Live Attenuated Vaccines against Tuberculosis: Targeting the Disruption of Genes Encoding the Secretory Proteins of Mycobacteria

Abstract

Tuberculosis (TB), a chronic infectious disease affecting humans, causes over 1.3 million deaths per year throughout the world. The current preventive vaccine BCG provides protection against childhood TB, but it fails to protect against pulmonary TB. Multiple candidates have been evaluated to either replace or boost the efficacy of the BCG vaccine, including subunit protein, DNA, virus vector-based vaccines, etc., most of which provide only short-term immunity. Several live attenuated vaccines derived from Mycobacterium tuberculosis (Mtb) and BCG have also been developed to induce long-term immunity. Since Mtb mediates its virulence through multiple secreted proteins, these proteins have been targeted to produce attenuated but immunogenic vaccines. In this review, we discuss the characteristics and prospects of live attenuated vaccines generated by targeting the disruption of the genes encoding secretory mycobacterial proteins.

1. Introduction

Tuberculosis (TB) is caused by a Gram-positive bacterial pathogen, Mycobacterium tuberculosis (Mtb), which is known for its thick cell wall made up of long-chain fatty acids called mycolic acids [1]. Despite effective control measures, neither the death rate nor the incidence rate of TB has shown any sign of decline in recent years, and they remain at 1.3 million and 10.6 million in 2022 [2]. In addition, a quarter of the world population is estimated to have latent TB infection (LTBI) [3]. This situation is further aggravated by the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains, which require second-line drugs and prolonged care. In addition, HIV-TB coinfection contributes to about 12.8% of the total deaths due to TB, because HIV-1 depletes the CD4⁺ T cells associated with Th1 immune response [2]. The goal of the World Health Organization (WHO) is to reduce the number of new TB cases by 90% in the year 2035, and achieving this goal requires the development of novel drugs and therapeutic agents for the treatment of the disease and, importantly, efficacious preventive and therapeutic vaccines.

Currently, Bacille Calmette–Guerin (BCG) is the only approved vaccine for TB. It was derived from the virulent Mycobacterium bovis, the pathogen that causes TB in cattle, when it was passaged in culture media over two hundred times [4,5]. An estimated four billion doses of BCG have been administered to infants with very few adverse incidences, indicating that it is the safest vaccine available [6]. However, a major weakness of BCG is its variable efficacy (0–80%) in different populations of different ethnicities [5,7]. Several reasons account for this discrepancy, including (a) the geographical location of the populations tested [8], (b) environmental mycobacteria that share antigens with BCG [7], and (c) variation in the antigenic profile of BCG sub-strains due to genetic differences [9]. In addition, it has been proposed that humans exposed to atypical mycobacteria [10] and helminthic infections will have increased Th2 response, which can also reduce the efficacy of BCG vaccination [11,12]. Regardless of these factors, the consensus is that BCG prevents TB meningitis in children but has no effect on the most common pulmonary TB in children and adults [5,7]. A recent meta-review also reiterates that infant vaccination using BCG can prevent tuberculosis in young children but is ineffective in adolescents and adults [13]. This underscores the need for a better primary vaccine and a booster that can induce BCG-induced primary immunity.

The past two decades have witnessed the development of many vaccines against TB, and some of these vaccines have advanced to clinical trials [14]. These include platforms based on protein subunits, viral vectors, recombinant mycobacterial live attenuated vaccines (LAVs), killed whole cell vaccines, and an mRNA-based vaccine [6,15,16,17,18,19,20,21]. Compared to other vaccines that transiently express antigens, LAVs are considered superior to others because they tend to persist for extended periods, encoding antigens and enabling the longer-lasting stimulation of immune cells to induce protective immune responses [22,23]. Further, the antigens produced by LAVs are closer to native antigens, which are properly folded proteins, carbohydrates, and lipids [24,25]. Moreover, these diverse antigens will likely stimulate multiple immune cell populations like subsets of T and B cells and phenotypes such as NK and innate T cells. Another advantage of using LAVs is that their cell wall components can stimulate innate immunity [26,27]. Thus, cell wall lipids such as trehalose dimycolate (TDM) can activate Mincle receptor-inducing trained immunity [28,29], whereas other glycolipids and several pathogen-associated molecular patterns (PAMPs) can trigger pattern recognition receptors (PRR) like TLR-2 and TLR-4 [30]. However, great caution should be exercised when considering lipids as inducers of the immune response because some essential Mtb lipids, like sulfoglycolipids, inhibit the innate immune response [31]. More importantly, the loss of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) in the BCG Pasteur strain reduces the efficacy of the BCG vaccine, highlighting the importance of PDIMs/PGLs [32]. Another attractive strategy for modifying live attenuated vaccines (LAVs) is the addition of major Mtb proteins to BCG to create recombinant BCG vaccines for better efficacy. However, our review is focused explicitly on Mtb or BCG knockouts as LAVs.

A major approach to derive LAVs against TB depends on a ‘rational deletion of genes’ in the chromosomes of Mtb and BCG [33]. Often, the target genes play a critical role in immune evasion by Mtb. Importantly, there is a strong link between immune evasion mechanisms and the ‘secretory proteins’ of mycobacteria, because many of these seem to be released into the host environment to modulate phagolysosomal (PL) fusion, autophagy, apoptosis, the modulation of cytokines, the intracellular survival of pathogens, and other related antimicrobial pathways [34,35,36,37] (Figure 1, Figure 2 and Figure 3). Coincidentally, many LAVs for TB are based on deleting genes encoding ‘secretory proteins’ or their regulators and transporters (Table 1 and Table 2). In this review, we describe the immunological parameters of LAVs deficient in secreted protein(s) and their efficacy compared with the BCG vaccine, which also secretes several antigenic proteins [38]. We specifically address LAVs deficient in secretory antigens, since other gene knockout mutants have been described by others [15,18,39,40].

2. Secretory Systems of Mycobacteria

To determine the significance of host immune modulation by Mtb, it is imperative to understand the secretory systems of mycobacteria. Bacteria generally have intriguing mechanisms to transport some of their proteins across the cytoplasmic membrane, which are called secretion systems or secretory pathways [41]. They transport proteins that need to be localized in the periplasm, outer membrane, or surface or released in the extracellular environment and into the host cells. The two major secretory pathways in bacteria are the general secretory pathway (or Sec pathway) and the twin-arginine translocation pathway or TAT pathway [41]. Both are highly conserved systems and are present in most bacteria [42,43]. In addition to Sec and TAT pathways, Gram-negative pathogenic bacteria have special pathways to transport virulence factors. There are six such special pathways, which are named type I through type VI secretory systems. Some of these systems, like the type III secretion system in Salmonella species, form microneedles to inject the effector proteins directly into the host cells [41]. The Gram-positive mycobacteria were initially thought to have only the Sec and TAT pathways to secrete proteins. However, a new special secretory system was later identified in mycobacteria [44] and was named the type VII secretion system [45], in line with the previously labeled secretion systems.

In contrast to other species, mycobacteria have two Sec pathways, SecA1 and SecA2 [44]. While transporting proteins through SecA1 requires a signal sequence, no signal sequence is required for transporting proteins through SecA2. The SecA2 pathway in Mtb appears to mainly transport proteins related to pathogenesis, such as SodA, SapM, and PknG [46]. On the other hand, the TAT pathway requires a TAT signal sequence to transport proteins [41]. In Mtb, the TAT pathway transports fewer proteins associated with pathogenesis, including phospholipase A and C [46].

Further, the Type VII secretion system in Mtb and related mycobacteria has five export systems, and they are named ESX-1 through ESX-5 [47,48,49]. Each system has a cluster of genes to encode the proteins, facilitated by the structural proteins required for transportation. The ESX systems recognize the substrates or the proteins to be transported by the YxxxD/E signal (amino acid) motif in their sequence [50]. Among the five ESX systems, ESX-4 seems to be the oldest system, and others appear to have evolved through duplication events [51,52]. Notably, the gene cluster for this system lacks genes encoding PE/PPE proteins, although the other four systems do have genes to encode these proteins.

