Chemical and Biological Characterization of Mycobacterium tuberculosis-Specific ESAT6-Like Proteins and Their Potentials in the Prevention of Tuberculosis and Asthma

Tuberculosis (TB) and asthma are two major infectious and non-infectious diseases, respectively, with worldwide prevalence. According to recent estimates at the global level, about 10 million people fell ill with TB and 1.5 million died in 2020 [1], and asthma made 262 million people sick with 0.455 million deaths in 2019 [2]. Most patients with TB (>85%) suffer from pulmonary disease. Both pulmonary TB and asthma affect the lungs.

The bacterium Mycobacterium tuberculosis which causes TB is an intracellular organism that lives in infected macrophages. M. tuberculosis infects macrophages in the lungs of pulmonary TB patients and results in the destruction of alveoli due to the influx of immune cells and the release of proinflammatory cytokines in response to the mycobacterial antigens. The type of immune response induced is pivotal to protection and susceptibility to TB. T helper (Th)1 cells and their cytokines, interleukin (IL)-2 and interferon-gamma (IFN-γ), provide protection, whereas Th2 cells and their cytokines (IL-4, IL-5, IL-10, and IL-13) are responsible for pathogenesis in TB [3]. A vaccine against TB is expected to induce primarily Th1 responses in immunized individuals [3].

The load of TB is more in developing and poor countries of the world [1], whereas asthma is more common in developed and rich countries [4]. This differential outcome has been suggested due to the varying hygienic conditions in rich and poor countries [5]. The “hygiene hypothesis” suggests that frequent exposure to microbes and higher rates of cross-infections due to unhygienic conditions in poor countries can activate Th1 cells early in life, thus inhibiting Th2-associated inflammatory responses that lead to the development of asthma. In contrast, improved hygienic conditions in rich countries lead to higher rates of asthma by reducing early exposure to infections and preferential activation of Th2 cells instead of Th1 cells [5]. The hygiene hypothesis is supported by studies like that of Bashir et al. [6], who have reported that TB infection significantly decreased the prevalence of asthma, severity of symptoms, and the number of attacks (Table 1). The findings of Bashir et al. [7] are supported by other studies which showed that M. tuberculosis infection reduced the occurrence of asthma in women. People living in an TB-endemic area with strong tuberculin skin test reactions (a sign of infection with M. tuberculosis) had inverse association with allergic disease symptoms [8]. Furthermore, a systematic review and meta-analysis of 20 studies with 222,928 participants has shown that early childhood immunization with mycobacterial antigens in the form of Mycobacterium bovis BCG vaccine reduced the risk of asthma significantly [9]. These studies suggest that the type of immune response induced by infection with M. tuberculosis or vaccination with Mycobacterium bovis BCG is protective against asthma. Hence, it is considered important to identify mycobacterial antigens that induce Th1 responses and can prevent both TB and asthma [10, 11].

Antigens actively secreted by M. tuberculosis have been considered important for the activation of immune responses and the development of vaccines because these are the antigens that will be encountered first by the immune system of an infected person [12, 13]. A study in 1995 to identify such antigens from the culture filtrates of M. tuberculosis [14] showed that several proteins were secreted early during the in vitro growth of M. tuberculosis and were recognized by immune cells of mice infected with M. tuberculosis [14]. A protein of low molecular weight, i.e., 6 KDa, was the most efficient in inducing the secretion of Th1 cytokine interferon-gamma (IFN-γ); it was named “6 KDa early secretory antigenic target” (ESAT6) [14]. Further work with peripheral blood mononuclear cells (PBMCs) of pulmonary tuberculosis (TB) patients with a battery of purified M. tuberculosis antigens showed that ESAT6 induced the highest concentrations of IFN-γ [15]. ESAT6 was also found to be highly immunogenic in several animal models of TB [16]. Similarly, a high proportion of healthy people exposed to M. tuberculosis responded to ESAT6, and low responses were observed in healthy people not exposed to M. tuberculosis [17]. These and other results suggested that EST6 was specific to M. tuberculosis [18]. Another study reported the presence of a second major antigen of M. tuberculosis present in culture filtrates, known as “culture filtrate protein 10” (CFP10) [19]. Studies in animal models have shown that immunization with these antigens protects a variety of animals against TB [20, 21]. Clinical trials in humans with vaccine preparations containing these antigens have shown promising results [22]. Hence, ESAT6 and CFP10 could be considered subunit vaccine candidates against TB.

The availability of the complete genome sequence of M. tuberculosis in 1999 and its comparisons with the genomes of other mycobacteria showed that ESAT6 and CFP10 are specific for M. tuberculosis and are deleted in all BCG vaccine sub-strains as well as in most non-tuberculous mycobacteria [23]. Hence these proteins were proposed for the specific diagnosis of TB as they will not interfere with BCG vaccination, as is the case with the purified protein derivative of M. tuberculosis (PPD) in the Tuberculin/Mantoux test [24, 25]. A mixture of these proteins and their peptides has been extensively used in the specific diagnosis of active and latent TB [26].

