Tuberculosis (TB) is an infectious disease generally transmitted through aerosols containing Mycobacterium tuberculosis. TB has been responsible for the death of one billion people in the last 200 years (1) and is one of the 10 leading causes of death in the world (including people infected with human immunodeficiency virus [HIV]) (2). In 2015, approximately 10.4 million people worldwide had TB and 1.8 million people died of TB (2). Key risk factors for TB include poverty, overcrowding, malnutrition, alcohol abuse, and HIV infection (345). Most infections caused by M. tuberculosis are latent TB without symptoms; however, approximately 12% of these cases develop into active TB (26). Prior to 2001, the tuberculin skin test was the only diagnostic test for TB; however, in the 2000s, 2 interferon (IFN)-γ release assays (IGRAs) were approved by the Food and Drug Administration (FDA). In IGRA, the concentration of IFN-γ secreted by T lymphocytes in patients exposed to M. tuberculosis is measured after stimulation with early secretory antigenic target-6 (ESAT-6), which is the main antigen produced by M. tuberculosis (7). The first licensed IGRA was the QuantiFERON-TB Gold In-Tube test. This method measures the concentration of IFN-γ secreted into the peripheral blood of a patient after exposure to ESAT-6, CFP-10, and TB7.7. The second licensed IGRA was the T-SPOT.TB test, which is performed on whole blood. First, peripheral mononuclear cells are extracted from the collected blood sample and exposed to ESAT-6 and CFP-10, after which the concentration of secreted IFN-γ is measured. According to FDA-approved indications, diagnostic tests use different antigens; therefore, test results may be different. Consequently, it is recommended that these assays are considered indirect tests and are performed together with radiology or other medical evaluations (7).

Typical anti-TB drugs such as isoniazid, also known as isonicotinylhydrazide, and rifampicin are not effective against multi-drug resistant tuberculosis (MDR-TB) (8). Research on new anti-TB drugs is ongoing. Currently, bedaquiline (Sirturo^®; Janssen Therapeutics, Titusville, NJ, USA) and delamanid (Deltyba^®; Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) are being developed as treatments for MDR-TB (9). Other possible treatments include repurposed drugs such as linezolid, imatinib, and metformin (1011121314).

Currently, the only licensed vaccine for TB is the Bacillus Calmette-Guérin (BCG) vaccine, which is made by attenuating Mycobacterium bovis (15). Although vaccination with BCG prevents against tuberculous meningitis and disseminated TB in children, it does not prevent the development of a primary infection. Furthermore, it is not effective in preventing the reactivation of latent pulmonary infection. The efficacy of BCG vaccination is reported to vary between 0% and 80% for the prevention of pulmonary TB. In addition, it has limited efficacy in adults (1617). Therefore, the development of new TB vaccines is necessary. In this review, we have summarized the findings of studies on newly developed TB vaccines, the status of adjuvant research, and novel approaches for developing new TB vaccines.

According to data from the World Health Organization (WHO), there were 10.4 million TB cases globally in 2015, which indicated an incidence of 142 cases per 100,000 people (2). The number of annual TB cases per country in 2015 was less than 10 per 100,000 in high-income countries and 150–300 per 100,000 in 30 countries considered as high-TB burden countries by WHO. Countries with an incidence rate of more than 500 per 100,000 people include Lesotho, Mozambique, and South Africa (2). The Republic of Korea ranks first in terms of TB incidence (80 persons per 100,000 people) and mortality (5.1 persons per 100,000 people) among the member countries of the Organization for Economic Cooperation and Development (OECD) (Fig. 1, Tables 1 and 2). These are above the OECD average TB incidence of 11.4 (per 100,000 population) and the average TB mortality of 1.0 (per 100,000 population) (Table 3). To solve this problem, the Korean government is conducting a national project to reduce TB incidence to 50 per 100,000 people by the year 2020.

Democratic People's Republic of Korea (DPR Korea) is a high-risk country for TB; therefore, it is included in the 30 high-MDR-TB burden countries and 30 high-TB burden countries. It is also among ‘The three TB-high-burden country lists’ (Fig. 2, Table 3) (2). In 2015, TB incidence in DPR Korea was 561 per 100,000 individuals, which was the third highest in the world (1st: South Africa [834 per 100,000 individuals], 2nd: Lesotho [788 per 100,000 individuals]). Based on the 2016 Global Summary reported by WHO, the national BCG vaccine coverage for newborns in DPR Korea was 98% from 2010 to 2014 and 97% in 2015. These data are not completely reliable because these were not directly examined by WHO.