Nonetheless, the ESX-1 system seems to be the most well-studied and is responsible for the secretion of EsxA (ESAT-6) and EsxB (CFP-10) proteins in Mtb and related pathogens [53,54,55,56]. These proteins are both virulence factors and immunodominant antigens, and coincidentally, their absence leads to an avirulent phenotype in Mtb [55]. ESAT-6 enables the bacteria to lyse the phagosomal membrane with the aid of the chaperone CFP-10, a strategy that is missing in BCG vaccine strains [57]. The ESX-1 system also constitutes the Region of Difference 1 (RD1), deleted in the BCG vaccine strains [54]. Indeed, the attenuation of BCG appears to be due to the absence of the RD1 region in its chromosome, because the complementation of BCG with the RD1 region restores its virulence [54]. Both ESX-1 and ESX-2 systems are intact in Mtb and other related pathogens, but little information is available about its secreted products and role in pathogenesis [58].

Further, ESX-3 and ESX-5 systems participate in the secretion of their proteins, and the latter plays a significant role in immune modulation or inflammasome activation [59,60,61,62,63]. ESX-4 is different in functionality, and unlike other systems, it plays a vital role in conjugation between bacteria [64]. Overall, mycobacteria use Sec, TAT, and ESX systems to transport proteins that regulate virulence and immunogenicity. However, some secreted proteins do not seem to have specific export signatures.

Figure 1

Subversion of phagocytosis and related processes by mycobacterial secreted proteins. Phagosome maturation: pattern recognition receptors (PRRs) of macrophages recognize mycobacteria through mycobacterial pathogen-associated molecular patterns (PAMPs), resulting in the engulfment of bacilli by a phagosome, an organelle derived from the plasma membrane. Phagosomes undergo a series of steps called phagosome maturation to digest the engulfed bacilli and present the antigens to the immune cells. Additionally, phagosomes fuse with early endosomes, late endosomes, and, subsequently, with lysosomes to acquire the materials/properties required for the killing/digestion of the pathogen. However, intracellular mycobacteria like M. tuberculosis (Mtb), M. bovis, M. marinum, and BCG have multiple strategies to protect them against the phagocytic processes. M. marinum secretes PPE38 [65] to block the phagocytosis of the bacilli. Mtb secretes several secretory proteins to inhibit the phagosome maturation process. SapM [34] and PtpB [66] dephosphorylate phosphatidylinositol-3-phosphate (PI3P) to inhibit phagosome maturation, whereas PtpA protein inhibits phagosome acidification by blocking Vacuolar-type ATPase (V-ATPase) [67]. TlyA inhibits Early Endosomal Antigen-1 (EEA1), Ras-related protein 5 (RAB5), and RAB7 recruitment [68]. NdkA inhibits RAB5 and RAB7 [69], while PknG inhibits RAB7L1 [70]. Additionally, Mtb secretes UreC to alkalize the phagosomes [71]. In addition, Mtb perforates the phagosome and escapes to the cytosol using the concerted action of phthiocerol dimycocerosates (PDIM) and ESX-1 system [72]. Host efforts to repair phagosomal rupture were blocked by the EsxH protein [59]. ROS/iNOS: Host NADPH oxidases (NOX) from the cytoplasm and mitochondrial electron transport chain are the primary sources of reactive oxygen species (ROS) production. ROS is blocked by Mtb proteins like Eis, ESAT-6/CFP-10, NuoG, NdkA, PPE2, SodA [73,74,75,76], and inducible nitric oxide synthase (iNOS), while the mediated production of NO is blocked by PtpB, PPE2, PE_PGRS62, PE5, PE15, and PE4 [77,78,79,80]. Epigenetic regulators: Mtb secretes proteins like Rv1988 [81] and Rv2966c [82] to methylate host DNA and proteins like Eis [83] and Rv3423.1 [84] to acetylate host DNA to manipulate the host immune response. Proinflammatory cytokines: Mtb secretes proteins like PtpA, PtpB, ESAT-6/CFP-10, Eis, EchA1, and PknG [77,85,86,87,88,89] to inhibit proinflammatory cytokines. Cell death pathways: Cytosolic escape of the pathogen leads to activation of various cell death pathways like necrosis mediated by Zmp1, CpnT, and PE25:PPE41 [90,91,92] or ferroptosis via PtpA which benefits the pathogen [93]. The cytosolic presence of bacilli DNA or RNA triggers various pathways like apoptosis, autophagy/xenophagy, and pyroptosis, which are detrimental to the pathogen. Mtb secretes NuoG, PtpA, PtpB, NdkA, Rv3654c, Rv3655c, and Rv3033 to block apoptosis [36,75,77,94,95,96]. Proteins like Zmp1, PknF, PtpB, and Rv3364c block inflammasome activation and/or pyroptosis [35,97,98,99]. Autophagy/xenophagy pathways are blocked by proteins like NuoG, Eis, SapM, PE_PGRS20, PE_PGRS47, PPE51, LprE, and PknG [86,100,101,102,103,104,105]; however, these proteins block autophagy indirectly by blocking early/late phagosome proteins. Note: Some live attenuated Mtb or BCG vaccines described in this review lack one or more secretory proteins mentioned above, and they are designated in this figure with rose-colored oval shapes.

[Figure omitted. See PDF]

3. Mycobacterial Vaccines Deficient in Secreted Protein(s)

As noted above, the preference for LAVs over others is due to their superior number of antigens and ability to stimulate immune response for a more prolonged period in vivo [22]. BCG, an excellent example of an LAV, is poorly effective in adults, requiring a replacement or a booster. Initial studies to improve BCG through the recombinant overexpression of antigenic genes did improve its efficacy against TB in the mouse model [106,107,108,109,110]. In particular rBCG30 and BCG85B vaccines showed remarkable efficacy against TB in mice but did not advance to clinical trials. One possible reason is that they lacked the immunogenic RD1 region that encodes ESAT6 and CFP10, which are paradoxically related to virulence in Mtb. An extensively explored approach is deleting genes in BCG and Mtb to derive LAVs. In the past two decades, over fifty mycobacterial mutant strains with deletion or disrupted gene(s) in the chromosome have been tested for their vaccine efficacy in animal models, regardless of their attenuation status [18,110]. Interestingly, a large proportion of the LAVs tested so far are those that lack secretory proteins (Table 1 and Table 2). The primary candidate LAVs lacking secretory proteins are discussed below.

3.1. Ag85 Complex

Mtb expresses three secreted fibronectin proteins (Fbp), namely FbpA (Rv3804), FbpB (Rv1886c), and FbpC (Rv0129c) [111,112]. All three of them are major antigens (Ag), and hence, they are also known as Ag85A (31 kDa), Ag85B (30 kDa), and Ag85C (31.5 kDa) and collectively as the Ag85 complex of proteins [111,112]. The amino acid sequences of these proteins are highly conserved among mycobacterial species and, in particular, the Mtb complex. Besides fibronectin-binding activity, these proteins show mycolyl transferase activity essential in assembling the mycobacterial cell walls [113]. Armitige et al. [114] disrupted the genes fbpA and fbpB in Mtb and assessed the mutant strains (ΔfbpA and ΔfbpB) for their growth and survival in culture, macrophages, and mice [114,115]. The ΔfbpA strain was later shown to protect C57BL/6 mice against challenges with an efficacy similar to BCG [115]. To our knowledge, this is the first study demonstrating that an Mtb mutant lacking a secretory protein antigen is effective as a vaccine against TB. The ΔfbpA candidate vaccine induced high levels of phagosome maturation, proinflammatory cytokines, and Th1 immune response and an increased expansion of CD4+ CXCR3+ IFN-γ+ cells in mice after vaccination [116]. Although recombinant mycobacterial strains over-expressing Ag85B (FbpB) and Ag85C (FbpC) also protect mice against TB [106,117], thus far, Mtb mutants ‘lacking’ these proteins have not been tested for vaccine efficacy. Notably, a BCG strain lacking the Ag85B protein showed efficacy against TB in mice, like BCG [118]. This is not surprising, because BCG naturally has a mutation in Ag85B, which affects the mycolyl transferase activity and its stability [119]. It is intriguing to note that Ag85 complex proteins are immunogenic in mice, guinea pigs, and human immune cells, and individual deletion yields mutants defective in cell wall lipids due to their mycology transferase activity. The ΔfbpA has reduced levels of trehalose dimycolate (TDM) in its cell wall [120] and TDM has been thought to be a virulence factor [121]. Therefore, we are pursuing the hypothesis that the selective deletion of Ag85 complex genes in combination with other functionally characterized genes will lead to markedly immunogenic and concurrently attenuated vaccines [122] (under submission). It should be noted that the MTBVAC vaccine, which is currently undergoing clinical trials, shows a higher secretion of Ag85 proteins, indicating its role in inducing an immune response [123].

