A Novel Therapeutic Approach Using Mesenchymal Stem Cells to Protect Against Mycobacterium abscessus.

Recent studies have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) for the treatment of acute inflammatory injury and bacterial pneumonia, but their therapeutic applications in mycobacterial infections have not been investigated. In this study, we demonstrated the use of MSCs as a novel therapeutic strategy against Mycobacterium abscessus (M. abscessus), which is the most drug-resistant and difficult-to-treat mycobacterial pathogen. The systemic intravenous injection of MSCs not only improved mouse survival but also enhanced bacterial clearance in the lungs and spleen. Additionally, MSCs enhanced IFN-γ, TNF-α, IL-6, MCP-1, nitric oxide (NO) and PGE2 production and facilitated CD4(+) /CD8(+) T cell, CD11b(high) macrophage, and monocyte recruitment in the lungs of M. abscessus-infected mice. To precisely elucidate the functions of MSCs in M. abscessus infection, an in vitro macrophage infection system was used. MSCs caused markedly increased NO production via NF-κB activation in M. abscessus-infected macrophages cultured in the presence of IFN-γ. Inhibiting NO or NF-κB signaling using specific inhibitors reduced the antimycobacterial activity of MSCs. Furthermore, the cellular crosstalk between TNF-α released from IFN-γ-stimulated M. abscessus-infected macrophages and PGE2 produced by MSCs was necessary for the mycobacterial-killing activity of the macrophages. Finally, the importance of increased NO production in response to MSC administration was confirmed in the mouse M. abscessus infection model. Our results suggest that MSCs may offer a novel therapeutic strategy for treating this drug-resistant mycobacterial infection by enhancing the bacterial-killing power of macrophages. Stem Cells 2016;34:1957-1970.

Essential engagement of Toll-like receptor 2 in initiation of early protective Th1 response against rough variants of Mycobacterium abscessus.

Although Mycobacterium abscessus (M. abscessus) is becoming more prevalent in patients without overt immunodeficiency, little is known about the factors that contribute to disease susceptibility. This study was undertaken to investigate how Toll-like receptor 2 (TLR2) functionally contributes to the generation of protective immunity against M. abscessus in a morphotype-specific manner. We found that Tlr2-/- mice were extremely susceptible to an intravenous (i.v.) model of infection by M. abscessus rough variants, displaying uncontrolled infection in the lungs and a significantly lower survival rate than with wild-type (WT) mice. This uncontrolled infection resulted from failures in the following processes: (i) production of the crucial cytokines gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 12p70 (IL-12p70); (ii) early infiltration of neutrophils, monocytes, and dendritic cells (DCs) in the lungs of Tlr2-/- mice; (iii) rapid influx of CD4+ and CD8+ T cells; and (iv) the expansion of memory/effector T cells. Notably, systemic administration of M. abscessus culture filtrate-treated syngeneic DCs from WT mice greatly strengthened immune priming in vivo, resulting in a dramatic reduction in bacterial growth and improved long-term survival in Tlr2-/- mice, with a recovery of protective immunity. Our findings demonstrate that TLR2 is an essential contributor to instructive and effector immunity during M. abscessus infection in a morphotype-specific manner.

Mycobacterium tuberculosis MmsA, a novel immunostimulatory antigen, induces dendritic cell activation and promotes Th1 cell-type immune responses.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is an outstanding pathogen that modulates the host immune response. This inconvenient truth drives the continual identification of antigens that generate protective immunity, including Th1-type T cell immunity. Here, the contribution of methylmalonate semialdehyde dehydrogenase (MmsA, Rv0753c) of Mtb to immune responses was examined in the context of dendritic cell (DC) activation and T cell immunity both in vitro and in vivo. The results showed that MmsA induced DC activation by activating the MAPK and NF-κB signaling pathways. Additionally, MmsA-treated DCs activated naïve T cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2, and induced T cell proliferation. These results indicate that MmsA is a novel DC maturation-inducing antigen that drives the Th1 immune response. Thus, MmsA was found to potentially regulate immune responses via DC activation toward Th1-type T cell immunity, enhancing our understanding of Mtb pathogenesis.

