Safety and immunogenicity of a thermostable ID93 + GLA-SE tuberculosis vaccine candidate in healthy adults.

Adjuvant-containing subunit vaccines represent a promising approach for protection against tuberculosis (TB), but current candidates require refrigerated storage. Here we present results from a randomized, double-blinded Phase 1 clinical trial (NCT03722472) evaluating the safety, tolerability, and immunogenicity of a thermostable lyophilized single-vial presentation of the ID93 + GLA-SE vaccine candidate compared to the non-thermostable two-vial vaccine presentation in healthy adults. Participants were monitored for primary, secondary, and exploratory endpoints following intramuscular administration of two vaccine doses 56 days apart. Primary endpoints included local and systemic reactogenicity and adverse events. Secondary endpoints included antigen-specific antibody (IgG) and cellular immune responses (cytokine-producing peripheral blood mononuclear cells and T cells). Both vaccine presentations are safe and well tolerated and elicit robust antigen-specific serum antibody and Th1-type cellular immune responses. Compared to the non-thermostable presentation, the thermostable vaccine formulation generates greater serum antibody responses (p < 0.05) and more antibody-secreting cells (p < 0.05). In this work, we show the thermostable ID93 + GLA-SE vaccine candidate is safe and immunogenic in healthy adults.

Vaccination inducing durable and robust antigen-specific Th1/Th17 immune responses contributes to prophylactic protection against Mycobacterium avium infection but is ineffective as an adjunct to antibiotic treatment in chronic disease.

Mycobacterium avium complex (MAC) causing pulmonary disease in humanshas emerged worldwide. Thus, effective strategies simultaneously aiming to prevent MAC infection and accelerate therapeutic efficacy are required. To this end, subunit vaccine-induced protection against a well-defined virulent Mycobacterium avium (Mav) isolate was assessed as a preventative and therapeutic modality in murine models. Mav-derived culture filtrate antigen (CFA) was used as a vaccine antigen with glucopyranosyl lipid A stable emulsion (GLA-SE) or GLA-SE plus cyclic-di-GMP (GLA-SE/CDG), and we compared the immunogenicities, protective efficacies and immune correlates. Interestingly, CFA+GLA-SE/CDG immunization induced greater CFA-specific Th1/Th17 responses in both the lung and spleen than among the tested groups. Consequently, protective efficacy was optimally achieved with CFA+GLA-SE/CDG by significantly reducing bacterial loads along with long-lasting maintenance of antigen-specific Th1/Th17 cytokine-producing multifunctional T cell responses and relevant cytokine productions. Thus, we employed this subunit vaccine as an adjunct to antibiotic treatment. However, this vaccine was ineffective in further reducing bacterial loads. Collectively, our study demonstrates that strong Mav CFA-specific Th1/Th17 responses are critical for preventative protection against Mav infection but may be ineffective or even detrimental in an established and progressive chronic disease, indicating that different approaches to combating Mav infection are necessary according to vaccination purposes.

Dynamics of Tuberculosis (TB) with Drug Resistance to First-Line Treatment and Leaky Vaccination: A Deterministic Modelling Perspective.

A deterministic model was formulated and employed in the analysis of the dynamics of tuberculosis with a keen emphasis on vaccination and drug resistance as the first line of treatment. It was assumed that some of the susceptible population were vaccinated but with temporal immunity. This is due to the fact that vaccines do not confer permanent immunity. Moreover, part of the infected individual after treatment grows resistance to the drug. Infective immigrants were also considered to be part of the population. The basic reproductive number for the model is estimated using the next-generation matrix method. The equilibrium points of the TB model and their local and global stability were determined. It was established that if the basic reproductive number was less than unity (R 0 < 1), then the disease free equilibrium is stable and unstable if R 0 > 1. Furthermore, we investigated the optimal prevention, treatment, and vaccination as control measures for the disease. As the objective functional was optimised, there have been a significant reduction in the number of infections and an increase in the number of recovery. The best control measure in combating tuberculosis infections is prevention and vaccination of the susceptible population.

T helper type 1 biased immune responses by PPE17 loaded core-shell alginate-chitosan nanoparticles after subcutaneous and intranasal administration.

