BCG-booster vaccination with HSP90-ESAT-6-HspX-RipA multivalent subunit vaccine confers durable protection against hypervirulent Mtb in mice.

The quest for effective and enhanced multiantigenic tuberculosis (TB) subunit vaccine necessitates the induction of a protective pathogen-specific immune response while circumventing detrimental inflammation within the lung milieu. In line with this goal, we engineered a modified iteration of the quadrivalent vaccine, namely HSP90-ESAT-6-HspX-RipA (HEHR), which was coupled with the TLR4 adjuvant, CIA09A. The ensuing formulation was subjected to comprehensive assessment to gauge its protective efficacy against the hypervirulent Mycobacterium tuberculosis (Mtb) Haarlem clinical strain M2, following a BCG-prime boost regimen. Regardless of vaccination route, both intramuscular and subcutaneous administration with the HEHR vaccine exhibited remarkable protective efficacy in significantly reducing the Mtb bacterial burden and pulmonary inflammation. This underscores its notably superior protective potential compared to the BCG vaccine alone or a former prototype, the HSP90-E6 subunit vaccine. In addition, this superior protective efficacy was confirmed when testing a tag-free version of the HEHR vaccine. Furthermore, the protective immune determinant, represented by durable antigen-specific CD4+IFN-γ+IL-17A+ T-cells expressing a CXCR3+KLRG1- cell surface phenotype in the lung, was robustly induced in HEHR-boosted mice at 12 weeks post-challenge. Collectively, our data suggest that the BCG-prime HEHR boost vaccine regimen conferred improved and long-term protection against hypervirulent Mtb strain with robust antigen-specific Th1/Th17 responses.

Immunogenicity and protective efficacy of RipA, a peptidoglycan hydrolase, against Mycobacterium tuberculosis Beijing outbreak strains.

Given that individuals with latent tuberculosis (TB) infection represent the major reservoir of TB infection, latency-associated antigens may be promising options for development of improved multi-antigenic TB subunit vaccine. Thus, we selected RipA, a peptidoglycan hydrolase required for efficient cell division of Mycobacterium tuberculosis (Mtb), as vaccine candidate. We found that RipA elicited activation of dendritic cells (DCs) by induction of phenotypic maturation, increased production of inflammatory cytokines, and prompt stimulation of MAPK and NF-κB signaling pathways. In addition, RipA-treated DCs promoted Th1-polarzied immune responses of naïve CD4+ T cells with increased proliferation and activated T cells from Mtb-infected mice, which conferred enhanced control of mycobacterial growth inside macrophages. Moreover, mice immunized with RipA formulated in GLA-SE adjuvant displayed remarkable generation of Ag-specific polyfunctional CD4+ T cells in both lung and spleen. Following an either conventional or ultra-low dose aerosol challenges with 2 Mtb Beijing clinical strains, RipA/GLA-SE-immunization was not inferior to BCG by mediating protection as single Ag. Collectively, our findings highlighted that RipA could be a novel candidate as a component of multi-antigenic TB subunit vaccines.

Bridging the gaps to overcome major hurdles in the development of next-generation tuberculosis vaccines.

Although tuberculosis (TB) remains one of the leading causes of death from an infectious disease worldwide, the development of vaccines more effective than bacille Calmette-Guérin (BCG), the only licensed TB vaccine, has progressed slowly even in the context of the tremendous global impact of TB. Most vaccine candidates have been developed to strongly induce interferon-γ (IFN-γ)-producing T-helper type 1 (Th1) cell responses; however, accumulating evidence has suggested that other immune factors are required for optimal protection against Mycobacterium tuberculosis (Mtb) infection. In this review, we briefly describe the five hurdles that must be overcome to develop more effective TB vaccines, including those with various purposes and tested in recent promising clinical trials. In addition, we discuss the current knowledge gaps between preclinical experiments and clinical studies regarding peripheral versus tissue-specific immune responses, different underlying conditions of individuals, and newly emerging immune correlates of protection. Moreover, we propose how recently discovered TB risk or susceptibility factors can be better utilized as novel biomarkers for the evaluation of vaccine-induced protection to suggest more practical ways to develop advanced TB vaccines. Vaccines are the most effective tools for reducing mortality and morbidity from infectious diseases, and more advanced technologies and a greater understanding of host-pathogen interactions will provide feasibility and rationale for novel vaccine design and development.

