Mycobacterial membrane protein large (MmpL) proteins are a family of VFs of the Mycobacterium genus, responsible for transport of lipids and siderophores to the periplasmic space (Melly and Purdy, 2019). These include complex lipids that are essential for the integrity of the mycobacterial envelope, which forms a barrier of protection from immune cells and chemotherapeutic agents (Bailo et al., 2015). Many of these lipids play prominent roles in modifying host–pathogen interactions, and as a result mutations within MmpL proteins can lead to alterations in mycobacterial virulence (Chalut, 2016).

The MmpL class of transport proteins is overrepresented in M. abscessus, with 31 identified MmpL proteins in the reference strain compared to 14 within M. tb (Deshayes et al., 2010). MmpL4 proteins have an important contribution to M. abscessus virulence through their transport of glycopeptidolipids (GPL) to the outer mycobacterial surface which gives colonies a glossy and smooth appearance on solid agar. Disruption of MmpL4 proteins results in the conversion from the smooth morphotype to a rough cording morphotype, the latter of which is associated with enhanced virulence due to large serpentine cord formation, triggering pro-inflammatory responses and apoptosis (Pawlik et al., 2013). Disruption of M. abscessus MmpL8 reduces the transport of glycosyl-diacylated-nondecyl-diols (GDND), a novel glycolipid that facilitates bacterial adhesion to macrophages and induces macrophage phagosomal rupture, and thereby reduces mycobacterial virulence (Halloum et al., 2016). MmpL proteins from mycobacterial species, such as Mycobacterium smegmatis, also confer resistance to some antibiotics, such as isoniazid, by acting as an efflux pump (Pasca et al., 2005; Viljoen et al., 2017). Mutations within transcriptional regulator tetR, which controls two MmpS-MmpL gene pairs, promote resistance to clofazimine and bedaquiline in M. abscessus (Richard et al., 2019), while MmpS5-MmpL5 are associated with efflux of thiacetazone derivatives (Halloum et al., 2017). Absence of either MmpL-S pair increases susceptibility of intracellular M. abscessus to bedaquiline, indicating the contribution of this protein to providing antibiotic resistance (Gutiérrez et al., 2019). Given the role of MmpL proteins in enhancing or attenuating virulence and conferring antimicrobial resistance, this a particularly noteworthy area of continued research for the development of M. abscessus vaccine candidates.

Members of the Mycobacterium genus possess an outer mycomembrane comprised of components that must be transported across the cell membrane to reach the extracellular space. Early secretory antigenic target (ESAT-6) secretion (ESX) systems, also known as Type VII secretion systems, are important players in facilitating transport of proteins across this outer membrane and are known to play a significant role in mycobacterial virulence, nutrient acquisition, and bacterial conjugation (Lagune et al., 2021). There are five characterized ESX secretion systems spread across the Mycobacterium genus, varying in complexity and in function (Gröschel et al., 2016). However, all ESX systems have a conserved set of genes that include the machinery for substrate secretion, accessory proteins, and ESX secreted proteins (Houben et al., 2014). The most extensively studied ESX system, ESX-1, facilitates M. tb intracellular survival via inhibition of phagosomal acidification and induces phagosome rupture and mycobacterial escape to the cytosol (Sturgill-Koszycki et al., 1994; van der Wel et al., 2007). The contribution of other ESX secretion systems to virulence is less well-characterized; however, the ESX-3 system of M. tb is known to be utilized for iron acquisition in low nutrient environments and the absence of this system attenuates growth in vivo (Siegrist et al., 2009; Tufariello et al., 2016). The role of the ESX-5 secretion system in slow-growing mycobacteria relates to the integrity of the mycobacterial capsule, with disruption of ESX-5 in M. marinum leading to inability of this pathogen to disrupt the cellular membrane (Ates et al., 2016). While attenuation of ESX-5 deficient M. marinum is evident in zebrafish embryos, the same mutant is hypervirulent in adult zebrafish (Weerdenburg et al., 2012). Given that zebrafish embryos solely possess innate immunity, while the adult zebrafish harbors both innate and adaptive immunity, these findings suggest that ESX-5 may play different roles in disease pathogenesis depending on whether innate or adaptive immunity is the major driver of cellular control of mycobacterial infection (Cronan and Tobin, 2014).