PURPOSE: Tuberculosis, a cost and life threatening disease, was being subjected for improving vaccine strategies beyond BCG. Thus, a novel particulate delivery system using alginate-coated chitosan nanoparticles including PPE17 protein and CpG were administered through intranasal (IN) and subcutaneous (SC) routes. METHODS: The encapsulated nanoparticles were first characterized for size, surface charge, encapsulation efficiency and in vitro release of PPE17 antigen. The nanoparticles were then administered intranasal and subcutaneously to evaluate the induction of systemic and/or mucosal immune responses in mice. RESULTS: According to our result, the mean size of nanoparticles was measured about 427 nm, and exhibited a negative zeta potential of -37 mV. Following subcutaneous and intranasal administration, the results from cytokines assay showed that an increasing in the level of IFN-γ, and adversely a decrease in the level of IL-4 (presumptive Th1 biased immune response) was happened and also a notable elicitation in IL-17 cytokine was observed. CONCLUSION: In conclusion, our study demonstrated that alginate-coated chitosan nanoparticles showed to be an effective way to improve BCG efficiency as booster strategy for subcutaneous vaccine, and also can induce strong immune responses as prime strategy through intranasal vaccination.

In Vivo Antigen Expression Regulates CD4 T Cell Differentiation and Vaccine Efficacy against Mycobacterium tuberculosis Infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

The safety and efficacy of BCG encapsulated alginate particle (BEAP) against M.tb H37Rv infection in Macaca mulatta : A pilot study.

Due to the limited utility of Bacillus Calmette-Guérin (BCG), the only approved vaccine available for tuberculosis, there is a need to develop a more effective and safe vaccine. We evaluated the safety and efficacy of a dry powder aerosol (DPA) formulation of BCG encapsulated alginate particle (BEAP) and the conventional intradermal BCG immunization in infant rhesus macaques (Macaca mulatta). The infant macaques were immunized intratracheally with DPA of BEAP into the lungs. Animals were monitored for their growth, behaviour, any adverse and allergic response. The protective efficacy of BEAP was estimated by the ex-vivo H37Rv infection method. Post-immunization with BEAP, granulocytes count, weight gain, chest radiography, levels of liver secreted enzymes, cytokines associated with inflammation like TNF and IL-6 established that BEAP is non-toxic and it does not elicit an allergic response. The T cells isolated from BEAP immunized animals' blood, upon stimulation with M.tb antigen, secreted high levels of IFN-γ, TNF, IL-6 and IL-2. The activated T cells from BEAP group, when co-cultured with M.tb infected macrophages, eliminated largest number of infected macrophages compared to the BCG and control group. This study suggests the safety and efficacy of BEAP in Non-human primate model.

The epidemiologic impact and cost-effectiveness of new tuberculosis vaccines on multidrug-resistant tuberculosis in India and China.

BACKGROUND: Despite recent advances through the development pipeline, how novel tuberculosis (TB) vaccines might affect rifampicin-resistant and multidrug-resistant tuberculosis (RR/MDR-TB) is unknown. We investigated the epidemiologic impact, cost-effectiveness, and budget impact of hypothetical novel prophylactic prevention of disease TB vaccines on RR/MDR-TB in China and India. METHODS: We constructed a deterministic, compartmental, age-, drug-resistance- and treatment history-stratified dynamic transmission model of tuberculosis. We introduced novel vaccines from 2027, with post- (PSI) or both pre- and post-infection (P&PI) efficacy, conferring 10 years of protection, with 50% efficacy. We measured vaccine cost-effectiveness over 2027-2050 as USD/DALY averted-against 1-times GDP/capita, and two healthcare opportunity cost-based (HCOC), thresholds. We carried out scenario analyses. RESULTS: By 2050, the P&PI vaccine reduced RR/MDR-TB incidence rate by 71% (UI: 69-72) and 72% (UI: 70-74), and the PSI vaccine by 31% (UI: 30-32) and 44% (UI: 42-47) in China and India, respectively. In India, we found both USD 10 P&PI and PSI vaccines cost-effective at the 1-times GDP and upper HCOC thresholds and P&PI vaccines cost-effective at the lower HCOC threshold. In China, both vaccines were cost-effective at the 1-times GDP threshold. P&PI vaccine remained cost-effective at the lower HCOC threshold with 49% probability and PSI vaccines at the upper HCOC threshold with 21% probability. The P&PI vaccine was predicted to avert 0.9 million (UI: 0.8-1.1) and 1.1 million (UI: 0.9-1.4) second-line therapy regimens in China and India between 2027 and 2050, respectively. CONCLUSIONS: Novel TB vaccination is likely to substantially reduce the future burden of RR/MDR-TB, while averting the need for second-line therapy. Vaccination may be cost-effective depending on vaccine characteristics and setting.

Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice.

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.

The TB vaccine development pathway - An innovative approach to accelerating global TB vaccine development.