Figure 2

Subversion of LC3-associated phagocytosis (LAP) by secreted proteins of mycobacteria. LC3-associated phagocytosis is initiated by macrophage via engaging specific receptors like TLR1/2, TLR2/6, TLR4, Fc receptors, CLEC7A/Dectin-1, and TIM4, and it also recognizes apoptotic, necrotic, and entotic cells [124]. Once the bacilli are engulfed within a single membraned phagosome, phosphatidylinositol-3-phosphate (PI3P) is recruited over the phagosome, which is generated by the PtdIns3K (Class lll phosphatidylinositol 3-kinase) complex. LAP and canonical autophagy share common features and unique features in their pathway. Both require PI(3)P production and common machinery like BECLIN, VPS 34, ATG5, ATG7, ATG16L, TSG101, and RAB7 to recruit LC3 over the phagosome [125]. Recruitment of LC3 over a single-layered membrane is called LAPosome. LAPosome subsequently fuses with lysosome to eliminate M. tuberculosis (Mtb). Some unique proteins involved in the LAP pathway are Rac, p47, p40, p67, p22, gp91phox, and Rubicon [125]. However, Mtb secretes multiple proteins to block the LAP pathway. Mtb proteins like CpsA, NdkA, and PPE2 inhibit the phagosomal recruitment of NADPH oxidase (NOX-2 complex) to the phagosome, whereas NuoG and KatG proteins neutralize reactive oxygen species (ROS) [126]. Note: some live attenuated Mtb or BCG vaccines described in this review lack one or more secretory proteins mentioned above, and they are designated in this figure with rose-colored oval shapes.

[Figure omitted. See PDF]

3.2. LpqH

Proteomics identified the LpqH (Rv3763) protein in the cell wall and the culture filtrates of Mtb. LpqH is a 19 kDa lipoprotein that plays multiple roles in the virulence of Mtb, such as TLR-2 interaction, the induction of humoral and T cell-mediated responses, and apoptosis [127,128]. Macrophages infected with a mutant strain of Mtb lacking this protein (Δ19) show a reduced surface expression of MHC-II molecules and the secretion of cytokines IL-1β, IL12p40, and TNF-α [129]. The Δ19 strain was highly attenuated for growth in C57BL/6 mice compared to wild-type Mtb H37Rv. Despite poor in vivo growth, in mice, LpqH elicited a protective efficacy equal to BCG (1 log¹⁰ reduction in Mtb counts in lungs) [130]. Reduced Mtb growth correlated with increased IFN-γ-secreting CD4+ and CD8+ T cells comparable to mice receiving the BCG vaccine, although the lung granulomas of Δ19 strain-vaccinated mice had more lymphocytes than BCG-vaccinated mice, which had more vacuolated foamy macrophages.

3.3. LprG

This is another mycobacterial lipoprotein agonist of TLR-2 receptors in macrophages [131], which, in combination with immunomodulatory lipids, seems to prevent PL fusion in macrophages and limit antigen presentation through the MHC-II pathway [132]. The Rv1411c gene encoding LprG is transcriptionally linked to Rv1410c, which encodes a transmembrane efflux pump, and the deletion of the 1411c-1410c locus in Mtb (ΔlprG) attenuates its growth in immunodeficient mice [133]. The growth attenuation appears to be due to the accumulation of intracellular triacylglycerides (TAG) and altered bacterial metabolism [133]. The ΔlprG mutant was evaluated for its immunogenicity and vaccine efficacy in three mouse strains [134]. While ΔlprG-induced protection was comparable to BCG in C57BL/6 and BALB/c mice, it showed a 0.9 log¹⁰ better decrease in Mtb load in the lungs of C3HeB/FeJ mice; the latter developed necrotizing TB granulomas, similar to humans [134]. Variable protection was also reflected in pathology as ΔlprG-vaccinated mice showed fewer granulomas than mice given BCG. Further, compared to BCG, increased Ag-specific CD4+ positive T cell responses, lower percentages of PD-1 positive T cells, and increased antigen-specific IL-17A-secreting T cells were found in the lungs of ΔlprG-immunized mice [134]. These data were confirmed by a recent study where increased protection was observed in ΔlprG-immunized C3HeB/FeJ mice given a low dose aerosol infection. Protection correlated with elevated serum levels of IL-17A, IL-6, CXCL2, CCL2, IFN-γ, and CXCL1 [135]. The ΔlprG mutant illustrates that LAVs may show variable protection in mouse strains of different genetic backgrounds.

3.4. BfrB

Iron limitation is a major factor affecting host–pathogen interactions [136]. Though Mtb has multiple mechanisms to sequester iron from the host, it uses bacterioferritin (BfrB, Rv3841), a secretory protein [137,138], to store the iron when it is abundant and to release it when required. A ΔbfrB mutant of Mtb could not establish a chronic infection in mice [139]. When it was used as a vaccine against Mtb H37Rv in mice, the mutant generated protection comparable to BCG, although they differed in organ pathology and lung transcriptomic signatures [140]. Further, at eight weeks post-vaccination using ΔbfrB, mice had reduced inflammation, smaller lung granulomas, and extensive fibrosis [140].

3.5. CpsA

Encoded by the gene Rv3484, CpsA belongs to the LytR-CpsA-Psr family of conserved proteins related to cell wall assembly in Gram-positive bacteria [141]. Koster et al. reported that Mtb lacking the CpsA protein (ΔcpsA) enhances LC3-associated phagocytosis (LAP) and the recruitment of NADPH oxidase to their phagosomes, suggesting that secreted CpsA protein inhibits these innate immune responses to survive inside the host [125]. In addition, the ΔcpsA strain showed defective growth in mice, which was restored by complementation using a functional cpsA gene [125]. Since LAP can increase antigen presentation through the MHC-II pathway, the authors proposed that the ΔcpsA could be a potential vaccine candidate and created a new mutant mc²6206ΔcpsA by deleting cpsA in mc²6206, which is an auxotrophic mutant strain (ΔleuCD and ΔpanCD) [142]. Despite increased LC3 trafficking in BMDMs, mc²6206ΔcpsA showed protection similar to BCG in C57BL/6 mice challenged with Mtb H37Rv [142]. Given its ability to increase LC3 trafficking, it is unclear why there was no better protection than BCG, which uses a sapM-dependent mechanism to evade autophagy. A deletion of cpsA in Mtb may generate a more protective phenotype.

3.6. BioA

The enzyme 7,8-diaminopelargonic acid synthase, also known as BioA (Rv1568), is one of the four enzymes associated with synthesizing biotin molecules in Mtb [143]. It appears to be critical for the acute and chronic infection of mice infected with Mtb [144]. Mtb ΔbioA was severely attenuated in guinea pigs regardless of aerosol or intradermal route of infection, and intriguingly, the lungs of ΔbioA-infected guinea pigs did not show live bacteria after six weeks post-infection [145]. Although vaccinating guinea pigs using this mutant significantly reduced the bacillary burden in the lungs and spleens, repeated vaccination before the Mtb challenge reduced its efficacy [145]. This study illustrates that the hyperattenuation of Mtb may not always correlate with vaccine efficacy.

3.7. Gln Proteins

Glutamine synthetase activity in the culture filtrate of Mtb has been reported [146]. The Mtb genome has multiple genes encoding glutamine synthetase, namely GlnA1 (Rv2220), GlnA2 (Rv2222c), GlnA3 (Rv1878), GlnA4 (Rv2860c), and a regulator protein GlnE (Rv2221c). However, only GlnA1, GlnA3, and GlnA4 were identified in the culture filtrate of Mtb using proteomics [138,147]. Lee et al. [148] characterized the GlnA1, GlnA2, GlnA3, and GlnA4 mutants and a triple mutant for GlnA1EA2. Of these, only glnA1 and glnA1EA2 were essential for the growth of Mtb, and they were auxotrophic to glutamine. Both ΔglnA1 and ΔglnA1EA2 showed attenuated growth in immunodeficient SCID mice and immunocompetent C57BL/6 mice [148]. Additional studies using C57BL/6 mice revealed that they protected mice like BCG [148]. The inability of these mutants to multiply in vivo appears to have discouraged their further validation. GlnA1 is abundant in mycobacterial extracellular vesicles (MEV), suggesting that it may serve as a diagnostic marker [149].