Although ESAT6 and CFP10 are highly immunogenic, M. tuberculosis specific, and provide protection against challenge with M. tuberculosis [27], they cannot be used for both diagnosis and vaccination. This is because immunized healthy people will show a positive response and will be falsely reported as having TB. As the combined use of ESAT6 and CFP10 in the diagnosis of TB has shown their value for world-wide application and several commercial kits have been developed and used in the field, the search for similar antigens in antigenicity but different in the sequence of amino acids and chemical nature has continued for use in vaccines [28‒30].

Analysis of M. tuberculosis genome sequence data showed that the genes for ESAT6 and CFP10 have DNA containing 288 and 303bp, respectively and encode proteins containing 95 aa and 100 aa, respectively [12]. Furthermore, the genes encoding ESAT6 and CFP10 are adjacent to each other in an operon known as ESAT6-operon, and both proteins are expressed by a single promotor (Fig. 1) [24].

The search for ESAT6-like genes in M. tuberculosis genome showed that there were 21 additional genes with similar organization in the M. tuberculosis genome [24, 31‒33]. All these genes encoded small molecular-weight proteins (ESXC to ESXW) containing 90 aa to 125 aa in their primary sequence (Table 2). Homology searches did not identify significant sequence identity between ESAT6/ESXA, CFP10/ESXB, and other ESAT-6-like proteins (Table 3), suggesting that these proteins were similar in organization but different in structure and function.

Although members of ESAT6-family proteins do not share significant sequence homology with ESXA and ESXB, 15 of the 23 proteins share significant sequence homology with other proteins of ESAT6-family, and such proteins could be divided into four subfamilies (1–4) based on the extent of their sequence identities (Table 4). The members of each subfamily are present in various locations in the M. tuberculosis genome and could have arisen due to gene duplication events.

To immunologically characterize the ESAT6-like proteins ESXC to ESXW, 25-mer peptides overlapping by 10 aa covering the sequence of each protein were synthesized using the standard fluorenylmethoxycarbonyl (Fmoc)-protecting group chemistry for solid phase synthesis, and the accuracy and purity of the synthesized peptides were checked by mass spectrometry [34, 35]. Testing of the pools of peptides of individual proteins with human PBMCs obtained from TB patients and healthy donors showed that all of them were immunologically reactive [36]. The results further showed that ESXL/Rv1198 and ESXF/Rv3905 are best suited for the diagnosis of active TB because they are preferentially recognized by the PBMCs of TB patients. Furthermore, ESXH/Rv0288 and proteins of subfamilies 1 and 2 are suited for use as candidate vaccines against TB because they are preferentially recognized by the PBMCs of healthy donors [36].

To further evaluate the immunogenicity of ESAT6-subfamilies 1 and 2, two representative proteins of each subfamily (ESXO/Rv2346 and ESXV/Rv3619 of subfamily 1 and ESXP/Rv2347 and ESXW/Rv3620 of subfamily 2) were selected. These proteins were selected because like the genes of ESAT6/ESXA/Rv3875 and CFP10/ESXB/Rv3874, which are located in the M. tuberculosis-specific genomic region of difference (RD)1, the genes for ESXO/Rv2346 and ESXV/Rv3619 are located in the M. tuberculosis-specific genomic region RD7, and the genes for ESXP/Rv2347 and ESXW/Rv3620 are located in the M. tuberculosis-specific genomic region RD9 [37].

The above-mentioned four ESAT6-like proteins were expressed in recombinant forms by cloning their genes in different vectors to express them in various expression systems, i.e., Escherichia coli, Mycobacterium smegmatis, Mycobacterium vaccae, and DNA plasmids [38, 39]. The antigens expressed in E. coli can be purified as recombinant proteins and used as immunizing agents along with appropriate adjuvants to induce Th1 responses [40‒42]. DNA plasmids used as vaccine vectors have in-built DNA sequences with strong adjuvant activity to induce the secretion of Th1 cytokines as well as Th1-like humoral immune responses [43]. The DNA plasmid to express the ESAT6-like antigens was pUMVC6 [44]. It contains the expression vector of CMV IE promoter at the 5′ end and a kanamycin marker. In addition, this expression vector contains human IL-2 secretory peptide, which acts as an adjuvant and allows the cloned gene to be secreted as a cytoplasmic protein to elicit Th1 immune responses in vivo [44]. Earlier studies have shown that immunization of mice with DNA plasmid pUMVC6 expressing M. tuberculosis-specific protein PE35 induced the secretion of antigen-specific humoral and cellular immune responses, including Th1 cytokine IFN-γ but not Th2 and Treg cytokines IL5 and IL-10, respectively [45, 46]. M. smegmatis and M. vaccae are non-pathogenic mycobacteria and have been used for expressing immunodominant M. tuberculosis antigens for use as recombinant TB vaccines in preclinical studies due to the rapid growth, genetic and structural homology of these mycobacteria to M. tuberculosis, and the induction of long-lasting immunity [47, 48]. In addition, the safety and efficacies of M. smegmatis and M. vaccae have been documented as vaccines and immunotherapeutic agent for the treatment of TB and other diseases [49, 50]. However, being non-tuberculosis mycobacteria/environmental mycobacteria, the use of M. vaccae and M. smegmatis-based vaccines in humans may have raised concerns in relation to potential immune interference by downregulating or interfering with the immune responses to M. tuberculosis-specific antigens [51].

