Adjunctive etanercept reduces TB-associated pathology.

<p>Hematoxylin and eosin staining (20x and 200x – inset) was performed on lung tissues. Images shown are representative of sections obtained from 4 animals per group. Each time-point is shown in weeks on the individual images. Lung pathology in animals receiving adjunctive etanercept (RHZ plus etanercept) (panel B) appeared to resolve earlier than those receiving standard treatment (RHZ alone) (panel A). Morphometric analysis confirmed these findings and demonstrated reduced lung involvement in mice treated with adjunctive etanercept (RHZ plus etanercept; blue x) versus standard treatment (RHZ alone; red □) (panel C). Results are represented as percentage lung involvement, calculated using ImageJ software.</p

Bacterial burden in the lungs of mice.

<p>Six weeks after an aerosol infection with <i>Mycobacterium tuberculosis</i>, C3HeB/FeJ mice were split among four treatment groups: Untreated (no treatment), standard TB treatment (RHZ), etanercept alone and standard TB treatment with adjunctive etanercept (RHZ + Etanercept). The number of viable bacteria in the lungs were estimated by determining colony-forming units (CFU). Results are shown for the duration of study (panel A) and also as individual dot plots for 8 (panel B) and 10 (panel C) weeks after starting TB treatment. Compared to standard TB treatment, the addition of etanercept resulted in a significantly lower pulmonary bacterial burden, corresponding to the phase when a significant proportion of bacteria are multiplying slowly (panels B, C). Results are presented as mean (±SD) CFU in the lungs, detected from a minimum of four mice at each time point and for each group. CFU are presented on a logarithmic scale (log₁₀) in panel A and on a linear scale for panels B and C and represent the same data.</p

Immune responses during TB treatments.

<p>Splenic recall assays were performed to monitor the immune responses during TB treatment in animals receiving adjunctive etanercept (RHZ plus etanercept) (blue x) and standard treatment (RHZ alone) (red □). Infected but untreated mice (black ○) and mice treated with etanercept alone (green Δ) were used as controls. High levels of IL-2, IFN-γ and CCL4 were noted in mice receiving standard TB treatment with or without adjunctive etanercept. Conversely, higher levels of IL-10 were only noted in the untreated mice. Overall, this represents a transition, from a regulatory to an effector phenotype, following the onset of treatment. Determinations were made from four mice, and each assay was performed in triplicate. Results are expressed as mean level (pg/ml) (±SD).</p

Immunogenicity without Efficacy of an Adenoviral Tuberculosis Vaccine in a Stringent Mouse Model for Immunotherapy during Treatment

<div><p>To investigate if bacterial persistence during TB drug treatment could be overcome by modulation of host immunity, we adapted a clinically-relevant model developed for the evaluation of new drugs and examined if immunotherapy with two adenoviral vaccines, Ad35-TBS (AERAS-402) and Ad26-TBS, could shorten therapy in mice. Even though immunotherapy resulted in strong splenic IFN-γ responses, no effect on bacterial replication in the lungs was seen. Multiplex assay analysis of lung samples revealed the absence of cytokine augmentation such as IFN-γ, TNF-α and IL-2, suggesting that immunization failed to induce immunity in the lungs. In this model, we show that IFN-γ levels were not associated with protection against disease relapse. The results obtained from our study raise questions regarding the traits of protective TB immunity that are relevant for the development of future immunotherapeutic and post-exposure vaccination strategies.</p></div

Immunogenic properties of the Rv2190c protein.

<p>N-terminal truncated Rv2190c was purified and analyzed for immunogenic properties by Dot-Blot ELISA against various sera. <b>A.</b> Immunogenic response in the human serum of an individual with latent TB (PPD-Pos.) (left panel) as opposed to a negligible response in a PPD negative human serum (right panel). <b>B.</b> Immunogenic response in serum from an <i>M. bovis</i> infected rabbit (left panel) and weak responses from sera raised against WhiB6 protein (middle panel) or pre-bleeds (right panel). i) 2ng of Rv2190c and ii) 2ng of BSA.</p

Effect of nitric oxide on <i>whiB1-7</i> expression.

<p><i>M.tb</i> CDC1551 was grown for 12 h in the presence of 500 µM DETA-NO. <i>whiB3, whiB6</i> and to a lesser extent, <i>whiB7</i> were upregulated.</p

Effect of db-cAMP on <i>whiB1-7</i> expression.

<p><i>M.tb</i> CDC1551 was grown 24h in the presence of 500 µM db-cAMP. <i>whiB1</i> and to a lesser extent <i>−2</i> and <i>−4</i> were up-regulated.</p

Relative abundance of <i>whiB1-7</i> transcripts.

<p>mRNA levels of the seven <i>M.tb whiB</i> genes at mid-logarithmic growth in 7H9 presented as fold change in relation to <i>sigA</i> (set to 0). <i>WhiB1</i> was highly expressed and <i>whiB5</i> was under-expressed compared to the other <i>whiB</i> genes, which exhibited an expression level very close to that of <i>sigA</i>.</p

The <i>M. tuberculosis Rv2190c</i> causes decreased lung histopathology in the mouse.

<p>Formalin-fixed lung sections from mice infected with WT, <i>Rv2190c</i> mutant and complement strains were stained with hematoxylin and eosin and visualized using light microscopy. Scale bar = 0.1 mm.</p

Schematic of study design.

<p>420 mice were divided into animals that were uninfected (NC-UU) (20 mice) and those that were infected with <i>M</i>.<i>tb</i> at week -2 (NC-IU) (400 mice). At week 0, 365 mice from NC-IU were removed to create the PC cohort that was initiated on therapy with R, H and Z via oral gavage, 7 days per week. The remaining NC-IU animals were left untreated up to week 12, when they became moribund and were sacrificed. At week 4, 284 animals from the PC cohort were randomized into groups NC-T4, NC-V and TV Ad35+. At this time point, mice in the NC-V and TV Ad35+ groups received intramuscular immunotherapy with 10¹⁰ viral particles of Ad35 empty vector or Ad35-TBS vaccine, which were respectively boosted at week 8. Animals in the PC, NC-T4, NC-V and TV Ad35+ groups were switched to therapy with R and H at week 8 which lasted to week 16, except for the PC group which received therapy up to week 24. At week 16, when therapy was shortened, 56 mice from the TV Ad35+ group were removed and divided into 2 further groups, TV Ad26+ and TV Ad26- which received immunotherapy with Ad26-TBS and Ad26, respectively. All animals were followed through for 3 months following therapy cessation at week 16 and week 24 (PC). Animals were subsequently sacrificed for the final lung CFU enumeration. Multiplex analysis on lung samples were carried out at times indicated for CFU counting, from week 4 onwards.</p