There are 2 types of TB vaccines: preventive and therapeutic (Fig. 3). The preventive vaccines include priming vaccines and boosting vaccines.

Therapeutic TB vaccines are vaccines that are administered along with the existing drug therapy (Fig. 3) to shorten the treatment period.

RUTI, a TB vaccine candidate, is based on detoxified liposomal fragments of M. tuberculosis. It is used in addition to chemotherapy for treating latent TB infection. It was developed as an immunotherapeutic agent to reduce the amount and duration of drug therapy in patients with active TB. A phase II trial on the RUTI vaccine has been recently completed in TB patients with HIV infection (44).

Vaccae (SRL-172) is a vaccine developed by Biologic Pharmacy Co., Ltd (Longcom; Hefei City, China). It is composed of whole heat-killed Mycobacterium vaccae and has already been approved for use as a therapeutic adjuvant for patients infected with M. tuberculosis (46). A phase III clinical trial has been completed in HIV-coinfected patients. Vaccae is currently undergoing clinical trials for stability and efficacy in people with high-risk TB who are positive for the purified protein derivative skin test (ClinicalTrials.gov identifier: NCT01979900). Mycobacterium indicus pranii, formally known as Mycobacterium w, is used to prepare a whole-cell vaccine that is currently undergoing a phase III clinical trial. M. indicus pranii is a cultivable, non-pathogenic, and killed Mycobacterium species that contains antigens similar to Mycobacterium leprae (47).

Here, we briefly describe the adjuvants that have been recently used for TB vaccine development (Table 5). IC31 is an adjuvant consisting of the immunostimulatory TLR9 ligand ODN1a, an oligodeoxynucleotide, and the synthetic antimicrobial peptide H-KLKL5KLK-OH. It is used in H4 and H56 vaccines (48). AS01 contains the TLR4 ligand 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and the saponin derivative QS-21 as a liposomal formulation (4950). AS01E is an adjuvant that reduces the capacity of AS01 for pediatric use. It is being used for M72 vaccine candidates. AS02 is also composed of MPL and QS-21; however, it is an o/w emulsion formulation rather than a liposomal formulation (51). CAF01 is formulated with an N,N′-dimethyl-N,N′-dioctadecylammonium (DDA) liposome and the synthetic mycobacterial immunomodulator α.α′-trehalose 6,6′-dibehenate (TDB) in a 5:1 ratio (52). It is used in H1 vaccines. GLA-SE is a stable emulsion of the synthetic TLR4 ligand glucopyranosyl lipid adjuvant, and is used in ID93 (37).

The effect of BCG may differ depending on the route of administration. Thus, changes and improvement in the administration route must be considered in vaccine development. In animals challenged with M. tuberculosis after vaccination with BCG, the colony-forming units of M. tuberculosis in the lungs vary according to the route of administration. Aerosolized BCG administered through the nasal cavity shows the best inhibitory effect, which may be because it activates both mucosal and systemic immunity. However, it is associated with side effects, including pulmonary inflammation, which causes lung tissue damage. Oral administration of a lipid-microencapsulated vaccine formulation to mice and guinea pigs resulted in improved outcomes by inducing the production of diverse and prolonged CD4⁺ T cell responses. The formulation was also associated with decreased side effects. Subcutaneous injection of the vaccine stimulated delayed hypersensitivity and cytokine production by Th cells (23).

VPM1002 (rBCGΔureC∷hly) is being developed as a priming vaccine. A study of VPM1002 has been conducted in a cell model lacking a virulence gene called nuoG, which is responsible for inhibiting the apoptosis of infected host cells (53). It has been reported that BCGΔureC::hlyΔunoG shows a higher immune response and stability than BCGΔureC::hly in mice; therefore, the former may be useful as a next-generation candidate (54).

Ag85B antigen is a mycolyl transferase that is conserved in mycobacterial species such as M. tuberculosis and BCG (55). Ag85B is involved in lipid accumulation and storage, which are important processes during dormancy in M. tuberculosis. It is also used in many vaccine candidates because it induces a strong Th1 immune response. However, 8 out of the 13 vaccine candidates are subunit vaccines, and 6 of them use Ag85 antigens (Ag85A, Ag85B). Therefore, it is necessary to find novel antigens that can be used as vaccines.