Two proof of concept clinical trials with TB vaccines demonstrate that new approaches can prevent sustained TB infection in adolescents (BCG revaccination) and TB disease in adults (M72/ASO1E) (Nemes et al., 2018; Tait et al., 2019) [1,2]. Both approaches are in late stage development and provide motivation and rationale to invest into a global TB vaccine pipeline. This pipeline needs to be diverse to address TB-specific challenges including variation in target populations, uncertainties in animal model predictivity and lack of immune correlates of protection. It requires that individual vaccine candidates must be advanced rationally and that the global pipeline must be managed in the most nimble and resource-efficient way, especially in the current constrained funding environment. The TB Vaccine Development Pathway is a webtool which has been developed as an offer to the field to provide a source of information and guidance covering vaccine development from discovery to implementation. It is underpinned by generic and TB vaccine-specific guidelines, regulatory frameworks and best practice, and was compiled by a multi-disciplinary team of scientific and technical experts with the input of the TB vaccine community. The Pathway is a unique tool to guide and accelerate the development of TB vaccine candidates and may be useful for other vaccine development fields.

Tuberculosis vaccination needs to avoid 'decoy' immune reactions.

Current search for a new effective vaccine against tuberculosis involves selected antigens, vectors and adjuvants. These are being evaluated usually by their booster inoculation following priming with Bacillus Calmette-Guerin. The purpose of this article is to point out, that despite being attenuated of virulence, priming with BCG may still involve immune mechanisms, which are not favourable for protection against active disease. It is postulated, that the responsible 'decoy' constituents selected during the evolution of pathogenic tubercle bacilli may be involved in the evasion from bactericidal host resistance and stimulate immune responses of a cytokine phenotype, which lead to the transition from latent closed granulomas to reactivation with infectious lung cavities. The decoy mechanisms appear as favourable for most infected subjects but leading in a minority of cases to pathology which can effectively transmit the infection. It is proposed that construction and development of new vaccine candidates could benefit from avoiding decoy-type immune mechanisms.

Combinational PRR Agonists in Liposomal Adjuvant Enhances Immunogenicity and Protective Efficacy in a Tuberculosis Subunit Vaccine.

Bacillus Calmette-Guerin (BCG) is the only licensed vaccine to prevent children from tuberculosis (TB), whereas it cannot provide effective protection for adults. Our previous work showed a novel vaccine candidate, liposomal adjuvant DMT emulsified with a multistage antigen CMFO, could protect mice against primary progressive TB, latency, and reactivation. To develop a more effective vaccine against adult TB, we aimed to further understand the role of pattern recognition receptor (PRR) agonists monophosphoryl lipid A (MPLA) and trehalose-6,6'-dibehenate (TDB) of the liposomal adjuvant DMT in the CMFO subunit vaccine-induced protection. Using C57BL/6 mouse models, the current study prepared different dimethyldioctadecylammonium (DDA)-based liposomal adjuvants with MPLA, TDB, or both (DMT), and then compared the immunogenicity and the protective efficacy among different liposomal adjuvanted CMFO subunit vaccines. Our study demonstrated that CMFO/DMT provided stronger and longer-lasting protective efficacy than the CMFO emulsified with adjuvants DDA or DDA/TDB. In addition, DDA/MPLA adjuvanted CMFO conferred a comparable protection in the lung as CMFO/DMT did. Higher levels of IFN-γ, IL-2, TNF-α, and IL-17A secreted by splenocytes were related with a more powerful and durable protection induced by CMFO/DMT through a putative synergistic effect of both MPLA and TDB via binding to TLR4 and Mincle. IL-2+ CD4+ T cells, especially IL-2+ CD4+ TCM cells, in the lung after infection were significantly associated with the vaccine-induced protection, whereas stronger IL-10 response and lower IL-2+ CD4+ T cells also contributed to the inferior protection of the DDA/TDB adjuvanted CMFO subunit vaccine. Given their crucial roles in vaccine-induced protection, combinational different PRR agonists in adjuvant formulation represent a promising strategy for the development of next-generation TB vaccine.

Integrating fish models in tuberculosis vaccine development.