There are currently three characterized secretion systems of M. abscessus—ESX-3, ESX-4, and ESX-P. Secretion targets of ESX-3 (EsxG/H proteins) induce pro-inflammatory cytokine production when co-cultured with bone marrow-derived macrophages, and mice infected with ESX-3-deleted-M. abscessus display a reduced inflammatory and granulomatous response coupled with decreased bacterial survival (Kim et al., 2017). However, this work did not elucidate specific mechanisms by which ESX-3 promotes M. abscessus growth in vivo. Furthermore, this study did not include complementation of ESX-3 function in M. abscessus, highlighting the need for further investigation into the role of ESX-3 in M. abscessus virulence. In contrast, the function of ESX-4 within M. abscessus was recently characterized in detail (Laencina et al., 2018). Through generation of an extensive mutagenesis library, Laencina et al. associated disruptions of the ESX-4 locus with a decrease in intramacrophage and amoeba survival. ESX-4 associated ATPase EccB4 was shown to contribute to virulence by limiting phagosomal acidification and facilitating cytosol contact through phagosomal membrane damage. Unlike ESX-3 and ESX-4, ESX-P is a plasmid-borne secretion system unique to a clinical isolate of M. bolletti (Dumas et al., 2016). More recent work has characterized plasmid-borne ESX components within M. abscessus clinical isolates, suggesting a more extensive presence of ESX systems in this species than previously thought (Dedrick et al., 2021). Whether these secretion systems are functional and contribute to M. abscessus virulence is an area of continued research, and may represent an unexplored means by which M. abscessus is able to acquire novel VFs that contribute to evolving pathogenicity.

Lsr2 is a histone-like protein possessed by several members of the Mycobacterium genus, with roles in transcriptional regulation and DNA damage protection. In M. tb, Lsr2 is required for normal growth in anaerobic and DNA damage-inducing conditions (Bartek et al., 2014). When functioning as a transcriptional regulator, the binding of Lsr2 to coding sequences (of which VF genes are common) may contribute to the transition to a latent metabolic state that occurs during chronic M. tb infection (Gordon et al., 2010; Bartek et al., 2014). In M. smegmatis, lsr2-knockout strains have decreased persistence within macrophages, an effect which is reversed following scavenger treatment to sequester free radicals (Colangeli et al., 2009). Mycobacterium smegmatis lsr2 knockouts also display a smooth, glossy phenotype and are defective in biofilm and pellicle formation (Chen et al., 2006). This is reflected in M. abscessus, where rough morphotypes display greater levels of Lsr2 expression than smooth morphotypes. While M. abscessus lsr2 knockouts are not affected in their glycopeptidolipids (GPL) profile, lsr2 knockout in the rough morphotype of M. abscessus show greater susceptibility to reactive oxygen intermediates and display reduced survival within macrophages, as well as in the zebrafish and murine model of infection, particularly at later time points during infection (Le Moigne et al., 2019). Given the importance of this transcriptional regulator for pathogenesis of M. abscessus, Lsr2 may be a promising target for drug development (Liu and Gordon, 2012).

Mycobacterium abscessus grown on solid agar medium displays distinct colony morphologies characterized as smooth (S) and rough (R) morphotypes, each with unique patterns of virulence. These are linked to the variable expression of GPL on the cell surface of M. abscessus, with expression of GPL within S morphotypes resulting in round, glossy colonies, while absence of GPL leads to R morphotypes resulting in a dry, corded appearance (Nessar et al., 2011). The localization of M. abscessus GPL has been shown to cluster to specific nanodomains on the bacterial surface, ultimately modulating surface hydrophobicity and likely influencing bacterial adhesion and virulence (Nessar et al., 2011; Viljoen et al., 2020). The conversion of S to R occur as a result of mutation within genes located within the GPL locus, often associated with GPL synthesis or secretion (Nessar et al., 2011; Pawlik et al., 2013; Park et al., 2015; Li et al., 2020). To date, the mechanisms responsible for S to R conversion are not known; however, this has been hypothesized to occur in response to stress (e.g., Antibiotic stress) which may manifest during host colonization (Bernut et al., 2016b; Gutiérrez et al., 2018). R morphotypes are more frequently isolated from CF patients with chronic infection, and display heightened virulence compared to smooth morphotypes (Jönsson et al., 2007; Kreutzfeldt et al., 2013). The differences in virulence can be linked to different interactions between S and R morphotypes with immune subsets following infection.

As surface molecules on M. abscessus, the absence of GPL on the surface of R morphotypes alters the initial interaction between bacteria and phagocytic cells. A lack of surface GPL exposes immunostimulatory ligands such phosphatidyl-inositol mannoside (PIM) moieties which activate Toll-like receptor (TLR) signaling, resulting in downstream pro-inflammatory cytokine production (Rhoades et al., 2009). While S morphotypes also express PIMs, the heightened expression of surface GPL masks these TLR ligands which acts to prevent innate cell activation (Davidson et al., 2011). Polar GPL themselves also act in an immunosuppressive manner, dampening pro-apoptotic signals induced by R morphotypes and limiting reactive oxygen species (ROS) production (Whang et al., 2017). It is likely that GPL also play an important role in cell–cell interactions, with post-translational modifications of GPL affecting adherence and invasion of M. abscessus to macrophages (Daher et al., 2020). Remodeling of surface GPL also occurs following growth in artificial CF sputum, although the biological significance of this is unknown (Wiersma et al., 2020). Together, these data point to the prominent role of GPL in defining early infection outcomes, with immunologically “silent” establishment of S morphotype M. abscessus in the upper respiratory tract likely facilitating initial colonization of the lung (Gutiérrez et al., 2018).