Induction of immune responses in mice and pigs by oral administration of classical swine fever virus E2 protein expressed in rice calli.

Classical swine fever (CSF), caused by the CSF virus (CSFV), is a highly contagious disease in pigs. In Korea, vaccination using a live-attenuated strain (LOM strain) has been used to control the disease. However, parenteral vaccination using a live-attenuated strain still faces a number of problems related to storage, cost, injection stress, and differentiation of CSFV infected and vaccinated pigs. Therefore, two kinds of new candidates for oral vaccination have been developed based on the translation of the E2 gene of the SW03 strain, which was isolated from an outbreak of CSF in 2002 in Korea, in transgenic rice calli (TRCs) from Oriza sativa L. cv. Dongjin to express a recombinant E2 protein (rE2-TRCs). The expression of the recombinant E2 protein (rE2) in rE2-TRCs was confirmed using Northern blot, SDS-PAGE, and Western blot analysis. Immune responses to the rE2-TRC in mice and pigs were investigated after oral administration. The administration of rE2-TRCs increased E2-specific antibodies titers and antibody-secreting cells when compared to animals receiving the vector alone (p < 0.05 and p < 0.01). In addition, mice receiving rE2-TRCs had a higher level of CD8+ lymphocytes and Th1 cytokine immune responses to purified rE2 (prE2) in vitro than the controls (p < 0.05 and p < 0.01). Pigs receiving rE2-TRCs also showed an increase in IL-8, CCL2, and the CD8+ subpopulation in response to stimulation with prE2. These results suggest that oral administration of rE2-TRCs can induce E2-specific immune responses.

Supplementation of dietary germanium biotite enhances induction of the immune responses by foot-and-mouth disease vaccine in cattle.

BACKGROUND: After the recent outbreak of foot-and-mouth disease (FMD) in Korea, a vaccination policy has been applied to control the disease. In addition, several non-specific immune stimulators have been used without any scientific evidence that they would enhance the immune response after FMD vaccination and/or protect against FMD. Based on the current situation, the aim of this study was to evaluate the effect of the non-specific immune stimulator germanium biotite on FMD vaccination and immune responses in cattle. To achieve our goal, immune responses to FMD vaccination, such as levels of IgG and IgA, antibody duration, and virus-neutralizing titers were investigated after germanium biotite feeding. The PBMC typing and proliferative response after stimulation with mitogens, the cytokines expression level of PBMC, and the lysozyme activity in the serum were measured to evaluate the immune enhancing effects of germanium biotite following its administration. RESULTS: Following the first vaccination, high level of IgG (at 4 weeks) and IgA (at 2 and 31 weeks) titers in serum and saliva were observed in the germanium biotite-feeding group (p < 0.05). The germanium biotite group also showed high and longstanding inhibition percentage value in ELISA assay at 31 weeks (p < 0.05). Generally, higher virus-neutralizing antibody titers were observed in the feeding group at 20 and 31 weeks after vaccination. Following the feeding germanium biotite, the germanium biotite group showed increased subpopulation of CD4+ lymphocytes and MHC I+II+ cells in PBMCs at 23 week, responding to stimulation of ConA. The levels of IFN-γ (at 3 and 8 weeks), IL-1α (at 3, 11, and 23 weeks), IL-1ß (at 3, 8, and 11 weeks), and IL-4 (at 8 and 11 weeks) gene expression were also significantly increased in the feeding group (p < 0.01 and p < 0.05). Feeding with germanium biotite increased the lymphocytes' proliferative response to the stimulation of ConA and LPS at 23 weeks and lysozyme activity at 9 weeks after feeding. CONCLUSIONS: These results suggest that germanium biotite feeding could increase the protection against FMD virus infection via the induction of higher humoral and cellular immune responses in cattle.

Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7.