A Structural View at Vaccine Development against M. tuberculosis.

Tuberculosis (TB) is still the leading global cause of death from an infectious bacterial agent. Limiting tuberculosis epidemic spread is therefore an urgent global public health priority. As stated by the WHO, to stop the spread of the disease we need a new vaccine, with better coverage than the current Mycobacterium bovis BCG vaccine. This vaccine was first used in 1921 and, since then, there are still no new licensed tuberculosis vaccines. However, there is extremely active research in the field, with a steep acceleration in the past decades, due to the advance of technologies and more rational vaccine design strategies. This review aims to gather latest updates in vaccine development in the various clinical phases and to underline the contribution of Structural Vaccinology (SV) to the development of safer and effective antigens. In particular, SV and the development of vaccine adjuvants is making the use of subunit vaccines, which are the safest albeit the less antigenic ones, an achievable goal. Indeed, subunit vaccines overcome safety concerns but need to be rationally re-engineered to enhance their immunostimulating effects. The larger availability of antigen structural information as well as a better understanding of the complex host immune response to TB infection is a strong premise for a further acceleration of TB vaccine development.

Structure based design of effective HtpG-derived vaccine antigens against M. tuberculosis.

Vaccine development against Tuberculosis is a strong need, given the low efficacy of the sole vaccine hitherto used, the Bacillus Calmette-Guérin (BCG) vaccine. The chaperone-like protein HtpGMtb of M. tuberculosis is a large dimeric and multi-domain protein with promising antigenic properties. We here used biophysical and biochemical studies to improve our understanding of the structural basis of HtpGMtb functional role and immunogenicity, a precious information to engineer improved antigens. We showed that HtpGMtb is a dimeric nucleotide-binding protein and identified the dimerisation interface on the C-terminal domain of the protein. We also showed that the most immunoreactive regions of the molecule are located on the C-terminal and middle domains of the protein, whereas no role is played by the catalytic N-terminal domain in the elicitation of the immune response. Based on these observations, we experimentally validated our predictions in mice, using a plethora of immunological assays. As an outcome, we designed vaccine antigens with enhanced biophysical properties and ease of production, albeit conserved or enhanced antigenic properties. Our results prove the efficacy of structural vaccinology approaches in improving our understanding of the structural basis of immunogenicity, a precious information to engineer more stable, homogeneous, efficiently produced, and effective vaccine antigens.

A Dendritic Cell-Activating Rv1876 Protein Elicits Mycobacterium Bovis BCG-Prime Effect via Th1-Immune Response.

The widely administered tuberculosis (TB) vaccine, Bacillus Calmette-Guerin (BCG), is the only licensed vaccine, but has highly variable efficiency against childhood and pulmonary TB. Therefore, the BCG prime-boost strategy is a rational solution for the development of new TB vaccines. Studies have shown that Mycobacterium tuberculosis (Mtb) culture filtrates contain proteins that have promising vaccine potential. In this study, Rv1876 bacterioferritin was identified from the culture filtrate fraction with strong immunoreactivity. Its immunobiological potential has not been reported previously. We found that recombinant Rv1876 protein induced dendritic cells' (DCs) maturation by MAPK and NF-κB signaling activation, induced a T helper type 1 cell-immune response, and expanded the population of the effector/memory T cell. Boosting BCG with Rv1876 protein enhanced the BCG-primed Th1 immune response and reduced the bacterial load in the lung compared to those of BCG alone. Thus, Rv1876 is a good target for the prime-boost strategy.

Mycobacterium tuberculosis RpfE-Induced Prostaglandin E2 in Dendritic Cells Induces Th1/Th17 Cell Differentiation.

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1ß was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling.

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

Recombinant Rv1654 protein of Mycobacterium tuberculosis induces mitochondria-mediated apoptosis in macrophage.