The S and R morphologies of M. abscessus interact differently with immune cells, leading to different patterns of growth in vivo. While S morphotypes exist largely as single bacilli, R morphotypes form clumps which are difficult for phagocytic cells to engulf (Roux et al., 2016). These aggregates remain adhered to phagocytic cups or, if small enough, are contained within “social” phagosomes containing multiple bacilli. Within the macrophage, R morphotypes appear resistant to lysosomal degradation, leading to extensive and rapid intracellular growth, followed by macrophage apoptosis (Aguilo et al., 2013; Kim et al., 2019). Following apoptosis, rough bacilli are then released into the extracellular space whereby they replicate freely and aggregate to form large extracellular serpentine cords which resist phagocytosis, causing excessive inflammation and abscess formation (Bernut et al., 2014a). Disruption of M. abscessus cording attenuates virulence in vivo, indicating the importance of this phenotype in dictating the outcome of infection (Bernut et al., 2014a; Halloum et al., 2016).

S morphotypes of M. abscessus also resist intracellular degradation, through more direct manipulation of macrophage effector function. Phagocytosed S bacilli often exist in “loner phagosomes,” and act to limit phagosomal acidification and disrupt phagosome membrane integrity, which may facilitate escape to the cytosol (Roux et al., 2016). While cytosol escape and blocking of phagosome acidification are associated with functional ESX-4 found in M. abscessus, it is unclear what role surface GPL may play in facilitating this effector function (Gutiérrez et al., 2018; Laencina et al., 2018). Given the integral link between mycobacterial virulence and macrophage function, understanding the mechanisms by which M. abscessus interacts with innate immune cells is critical in our understanding of M. abscessus virulence (Feng et al., 2020).

The structure of the mycobacterial cell wall and outer envelope is comprised of a complex array of lipoproteins, glycolipids, and glycoproteins, many of which take part in host–pathogen interactions (Jackson, 2014). These may directly modulate immune responses, or improve the structural integrity of the mycomembrane which renders M. abscessus more resistant to immune mediators (Karakousis et al., 2004). For example, succinylation of M. abscessus surface polysaccharides alters intracellular survival by an uncharacterized mechanism, possibly in relation to altered cell surface hydrophobicity and charge (Palčeková et al., 2020). Other VFs may arise from proteins associated with the modification or transport of cell surface molecules. Knockout of pmt, a protein-O-mannosyltransferase which is responsible for glycosylation of lipoproteins in the mycobacterial cell envelope, increases cell wall permeability which may enhance susceptibility to innate cellular defenses, such as ROS (Becker et al., 2017). Similarly, probable N-acetyl transferase Eis2 is thought to play a role in cell wall biogenesis or transport of cell wall components; deletion of this VF increases M. abscessus susceptibility to ROS and H₂O₂ and reduces intracellular survival (Dubois et al., 2019). Expression of some surface components, such as trehalose polyphelates (TPPs), does not directly affect macrophage viability, but promote virulence by facilitating M. abscessus cording (Llorens-Fons et al., 2017). Mycobacterium abscessus cell envelope-derived lipids, such as cardiolipin or I-mannosides, are also effective at neutralizing LL-37, an antimicrobial peptide produced by neutrophils and macrophages (Honda et al., 2015, 2020). It has been established that different growth conditions alters the cell wall composition of M. abscessus; whether this occurs in an infection setting and how this impacts on the outcome of infection remains to be established (Hunt-Serracin et al., 2019).