BACKGROUND: Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. RESULTS: Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. CONCLUSIONS: In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further.

Oral immunization of mice with Saccharomyces cerevisiae expressing a neutralizing epitope of ApxIIA exotoxin from Actinobacillus pleuropneumoniae induces systemic and mucosal immune responses.

An oral delivery system based on ApxIIA#5-expressed on Saccharomyces cerevisiae was studied for its potential to induce immune responses in mice. Murine bone marrow-derived dendritic cells (DCs) stimulated in vitro with ApxIIA#5-expressed on S. cerevisiae upregulated the expression of maturation and activation markers, leading to production of tumor necrosis factor-α, interleukin (IL)-1ß, IL-12p70 and IL-10. Presentation of these activated DCs to cluster of differentiation CD4+ T cells collected from mice that had been orally immunized with the ApxIIA#5-expressed on S. cerevisiae elicited specific T-cell proliferation. In addition, the orally immunized mice had stronger antigen-specific serum IgG and IgA antibody responses and larger numbers of antigen-specific IgG and IgA antibody-secreting cells in their spleens, Peyer's patches and lamina propria than did those immunized with vector-only S. cerevisiae or those not immunized. Furthermore, oral immunization induced T helper 1-type immune responses mediated via increased serum concentrations of IgG2a and an increase predominantly of IFN-γ-producing cells in their spleens and lamina propria. Our findings suggest that surface-displayed ApxIIA#5-expressed on S. cerevisiae may be a promising candidate for an oral vaccine delivery system for eliciting systemic and mucosal immunity.

A novel defined TLR3 agonist as an effective vaccine adjuvant.

Synthetic double-stranded RNA analogs recognized by Toll-like receptor 3 (TLR3) are an attractive adjuvant candidate for vaccines, especially against intracellular pathogens or tumors, because of their ability to enhance T cell and antibody responses. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been limited due to heterogeneous molecular size, inconsistent activity, poor stability, and toxicity. To overcome these limitations, we developed a novel dsRNA-based TLR3 agonist named NexaVant (NVT) by using PCR-coupled bidirectional in vitro transcription. Agarose gel electrophoresis and reverse phase-HPLC analysis demonstrated that NVT is a single 275-kDa homogeneous molecule. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without any signs of serious toxicity. NVT stimulated TLR3 and increased the expression of viral nucleic acid sensors TLR3, MDA-5, and RIG-1. When intramuscularly injected into C57BL/6 mice, ovalbumin (OVA) plus NVT highly increased the migration of dendritic cells (DCs), macrophages, and neutrophils into inguinal lymph node (iLN) compared with OVA alone. In addition, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-ß production. Furthermore, NVT exhibited an appropriate adjuvanticity because it elevated OVA-specific IgG, in particular, higher levels of IgG2c (Th1-type) but lower IgG1 (Th2-type). Concomitantly, NVT increased the levels of Th1-type T cells such as IFN-γ+CD4+ and IFN-γ+CD8+ cells in response to OVA stimulation. Collectively, we suggest that NVT with appropriate safety and effectiveness is a novel and promising adjuvant for vaccines, especially those requiring T cell mediated immunity such as viral and cancer vaccines.

Plasmid typing and resistance profiling of Escherichia fergusonii and other Enterobacteriaceae isolates from South Korean farm animals.

In this study, we focused on determining the distribution and prevalence of major plasmid replicons in ß-lactam-resistant Escherichia fergusonii and Enterobacteriaceae of animal and human origin. A high degree of plasmid variability and multiple plasmid replicons were observed among the isolates. The IncF and IncI1 replicons were the most prevalent in E. fergusonii and Salmonella enterica serovar Indiana isolated from swine and poultry in South Korea, respectively. The presence of broad-host-range plasmid replicons such as IncN, IncA/C, IncHI1, and IncHI2 that are associated with important virulence genes and toxins as well as antimicrobial resistance determinants indicates that E. fergusonii has the potential to become an important pig pathogen and possible emerging opportunistic zoonotic pathogen.