Mycobacterium tuberculosis contains diverse immunologically active components. This study investigated the biological function of a newly identified component, Rv1654, with the potential to induce apoptosis in macrophages. Recombinant Rv1654 induced macrophage apoptosis in a caspase-9/3-dependent manner through the production of reactive oxygen species (ROS) and interaction with Toll-like receptor 4. In addition, Rv1654 induced the production of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 through the mitogen-activated protein kinase pathway. Furthermore, Rv1654-induced c-Jun N-terminal kinase (JNK) activation was inhibited by the ROS scavenger and Rv1654-induced apoptosis was inhibited by the JNK inhibitor. Moreover, it was found that treatment of macrophages with Rv1654 led to the loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and translocation of Bax into the mitochondria. Finally, Rv1654-mediated apoptosis was inhibited in macrophages transfected with Bax siRNA. These results suggest that Rv1654 induces macrophage apoptosis through a mitochondrial-dependent pathway and ROS-mediated JNK activation.

Fusion of Dendritic Cells Activating Rv2299c Protein Enhances the Protective Immunity of Ag85B-ESAT6 Vaccine Candidate against Tuberculosis.

In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity. In this study, to prove that Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens, Ag85B-ESAT6, a well-known vaccine candidate, was selected as a fusion partner of Rv2299c. Recombinant Rv2299c-Ag85B-ESAT6 protein induced DC maturation and activation. Furthermore, fusion of Rv2299c enhanced the protective efficacy of the Ag85B-ESAT6 vaccine in a mouse model and significantly higher production of TNF-α and IL-2 was detected in the lungs, spleen, and lymph nodes of the group immunized with the Rv2299c-fused protein than with Ag85B-ESAT6. In addition, fusion of Rv2299c enhanced the Ag85B-ESAT6-mediated expansion of multifunctional CD4+ T cells. These data suggested that the DC-activating protein Rv2299c may potentiate the protective immunity of the vaccine candidate comprising T cell antigens.

Mycobacterium tuberculosis Rv2005c Induces Dendritic Cell Maturation and Th1 Responses and Exhibits Immunotherapeutic Activity by Fusion with the Rv2882c Protein.

Immunotherapy represents a promising approach for improving current antibiotic treatments through the engagement of the host's immune system. Latency-associated antigens have been included as components of multistage subunit tuberculosis vaccines. We first identified Rv2005c, a DosR regulon-encoded protein, as a seroreactive protein. In this study, we found that Rv2005c induced dendritic cell (DC) maturation and Th1 responses, and its expression by Mycobacterium tuberculosis (Mtb) within macrophages was enhanced by treatment with CoCl2, a hypoxia-mimetic agent. T cells activated by Rv2005c-matured DCs induced antimycobacterial activity in macrophages under hypoxic conditions but not under normoxic conditions. However, Rv2005c alone did not exhibit any significant vaccine efficacy in our mouse model. The fusion of Rv2005c to the macrophage-activating protein Rv2882c resulted in significant activation of DCs and antimycobacterial activity in macrophages, which were enhanced under hypoxic conditions. Furthermore, the Rv2882c-Rv2005c fusion protein showed significant adjunctive immunotherapeutic effects and led to the generation of long-lasting, antigen-specific, multifunctional CD4+ T cells that coproduced TNF-α, IFN-γ and IL-2 in the lungs of our established mouse model. Overall, these results provide a novel fusion protein with immunotherapeutic potential as adjunctive chemotherapy for tuberculosis.

Recombinant Rv3261 protein of Mycobacterium tuberculosis induces apoptosis through a mitochondrion-dependent pathway in macrophages and inhibits intracellular bacterial growth.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.

Antigen-Specific IFN-γ/IL-17-Co-Producing CD4+ T-Cells Are the Determinants for Protective Efficacy of Tuberculosis Subunit Vaccine.

The antigen-specific Th17 responses in the lungs for improved immunity against Mycobacterium tuberculosis (Mtb) infection are incompletely understood. Tuberculosis (TB) vaccine candidate HSP90-ESAT-6 (E6), given as a Bacillus Calmette-Guérin (BCG)-prime boost regimen, confers superior long-term protection against the hypervirulent Mtb HN878 infection, compared to BCG or BCG-E6. Taking advantage of protective efficacy lead-out, we found that ESAT-6-specific multifunctional CD4+IFN-γ+IL-17+ T-cells optimally correlated with protection level against Mtb infection both pre-and post-challenge. Macrophages treated with the supernatant of re-stimulated lung cells from HSP90-E6-immunised mice significantly restricted Mtb growth, and this phenomenon was abrogated by neutralising anti-IFN-γ and/or anti-IL-17 antibodies. We identified a previously unrecognised role for IFN-γ/IL-17 synergism in linking anti-mycobacterial phagosomal activity to enhance host control against Mtb infection. The implications of our findings highlight the fundamental rationale for why and how Th17 responses are essential in the control of Mtb, and for the development of novel anti-TB subunit vaccines.