Phospholipases are virulence determinants found in a diverse range of mycobacterial and non-mycobacterial species, including P. aeruginosa, Staphylococcus aureus, and Listeria monocytogenes (Ghannoum, 2000). Phospholipases promote virulence by allowing escape from the phagosome and cell-to-cell spread, which is thought to be mediated by cleavage of phospholipids that form part of the cell membrane (Camilli et al., 1991; Smith et al., 1995; Ghannoum, 2000). Expression of plc genes by P. aeruginosa is associated with phosphate limiting conditions, and disruption of plcC results in a reduction of P. aeruginosa virulence in the murine model of infection (Ostroff et al., 1989). Similarly, phospholipase C is upregulated by virulent M. tb in phosphate limiting conditions, such as those that may occur in an intracellular environment (Le Chevalier et al., 2015). PlcC isolated from M. abscessus induces macrophage lysis, and plcC knockout strains display reduced survival within amoeba (N’Goma et al., 2015). Given the sequence similarity between plcC from M. abscessus and other CF pathogens (N’Goma et al., 2015), PlcC is a promising antigenic target for vaccine development. CF patients with pulmonary NTM infection or P. aeruginosa infection have higher titers of anti-PlcC antibodies in serum than uninfected individuals, indicating immune exposure to the PlcC antigen following infection (Le Moigne et al., 2015). Vaccination with M. abscessus plcC DNA induces potent antibody responses and a moderate reduction in bacterial burden in ΔF508 mice, which possess the most common CF gene mutation (Cutting, 2015; Le Moigne et al., 2015). However, the role of PlcC in nutrient acquisition in the lung, and if it enables M. abscessus to maintain long-term infection is unclear (Raynaud et al., 2002).

MgtC plays an essential role in the survival of pathogens such Salmonella enterica subsp. typhimurium and CF pathogens B. cenopacea and P. aeruginosa (Alix and Blanc-Potard, 2007; Belon et al., 2015). While the physiological function of this protein has not been fully elucidated, MgtC is a membrane protein that confers survival benefits for both intracellular and extracellular pathogens (Belon et al., 2015). Expression is essential for growth in magnesium poor conditions for P. aeruginosa and B. cenopacea, while upregulation of MgtC following phagocytosis provides enhanced intracellular survival and resistance to low pH for intracellular pathogen Salmonella spp. (Maloney and Valvano, 2006; Rang et al., 2007; Lee and Lee, 2015). MgtC appears to perform a similar role in M. tb, as the protein contributes to improved survival in low magnesium and pH conditions and enhanced virulence in mice (Buchmeier et al., 2000). Mycobacterium abscessus has two sequences encoding proteins homologous to MgtC, MAB_3953, and MAB_0146, both of which bear sufficient sequence similarity to S. typhurmium MgtC, as they can partially restore its function in an mgtC-deficient strain (Le Moigne et al., 2016). Mycobacterium abscessus MgtC is induced in low Mg²⁺ conditions and within the macrophage, mgtC-encoding DNA provides protection against M. abscessus challenge (Le Moigne et al., 2015, 2016). Of note, M. abscessus lacking functional MgtC does not show a significant impairment in macrophage survival, which may indicate a role of this VF in the extracellular stage of M. abscessus growth (Bernut et al., 2014a).

Porins are a diverse class of pore proteins that play an important role in the transport of hydrophilic molecules into the bacterial cell. Porins are expressed widely among gram-positive and negative virulence of different species (Achouak et al., 2001). The contribution of porins to the virulence of different species of pathogenic bacteria is extremely diverse and includes facilitating cell adherence, inducing host cell apoptosis and transporting surface proteins associated with virulence (Müller et al., 1999; Azghani et al., 2002; Brunson et al., 2019). Within the context of mycobacteria, M. smegmatis has a collection of pore-forming proteins that regulate nutrient influx which is important for maintaining normal growth rates (Stephan et al., 2005). Deletion of M. smegmatis porin MspA, MspC, and MspD improves M. smegmatis survival in macrophages, by providing greater resistance of M. smegmatis to host-produced nitric oxide (NO; Fabrino et al., 2009). Similarly, deletion of porins mmpA/B in M. abscessus also improves M. abscessus intracellular survival within phagocytic cells and bacterial persistence in SCID mice (de Moura et al., 2021). Porin knockout strain ΔmmpA displays reduced uptake of glucose, but mutants did not display varied uptake by macrophages, susceptibility to NO, or cell cytotoxicity. Of note, sequential samples taken from different early and late M. abscessus infections in CF patients show mutations within the mmp porin genes (Lewin et al., 2021). However, phylogenetic analysis by de Moura et al. could not establish a contributing role of porin mutations to enhanced virulence or transmission of dominant clinical M. abscessus isolates (de Moura et al., 2021). This suggests that while this class of proteins are significant VFs of other species, porins possessed by mycobacterial species including M. abscessus may perform a role unrelated to virulence.

A thorough understanding of the immune correlates of protection against M. abscessus infection is critical for the development of an effective M. abscessus vaccine. This is an area of ongoing research, primarily focused on the use of in vitro, zebrafish, and murine models (Bernut et al., 2015, 2017; Caverly et al., 2015; Meir et al., 2018). Incorporating these different approaches has led to a greater knowledge of immune mechanisms involved in protection against this pathogen.