ESAT-6 is a protein encoded by a gene located in the region of difference 1, which is expressed in M. tuberculosis but is not found in BCG. It has sufficient immunogenicity in mice and humans after M. tuberculosis infection. However, inducing an immune response for the prophylactic vaccine using ESAT-6 protein requires the use of an additional adjuvant to increase the protective or vaccination effect. In a previous study, ESAT-6 showed a potent immune response when it was combined with adjuvants such as dimethyl dioctadecyl ammonium bromide and MPL (56). Furthermore, a recombinant adenovirus expressing ESAT-6 with calreticulin showed an increased immune response to ESAT-6 by secreting more IFN-γ and TNF-α; however, it could not decrease M. tuberculosis burden in the lungs (57). In addition, DNA-based vaccines prepared using the T cell epitope of M. tuberculosis are known to induce immune responses with high safety and stability (58). However, ESAT-6 itself could pose a hurdle in the process of antigen presentation. Thus, for a protective effect, modified DNA vaccines have been prepared using nano-chitosan. A previous study showed that nano-Esat-6/3eFL (nano-chitosan-based DNA vaccine prepared using fms-like tyrosine kinase 3 ligand) induced a strong immune response and exhibited a protective effect by upregulating Th1 and cytolytic T cell (CTL) responses against M. tuberculosis (59). In contrast, the Ag85A/ESAT-6 chimeric DNA vaccine induced a hypersensitivity response and worsened the severity of M. tuberculosis infection in mice (60). Recently, it was suggested that novel cell-based vaccine candidates prepared using antigen-presenting cells (APCs) such as dendritic cells and B cells can induce strong antigen-specific T cell immunity (6162). Furthermore, ESAT-6-expressing adenovirus or vaccinia virus can deliver ESAT-6 antigen to APCs, which consequently upregulate the expression of MHC and co-stimulatory molecules. However, antigen delivery using a viral vector showed dominant expression of MHC class I, and its potency of vaccine efficacy might be CD8⁺ T cell-dependent. Thus, an alternative method to increase MHC class II expression is required (63). Activated natural killer T (NKT) cells can increase the immunogenicity, including CD4⁺ T cell responses, of cell-based vaccines when α-galactosylceramide (α-GalCer) is added as a ligand (61646566). Thus, using recent approaches for the development of TB vaccines, an APC-based cell vaccine expressing ESAT-6 and loaded with α-GalCer could be formulated as a novel vaccine candidate for the prevention and treatment of M. tuberculosis infection (Fig. 4).

ESAT-6 forms a heterodimeric complex with CFP-10 in the phagosome lumen and is secreted through the ESX-1 secretion system (67). The heterodimer inhibits the intracellular antimicrobial response by inducing oxidative burst by downregulating the level of inducible nitric oxide synthase, which decreases the levels of nitric oxide and reactive oxygen species (68). ESAT-6 also suppresses T cell function and inhibits the intracellular defense system. In mouse neutrophils, ESAT-6 acts as a leukocidin, a bacterial enzyme that kills neutrophils and increases calcium ion influx (69). It has been suggested that a high burden of ESAT-6-expressing M. tuberculosis leads to macrophage necrosis via caspase-1- and cathepsin-B-independent pathways (70). In addition, ESAT-6 inhibits autophagy through mechanistic target of rapamycin activation, suggesting that vaccination with BCG together with ESAT-6 may increase BCG survival (7172). ESAT-6 expression is rapidly increased in host cells infected with M. tuberculosis. Furthermore, it triggers phagosomal rupture since it forms a membrane-spanning channel. ESAT-6 consists of an N-terminal, C-terminal, helix1, and helix2. The N-terminal and C-terminal are involved in cell membrane interactions, whereas helix1 and 2 are involved in membrane-spanning channel formation (73). ESAT-6 may disrupt the artificial bilayer (74) and plasma membrane of red blood cells, and play a role in the lysis of liposomes (75). In addition to causing phagosomal rupture through the formation of phagosomal membrane-spanning channels, ESAT-6 also interacts with beta 2 microglobulin (β2M), a constituent of the MHC class I molecule in all nucleated cells. ESAT-6 and ESAT-6:CFP-10 are secreted through the ESX-1 secretion system, after which they migrate to the endoplasmic reticulum in the host cell to form a complex with β2M. Consequently, cell surface expression of the MHC class I-β2M complex is inhibited and MHC-I-dependent antigen presentation is downregulated (76). M. tuberculosis infection also causes the downregulation of class II transactivator and MHC II by inducing hypermethylation of histone H3 lysine9 (H3K9me2/3). This is mediated by EsxL, one of the 23 ESAT-6 family proteins in M. tuberculosis (77).