3.8. SapM

Among the three known secreted phosphatases (SapM, PtpA, and PtpB) of Mtb, SapM (Rv3310) is the first to be identified as an acid phosphatase [150]. After Mtb infects human macrophages, its survival within macrophages depends upon the inhibition of phagosome maturation. SapM is one of the key enzymes implicated in phagosomal maturation arrest during Mtb–macrophage interactions [46,151]. SapM dephosphorylates phagosomal membrane-bound phosphatidylinositol 3-phosphate (PI3P), a lipid molecule associated with the recruitment of downstream effector Rab proteins essential for phagosomal maturation, also called phagosome–lysosome (PL) fusion [34]. Essentially, the SapM-mediated removal of a phosphate molecule from PI3P affects its structure and reduces the recruitment of effector proteins, inhibiting PL fusion. Further, SapM also inhibits autophagy by blocking Rab7, a small GTPase required for lysosomal fusion [100]. Two independent studies used MtbΔsapM to define the role of SapM during PL fusion [152,153]. Upon the infection of macrophages, these mutants were seen more frequently in the matured phagolysosomal compartments enriched with lysosomal markers like LAMP1 when compared to macrophages infected with wild-type Mtb [152,153]. The MtbΔsapM mutant also showed attenuated growth in macrophages and in the lungs and spleen of guinea pigs [152,153]. Consistent with PL fusion competence and attenuation, the mutant showed increased in vitro and in vivo immunogenicity; its immunogenicity was further increased when fbpA was deleted, yielding an MtbΔfbpA-ΔsapM double knockout (DKO) mutant [152]. Notably, mice vaccinated with the DKO strain and challenged with Mtb showed better protection (>1 log¹⁰) than mice receiving the BCG vaccine [122]. Others reported similar results when guinea pigs were vaccinated using a triple knockout strain of Mtb (MtbΔmms) that lacked three phosphatases (PtpA, PtpB, and SapM) [154]. MtbΔmms was more effective in decreasing Mtb CFUs in the lungs of guinea pigs (3.60 log10) compared to the lungs of guinea pigs receiving BCG vaccination (4.43 log10) [154]. These two studies suggest that the deletion of the same gene increases the vaccine efficacy in the KO mutants, even if the other deleted genes are functionally different, mainly because the deletion of the bioA gene in MtbΔmms (MtbΔmmsb) had no additional benefit [155]. It is relevant to recall here that a BCG sapM mutant (BCGΔsapM) transposon mutant also shows enhanced protection as a vaccine against Mtb in BALB/c mice [156]. BCGΔsapM enriched CD11c+MHC-II intCD40int dendritic cells (DCs) in the draining lymph nodes and was found to be safer than the BCG in SCID mice [157]. Since sapM deletion increased the vaccine efficacy of both Mtb- and BCG-derived mutants, it may be an essential target for more effective vaccines.

3.9. Ptp

PtpA (Rv2234) and PtpB (Rv0153c) are the only two secreted phosphotyrosine protein phosphatases (Ptp) identified in Mtb [158]. By interacting with host signaling partners, they can modulate the cellular pathways of the host cells [159]. The genes encoding PtpA and PtpB have been disrupted in the chromosome of Mtb, and their roles in pathogenicity are reported [160,161]. PtbA primarily affects phagosomal maturation by interacting with H subunit V-ATPase, an enzyme required for phagosomal lumen acidification, and PL fusion by dephosphorylating the vacuolar protein sorting-associated protein 33B (VPS33B), a late endosomal molecule [67,160]. In addition, PtpA plays a critical role in suppressing innate immune responses by interacting with ubiquitin, dephosphorylating JNK, and regulating host genes such as GADD45A [85,158]. Similarly, PtpB has been reported to promote the survival of Mtb H37Rv by suppressing iNOS, IL-1β, and IL-6 [77], thus suppressing innate immune responses through ERK1/2 and Akt pathways [162] and interacting with ubiquitin and inhibiting host cell pyroptosis [97]. Coincidentally, the ΔptpB strain is attenuated for growth in macrophages and guinea pigs compared to wild-type Mtb [161]. Further, an Mtb strain with triple deletions (ptpA, ptpB, and sapM) protected against TB in a guinea pig challenge model better than BCG. [154]. Although the individual roles of ΔptpA or ΔptpB in contributing to vaccine efficacy remain unclear, the fact that they affect multiple host processes justifies their deletion as a strategy to derive vaccines.

3.10. Zmp1

Zinc-containing metalloprotease 1, or Zmp1, is encoded by the gene Rv0198c. Masters et al. [35] first generated a zmp1 deletion mutant in Mtb and BCG, showing that it is crucial in preventing inflammasome activation in macrophages and phagosomal maturation. Macrophages infected with an MtbΔzmp1 mutant not only secreted more IL-1β but also enhanced PL fusion, indicating that it is a key virulence factor [35]. In addition, Zmp1 causes necrotic cell death and the dissemination of Mtb [90]. Interestingly, the deletion of zmp1 has similar effects in both Mtb and BCG fields [35]. This led Sanders et al. to evaluate zmp1 deletion in the BCG vaccine [163,164]. Mouse bone marrow-derived dendritic cells (DCs) infected with BCG and BCGΔzmp1 were compared for their antigen presentation to T cells using Mtb Ag85A-specific MHC-II restricted hybridoma T cells [163]. As expected, DCs infected with BCGΔzmp1 displayed enhanced antigen presentation, suggesting that the BCGΔzmp1 strain is immunogenic [163]. BCGΔzmp1-vaccinated mice showed a stronger delayed-type hypersensitivity (DTH) reaction, and splenocytes showed heightened IFN-γ levels in response to PPD stimulation when compared to splenocytes from BCG-immunized mice [163].

Further, BCG Pasteur lacking Zmp1 and BCG Denmark strain were compared with wild-type BCG Denmark (Danish) for efficacy against TB in guinea pigs [164]. Both mutants showed impressive protection against the Mtb challenge and reduced the lung Mtb burden by approximately 0.5 log¹⁰ CFU compared to BCG Denmark. This observation is remarkable because BCG Denmark, on its own, showed about 1.8 log¹⁰ CFU reduction compared to unvaccinated controls [164]. BCGΔzmp1 has a high safety profile in SCID mice, particularly the Danish BCGΔzmp1, which is hyper-attenuated [164]. Although the mechanisms underlying protection are unclear, BCGΔzmp1 strain was about to enter into a phase I clinical trial in 2017 [21].

3.11. Eis

The enhanced intracellular survival (EIS) protein is a secretory protein of Mtb encoded by the gene Rv2416c [165]. The name EIS is because it enhances the survival of M. smegmatis within macrophages [166]. Paradoxically, an eis deletion mutant of Mtb (MtbΔeis) showed no defect in intracellular survival within macrophages but induced higher levels of proinflammatory cytokines [167]. Such macrophages also showed increased reactive oxygen species (ROS) generation, autophagy, and cell death [86]. The Eis protein is an enzyme with aminoglycoside N-acetyltransferase activity. Thus, it seems capable of modulating or inhibiting proinflammatory responses, JNK-dependent autophagy, ROS generation, and, to some extent, phagosome maturation by acetylating the host phosphatase protein DUSP16/MKP-7 [73]. The inhibition of autophagy by Eis also seems to be due to the acetylation of histone H3 (Ac-H3), which can upregulate the expression of IL-10 and, as a consequence, activate the Akt/mTOR/p70S6K pathway [83]. Eis is the first secreted protein of Mtb to epigenetically modify macrophages. Recently, its homolog from BCG (BCG_2432c) was knocked out in BCG (China sub-strain), and the mutant ΔBCG_2432c was tested as a vaccine against TB in a C57BL/6 mice [168]. Remarkably, ΔBCG_2432c-immunized mice showed approximately a 2.0 log¹⁰ reduction in CFU in the lungs compared to mice immunized with wild-type BCG (China sub-strain). This enhanced protection was likely due to elevated levels of IFN-γ+ CD4+ TEM (effector memory T cells) and IL2+CD4+TCM (Central memory T cells) in the lungs and spleens of ΔBCG_2432c-immunized mice. This is the first study demonstrating a significant reduction in Mtb CFU in mice by a BCG vaccine with a single gene deletion in the chromosome. Because of its effect in mice, the deletion of the eis gene in Mtb appears to be a promising approach for TB vaccines.