As with other members of the Mycobacterium genus, early and robust innate immune responses appear to play a critical role in shaping immune control of M. abscessus infection. Monocytes and macrophages are the most common cell subset infected by M. abscessus in human lung tissue (Ganbat et al., 2016). Both S and R morphotypes of M. abscessus are rapidly phagocytosed by macrophages early after infection and depletion of this subset substantially increases bacterial burden in zebrafish (Bernut et al., 2014a 2019). ROS and NO are major bactericidal mechanisms employed by macrophages to kill M. abscessus, with production of NO and ROS strongly correlated with a protective effect across in vitro and animal models of M. abscessus infection (Kim et al., 2014a; Lee et al., 2017). Interestingly, NO production is enhanced by a type I interferon (TI IFN) response and prophylactic administration of rIFN-Β can promote bacterial clearance (Lee et al., 2017). This is contrary to recent work on M. abscessus suggesting that TI IFNs promote macrophage apoptosis and bacterial spread from cell-to-cell (Zhang et al., 2019). In humans, the role of NO and ROS in providing protection against M. abscessus is less clear. When used as an inhaled therapeutic, NO improves lung function and quality of life in CF patients, but this has not been definitively linked to reduction in M. abscessus burden (Bentur et al., 2020). An oxidative environment within the macrophage also appears to enhance M. abscessus growth in an ex vivo setting (Oberley-Deegan et al., 2010). Moreover, NOS2 knockout (Nos2^−/−) mice do not display exacerbated bacterial burden compared to wild type (Obregón-Henao et al., 2015). Taken together, these findings suggest a redundant or possibly detrimental role of ROS in protection against M. abscessus infection.

As early responders to pulmonary infection, neutrophils are a prominent component of the immune response against M. abscessus. Neutrophil accumulation is driven by TNF and IL-8 production by macrophages and alveolar epithelial cells, and this subset congregates within murine lungs following infection with both S and R morphotypes (Caverly et al., 2015; Bernut et al., 2016a; Malcolm et al., 2018). Despite the integral role of neutrophils in the formation of the granuloma, the efficacy of this subset in reducing bacterial burden remains unclear. Neutrophils phagocytose bacteria and employ neutrophil extracellular traps (NETs) and ROS production to eliminate M. abscessus, but neutrophil-derived ROS and LL-37 has limited killing activity against M. abscessus (Honda et al., 2015; Malcolm et al., 2018). Extracellular DNA released in response to M. abscessus during NETosis may also contribute to biofilm formation in the CF lung (Malcolm et al., 2013). Pulmonary infection with the more virulent R morphotype of M. abscessus is also accompanied by neutrophil accumulation in the bronchoalveolar lavage fluid, to a greater extent than that induced by the S morphotype (Caverly et al., 2015). Given the inflammatory nature of this immune cell subset, excessive neutrophilia can be extremely damaging to lung tissue and is a primary cause of tissue damage in CF (Downey et al., 2009). How exactly this subset contributes to protection or pathology following chronic infection is an area of continued research.

A cornerstone of the immune response to mycobacterial infection is the formation of the granuloma, a cluster of recruited host immune cell subsets which form a physical structure to restrict bacteria (Figure 2A). In zebrafish, M. abscessus granulomas are primarily comprised of neutrophils and macrophages. The accumulation of these subsets is dependent on TNF, which is required for development of the granuloma structure and the containment of bacterial growth (Bernut et al., 2016a). However, granuloma formation is not always sufficient in containing M. abscessus infection—the induction of macrophage cell death by M. abscessus releases extracellular bacteria, which form large serpentine cords that evade phagocytosis and resist immune defenses (Figure 2B; Bernut et al., 2014a; Halloum et al., 2016). Currently, the specific immunological factors differentiating protective granuloma formation from unrestrained bacterial growth are not known (Johansen et al., 2020). Murine models of M. abscessus infection also display granuloma formation in the lungs and spleen after aerosol or intravenous infection, respectively, and studies using TNF knockout (Tnf^−/−) and IFN-γ knockout (Ifngr1^−/− and GKO^−/−) mice highlight the importance of these cytokines for maintenance of granuloma structure (Rottman et al., 2007; Ordway et al., 2008). Nude, severe combined immunodeficiency (SCID) and GMCSF knockout (GMCSF^−/−) mice also develop progressive granulomatous lesions, making these useful models for preclinical drug testing but less suitable for understanding immune factors involved in granuloma formation (Obregón-Henao et al., 2015). While evidence from animal models points to a significant role of this structure in control of M. abscessus infection, the importance of granuloma formation in humans is less clear. Granuloma formation is well documented following cutaneous M. abscessus infection (Bartralot et al., 2000; Kwon et al., 2009), but fewer studies characterize the granulomatous response following pulmonary infection (Jeong et al., 2004; Okazaki et al., 2013). Mycobacterium abscessus results in heterogeneous changes to pulmonary tissue following infection, including inflammatory infiltrate which may be accompanied by the formation of necrotizing or non-necrotizing granulomas (Jeong et al., 2004). Based on what is known about other NTM infections, host immune responses, clinical isolate heterogeneity, and pathogen virulence may all contribute to the extent of granuloma formation and its ability to contain M. abscessus infection (Lammas et al., 2002).

