Association of IS<i>Mav6</i> with the Pattern of Antibiotic Resistance in Korean <i>Mycobacterium avium</i> Clinical Isolates but No Relevance between Their Genotypes and Clinical Features

<div><p>The aim of this study was to genetically characterize clinical isolates from patients diagnosed with <i>Mycobacterium avium</i> lung disease and to investigate the clinical significance. Multi-locus sequencing analysis (MLSA) and pattern of insertion sequence analysis of <i>M</i>. <i>avium</i> isolates from 92 Korean patients revealed that all isolates were <i>M</i>. <i>avium</i> subspecies <i>hominissuis</i>. In <i>hsp65</i> sequevar analysis, codes 2, 15, and 16 were most frequently found (88/92) with similar proportions among cases additionally two isolates belonging to code N2 and an unreported code were identified, respectively. In insertion element analysis, all isolates were IS<i>1311</i> positive and IS<i>900</i> negative. Four of the <i>M</i>. <i>avium</i> subsp. <i>hominissuis</i> isolates did not harbor IS<i>1245</i> and 1 of the <i>M</i>. <i>avium</i> isolates intriguingly harbored DT1, which is thought to be a <i>M</i>. <i>intracellulare</i>-specific element. <i>M</i>. <i>avium</i> subsp. <i>hominissuis</i> harboring IS<i>Mav6</i> is prevalent in Korea. No significant association between clinical manifestation and treatment response has been found in patients with the <i>hsp65</i> code type and IS<i>Mav6</i>, indicating that no specific strain/genotype among <i>M</i>. <i>avium</i> subsp. <i>hominissuis</i> organisms was a major source of <i>M</i>. <i>avium</i> lung disease. Interestingly, the presence of IS<i>Mav6</i> was correlated with greater resistance to moxifloxacin. Conclusively, the genotype of Korean <i>M</i>. <i>avium</i> subsp. <i>hominissuis</i> isolates is not a disease determinant responsible for lung disease and specific virulent factors of <i>M</i>. <i>avium</i> subsp. <i>hominissuis</i> need to be investigated further.</p></div

Phylogenetic tree based on concatenated <i>rpoB</i> and <i>hsp65</i> sequences using the neighbor-joining method with Kimura’s two-parameter distance correction model.

<p>Bootstrap analyses determined from 1,000 replicates are indicated at the nodes. Bar, 0.5% difference in nucleotide sequence. GenBank accession numbers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148917#pone.0148917.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148917#pone.0148917.s001" target="_blank">S1 Fig</a>.</p

Immunogenicity in the lungs of immunized mice.

<p>Three weeks after the final immunization, mice from each group (n = 5) were sacrificed and lung cells were prepared as described in the materials and methods section. The percentages of CD4⁺, CD8⁺ central memory (CD44^hiCD62L⁺CD127⁺), effector memory (CD44^hiCD62L^-CD127⁺), effector (CD44^hiCD62L^-CD127^-), and naïve (CD44^loCD62L⁺CD127⁺) T cells were analyzed by flow cytometry (A). A total of 2 × 10⁶ cells were added to each well of microtiter plates and incubated with PPD (2 μg/ml) or Ag85A-specific CD4 or CD8 T-cell peptides (2 μg/ml) for 24 h at 37°C. The IFN-γ concentrations in the suspension were detected using commercial ELISA kits (B). The induction of PPD-specific IgG2c antibodies in the serum from each group of mice (C). The data are presented as the means ± SD from five mice in each group. An unpaired <i>t</i>-test was used to determine the significance of differences. A value of <i>p</i><0.05 was considered to be statistically significant. ^#<i>p</i><0.05, ^##<i>p</i><0.01, and ^###<i>p</i><0.001 compared to G1. * <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001 compared to G3. <i>n</i>.<i>s</i>.: not significant.</p

Induction of multifunctional T cells in the lungs of mice after challenge with the <i>M</i>. <i>tuberculosis</i> HN878 strain.

<p>Five and ten weeks post-infection, mice from each group (n = 5) were euthanized and their lung cells (2 × 10⁶ cells) were stimulated with PPD (2 μg/ml) for 12 h at 37°C in the presence of GolgiStop. The percentage of antigen-specific CD4⁺CD62L^- and CD8⁺CD62L^- T cells producing IFN-γ, TNF-α, and/or IL-2 in the cells isolated from the lungs of each group of mice were analyzed by multicolor flow cytometry by gating for CD4⁺ and CD8⁺ lymphocytes (A). Pie charts (B) show the mean frequencies of cells coexpressing IFN-γ, TNF-α, and/or IL-2. The data are presented as the mean ± SD from five mice in each group. An unpaired <i>t</i>-test was used to determine the significance of differences. A value of <i>p</i><0.05 was considered to be statistically significant. ^#<i>p</i><0.05, ^##<i>p</i><0.01, and ^###<i>p</i><0.001 compared to G2. * <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001 compared to G3.</p

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

<div><p><i>Mycobacterium bovis</i> bacillus Calmette-Guerin (BCG), the only licensed vaccine, shows limited protection efficacy against pulmonary tuberculosis (TB), particularly hypervirulent <i>Mycobacterium tuberculosis</i> (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 <i>Mycobacterium tuberculosis</i> 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 <i>Mycobacterium tuberculosis</i> HN878 infection in a boosting number-dependent manner.</p></div

Histopathological lesions of the lungs.