Tuberculosis is a chronic infection by Mycobacterium tuberculosis that results in over 1.5 million deaths worldwide each year. Currently, there is only one vaccine against tuberculosis, the Bacillus Calmette-Guérin (BCG) vaccine. Despite widespread vaccination programmes, over 10 million new M. tuberculosis infections are diagnosed yearly, with almost half a million cases caused by antibiotic-resistant strains. Novel vaccination strategies concentrate mainly on replacing BCG or boosting its efficacy and depend on animal models that accurately recapitulate the human disease. However, efforts to produce new vaccines against an M. tuberculosis infection have encountered several challenges, including the complexity of M. tuberculosis pathogenesis and limited knowledge of the protective immune responses. The preclinical evaluation of novel tuberculosis vaccine candidates is also hampered by the lack of an appropriate animal model that could accurately predict the protective effect of vaccines in humans. Here, we review the role of zebrafish (Danio rerio) and other fish models in the development of novel vaccines against tuberculosis and discuss how these models complement the more traditional mammalian models of tuberculosis.

Methylation in Mycobacterium-host interaction and implications for novel control measures.

Methylation epigenetically regulates many pivotal biological processes. Mycobacterium tuberculosis, the pathogen of tuberculosis, can modulate host methylome. The methylated genes, sites, signaling pathway, chromatin remodeling, especially the immune related genes such as cytokines and chemokines, drug resistance and vaccines efficacy relevant genes were summarized in this paper. The results showed that methylation plays important roles in immune evasion, pathogenesis, persistence, disease progression, active, drug responder and non-responder. This will inform better practice for the development of new drugs and vaccines to eradicate tuberculosis.

A review on bovine tuberculosis in India.

Bovine tuberculosis (bTB) is a chronic illness in animals, especially in cattle, leading to loss in the productivity and signifies a crucial public health risk. Regardless of the zoonotic threat and significant economic costs associated with the disease, precise estimates of bTB prevalence are deficient in many countries, including India, where national control programs are yet to be instigated. The true burden of the disease remains unknown due to lack of routine surveillance data from most of the developing countries. India is progressing well towards attaining the End TB goal, yet bTB continues to remain largely hidden. Moreover, the paucity of literature on bTB in India might lead to undue complacency and hence has to be scrupulously guarded and prevented from gaining any misconceptions in the minds of the common people. Preventing and controlling bTB at the animal interface is pivotal to evade transmission to human, increase food safety and guard the livelihood of the people. To attain this goal, implementation of strategies based on international norms and a multi-sectoral approach will empower enhanced surveillance and diagnosis of disease in animals and subsequently reduce the risk for humans. As an initiative, we step forward to address this review which briefly summarizes the available data in the literature from early 20th century to date to assess the status of bTB in India. We have discussed in detail, the epidemiology, transmission and diagnosis pertaining to bTB. The review also focuses on the interconnection between the health of people and animal, discuss the preventions and control strategies and recommend the use of vaccination in cattle to reduce the spread of infection among other animals and humans. Implementing One Health approach in India, which recognizes the interdependence of the health of people and animals will help the nation in the fight against TB.

Whole genome profiling refines a panel of correlates to predict vaccine efficacy against Mycobacterium tuberculosis.

New vaccines are needed to combat the public health threat posed by M. tuberculosis (M. tb), but no correlates have been defined to aid vaccine development. Using mouse models, we previously developed an in vitro system that measures the ability of M. tb-immune lymphocytes to control bacterial replication during co-culture with M. tb-infected macrophages. We demonstrated that the degree of in vitro growth control by lymphocytes from mice given vaccines of varying efficacy reflected the relative degree of in vivo protection against lethal challenge. Further, using targeted analyses of gene expression in lymphocytes recovered from co-cultures, we found mediators whose relative expression also correlated with in vitro and in vivo outcomes. Here we advanced those findings by employing genome-wide expression analyses. We first screened splenocytes recovered from co-cultures by microarray, revealing additional genes whose expression correlated with protection. After applying pathway analyses to down-select gene candidates, we used both splenocytes and peripheral blood lymphocytes to validate microarray findings by qRT-PCR. We then subjected data from top candidates to rigorous statistical analyses. Resulting correlate candidates, including CXCL9, IFN-γ, and CCL5, significantly predicted protection with high specificity. These findings therefore refine and extend a panel of relevant immune correlates to advance vaccine development.

Metabolic Profiling and Compound-Class Identification Reveal Alterations in Serum Triglyceride Levels in Mice Immunized with Human Vaccine Adjuvant Alum.