Most relevant for vaccine design is the contribution of adaptive immune subsets to M. abscessus pulmonary infection. CD4⁺ T helper (T_H) cells appear to be a key component of the protective response, with T_H1 responses enabling control of mycobacterial dissemination and granuloma formation through their stimulation of innate immune subsets, such as macrophages and neutrophils (Rottman et al., 2007; Bernut et al., 2016a). In a murine model of M. abscessus infection, bacterial clearance is preceded by an influx of IFN-γ producing CD4⁺ T cells, and it has been shown that M. abscessus infection persists in IFN-γ knockout GKO^−/− mice (Ordway et al., 2008; Obregón-Henao et al., 2015). High-dose infection models in GKO^−/− mice display a heightened T_H2 response resulting in an immunosuppressive phenotype, supporting the notion that T_H1 polarization is ideal for protection against intracellular mycobacteria (Ordway et al., 2008). Similarly, individuals with deficiencies affecting T-cell function appear predisposed to M. abscessus infection (Lutzky et al., 2018). Patients with active M. abscessus infection also display a dampened T_H1/T_H2 and heightened T_H17 cytokine profile compared to healthy controls, suggestive of a link between T_H1 polarization and a protective phenotype (Kim et al., 2014b; Lake et al., 2016). This is an especially important consideration in the design of subunit M. abscessus vaccines, where adjuvant choice affects T-cell polarization and thus resulting protection (Knudsen et al., 2016).

While robust T-cell responses are clearly essential for protection against M. abscessus, the contribution of the humoral response to protection against pulmonary M. abscessus infection is less established. Antibodies to M. abscessus are generated following infection or vaccination (Jeon et al., 2009; Le Moigne et al., 2015) and B-cell deficiency in the murine model of infection promotes bacterial growth (Rottman et al., 2007). While humoral responses in healthy individuals are considered a critical components for extracellular pathogens, such as P. aeruginosa, their efficacy appears limited in preventing bacterial colonization or eradicating infection in CF patients (Yonker et al., 2015; Sainz-Mejías et al., 2020). Humoral responses that target surface GPL of M. abscessus S morphotypes may also encourage the conversion to the more virulent R morphotype, thus proving detrimental to the host (Gutiérrez et al., 2018). It is also unclear whether the extracellular stages of M. abscessus growth, such as biofilm formation during early infection, and extracellular cording following phagosome rupture would be effectively disrupted by neutralizing antibodies. Most vaccines currently approved for human use focus on the effective development of humoral responses, and development of vaccines targeting cell-mediated immunity appear more difficult to produce. As such, the contribution of this subset to M. abscessus protection will require continued investigation to inform development of an effective M. abscessus vaccine.

Mycobacterium abscessus infection primarily occurs in individuals with reduced pulmonary immune responses, including those with CF, COPD, and non-CF bronchiectasis (Ratnatunga et al., 2020). The development of novel immunization strategies for this pathogen has the potential to dramatically reduce the M. abscessus burden and improve quality of life for these susceptible populations. However, progress toward novel vaccines has been limited by an incomplete understanding of immune responses in these groups that perpetuate M. abscessus infection, and how this may impact the outcome of vaccination. While populations with COPD and non-CF bronchiectasis make up a significant proportion of M. abscessus cases, factors resulting in enhanced susceptibility of these populations to NTM infection have not yet been established (Griffith et al., 2007; Ratnatunga et al., 2020). It is speculated that structural damage within the lung in non-CF bronchiectasis permits bacterial colonization, but immune dysfunction, such as aberrant neutrophil migration and effector function, also perpetuates infection by preventing optimal clearance (Chalmers and Hill, 2013). Populations susceptible to NTM infection with no clear risk factors display reduced IFN in serum and altered adipokine levels, further suggesting an underlying link between immune responses and risk of infection (Kartalija et al., 2013).