The M. tuberculosis DNA that is exposed to the cytoplasm after phagosomal rupture is recognized by cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS), which induces the synthesis of cyclic GMP-AMP (cGAMP) as a cellular secondary messenger. cGAMP moves to the cytosol and activates the stimulator of interferon genes (STING) to activate TANK-binding kinase (TBK) 1 (a serine/threonine-protein kinase)-interferon regulatory factor 3 (IRF3) (Fig. 5) (7879). The activated cGAS/STING pathway finally triggers the expression of type I interferons such as IFN-α to suppress M. tuberculosis in the infected cells (8081). Additionally, TBK-1 activity and bacterial ubiquitination lead to selective autophagy of the pathogen (Fig. 5). The lipidated isoform of the microtubule-associated protein 1 light chain is involved in the ubiquitination of mycobacterial and/or phagosomal membrane fragments (82). In addition, mycobacterial DNA is recognized by absent in melanoma 2 (AIM2) and cGAS; however, NLRP3 and caspase-1 may not be involved in this process (83). It has been suggested that intracellular M. tuberculosis DNA sufficiently activates the AIM2 inflammasome, which releases IL-1β and IL-18 (84). However, another study has shown that M. tuberculosis inhibits IL-1β secretion via the ESX-1 secretion system (81). Furthermore, until date, the relationship between the cGAS and AIM2 pathways is not clear. However, it appears that components derived from M. tuberculosis, especially bacterial DNA, might play a critical role in host defense mechanisms. This consequently induces the activation of inflammasomes and the cGAS/STING pathway at the infection site to help reduce M. tuberculosis burden.

ESAT-6 downregulates the expression of MHC molecules and induces the migration of M. tuberculosis to the cytoplasm through host cell phagosomal rupture, which may be an adverse effect of using ESAT-6 as a vaccine. However, phagosomal rupture by ESAT-6 also induces the activation of the AIM2 inflammasome and the STING-TBK-1-IRF3 pathway, which triggers strong immune activation to eliminate M. tuberculosis infection.

In this review, we discuss the current progress in TB vaccine development. Despite the limitations of BCG, a novel TB vaccine that can replace it has not yet been developed. However, efforts to develop new TB vaccines have resulted in the production of a number of TB vaccine candidates. Here, we summarize the data on vaccine candidates that have been studied or are being investigated in clinical trials so that researchers can easily understand the current status of TB vaccine development.

We also discuss the role of ESAT-6, which is widely used as a target antigen in TB vaccine development, and question whether developing a TB vaccine with ESAT-6 is an effective strategy. ESAT-6 induces phagosome rupture in cells, which results in regulation of the migration of M. tuberculosis to the cytoplasm and is associated with the inhibition of MHC I and II. These characteristics of ESAT-6 are considered inappropriate for a TB vaccine to induce an immune response. In fact, many TB vaccine candidates that are formulated based on ESAT-6 induction have not progressed in clinical trials. In addition, problems arise when performing IGRA diagnostic tests using ESAT-6, as the results of the test cannot be attributed to the effects of the ESAT-6-based TB vaccine. To solve this problem, a study on ESAT-6-free IGRA assays is underway. In this study, EspC, EspF, and Rv2348c were screened as potent antigens and mixed with CFP-10 to produce a cocktail, which showed a diagnostic performance similar to that of QuantiFERON. As the development of vaccines using ESAT-6 proceeds, it is necessary to continue research and development in diagnosis of TB (85).

Despite the abovementioned limitations of ESAT-6, it presents several benefits as a vaccine antigen. According to previous reports on the development of ESAT-6 as a vaccine antigen, ESAT-6 is highly potent and can induce good CD4⁺ T cell and CTL responses to protect hosts against TB. The difficulty associated with the use of ESAT-6 appears to be the possibility of toxicity due to the immunization method. We propose the development of a novel cell-based vaccine that can overcome the low immunogenicity of ESAT-6. It appears that it is better to use a viral vector to deliver ESAT-6 antigen to APCs since ESAT-6 can suppress APC function.

Although dendritic-cell-based vaccines are well-known, we suggest the development of B-cell-based vaccines because they can sufficiently activate CD4⁺ T cell and CTL responses. Moreover, it is reported that an α-GalCer-loaded B cell-based vaccine induced NKT cell activation to boost immune responses elicited by a B-cell-based vaccine. Thus, a cell-based vaccine containing ESAT-6 could be a novel prophylactic and therapeutic vaccine that can enhance protection against M. tuberculosis infection.