculosis protects children against asthma. Several laboratories have shown that, in mouse models of allergic inflammation, administration of the whole live tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), prevents ovalbumin (OVA)-induced pulmonary eosinophilia. The aim of this study was to characterize specific M. tuberculosis molecules that are known to modulate immune responses to see if they affected pulmonary eosinophilia and bronchial hyper-responsiveness. C57Bl/6 mice were sensitized to OVA on days 0 and 7 and subsequently challenged with OVA on day 14 over a 3-day period. Pulmonary eosinophilia and bronchial hyper-responsiveness were measured 24 h following the last antigen challenge. In some groups, mice were pre-treated with M. tuberculosis or M. tuberculosis chaperonins (Cpns)60.1, 60.2 and 10, and the effect of this treatment on the allergic inflammatory response to aerosolized OVA was established. We show that M. tuberculosis Cpns inhibit allergen-induced pulmonary eosinophilia in the mouse. Of the three Cpns produced by M. tuberculosis, Cpn60.1, Cpn10 and Cpn60.2, the first two are effective in preventing eosinophilia when administered by the intra-tracheal route. Furthermore, the increase in airways sensitivity to inhaled methacholine following OVA challenge of immunized mice was suppressed following treatment with Cpn60.1. The allergic inflammatory response was also characterized by an increase in Th2 cytokines IL-4 and IL-5 in bronchoalveolar lavage fluid, which was also suppressed following treatment with Cpn60.1. These data show that bacterial Cpns can suppress eosinophil recruitment and bronchial hyper-responsiveness in a murine model of allergic inflammation.

Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis.

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6-dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.

Vaccine and skin testing properties of two avirulent Mycobacterium bovis mutants with and without an additional esat-6 mutation

Setting: Molecular techniques are now available to develop new live tuberculosis vaccines by producing avirulent strains of the Mycobacterium tuberculosis complex with known genes deleted. Objectives: Determine if removal of esat-6 from new live tuberculosis vaccines with known attenuating mutations affects their vaccine efficacy and if it could enable the development of discriminating diagnostic tests. Design: Remove the esat-6 gene by allelic exchange from two illegitimate mutants of Mycobacterium bovis that had previously been shown to have similar vaccine efficacy to BCG in a guinea pig vaccination model. Determine the effect this removal has on virulence, vaccine efficacy and skin test reactivity in guinea pigs. Results: Two double knockout strains of M. bovis were produced and their virulence and vaccine efficacy were compared to their parent strains. Removal of the esat-6 gene had no significant effect on vaccine efficacy. In skin tests, animals inoculated with the double knockout strains reacted to PPD but not ESAT-6, whereas those inoculated with the parent strains had similar skin test reactivity to both PPD and esat-6. Conclusion: Removal of esat-6 from new live tuberculosis vaccine candidates has no significant effect on vaccine properties but does enable the use of skin tests to distinguish between vaccination and infection.

Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti.

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.

An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine

Setting: Strains of the Mycobacterium tuberculosis complex are being rationally attenuated in order to develop better tuberculosis vaccines than BCG, and it would be helpful if new vaccines lacked an immunogenic protein which could be used as a skin test reagent for determining infection status.Objective: To delete theesat6 gene from a virulent Mycobacterium bovis strain and determine (i) whether this mutant sensitizes guinea pigs to a skin test based on ESAT6 and (ii) what effect this has on the virulence of M. bovis.Design: An homologous recombination technique was used to produce an esat6 knockout mutant of a virulent strain of M. bovis. Guinea pigs were inoculated with either the mutant or parent strain and their reactivity in intradermal skin tests was determined to bovine purified protein derivative (PPD) and recombinant ESAT6 protein.Results: Production of an esat6 knockout strain was demonstrated by Southern blot hybridization and the polymerase chain reaction. Guinea pigs inoculated with either the esat6 knockout strain or its virulent parent had positive skin test reactions to PPD but only animal inoculated with the parent strain had positive skin test reactions to ESAT6. Gross pathology, histopathology and mycobacterial culture of tissues indicated that the knockout strain was less virulent than its parent.Conclusion: If an effective live tuberculosis vaccine can be produced by inactivation of virulence genes in M. bovis, then prior or subsequent knockout of the esat6 gene could contribute to the loss of virulence and enable the development of a test to distinguish between vaccinated and infected animals.