Given the frequency with which CF populations become infected with NTM, the consideration of immune responses in CF populations is of utmost importance when developing a vaccine for M. abscessus. As research into the area continues, we are beginning to appreciate the profound impact of CFTR mutations within CF patients on immune cell functionality, and the implications of this for pulmonary bacterial clearance (Hartl et al., 2012). The immune landscape of CF patients includes macrophages with a skewed hyperinflammatory profile (Bruscia and Bonfield, 2016), neutrophils with dysfunctional phagosomal maturation and effector functions (Zhou et al., 2013; Gifford and Chalmers, 2014), and an inflammatory milieu that may limit the ability of innate cells to clear infection (Roghanian et al., 2006; Cockx et al., 2018). Recent work by Bernut et al. also showed impaired ROS production in professional phagocytic cells following M. abscessus infection, using a zebrafish model of CF (Bernut et al., 2019). While less is known about the implications of CFTR mutations on adaptive immune responses, lymphocytes from murine models of CF also display a predilection to the development of T_H2 and T_H17 responses (Mueller et al., 2011; Tiringer et al., 2013; Mulcahy et al., 2015). Cftr^−/− mice also display dysregulated B-cell follicle formation, activation, and accumulation in the lung (Polverino et al., 2019). This adds to the complexity of vaccine development for M. abscessus, as immune responses may be harder to predict in target populations and could potentially be naturally skewed toward non-protective phenotypes (Kartalija et al., 2013). The quality of immune responses within these populations will play a significant role in shaping the outcome of vaccination with an M. abscessus vaccine and is therefore a key area of continued research.

Research of potential therapeutic agents to protect against M. abscessus infection may greatly benefit from effective animal models that recapitulate the clinical presentation of M. abscessus lung disease. This has been a continual challenge for researchers, as animal genotypes that possess common CF mutations do not display the same pathological symptoms as humans (Guilbault et al., 2007). For example, the ΔF508 mouse model possesses the most common mutation in the CFTR gene in humans but does not recapitulate the disease pathophysiology (Scholte et al., 2004). Although some knockout strains show greater persistence of bacterial load compared to wild-type littermates, none of these models develop spontaneous colonization or persistent infection by CF pathogens in the lung, such as in human patients (McMorran et al., 2001; Guilbault et al., 2005, 2007; Ordway et al., 2008; Bernut et al., 2014b). This problem has been circumvented by the use of immunocompromised strains (Lerat et al., 2014), corticosteroid administration (Maggioncalda et al., 2020), or infection with thrombin and fibrinogen plugs; a strategy similar to the agarose bead infection model used for P. aeruginosa (Facchini et al., 2014; Caverly et al., 2015). In addition to zebrafish and A. castellanni models, these have provided us with snapshots of the immune response to M. abscessus infection; however, an in-depth understanding of correlates of protection is best achieved with immune competent models which recapitulate the immune response to M. abscessus infection in humans.

The discovery of VF acquisition by M. abscessus from other CF pathogens highlights the continued evolutionary potential of this pathogen and reinforces the urgent need for vaccines to provide robust protection to susceptible groups. Suggestions that the CF lung provides an environment that may enhance M. abscessus virulence is particularly worrisome; however, shared VFs across different bacterial species could allow the development of protein vaccine candidates that target multiple CF pathogens (Ripoll et al., 2009; Bryant et al., 2021). For instance, PlcC antibodies from CF patients infected with P. aeruginosa are cross-reactive with M. abscessus PlcC (Le Moigne et al., 2015). Cross-protective vaccines are clearly advantageous in terms of research and development costs; however, no M. abscessus vaccine study to date has confirmed a protective effect of cross-reactive immune responses induced by vaccination (Le Moigne et al., 2016). Given the number of VFs acquired from non-mycobacterial species in the M. abscessus genome, this is a noteworthy area of continued research (Ripoll et al., 2009).

In addition to the development of vaccines for CF pathogens, there is interest in development of an M. abscessus vaccine to provide protection against different mycobacterial species. Lower incidence rates of Mycobacterium avium infection in HIV-positive individuals with prior M. tb infection suggest a degree of protection afforded by mycobacterial exposure (Horsburgh et al., 1996). This has encouraged interest in repurposing the current M. tb vaccine, Mycobacterium bovis Bacille Calmette–Guérin (BCG), to be used for protection against NTM infection in susceptible populations (World Health Organization, 2020). While lower incidences of NTM infection in BCG-vaccinated children support the notion of BCG vaccine-induced cross-protection, there is a paucity of information in the literature specifically referring to protection against M. abscessus (Trnka et al., 1994; Zimmermann et al., 2018). T cells isolated from peripheral blood of latent-TB-infected or BCG-vaccinated individuals produce IFN-γ and granzyme A in response to M. avium restimulation, and these cross-reactive T cells restrict growth of M. avium or M. abscessus in monocytes. BCG vaccination of mice also generates a population of T cells that secrete cytokines upon NTM restimulation; however, the effect of vaccination on bacterial burden was not determined in this study (Abate et al., 2019). As there are clear advantages of repurposing a currently approved and widely distributed vaccine, the role of BCG vaccination in providing protection against M. abscessus infection requires further investigation.