3.12. Esx5

The products of the Esx5 system activate the inflammasome pathway in the host cells, facilitating the death of the cells and escape of the Mtb [49,61]. This system comprises 17 genes, including five encoding PPE25, PE18, PPE26, PPE27, and PE19 proteins, all containing strong T cell epitopes showing cross-reactivity with other non-Esx PE/PPE proteins [63,169]. Deleting the five ppe-pe genes (from ppe25 to pe19) of the esx5 renders the Mtb attenuated for growth in immunocompetent mice [169]. Further, C57BL/6 mice immunized with an Mtb Δppe25-pe19 and challenged with H37Rv had a reduced bacterial load in the lungs and spleen compared to BCG-vaccinated mice [169], indicating moderately better protection. In contrast, mice and guinea pigs immunized with the Δesx5 strain, which has a deletion of 17 genes in the esx5 locus (Rv1782-Rv1798), showed similar levels of protection to BCG-vaccinated mice exposed to a virulent HN878 strain of Mtb [170]. Better protection by the Δesx5 vaccine was noted only when administered using a prime-boost strategy with BCG as the prime vaccination [170]. Enhanced protection correlated with increased numbers of activated monocytes, central memory T cells (TCM), and follicular T cells (TFH) [170], although the mechanisms behind the increased protection by prime-boost vaccination remain unclear. In the same study, an MtbΔesx-3 mutant, which has a deletion of 11 genes, was also tested, but its protection seems to be lower than that of the Δesx5 strain. The authors of this study opined that the large-scale deletion of genes in the Δesx5 vaccine could safeguard its potential reversion to virulence. However, this may lead to the loss of protective T cell epitopes in proteins encoded by the deleted genes. Since Δesx5 is highly attenuated in immunocompromised SCID mice [170], it has an interesting potential to be developed as a vaccine for HIV-infected children who are susceptible to TB.

Figure 3

Subversion of xenophagy/autophagy by secreted proteins of mycobacteria. M. tuberculosis (Mtb) is well-known for inhibiting the maturation of phagosomes and their subsequent fusion with lysosomes. Through the ESX-1 secretion system and the cell envelope lipid PDIM, Mtb perforates the phagosome membrane and escapes to the cytosol. The first defensive step of a host is initiating the autophagy pathway by successfully binding ubiquitin to bacteria, followed by the recruitment of autophagy adaptors such as p62, OPTN, TAX18P1, NBR1, and TOLLIP. Subsequently, these autophagy adaptors engross with microtubule-associated-protein-1 light chain 3 (LC3) to deliver Mtb to autophagosomes. However, Mtb has multiple evasion strategies to escape from the host autophagic pathway via the secretion of various protein effectors. Mtb SapM inhibits Rab7, a late endosome marker required for autophagosomes to fuse with the lysosomes [100]. Mtb protein Eis inhibits autophagy via suppression of c-Jun N-terminal kinase (JNK)-mediated reactive oxygen species (ROS) signaling [86] or through the acetylation of host histone, which upregulates IL-10 and activates the Akt/mTOR/p70S6K pathway [83]. Mtb secretes LprE to suppress autophagy by inhibiting the expression of cathelicidin antimicrobial peptide (CAMP) via the p38 MAPK pathway [104]. Mtb protein PE_PGRS47 suppresses autophagy by inhibiting LC3 colocalization [171]. PknG blocks Rab14 to inhibit the autophagosome maturation [103]. PE_PGRS20 and PE_PGRS47 inhibit autophagy by interacting with RAB1A, which recruits the ULK1 (unc-51-like autophagy activating kinase 1) complex to the pre-autophagosome [102]. PPE51 inhibits autophagy by blocking the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway [105]. ESAT-6 released by Mtb perforates the lysosomes and autophagosomes, leading to perturbation of membranes. Damage to membrane releases cathepsin B in the cytosol and causes the subsequent activation of NLRP3-inflammasome and release of matured IL-1β [172]. Secreted ESAT-6 also affects autophagy flux in dendritic cells [173]. Note: Some live attenuated Mtb or BCG vaccines described in this review lack one or more secretory proteins mentioned above, and they are designated in this figure with rose-colored oval shapes.

[Figure omitted. See PDF]

3.13. UreC

Secreted urease C (Rv1850) of mycobacteria neutralizes the acidic environment of the phagosome of the macrophages, contributing to phagosomal maturation arrest [174,175]. Although BCG lacks the RD1 locus and is attenuated, it retains several genes related to phagosomal maturation arrest, including ureC. Consequently, BCG is sequestered within the neutral pH phagosome [176]. To nullify this effect, Kaufmann and his colleagues used a novel strategy of integrating the gene hly, which encodes the Listeria monocytogenes derived listeriolysin (LLO) toxin, into the chromosome of BCG, concurrently deleting ureC in its chromosome [177]. They proposed that BCG-secreted LLO could perforate the phagosomal membrane and allow the bacteria into the cytosol for bacterial antigen processing through the MHC-I pathway. The deletion of ureC, on the other hand, could favor the vATPase-mediated acidic pH required for LLO activity. They constructed two isogenic strains, BCG::hly and BCG::hlyΔureC, and assessed their vaccine efficacy in BALB/c mice [177]. Both showed better protection over the parental strain, although protection by BCG::hlyΔureC was superior [177]. This was due to the induction of apoptosis, increased antigen processing through MHC-I, the elicitation of T cells like TCM, TFH, and Th17, and high IgG antibody levels produced by BCG::hlyΔureC [177,178]. Further, the vaccine had an excellent safety profile in mice, guinea pigs, newborn rabbits, and non-human primates [179]. Currently named VPM1002, it is one of the few live mycobacterial vaccines undergoing clinical trials in sub-Saharan Africa and India [14].

3.14. NuoG

This protein is one of the subunits of type-I NADH dehydrogenase of Mtb and BCG and was identified by screening for anti-apoptotic genes in Mtb [36]. Mtb uses NuoG (Rv3151) to inhibit host apoptosis by neutralizing the ROS derived from NOX2 [74]. Since the vaccine-induced apoptosis of macrophages increases vaccine efficacy [177], nuoG gene deletion in BCG and BCG::hlyΔureC was created to improve vaccine efficacy [101]. Both BCGΔnuoG and BCG::hlyΔureCΔnuoG seem to enhance apoptosis in murine lymph nodes and improve autophagy activity in macrophages [101]. Consequently, both strains showed enhanced immunogenicity and efficacy against TB in mice challenged with Mtb H37Rv [101]. Specifically, the lung CFUs of BCGΔnuoG- and BCG::hlyΔureCΔnuoG-immunized mice demonstrated significant protection against TB. Protection against the more virulent Mtb Bejing W strain was also observed in mice immunized with BCG::hlyΔureCΔnuoG after 90 and 180 days post-challenge. The microarray analysis of draining lymph nodes from vaccinated mice showed an increased expression of genes related to GTPase activity, inflammatory responses, cell activation, and cell proliferation [101].

Additionally, the vaccine increased CD4+ TEM cells, TFH cells, germinal center B cells, and CD4+ TCM cells. Paradoxically, a nuoG mutant made in BCG China sub-strain had no significant protection in the Mtb-challenged mice [168]. These data suggest the possibility that the deletion of a gene in BCG sub-strains may give different effects, and this seems to be important because at least five major sub-strains of BCG (Copenhagen/Danish, Russian, Shanghai/China, Japan, Moreau) are used around the world for the primary immunization of infants.

Table 1

Efficacy of live Mycobacterium tuberculosis vaccines with gene(s) deleted for secreted protein(s).