Structural features of HtpGMtb and HtpG-ESAT6Mtb vaccine antigens against tuberculosis: Molecular determinants of antigenic synergy and cytotoxicity modulation.

Vaccine development against tuberculosis is an urgent need as the only available vaccine, M. bovis Bacillus Calmette Guerin (BCG), is unable to provide significant protection in adults. Among newly identified antigens, Rv2299c is an excellent candidate for the rational design of an effective multi-antigenic TB vaccine. Also, when fused to the T cell antigen ESAT6, it becomes highly effective in boosting BCG immunization and it adopts low cytotoxicity compared to ESAT6. We here characterize these proteins by coupling various biophysical techniques to cytofluorimetry and computational studies. Altogether, our data provide an experimental evidence of the role of Rv2299c as a dimeric and highly thermostable molecular chaperone, here denoted as HtpGMtb. Molecular dynamics simulations show that ATP rigidly anchors the ATP-binding loop in a conformation incompatible with the structure of the free enzyme. We also show that HtpGMtb dimeric state is an important molecular feature for the improved antigenic and cytotoxic properties of HtpG-ESAT6Mtb. Indeed, structural features of HtpG-ESAT6Mtb show that not only does this molecule combine the antigenic properties of HtpGMtb and ESAT6, but HtpGMtb locks ESAT6 in a dimeric state, thus improving its cytotoxicity properties. The data presented here provide solid basis for the rational design of upgraded antigens.

Cell wall skeleton of Mycobacterium bovis BCG enhances the vaccine potential of antigen 85B against tuberculosis by inducing Th1 and Th17 responses.

A safe and effective adjuvant is necessary to induce reliable protective efficacy of the protein-based vaccines against tuberculosis (TB). Mycobacterial components, such as synthetic cord factor and arabinogalactan, have been used as one of the adjuvant components. Mycobacterium bovis bacillus Calmette- Guérin cell-wall skeleton (BCG-CWS) has been used as an effective immune-stimulator. However, it is not proven whether BCG-CWS can be an effective adjuvant for the subunit protein vaccine of TB. In this study, we demonstrated that the BCG-CWS effectively coupled with Ag85B and enhanced the conjugated Ag85B activity on the maturation of dendritic cells (DCs). Ag85B-BCG-CWS-matured DCs induced significant Th1 and Th17 responses when compared to BCG-CWS or Ag85B alone. In addition, significant Ag85B-specific Th1 and Th17 responses were induced in Ag85B-BCG-CWS-immunized mice before infection with M. tuberculosis and maintained after infection. Moreover, Ag85B-BCG-CWS showed significant protective effect comparable to live BCG at 6 weeks after infection and maintained its protective efficacy at 32 weeks post-challenge, whereas live BCG did not. These results suggest that the BCG-CWS may be an effective adjuvant candidate for a protein-based vaccine against TB.

Mycobacterium tuberculosis Rv3463 induces mycobactericidal activity in macrophages by enhancing phagolysosomal fusion and exhibits therapeutic potential.

Macrophages are responsible for innate and adaptive immune response activation necessary for eliminating infections. Optimal activation of macrophages to phagocytize Mycobacterium tuberculosis is critical in anti-mycobacterial defense. Here, we identified a novel Rv3463 hypothetical protein that induces macrophage activation in Mtb culture filtrate. Recombinant Rv3463 activated mouse bone marrow-derived macrophages to induce the expression of surface molecules and secrete pro-inflammatory cytokines via the TLR2 and TLR4 pathways. Mitogen activated protein kinase, phospatidylinositol-4,5-bisphosphate 3-kinases, and the NF-κB signaling pathways are involved in Rv3463-mediated macrophage activation. Furthermore, Rv3463 induced bactericidal effects in Mtb-infected macrophages through phagosome maturation and phagolysosomal fusion enhanced by phospatidylinositol-4,5-bisphosphate 3-kinases and Ca2+ signaling pathways and exhibited therapeutic effects in a short-term Mtb-infection mouse model. Overexpression of Rv3463 in M. smegmatis caused rapid clearance of bacteria in macrophages and mice. Our study suggests that Rv3463 is a promising target for the development of post-exposure tuberculosis vaccines or adjunct immune-therapy.