Alum has been widely used as an adjuvant for human vaccines; however, the impact of Alum on host metabolism remains largely unknown. Herein, we applied mass spectrometry (MS) (liquid chromatography-MS)-based metabolic and lipid profiling to monitor the effects of the Alum adjuvant on mouse serum at 6, 24, 72, and 168 h post-vaccination. We propose a new strategy termed subclass identification and annotation for metabolomics for class-wise identification of untargeted metabolomics data generated from high-resolution MS. Using this approach, we identified and validated the levels of several lipids in mouse serum that were significantly altered following Alum administration. These lipids showed a biphasic response even 168 h after vaccination. The majority of the lipids were triglycerides (TAGs), where TAGs with long-chain unsaturated fatty acids (FAs) decreased at 24 h and TAGs with short-chain FAs decreased at 168 h. To our knowledge, this is the first report on the impact of human vaccine adjuvant Alum on the host metabolome, which may provide new insights into the mechanism of action of Alum.

Enhancement of immune response against Mycobacterium tuberculosis HspX antigen by incorporation of combined molecular adjuvant (CASAC).

Tuberculosis (TB) is one of the deadliest human diseases worldwide caused by mycobacterial infection in the lung. Bacillus Calmette-Guerin (BCG) vaccine protects against disseminated TB in children, but its effectiveness is still questionable due to highly variable protections in adolescence and elderly individuals. Targeting the latency M.tb antigen is a recent therapeutic approach to eradicate dormant pathogen that could possibly lead to disease activation. In this study, we aimed to potentiate immune responses elicited against 16 kDa α-crystalline (HspX) tuberculosis latency antigen by incorporation of Combined Adjuvant for Synergistic Activation of Cellular immunity (CASAC). Histidine-tagged recombinant HspX protein was initially produced in Escherichia coli and purified using Ni-NTA chromatography. To evaluate its adjuvanticity, C57BL/6 mice (n = 5) were initially primed and intradermally immunised in 2-weeks interval for 4 rounds with recombinant HspX, formulated with and without CASAC. Humoral and cell-mediated immune responses elicited against HspX antigen were evaluated using ELISA and Flow Cytometry. Our findings showed that CASAC improved humoral immunity with increased antigen-specific IgG1 and IgG2a antibody response. Stronger CD8+ and Th1-driven immunity was induced by CASAC formulation as supported by elevated level of IFN-γ, TNF-α, IL-12 and IL-17A; and with low IL-10 secretion. Interestingly, adjuvanted HspX vaccine triggered a higher percentage of effector memory T-cell population than those immunised with unadjuvanted vaccine. In conclusion, CASAC adjuvant has great potential to enhance immunogenicity elicited against HspX antigen, which could be an alternative regimen to improve the efficacy of future therapeutic vaccine against Mycobacterium tuberculosis.

The Future of Vaccines for Tuberculosis.

Exciting clinical results from 2 clinical TB vaccine trials were published in 2018. These, plus promising preclinical candidates form a healthy pipeline of potential vaccines against the leading cause of death from a single infectious agent. The only licensed vaccine, the BCG, continues to be an important tool in protecting against severe forms of TB in children, but has not stopped the diseases causing 1.3 million deaths per year. This review provides an overview of the current TB vaccine pipeline, highlighting recent findings, describes work relating to epidemiologic impact of vaccines, and discusses the future of TB vaccine development.

Long-term protective efficacy with a BCG-prime ID93/GLA-SE boost regimen against the hyper-virulent Mycobacterium tuberculosis strain K in a mouse model.

Since ID93/GLA-SE was developed as a targeted BCG-prime booster vaccine, in the present study, we evaluated the protective efficacy of ID93/GLA-SE as a boost to a BCG-prime against the hypervirulent Mycobacterium tuberculosis (Mtb) K challenge to provide further information on the development and application of this vaccine candidate. Boosting BCG with the ID93/GLA-SE vaccine significantly reduced bacterial burden at 16 weeks post-challenge while the BCG vaccine alone did not confer significant protection against Mtb K. The pathological analysis of the lung from the challenged mice also showed the remarkably protective boosting effect of ID93/GLA-SE on BCG-immunised animals. Moreover, qualitative and quantitative analysis of the immune responses following ID93/GLA-SE-immunisation demonstrated that ID93/GLA-SE was able to elicit robust and sustained Th1-biased antigen-specific multifunctional CD4+ T-cell responses up to 16 weeks post-challenge as well as a high magnitude of an antigen-specific IgG response. Our findings demonstrate that the ID93/GLA-SE vaccine candidate given as a BCG-prime boost regimen confers a high level of long-term protection against the hypervirulent Mtb Beijing infection. These findings will provide further and more feasible validation for the potential utility of this vaccine candidate particularly in East-Asian countries, with the predominance of the Beijing genotype, after BCG vaccination.