Vaccine Name	Vaccine Components	Secreted Protein(s)Absent	Immunization Route/Dose	Challenge Mtb Strain	Challenge Route/Dose	Animal Model (Strain)	Efficacy in Relation to BCG	log10 CFU/LUNGS Reduction Than BCG	Ref.
ΔfbpA	H37Rv strain with single gene (fbpA) knockout.	FbpA or Ag85A	SC/105 CFU/mouse	Erdman	Aerosol/2.5 log10 CFU per mouse	Mouse (C57BL/6)	Better than BCG	~1.5	[115]
ΔglnA1	H37Rv strain with single gene (glnA1) knockout.	Glutamine synthetase A1	SC/106 CFU/mouse	Erdman	Aerosol/200 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[148]
ΔglnA1EA2	H37Rv strain with 3 genes (glnA1, glnE, and glnA2) knockout.	Glutamine synthetase A, E and A2	SC/106 CFU/mouse	Erdman	Aerosol/200 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[148]
Δ19	H37Rv strain with single gene (lpqH) knockout.	Lipoprotein LpqH	SC/106 CFU/mouse	H37Rv	Aerosol/100 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[130]
Δmms	H37Rv strain with 3 genes (ptpA, ptpB, and sapM) knockout.	Phosphatases PtpA, PtpB, and SapM	ID/5 × 105 CFU/guinea pig	H37Rv	Aerosol/10–30 CFU per guinea pig	Guinea pigs	Better than BCG	0.83–4 weeks post-challenge;1.41–12 weeks post-challenge	[154]
ΔbfrB	H37Rv strain with single gene (bfrB) knockout	Bacterio-ferritin B	SC/106 CFU/mouse	H37Rv	Aerosol/100 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[140]
ΔbioA	H37Rv strain with single gene (bioA) knockout	BioA or 7,8-diaminopelargonic acid synthase	ID/106 CFU/guinea pig (single or double dose with 6 week interval)	Erdman	Aerosol/50 CFU per guinea pig	Guinea pigs	Equal to BCG	-	[145]
Δmmsb	H37Rv strain with 4 genes (ptpA, ptpB, sapM, and bioA) knockout.	Phosphatases PtpA, PtpB, SapM and BioA	ID/5 × 105 CFU/guinea pig	H37Rv	Aerosol/10–30 CFU per guinea pig	Guinea pigs	Less than BCG	-	[155]
mc26206 ΔcpsA	H37Rv strain with 3 genes (leuD, panCD, and cpsA) knockout.	CpsA	SC/106 CFU/mouse	H37Rv	Aerosol/400 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[142]
ΔlprG	H37Rv strain with two genes (lprG and Rv1410c) knockout.	Lipoprotein LprG	SC/~106 CFU/mouse	H37Rv and Erdman	Aerosol/75 CFU per mouseor Aerosol/1 Median Infectious Dose (1MID50).	Mouse (C57BL/6, BALB/c and C3HeB/FeJ)	Equal or better than BCG	0.67–0.9 (in C3HeB/FeJ mice);	[134,135]
SO2	MT103 strain with single gene (phoP) knockout.	All secreted proteins that are affected by PhoP	SC/107 CFU/mouse	H37Rv	IV/2.5 × 105 CFU per mouse	Mouse (BALB/c)	Equal to BCG	-	[180,181]
			SC/5 × 104 CFU/ guinea pig	H37Rv	Aerosol/10–50 CFU or 500 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Better than BCG in high-dose challenge	>1
			ID/5 × 105 CFU/macaques	Erdman	IT/1000 CFU per macaques	Rhesus macaques (Macaca mulatta)	Better than BCG	0.77
Δppe25-pe19	H37Rv strain with 5 genes (ppe25, pe18, ppe26, ppe27, and pe19) knock out.	PPE25, PE18, PPE26, PPE27 and PE19	SC/106 CFU/mouse	H37Rv	Aerosol/100 CFU per mouse	Mouse (C57BL/6)	Better than BCG	~0.5	[169]
ΔsecA2	mc23112 strain with single gene (secA2) knock out.		SC/106 CFU/mouse	Beijing/W (HN878) or Erdman	Aerosol/50–100 CFU per mouse	Mouse (C57BL/6)	Better than BCG	0.72	[182]
			ID/103 CFU/guinea pig	H37Rv	Aerosol/10–30 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Better than BCG in lymph node but not in lungs	-
ΔsecA2ΔlysA	mc23112 strain with double gene (secA2 and lysA) knockout.	Proteins secreted by SecA2 secretion system	SC/106 CFU/mouse	Erdman	Aerosol/50–100 CFU per mouse	Mouse (C57BL/6)	Better than BCG	0.66	[183]
MTBVAC	MT103 strain with double gene (phoP and fadD26) knockout.	All secreted proteins affected by PhoP	SC/5 × 105 CFU/mouse	H37Rv	IN/100 CFU per mouse	Mouse (C57BL/6)	Better than BCG	~0.5	[184,185]
			SC/5 × 103 − 5 × 105 − CFU/guinea pig	H37Rv	Aerosol/10–50 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Equal to BCG	-
			ID/8.2 × 105 CFU/macaques	Erdman	Aerosol/14–30 CFU per macaques	Rhesus macaques (Macaca mulatta)	Better than BCG but not in CFU	-
MTB VAC erp-	MT103 strain with triple gene (phoP, fadD26, and erp) knock out.	All secreted proteins which are affected by PhoP and Erp	ID/105 CFU/mouse	H37Rv	IT/103 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[186]
Δesx-5	H37Rv strain with 17 genes (eccB5, eccc5, cyp143, Rv1786, ppe25, pe18, ppe26, ppe27, pe19, esxM, esxN, ncRv11793, Rv1794, eccD5, mycP5, eccE5, and eccA5) knock out.	ECCB5, ECCC5, CYP143, RV1786, PPE25, PE18, PPE26, PPE27, PE19, ESXM, ESXN, NCRV11793, RV1794, ECCD5, MYCP5, ECCE5, and ECCA5	IM/106 CFU/mouse (2 dose with 6 week interval)	HN878, and H37Rv	Aerosol/40–100 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[170]
			IM/104 CFU/guinea pig	Beijing 212	Aerosol/10–20 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Equal to BCG	-
Δesx-3	H37Rv strain with 11 genes (eccA3, eccB3, eccC3, pe5, ppe4, esxG, esxH, espG3, eccD3, mycP3, and eccE3) knock out	ECCA3, ECCB3, ECCC3, PE5, PPE4, ESXG, ESXH, ESPG3, ECCD3, MYCP3, and ECCE3	IM/104 CFU/guinea pig	Beijing 212	Aerosol/10–20 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Not mentioned	-	[170]

Note: subcutaneous (SC); intradermal (ID); intramuscular (IM); intravenous (IV); intranasal (IN); intratracheal (IT). Efficacy was determined based on lung CFU load in comparison with the wild-type BCG strain at least at one-time point. Log₁₀ CFU was not mentioned for some of the studies for which we stated the approximate values based on bar graphs.

3.15. SecA2

As discussed above, Mtb possesses two secretory transport systems: the primary SecA1 (Rv3241c) and the accessory SecA2 (Rv1821). The latter is predicted to be a unique system in Mtb to transport a small subset of Mtb proteins related to its pathogenicity. Consistent with this prediction, an secA2 deletion mutant in Mtb H37Rv revealed its role in pathogenicity [187]. An secA2 mutant showed diminished SodA expression and relatively attenuated growth in immunocompetent and SCID mice and in mouse macrophages derived from Phox(−/−) and Nos2(−/−) mice [188]. Further, macrophages infected with this strain induced relatively higher levels of TNF-α, IL-2, IFN-γ, and reactive nitrogen intermediates (RNI) than macrophages infected with Mtb H37Rv, suggesting an immunosuppressive role for SecA2 [188].

Interestingly, the secA2 mutant enhanced apoptosis in macrophages and the priming of antigen-specific CD8+ T cells in mice [182], which led to its evaluation as a vaccine in mice and guinea pigs. C57BL/6 mice and guinea pigs immunized with a ΔsecA2 strain and challenged with HN878 and H37Rv showed a better reduction in CFUs against TB compared to BCG [182]. The lung CFUs of ΔsecA2-immunized mice were 0.72 log¹⁰ lower than those lung CFUs of mice vaccinated with BCG. This reduction in CFUs was also accompanied by a decrease in histopathological scores for the lungs in ΔsecA2-immunized mice [182]. Interestingly, it was proposed that ΔsecA2 mutant effects were due to SodA, the only enzyme affected by secA2 deletion at that time. However, current literature reveals that SecA2 is responsible for transporting several host effector proteins released by Mtb, including SapM, Ndk, PknG, LdpC, and others [46]. Thus, secA2 deletion is likely to decrease the expression of multiple proteins.