<p>Mice were infected with 200 CFU of the <i>M</i>. <i>tuberculosis</i> HN878 strain via the aerosol route, and the lungs were removed at 5 weeks (A) and 10 weeks (B) post-infection. Representative lung pathological changes from the different groups and the percentages of inflamed areas in lung section are also shown. The data regarding the percentages of the inflamed areas are presented as Whisker box plots (Whiskers represent minimum and maximum values) (n = 5), and a one-way ANOVA followed by Dunnett’s test was used to determine the significance of the findings. A value of <i>p</i><0.05 was considered to be statistically significant. ^#<i>p</i><0.05, ^##<i>p</i><0.01 and ^###<i>p</i><0.001 compared to G2. * <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001 compared to G3.</p

Induction of antigen-specific multifunctional T cells in the lungs of immunized mice.

<p>Each group of mice was immunized and sacrificed as described in the materials and methods section. Three weeks after the final immunization, mice from each group (n = 5) were euthanized and their lung cells (2 × 10⁶ cells) were stimulated with PPD (2 μg/ml) or Ag85A-specific CD4 or CD8 T cell peptides (2 μg/ml) for 12 h at 37°C in the presence of GolgiStop. The percentages of antigen-specific CD4⁺CD62L^- and CD8⁺CD62L^- T cells producing IFN-γ, TNF-α, and/or IL-2 in the cells isolated from the lungs of each group of mice were analyzed by multicolor flow cytometry by gating for CD4⁺ and CD8⁺ lymphocytes (A). Pie charts (B) show the mean frequencies of cells coexpressing IFN-γ, TNF-α, and/or IL-2. The data are presented as the mean ± SD from five mice in each group. An unpaired <i>t</i>-test was used to determine the significance of differences. A value of <i>p</i><0.05 was considered to be statistically significant. ^#<i>p</i><0.05, ^##<i>p</i><0.01, and ^###<i>p</i><0.001 compared to G1. * <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001 compared to G3.</p

Histopathological analysis of lungs infected with different strains of virulent Mtb.

<p>(A) Inflammatory scores of H&E-stained sections of lungs during Mtb infection. At 28, 56, and 112 days post-infection, mice were sacrificed, and lung sections were stained with H&E (<i>n</i> = 5 per group per designated time point). Ten pictures from each group were randomly selected and analyzed (2 pictures per mouse × 5 mice in each group). Lung inflammation scores are presented as the median percentage (± IQR) of inflammation for each mouse. Lung sections were stained with H&E (bar, 500 μm). A <i>p</i>-value ≤ 0.05 was considered significant: *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001. (B) Representative histopathology and gross pathology of mouse lungs infected with Mtb strains with different virulence levels at 112 days post-infection.</p

Analysis of innate immune cell kinetics of mice infected with Mtb with different virulence levels.

<p>Single-cell suspensions prepared from lung tissues of mice infected with Mtb strains at days 2, 5, 7, 14, 56 and 112 days post-infection were stained with the indicated antibodies and analyzed by flow cytometry (<i>n</i> = 4 per group per designated time point). (A) Gating strategy for the analysis of innate immune cells present in the in lungs. All surface-stained samples were primarily gated on forward scatter (FSC)^mid/^high and side scatter (SSC)^mid/^high and secondarily gated on F4/80^-/⁺ and CD11c^low. The cells were analyzed according to the expression of Gr-1 versus CD11b. F4/80^-/CD11c^-/CD11b^high/Gr-1^high cells were designated as neutrophils (a). The lower population was designated as CD11c^-/CD11b⁺/Gr-1^int cells (b). Next, dot-blots of lung cells were primarily gated on FSC^mid/^high and SSC^mid/^high and secondarily gated on F4/80^- and F4/80⁺. The cells were analyzed according to the expression of CD11b versus CD11c. F4/80⁺/CD11c⁺/CD11b^- cells (c), F4/80⁺/CD11c^-/CD11b⁺ cells (d), F4/80^-/CD11c^int/CD11b^int cells (e), F4/80^-/CD11c⁺/CD11b⁺ cells (f) and F4/80^-/CD11c⁺/CD11b^-/Siglec-H⁺/PDCA-1⁺ cells (g) were designated as alveolar macrophages, CD11b⁺ macrophages, monocytes, CD11b^high DCs and pDCs, respectively. (B) The line graphs display the absolute numbers of cells in (A) at various time points during Mtb lung infection. *<i>p</i> < 0.05, ***<i>p</i> < 0.001, Mtb K-infected <i>vs</i>. Mtb H37Rv-infected groups. (C) Bar graphs shows the percentage of infiltrated cells in the lungs. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, non-infected <i>vs</i>. Mtb-infected groups. The data are presented as the mean (± SD) or four mice per group at each time point from one representative experiment out of two independent experiments. NI: Non-infected; Ra: H37Ra-infected lung; Rv: H37Rv-infected lung; K: Beijing-K-infected lung.</p

PPD-specific cytokine response in lung cells from Mtb-infected mice.

<p>The amount of IFN-γ, IL-5, IL-10, IL-17A (A) and IFN-α (B) produced by lung cells (14, 28, 56 and 112 days post-infection) in response to PPD (10 μg/ml) stimulation for 24 h was measured by ELISA. All data are expressed as the mean ± SD (<i>n</i> = 4 per group per designated time point) of one representative experiment out of two independent experiments.</p