Despite being heavily researched since its discovery, M. tb is still a leading cause of death by infectious disease worldwide. In 2020, a quarter of the world’s population was estimated to be infected with M. tb with 10 million new infections in 2019 alone (Chakaya et al., 2021). The protection afforded by the current vaccine available, BCG, is extremely variable across different age groups, latitudes, and to those with previous infection or mycobacterial exposure (Mangtani et al., 2014; Ahmed et al., 2021). While numerous candidates for novel TB vaccines are currently in the developmental pipeline, none have been able to provide sufficient protection to replace BCG. However, we can learn from decades of TB research and vaccine development about factors to consider in the pursuit of an M. abscessus vaccine, in addition to identifying unique challenges specific to this pathogen.

A robust understanding of immune responses required for protection is essential in the development of novel M. abscessus vaccines. In the case of vaccines against M. tb, efforts have been consistently stalled by our incomplete knowledge of immune correlates of protection against M. tb (Counoupas et al., 2019). This was exemplified in the results of the Modified Vaccinia Ankara 85A (MVA85A) vaccine in Phase IIb clinical trials, which had been a leading candidate to replace BCG. While earlier studies had shown strong T-cell-mediated immune responses following vaccination, this vaccine did not show protective efficacy in infants (Scriba et al., 2010; Tameris et al., 2013). T_H1 helper subsets and cytokines IFN-γ and TNF also appear to be important for protection against M. abscessus, but whether these subsets are predictive of protection afforded by vaccination has not yet been established (Rottman et al., 2007; Bernut et al., 2016a). The difficulty in determining immune correlates of protection against M. abscessus is exacerbated by the diverse growth stages of this microorganism, with extracellular biofilm formation and colonization preceding the emergence of invasive intracellular variants which form chronic infection in the lung (Howard et al., 2006; Gutiérrez et al., 2018; Bryant et al., 2021). There is currently a paucity of information on how different branches of the immune response (namely, humoral and cell-mediated subsets) contribute to effective bacterial clearance across different infection stages. Diverse vaccination strategies can be aimed at preventing colonization by inducing a strong humoral response or at enhancing clearance of persistent infection by inducing a strong cell-mediated immune response. As with M. tb infection, it is also unclear what impact natural immunity from prior M. abscessus infection has on the risk of reinfection (Verver et al., 2005; McIvor et al., 2017). Recurrence of M. abscessus infection has been attributed to poor antimicrobial efficacy, particularly the inefficacy of macrolide treatments to fully eradicate infection (Pasipanodya et al., 2017). However, it has been noted that reinfection with NTM, such as M. abscessus, frequently occurs with strains of different genotype, suggesting that M. abscessus antigenic variability has an impact on protective immune responses (Koh et al., 2017). A comprehensive understanding of the importance of natural immunity over the duration of M. abscessus infection will be critical to dictate the trajectory of novel vaccine development.

As the field of vaccinology has advanced, so too has the use of bioinformatics approaches to aid vaccination efforts. Increases in genomic sequencing of pathogenic species has given rise to the field of “reverse vaccinology” whereby potential VFs and antigenic targets are discovered through in silico analysis of a pathogen’s genome and predicted proteome (Rappuoli, 2000). This method has been relatively underutilized for M. tb, likely due to the wealth of in vitro knowledge that has emerged from decades of research. A pathogen, such as M. abscessus, is an excellent candidate for reverse vaccinology approaches to streamline the identification of potential vaccine candidates, given the continual and growing need for a vaccine against this pathogen. There are numerous studies using pangenome analysis to identify shared VFs across clinical isolates of M. abscessus, and this is an ideal starting point for a reverse vaccinology approach (Choo et al., 2014; Davidson et al., 2014; Bryant et al., 2021; Lewin et al., 2021). There is also an extensive range of protein pathway mapping, MHC-II binding prediction, and epitope mapping software currently available which provides greater insight into those proteins likely to yield immunogenic vaccine candidates (Heinson et al., 2015). The reverse vaccinology approach has most recently been used to identify potential VFs and therapeutic targets of M. abscessus by development of a novel hierarchical approach; the development of such workflows is beneficial because they may be applied to other pathogens in future (Dar et al., 2021). There is also enormous advantage in a synergistic approach using wet-lab based and in silico techniques as a starting point for vaccine candidate identification, as this is based upon the confirmed proteome or secretome of different species. This was recently used by Steindor et al. to identify immunogenic proteins across different clinical isolates of M. abscessus and yielded many potential VFs that could be suitable vaccine targets (Steindor et al., 2019). However, current studies on M. abscessus reverse vaccinology lack experimental verification of protein immunogenicity; this will be a critical area of future research to determine the best candidates for progression. As we continue to build on our knowledge of this emerging pathogen, reverse vaccinology will become an essential tool in antigenic discovery for the development of subunit vaccines.