Nonetheless, an Mtb mutant lacking both SecA2 and LysA proteins showed increased protection against TB in mice compared to BCG [183]. Despite the efficacy of the ΔsecA2/ΔlysA mutant, it has not advanced as a vaccine candidate, which is slightl surprising. In addition, secA2 deletion in BCG had no impact on its immunogenicity [183], suggesting that secA2 may differently affect the genes of Mtb and BCG.

3.16. PhoP

PhoP is a sensor component of the PhoP–PhoR two-component regulatory system and an important virulence factor [189], regulating approximately 2% of the genes in the Mtb genome [190]. The loss of virulence in Mtb H37Ra (avirulent strain) is partly related to a point mutation in the phoP gene (Rv0757) [191]. Although PhoP is not a secretory protein, evidence indicates that it indirectly controls the translocation of the secreted proteins ESAT-6 and CFP-10 of the ESX1 secretion system [192]. It appears that the translocation of ESAT-6 and CFP-10 to the bacterium’s surface requires the products of espACD genes located within the ESX1 system, whose expression is controlled by phoP [193]. Thus, the deletion of the phoP gene could affect the expression of the espACD genes and, consequently, the translocation of ESAT-6 and CFP-10 [194,195]. The absence of phoP results in the secretion of CFP-10 independently of ESAT-6, eliciting immune responses to CFP-10 in both mice and non-human primates upon MTBVAC exposure [184,196]. A ΔphoP vaccine SO2 developed from a clinical strain of Mtb MT103 belongs to this category [180]. The SO2 vaccine was severely attenuated in SCID mice and conferred superior protection against TB compared to BCG in mice, guinea pigs, and non-human primates [180]. Later, to meet the stipulations of the Geneva Consensus [197], the SO2 vaccine was genetically modified as a marker-less double mutant with deletions in phoP and fadD26, and this new construct was named the MTBVAC vaccine [185]. Compared to BCG, MTBVAC showed enhanced safety, increased immunogenicity, and efficacy in animal models such as mice, guinea pigs, and non-human primates [184,185,196,198]. The MTBVAC vaccine protects macaques better than BCG by reducing disease pathology measured by in vivo imaging using CT scans, macroscopic pathological lesions examined at necropsy, and studying the frequency and severity of pulmonary granulomas [184]. The immune signatures after MTBVAC vaccination also included higher levels of Th1 cytokines response, especially poly- (IFN-γ+TNF-α+IL2+) and multi-(IFN-γ+TNF-α+) functional CD4+ T cells. After rigorous preclinical studies, the MTBVAC vaccine entered a clinical trial in Africa [199]. MTBVAC is an example of a vaccine developed through the rational deletion of genes in Mtb for eventual human application.

3.17. Mpt

Two homologous secreted proteins, namely Mpt70 (Rv2875) and Mpt 83 (Rv2873), were found to be highly immunogenic in humans and mice [200]. The genes encoding these proteins are conserved in both Mtb and BCG. A recent study evaluated a mutant in which these two genes and esxS, espC, and espA of the ESX system in BCG create a five-gene knockout ΔBCG-TK [201]. Interestingly, ΔBCG-TK showed vaccine efficacy similar to that of wild-type BCG in mice, and one can propose a contradictory observation that knocking out genes encoding secretory proteins may not affect vaccine efficacy. However, as noted above, with ΔlprG and ΔcpsA mutants, the genetic background of BCG or Mtb used to create a mutant may decide the vaccine’s immunogenicity.

3.18. Erp

Mtb encodes a 28 kDa secretory protein named exported repeated protein (Erp; Rv3810) that contains 11 proline–glycine–leucine–threonine–serine (PGLTS) repeats [202]. Mycobacterial erp mutants are attenuated in macrophages, zebrafish embryos, mice, and leopard frogs [203,204]. Erp is found to interact with another gene called Rv2212, an adenylyl cyclase. Through this interaction, Erp enhances Rv2212-mediated cyclic AMP (cAMP) production, which seems to lower the intracellular survival of Mtb Δerp. We note here that the deletion of the erp gene in the MTBVAC strain (MTBVAC erp (-) further attenuated its growth in SCID mice compared to MTBVAC and BCG. Although MTBVACΔerp generates protection similar to BCG, because of its higher safety profile, MTBVAC erp (-) has been recommended for the vaccination of immune-suppressed populations, such as people with HIV, where BCG causes disseminated disease, also known as BCGosis [186].

3.19. BCG_1419c

BCG_1419c is a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE) protein with phosphodiesterase activity [205]. This protein is encoded by gene BCG_1419c in the BCG Pasteur strain and by the gene Rv1357c in Mtb H37Rv and it was reported to degrade bis-(30–50)-c-di-GMP, which is linked to biofilm formation and virulence [205]. BCG_1419c is not a secretory protein. However, similar to the phoP mutant, which affects the secretion of ESAT-6 and CFP10, the BCG∆BCG1419c mutant vaccine increases the expression of secreted proteins like Tuf, GroEL1, DnaK, and GroES, while showing reduced levels of GroEL2 and AhcY/SahH [206]. Remarkably, the BCGΔBCG1419c vaccine strain demonstrates a better control of both active and chronic TB in murine and guinea pig models compared to saline control [207,208,209,210,211,212,213]. However, it does not provide better protection for animals in terms of reducing lung CFU compared to BCG. Interestingly, in chronic type 2 diabetes (T2D), murine model BCGΔBCG1419c effectively reduces pneumonia in comparison to BCG-vaccinated mice [213].

Table 2

Efficacy of live BCG vaccines with gene(s) deleted for secreted protein(s).

Vaccine Name	Vaccine Components	Secreted Protein(s)Absent	Immunization Route/Dose	Challenge Mtb Strain	Challenge Route/Dose	Animal Model (Strain)	Efficacy in Relation to BCG	log10 CFU/LUNGS Reduction Than BCG	Ref.
VPM1002 ( ΔureC::hly)	BCG Pasteur strain with single gene (ureC) knockout, which expresses listeriolysin (hly).	Urease C	IV/106 CFU/mouse	H37Rv or Beijing/W	Aerosol/30 or 200 CFU per mouse	Mouse (BALB/c)	Better than BCG	~0.5–2	[177]
sapM::T	BCG 1721 strain with single gene (sapM) knockout.	SapM phosphatase	SC/105 CFU/mouse	H37Rv	IV/5 × 104 CFU per mouse(or)IT/ 2 × 105 CFU per mouse	Mouse (BALB/c)	Better than BCG	~0.5 (Luminescence)	[156]
Δzmp1	BCG Pasteur or Denmark strain with single gene (zmp1) knockout.	Zmp1 or Zinc containing metalloprotease 1	SC/5 × 104 CFU/ guinea pig	H37Rv	Aerosol/10–50 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Better than BCG	~0.91	[164]
BCG: Δ85B	BCG Pasteur strain with single gene (fbpB) knockout.	FbpB/Ag85B	SC/5 × 105 CFU/mouse	H37Rv	Aerosol/100 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[118]
ΔnuoG	BCG Pasteur strain with single gene (nuoG) knockout.	NuoGtype-I NADH dehydrogenase subunit G	SC/106 CFU/mouse	H37Rv	Aerosol/100–200 CFU per mouse	Mouse (C57BL/6)	Better than BCG	~0.5	[101]
ΔureC::hly ΔnuoG	BCG Pasteur strain with double gene (ureC, nuoG) knockout, which expresses listeriolysin.	UreC and NuoG	SC/106 CFU/mouse	H37Rv or Beijing/W	Aerosol/100–200 CFU per mouse	Mouse (C57BL/6)	Better than BCG	~0.8–2	[101]
ΔBCG TK (triple knock-out)	BCG Danish strain with five gene (esxS, mpt70, mpt83, espC and espA) knockout	EsxS, Mpt70, Mpt83, EspC, and EspA	SC/5 × 104 CFU/guinea pig	M. bovis AF2122/97	Aerosol/10–20 CFU per guinea pig	Guinea pigs (Dunkin Hartley)	Equal to BCG	-	[201]
ΔBCG2432c	BCG China strain with eis gene (BCG2432c) knockout	EIS or Enhanced Intracellular Survival protein	SC/106 CFU/mouse	H37Rv	IN/100 CFU per mouse	Mouse (C57BL/6)	Better than BCG	~1–2	[168]
ΔBCG3174	BCG China strain with nuoG gene knockout (BCG3174)	NuoG	SC/106 CFU/mouse	H37Rv	Intranasal/100 CFU per mouse	Mouse (C57BL/6)	Equal to BCG	-	[168]
ΔBCG 1419c	BCG Pasteur strain with single gene (c-di-GMP phosphodiesterase) knockout.	All secreted proteins affected by (c-di-GMP phosphodiesterase).	SC/8 × 103 or 2.5 × 102 or 5 × 104 or 106 or ~105 or 107 CFU/mouse	H37Rv or M2 or HN878	IT/2.5 × 105 or 103 or ~170 CFU per mouse or Aerosol/100–200 CFU per mouse.	Mouse (BALB/c, B6D2F1, C57BL/6, I/StSnEgYCit)	Equal to BCG or Better than BCG in chronic infection model	~0.8 (chronic infection model)	[207,208,209,210,211,212,213]
			ID/103 CFU per guinea pig	H37Rv	Aerosol/10–20 CFU per guinea pig	Guinea pigs	Equal to BCG	-