All the recombinant proteins were purified from recombinant E. coli cultures and used as antigens for immunization [38]. The recombinant proteins along with two chemical adjuvants (Incomplete Freund’s Adjuvant and aluminum hydroxide), and the recombinant M. smegmatis, M. vaccae, and DNA plasmids, were injected into mice to determine the best delivery system for the induction of immunological responses against the immunizing antigens. The results showed that the protein ESXV/Rv3619c was the only protein immunogenic in mice using all the delivery systems by inducing the protective Th1 responses and IgG antibodies without inducing pathologic Th2, Th17, and Treg responses [38]. Further experiments showed that ESXV/Rv3619c induced broader immune responses because multiple epitopes of ESXV/Rv3619c were recognized by the spleen cells of immunized mice in Th1 responses, whereas none of the peptides induced Th2 and Treg responses [39].

ESAT6-like proteins have been evaluated as vaccines for protection against TB and asthma [10, 52]. Multimeric DNA vaccine preparations containing ESAT6-family genes generated potent and broad antigen-specific T cell responses in mice as compared to vaccination with BCG [52]. The earliest protection studies conducted in mice by Ansari et al. [53] showed that immunization with ESXV/Rv3619c induced memory responses and provided long-term protection against challenge with M. tuberculosis. The genes for ESXV/Rv3619c and ESXW/Rv3620 have also been incorporated in a subunit multi-protein anti-TB vaccine candidate known as ID93 [54]. Immunization of guinea pigs with this subunit vaccine did not induce sensitivity to purified protein derivative (PPD) of M. tuberculosis, suggesting that its use as a vaccine will not interfere with the use of PPD as a skin test reagent for the diagnosis of tuberculosis [55]. Immunization of mice with ID93 in combination with glucopyranosyl lipid adjuvant (GLA) in a stable nano-emulsion (SE) known as ID93/GLA-SE provided protection and long-lived immunity when infected with a clinical isolate of M. tuberculosis [56]. Furthermore, it provided cross-protection against a challenge with another mycobacterial pathogen causing leprosy, i.e., Mycobacterium leprae [57]. The candidate vaccine ID93/GLA-SE has also been tested in human clinical trials, where it was found to be safe and well-tolerated; it did not cause serious vaccine-related adverse reactions [58] and induced long-lasting antibody and Th1-type cellular immune responses [59]. Currently, this subunit vaccine candidate is undergoing a Phase 2 clinical trial in humans [60].

In experiments on protection against asthma, mice were immunized with the recombinant ESXV/Rv3619c protein emulsified in IFA, followed by an ovalbumin (OVA) challenge. Airway inflammation was assessed by quantifying airway cytology, histological changes, and secretion of the Th2 cytokine IL-5 by spleen cells. Levels of OVA-specific IgE, IgG, and IgG1 in sera of mice were also determined. The results showed that immunization of mice with recombinant ESXV/Rv3619c inhibited the phenotypes of asthma, i.e., OVA-induced increase in total cell counts, eosinophil airway cell infiltration in BAL fluid, perivascular and peribronchial inflammation and fibrosis, and goblet cell hyper/metaplasia. In addition, immunization with ESXV/Rv3619c inhibited the OVA-induced IL-5 secretion by spleen cells and decreased the production of OVA-specific IgE, IgG, and IgG1 in sera [61]. These results suggest that, in addition to TB and leprosy, immunization with ESXV/Rv3619c may be a suitable strategy for the prevention of asthma. However, studies with respect to protection against asthma using immunization with ESXV/Rv3619c are limited to a mouse model of asthma. The efficacy and safety of human subjects require further investigation. Additionally, potential adverse effects and long-term immunity need to be carefully addressed.

ESXV/Rv3619c is an ESAT6-like low-molecular-weight protein. It is a major antigen of M. tuberculosis recognized by the immune systems of humans and several animal models of TB. Immunization with subunit vaccine preparations protects animals against challenges with virulent M. tuberculosis and allergic asthma. These results suggest that ESXV/Rv3619c has potentials to protect against two major diseases in the world, i.e., tuberculosis and asthma, and hence may be used as a common vaccine against both diseases.

This review article did not require any ethical clearance because no unpublished data related to experiments done on humans or animals are presented.

The author declares no conflict of interest.

The study was supported by grants from the Research Sector (grants No. RM01/13 and SRUL02/13), Kuwait University, Kuwait.

All contributions are by Abu Salim Mustafa only.

Data supporting the findings of this study are available upon reasonable request.