Enzymatic analysis of the effect of naturally occurring Leu138Pro mutation identified in SHV ß-lactamase on hydrolysis of penicillin and ampicillin.

BACKGROUND: The aim of this study was to analyze the significance of leucine to proline substitution at position 138(Leu138Pro) on the hydrolysis of penicillin and ampicillin that we identified in the blaSHV gene of clinical Escherichia coli swine isolate. RESULTS: Kinetic analysis of the mutant proteins showed that K(m) value of the purified L138P mutant was comparatively higher than SHV-1, SHV-33 and SHV-33(L138P) enzyme for penicillin and ampicillin. Docking simulation of the SHV-1 and SHV-(L138P) enzymes also confirmed that ß-lactamases preferred penicillin to ampicillin and the SHV-1 had a higher binding affinity for antibiotics compared to the SHV-(L138P) and other mutants. CONCLUSIONS: Our result demonstrated that L138P has a reduced role in penicillin and ampicillin hydrolyzing properties of SHV ß-lactamases. These naturally occurring mutations rendering reduced function of the existing protein could trigger the emergence or acquisition of more effective alternative mechanisms for ß-lactam hydrolysis.

Antibiotic resistance patterns and detection of blaDHA-1 in Salmonella species isolates from chicken farms in South Korea.

Fifteen nonrepetitive ampicillin-resistant Salmonella spp. were identified among 91 Salmonella sp. isolates during nationwide surveillance of Salmonella in waste from 131 chicken farms during 2006 and 2007. Additional phenotyping and genetic characterization of these 15 isolates by using indicator cephalosporins demonstrated that resistance to ampicillin and reduced susceptibility to cefoxitin in three isolates was caused by TEM-1 and DHA-1 beta-lactamases. Plasmid profiling and Southern blot analysis of these three DHA-1-positive Salmonella serovar Indiana isolates and previously reported unrelated clinical isolates of DHA-1-positive Salmonella serovar Montevideo, Klebsiella pneumoniae, and Escherichia coli from humans and swine indicated the involvement of the large-size plasmid. Restriction enzyme digestion of the plasmids from the transconjugants showed variable restriction patterns except for the two Salmonella serovar Indiana isolates identified in this study. To the best of our knowledge, this is the first report of the presence of the DHA-1 gene among Salmonella spp. of animal origin.

Inter- and intraspecies plasmid-mediated transfer of florfenicol resistance in Enterobacteriaceae isolates from swine.

Florfenicol resistance was analyzed in 230 enteric pig isolates collected between 1998 and 2006. PCR, plasmid profiling, Southern blot hybridization, and a mixed-broth conjugation assay suggested the intra- and interspecies plasmid-mediated transfer of florfenicol resistance among the isolates that exhibited MICs for florfenicol between 4 to 128 mg/liter.

Analysis of the helicase gene of Korean swine hepatitis E virus isolates and trends in viral infection.

Hepatitis E virus (HEV) has been considered to be a zoonotic agent and an important public concern worldwide. In this study, a nested RT-PCR was developed to detect the helicase gene of swine HEV (sHEV) from sera of pigs. Using this RT-PCR, 16 out of 821 Korean isolates of sHEV were identified, with 1.9% prevalence. An age-specific prevalence was demonstrated with the highest prevalence in growing pigs (5.4%). Phylogenetic analysis of sHEV Korean isolates identified genotype 3, with 89.4-99.9% nucleotide sequence identity. The viruses were closely related to US and Japanese HEV isolates from swine and humans (89.4-93.1%). The prevalence of anti-HEV IgG in individual pigs and swine herds was 39.5 and 80%, respectively. The seroprevalence rate increased in proportion to the age of the swine. The seroprevalence of HEV was higher than previously reported. These results indicate that sHEV is widespread in Korean swine herds and further raise concerns about possible zoonosis.

Genotypes, antibiogram, and pulsed-field gel electrophoresis profiles of Escherichia coli strains from piglets in Korea.