Note: subcutaneous (SC); intradermal (ID); intramuscular (IM); intravenous (IV); intranasal (IN); intratracheal (IT). Efficacy was determined based on lung CFU load in comparison with the wild-type BCG strain at least at one time point. Log₁₀ CFU was not mentioned for some of the studies for which we stated the approximate values based on bar graphs.

4. Status of Mycobacterial Vaccines Deficient in Secreted Protein(s)

It is apparent from these studies that BCG is a ‘natural’ vaccine arising out of the deletion of genes encoding secretory proteins, as it lacks the Region of Difference 1 (RD1) that encodes several secretory proteins, including the immunodominant antigens ESAT-6 and CFP-10 [54]. Studies discussed above specifically targeted genes that played a role during pathogenesis for developing vaccines. Surprisingly, only a few Mtb-derived vaccines showed higher efficacy than BCG, and most were comparable to BCG. In contrast, many BCG mutants with deletions of secretory genes showed increased efficacy compared to the wild type. We recall here that Mtb- or BCG-derived vaccines that showed higher efficacy than BCG had deletions in fpbA, sapM, zmp1, ureC, nuoG, secA2, and eis genes. Intriguingly, except for the fbpA-encoded product, other genes modulated PL fusion, autophagy, apoptosis, and inflammasomes in the antigen-presenting cells (APCs) [34,35,36,61,83,214]. Although the disruption of fbpA in Mtb also led to increased PL fusion in APCs, the underlying mechanism remains unclear [215].

Because PL fusion, autophagy, apoptosis, and inflammasome activation ensure the efficient processing and presentation of vaccine antigens to the T cells by the APCs [216,217,218], it is apparent that the deletion of these genes enhanced the efficacy of the mutants against tuberculosis, justifying the strategy of secretory protein gene knockout. However, we need to recognize the caveat that these deletions of proteins that contain potent T and B cell epitopes like ESAT6 and CFP10 may lead to reduced immunogenicity. In addition, an in vitro phenotype may not always lead to increased immunogenicity. An example is Mtb lacking cpsA; although MtbΔcpsA enhanced autophagy in APCs, an increase in vaccine efficacy was not observed [142]. In this regard, deletion strategies should focus on those that interfere with PL fusion and autophagy (SapM, Zmp, PtpA, and PtpB).

It also appears that gene knockouts in BCG and Mtb will likely have different consequences because of the genetic background of the attenuated vaccine vs. virulent pathogen. Because BCG has an excellent safety record, the deletion of sapM, zmp1, and eis is likely to improve immunogenicity, since BCG contains multiple immunogenic genes, an Antigen85 complex, and others listed above. Intriguingly, eis deletion in BCG markedly reduced the Mtb load in the lungs of nasally challenged mice [168]. Further, the double deletion of ureC and nuoG synergistically enhanced the efficacy of BCG [101], suggesting that highly effective BCG mutants can be produced through multiple deletions of genes selected based on their functions. Herein, we again emphasize the need to exercise caution in selecting the parent platform, since the ΔnuoG BCG Pasteur strain was effective against TB [101] but not the ΔnuoG BCG China sub-strain [168].

Similar to BCG vaccine manipulations, using the Mtb platform to derive vaccines deleting secretory products appears encouraging. The ΔsecA-ΔlysA and ΔfbpA-sapM and mutants were more effective than BCG [116,152,183] and are potential booster vaccines for BCG-vaccinated infants. Among these, the ΔsecA-lysA strain has an excellent safety profile in mice [183] compared to ΔfbpA-sapM, qualifying it for clinical trials. Our unpublished observations indicate that the safety profile of the ΔfbpA-sapM mutant in mice is lower than that of BCG and may need additional gene deletions. Although ΔlprG derived from Mtb also seems to be a strong candidate vaccine, its superior efficacy over BCG is apparent only in TB-susceptible C3HeB/FeJ mice but not in the TB-resistant C57/BL6 mice [134].

5. Future Directions and Conclusions

A significant impediment to developing vaccines against TB is the lack of reliable immune correlates of protection [219,220,221,222]. Studies with mice, guinea pigs, and rabbits revealed that CD4+ T cells secreting IFN-γ, TNF-γ, and IL-1β and CD8+ T cells secreting granulysin and perforin play a critical role in defending Mtb infection [223,224]. Recent studies show that TH17 T cells secreting IL-17 are also a protective parameter [225].

All new TB vaccines aim to enhance T cell-dependent immune responses in the host. Interestingly, the role of innate immunity in designing more efficacious vaccines has received less attention [226]. An intriguing example is the induction of trained immunity by the BCG vaccine, which activates dectin signaling, generating protection against TB through epigenetic modifications of macrophages, neutrophils, and DCs [227]. Although BCG-induced trained immunity seems more effective against nontuberculous infections, it remains unclear to what extent trained immunity affects adaptive immunity during tuberculosis vaccination. It has been reported that BCG-induced protection wanes by year 5 in children, and TB continues to occur despite vaccination [228].

In this context, we noted that deleting multiple secretory proteins led to enhanced autophagy and inflammasome activation that delivered vaccines to lysosomes for better immune responses (our unpublished work). Autophagy and inflammasome pathways are major innate immunity pathways triggered by multiple mechanisms of mycobacteria, including TLR, NLR, and C-type lectin (also known as dectins) signaling. Intriguingly, dectin-dependent trained immunity induced by BCG cell wall components is regulated by autophagy [229]. There is, therefore, a pressing need to investigate whether new-generation tuberculosis vaccines can be genetically manipulated to activate both innate (autophagy and inflammasome) and adaptive T cell-dependent arms of immunity. For example, Mtb was reported to secrete a lysine acetyltransferase that epigenetically modified the ability of macrophages to secrete anti-inflammatory cytokines like IL-10.

Moreover, the acetylation of histones associated with the genes regulating autophagy regulates the induction of autophagy [230]. A lysine acetyltransferase mutant of Mtb may induce robust autophagy and show better protection. Further, Mtb-derived methyl transferases hyermethylate the DNA of tuberculosis patients, reducing their immune responses [231]. We propose that deletion mutants of Mtb that lack acetylase, methylase, or both may serve as promising vaccine candidates.

An attractive alternative approach to enhance innate immunity is to integrate ‘adjuvant’ active molecules such as TLR agonists into candidate vaccines to overcome immune-suppressing proteins. We recently fused a TLR2-activating CFP-10-derived peptide C5 with Ag85B and expressed it in BCG through a plasmid. The recombinant BCG85BC5 vaccine enhanced protection against TB and induced significant levels of T-effector and T-central memory cell response in mice [108]. We propose that integrating adjuvant constructs like this into ΔsapM or Δzmp1 mutants will markedly improve protection associated with long-term memory.

Finally, recent reports indicate that the humoral immune response against Mtb may also play a significant role in protection against TB [101,184]. In this direction, we propose that deletion mutants can be made to express antibody-inducing peptide epitopes.

Author Contributions

S.D. and C.J. conceived the project; R.V., S.S.G., C.J. and S.D. wrote and edited the manuscript; R.V. created the figures. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

All figures in this manuscript were created using www.BioRender.com.

Conflicts of Interest

The authors declare no competing interests.

Footnotes

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