Lot release and stability summary: MLSA-LAM and MLCwA Batch No. 23, Lot No. 051297.

<p>Lot release and stability summary: MLSA-LAM and MLCwA Batch No. 23, Lot No. 051297.</p

Bacterial fractionation flow chart.

<p>Bacteria were sonicated and fractionated into subcellular components: cell wall, cytosol, and membrane. The membrane antigen preparation was not further pursued. Cell wall associated proteins were extracted with SDS and both the cytosol and cell wall fractions were then extracted with TX-114 to remove non-specific hyporesponsive antigens, mostly LAM, LM, and PIM's. Residual detergent was removed by affinity chromatography. Antigens were diluted to prescribed concentrations, vialed, labeled, and autoclaved.</p

Leprosy skin test antigen pilot facility.

<p>In preparation for manufacturing, the five room cGMP suite consisted of: a Gowning and Material Transfer (GMT) ISO8 clean room; a Manufacturing Suite A (MF A) ISO7 clean room; a Manufacturing Suite B (MF B) ISO7 clean room; a Quarantine/Release Goods Room (Q/RG) clean/non-classified clean room; and, a Quality Control Laboratory (QC) ISO8 clean room.</p

Leprosy skin test antigen purification yields.

a<p>Liver tissues were divided into three sections with an average weight of 32 g±0.9 g/run.</p

Tissue fractionation flow chart.

<p><i>M. leprae</i> was purified from the tissues of experimentally infected armadillos. A total of seven batches were prepared to generate an adequate quantity of bacteria (128.4 mg) for bacterial fractionation.</p

Protein profile of MLSA-LAM and MLCwA.

<p>Unstained molecular weight markers (lane M), and 2 µg of pre-autoclaved leprosy skin test antigens MLSA-LAM (lane 1) and MLCwA (lane 2) were separated on a 15% reduced polyacrylamide gel and visualized by staining with silver nitrate. The depicted proteins were located by immunoblot with the corresponding monoclonal antibodies as described in the materials and methods. The SOD protein is a 23 kDa protein based on amino acid sequence, but resolves at 28 kDa under reduced gel electrophoresis conditions <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002791#pntd.0002791-Marques2" target="_blank">[50]</a>.</p

The <i>M. tuberculosis</i> RNA's activity is caspase-8 dependent.

<p><i>A.</i> Flow cytometry demonstrated <i>M. tuberculosis</i> H37Rv RNA (gpRNA) activated caspase-8. Human monocytes were stimulated with 1 µg/ml of purified <i>M. tuberculosis</i> H37Rv RNA gpRNA (open histogram with dotted line) or PPD (open histograms with solid line) for 48 h and caspase activity was determined by flow cytometry after staining with FLICA-YVAD-FMK (caspase-1), FLICA-DEVD-FMK (caspase-3), or FLICA-LETD-FMK (caspase-8). Tissue culture medium without RNA or PPD was used as the control (closed histogram). <i>B.</i> Stimulation of apoptosis based on annexinV staining was measured for monocytes stimulated with gpRNA or PPD (top histogram) and when the monocytes were pretreated for 1 h with 10 nM caspase-1 (YVAD-FMK), caspase-3 (DEVD-FMK), or caspase-8 (LETD-FMK) inhibitors (lower histograms).</p

Human monocyte apoptosis is specifically induced by gel-purified mycobacterial RNA.

<p><i>A.</i> SDS-PAGE with ethidium bromide (left) and silver stained (right) gel-purified RNA (gpRNA) from F7 and treated with RNaseV1 (gpRNA+RNaseV1). <i>B.</i> Monocyte apoptosis induced by gpRNA untreated or treated with RNaseV1, anti-CD95 untreated or treated with RNaseV1 (anti-CD95+RNaseV1) and rabbit mRNA. Significance as described above.</p

RNA in DEAE-Sepharose Fraction 7 induces apoptosis in human monocytes.

<p><i>A.</i> Apoptosis induced by DEAE-Sepharose Fraction 7 (F7), F7 treated with proteinase K (F7+ProtK), F7 treated with DNase1 (F7+DNase1), F7 treated with RNaseV1 (F7+RNaseV1), CF, CF treated with RNaseV1 (CF+RNaseV1), CF-Man, CF-Man treated with RNaseV1 (CF-Man+RNaseV1). Apoptosis was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive. *** significance p<0.001, ** significance p<0.01 as compared to unstimulated control or between treated samples. <i>B.</i> SDS-PAGE with ethidum bromide staining and <i>C.</i> silver staining of F7 and F7 treated with proteinase K, DNase1, or RNaseV1.</p

Separation of CF by ConA affinity chromatography and DEAE-Sepharose chromatography yielded a fraction with potent apoptotic activity in human monocytes.

<p><i>A.</i> Apoptosis induced by DEAE-Sepharose fractions 1 to 7 (F1 to F7) and unfractionated CF was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive *** significance p<0.001, ** significance p<0.01, * significance p<0.05 as compared to unstimulated control. <i>B.</i> SDS-PAGE and silver staining of fractions of CF after ConA affinity (CF-Man) and DEAE-Sepharose chromatography (F1 to F7). Mw denotes the molecular mass standards.</p