Mycobacterium tuberculosis Protein Rv3841 Activates Dendritic Cells and Contributes to a T Helper 1 Immune Response.

The attenuated vaccine Mycobacterium bovis BCG (Bacille Calmette Guerin) has limited protective efficacy against TB. The development of more effective TB vaccines has focused on the mycobacterial antigens that cause strong T helper 1 (Th1) responses. Mtb protein Rv3841 (bacterioferritin B; BfrB) is known to play a crucial role in the growth of Mtb. Nonetheless, it is unclear whether Rv3841 can induce protective immunity against Mtb. Here, we studied the action of Rv3841 in maturation of dendritic cells (DCs) and its engagement in the development of T-cell immunity. We found that Rv3841 functionally activated DCs by upregulating costimulatory molecules and increased secretion of proinflammatory cytokines. Activation of DCs by Rv3841 was mediated by Toll-like receptor 4 (TLR4), followed by triggering of mitogen-activated protein kinase and nuclear factor-κB signaling pathways. In addition, Rv3841-matured DCs effectively proliferated and polarized Th1 immune response of naïve CD4+ and CD8+ T-cells. Moreover, Rv3841 specifically caused the expansion of CD4+CD44highCD62Llow T-cells from Mtb-infected mice; besides, the T-cells activated by Rv3841-matured DCs inhibited intracellular mycobacterial growth. Our data suggest that Rv3841 induces DC maturation and protective immune responses, a finding that may provide candidate of effective TB vaccines.

Mycobacterium tuberculosis protein Rv2220 induces maturation and activation of dendritic cells.

Tuberculosis remains a serious health problem worldwide. Characterization of the dendritic cell (DC)-activating mycobacterial proteins has driven the development of effective TB vaccine candidates besides improving the understanding of immune responses. Some studies have emphasized the essential role of protein Rv2220 from M. tuberculosis in mycobacterial growth. Nonetheless, little is known about cellular immune responses to Rv2220. In this study, our aim was to test whether protein Rv2220 induces maturation and activation of DCs. Rv2220-activated DCs appeared to be in a mature state with elevated expression of relevant surface molecules and proinflammatory cytokines. DC maturation caused by Rv2220 was mediated by MAPK and NF-κB signaling pathways. Specifically, Rv2220-matured DCs induced the expansion of memory CD62LlowCD44highCD4+ T cells in the spleen of mycobacteria-infected mice. Our results suggest that Rv2220 regulates host immune responses through maturation of DCs, a finding that points to a new vaccine candidate against tuberculosis.

Rv2299c, a novel dendritic cell-activating antigen of Mycobacterium tuberculosis, fused-ESAT-6 subunit vaccine confers improved and durable protection against the hypervirulent strain HN878 in mice.

Understanding functional interactions between DCs and antigens is necessary for achieving an optimal and desired immune response during vaccine development. Here, we identified and characterized protein Rv2299c (heat-shock protein 90 family), which effectively induced DC maturation. The Rv2299c-maturated DCs showed increased expression of surface molecules and production of proinflammatory cytokines. Rv2299c induced these effects by binding to TLR4 and stimulating the downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. The Rv2299c-maturated DCs also showed an induced Th1 cell response with bactericidal activity and expansion of effector/memory T cells. The Rv2299c-ESAT-6 fused protein had greater immunoreactivity than ESAT-6. Furthermore, boosting BCG with the fused protein significantly reduced hypervirulent Mycobacterium tuberculosis HN878 burdens post-challenge. The pathological study of the lung from the challenged mice assured the efficacy of the fused protein. The fused protein boosting also induced Rv2299c-ESAT-6-specific multifunctional CD4+ T-cell response in the lungs of the challenged mice. Our findings suggest that Rv2299c is an excellent candidate for the rational design of an effective multiantigenic TB vaccine.

Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth.

Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.