Adherence factors and enterotoxins are major virulence factors of enterotoxigenic Escherichia coli (ETEC). Antibiotics have been used frequently for the treatment and prevention of ETEC infection in piggeries worldwide, including Korea. Therefore, data on both virulence profiles and antibiotic resistance patterns are useful in the epidemiological study of ETEC. A total number of 198 E. coli field isolates were examined. The most prevalent pathotype was F1, followed by a combination of F1 and EAST1. All of the 71 isolates were resistant to more than 2 antibiotics used in a disk diffusion test, and 87.94% of the isolates were found to be resistant to more than 4 antibiotics. Investigations were also conducted to correlate the virulence gene profiles with antibiogram and pulsed-field gel electrophoresis (PFGE). Although a high degree of polymorphism was noted among strains having the same virulence patterns, the highest similarity pattern was observed carrying the same virulence profiles and similar antibiogram. Thus, investigation of both virulence profiles and antibiogram is essential to the epidemiological study of ETEC. Moreover, the PFGE method might be applicable as a tool to reveal genetic relatedness among E. coli strains from piggeries in Korea.

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 tuberculosis PE27 activates dendritic cells and contributes to Th1-polarized memory immune responses during in vivo infection.

A gradual understanding of the proline-glutamate (PE) and proline-proline-glutamate (PPE) families, which compromise 10% of the coding regions in the Mycobacterium tuberculosis (Mtb) genome, has uncovered unique roles in host-pathogen interactions. However, the immunological function of PE27 (Rv2769c), the largest PE member, remains unclear. Here, we explored the functional roles and related signaling mechanisms of PE27 in the interaction with dendritic cells (DCs) to shape the T cell response. PE27 phenotypically and functionally induces DC maturation by up-regulating CD80, CD86, MHC class I and MHC class II expression on the DC surface to promote the production of TNF-α, IL-1ß, IL-6, and IL-12p70 but not IL-10. Additionally, we found that PE27-mediated DC activation requires the participation of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) signaling pathways. Interestingly, PE27-treated DCs directed naïve CD4(+) T cells to secrete IFN-γ and activate T-bet but not GATA-3. PE27 also induced IFN-γ-producing memory T cell responses in Mtb-infected mice, indicating that PE27 contributes to Th1-polarization. Taken together, these findings suggest that PE27 possesses Th1-polarizing potential through DC maturation and could be useful in the design of TB vaccines.

Pulmonary immunity and durable protection induced by the ID93/GLA-SE vaccine candidate against the hyper-virulent Korean Beijing Mycobacterium tuberculosis strain K.

The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4(+) T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFN-γ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8(+) central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent.

Mycobacterium tuberculosis Rv3628 drives Th1-type T cell immunity via TLR2-mediated activation of dendritic cells and displays vaccine potential against the hyper-virulent Beijing K strain.

Identification of vaccine target antigens (Ags) that induce Ag-specific Th1 immunity is the first step toward the development of a tuberculosis vaccine. Here, we evaluated the Mycobacterium tuberculosis (Mtb) protein Rv3628, a soluble inorganic pyrophosphatase, as a vaccine target and characterized the molecular details of its interaction with dendritic cells (DCs). Rv3628 activated DCs, increasing their expression of cell surface molecules and augmenting their production of TNF-α, IL-1ß, IL-6, and IL-12p70. Rv3628 mediated these effects by binding to TLR2 and activating downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. Rv3628-stimulated DCs induced the expansion of OVA-specific CD4+ and CD8+ T cells, which secreted IFN-γ and IL-2. Rv3628-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 Ag in samples of lung and spleen cells collected from Mtb-infected mice. Finally, an Rv3628 subunit vaccine adjuvanted with dimethyldioctadecylammonium liposomes containing monophosphoryl lipid-A caused significant reductions in bacterial counts and lung inflammation after challenge with the hyper-virulent Mtb K strain. Importantly, protective efficacy was correlated with the generation of Rv3628-specific CD4+ T cells co-producing IFN-γ, TNF-α and IL-2 and exhibiting an elevated IFN-γ recall response. Thus, Rv3628 polarizes DCs toward a Th1 phenotype and promotes protective immunity against Mtb infection.

Repeated Aerosolized-Boosting with Gamma-Irradiated Mycobacterium bovis BCG Confers Improved Pulmonary Protection against the Hypervirulent Mycobacterium tuberculosis Strain HN878 in Mice.

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent Mycobacterium tuberculosis (Mtb) strains, suggesting that a logistical and practical vaccination strategy is urgently required. Boosting the BCG-induced immunity may offer a potentially advantageous strategy for advancing TB vaccine development, instead of replacing BCG completely. Despite the improved protection of the airway immunization by using live BCG, the use of live BCG as an airway boosting agent may evoke safety concerns. Here, we analyzed the protective efficacy of γ-irradiated BCG as a BCG-prime boosting agent for airway immunization against a hypervirulent clinical strain challenge with Mycobacterium tuberculosis HN878 in a mouse TB model. After the aerosol challenge with the HN878 strain, the mice vaccinated with BCG via the parenteral route exhibited only mild and transient protection, whereas BCG vaccination followed by multiple aerosolized boosting with γ-irradiated BCG efficiently maintained long-lasting control of Mtb in terms of bacterial reduction and pathological findings. Further immunological investigation revealed that this approach resulted in a significant increase in the cellular responses in terms of a robust expansion of antigen (PPD and Ag85A)-specific CD4+ T cells concomitantly producing IFN-γ, TNF-α, and IL-2, as well as a high level of IFN-γ-producing recall response via both the local and systemic immune systems upon further boosting. Collectively, aerosolized boosting of γ-irradiated BCG is able to elicit strong Th1-biased immune responses and confer enhanced protection against a hypervirulent Mycobacterium tuberculosis HN878 infection in a boosting number-dependent manner.

Virulence-Dependent Alterations in the Kinetics of Immune Cells during Pulmonary Infection by Mycobacterium tuberculosis.

A better understanding of the kinetics of accumulated immune cells that are involved in pathophysiology during Mycobacterium tuberculosis (Mtb) infection may help to facilitate the development of vaccines and immunological interventions. However, the kinetics of innate and adaptive cells that are associated with pathogenesis during Mtb infection and their relationship to Mtb virulence are not clearly understood. In this study, we used a mouse model to compare the bacterial burden, inflammation and kinetics of immune cells during aerogenic infection in the lung between laboratory-adapted strains (Mtb H37Rv and H37Ra) and Mtb K strain, a hyper-virulent W-Beijing lineage strain. The Mtb K strain multiplied more than 10- and 3.54-fold more rapidly than H37Ra and H37Rv, respectively, during the early stage of infection (at 28 days post-infection) and resulted in exacerbated lung pathology at 56 to 112 days post-infection. Similar numbers of innate immune cells had infiltrated, regardless of the strain, by 14 days post-infection. High, time-dependent frequencies of F4/80-CD11c+CD11b-Siglec-H+PDCA-1+ plasmacytoid DCs and CD11c-CD11b+Gr-1int cells were observed in the lungs of mice that were infected with the Mtb K strain. Regarding adaptive immunity, Th1 and Th17 T cells that express T-bet and RORγt, respectively, significantly increased in the lungs that were infected with the laboratory-adapted strains, and the population of CD4+CD25+Foxp3+ regulatory T cells was remarkably increased at 112 days post-infection in the lungs of mice that were infected with the K strain. Collectively, our findings indicate that the highly virulent Mtb K strain may trigger the accumulation of pDCs and Gr1intCD11b+ cells with the concomitant down-regulation of the Th1 response and the maintenance of an up-regulated Th2 response without inducing a Th17 response during chronic infection. These results will help to determine which immune system components must be considered for the development of tuberculosis (TB) vaccines and immunological interventions.