Treatment of tumor bearing mice.

<p>C57BL/6 mice were injected s.c. in one flank with 640,000 wild type Panc02 cells. At 11, 18, and 25 days (arrows) post tumor injection, mice were injected i.m. contralaterally with PBS or 7.6 x 10⁷ pfu/mouse of MVA, MVAmeso (Meso) or MVAmesoA35Del (A35D) viruses in 25 ul. (A) Tumor volumes were measured 3 times per week and survival (B) was monitored.</p

Pretreatment and intratumoral injections.

<p>(A) Mice were vaccinated 2 times and then injected with tumor cells 2 weeks later. (B) Tumors were allowed to grow to 100 mm³, and 3.5 x 10⁶ pfu/mouse in 25 ul of MVA, MVAmeso (meso) or MVAmesoA35Del (A35D) viruses were injected directly into tumors 3 times.</p

MVA kills Panc02 cells.

<p>Panc02 cells (10,000 cells/well) were plated in triplicate in a 96-well plate. MVA was added to the wells at an MOI of 0.5 or 5.0 pfu/cell. After 24 h, 10 ul MTS/PMS was added to each well, and the absorbance was read at 492 nm. Averages <u>+</u> SD error bars are shown. * p< 0.005. Media with no cells was used to define the background control level.</p

Mesothelin is expressed from MVAmeso viruses.

<p>BHK cells (2x10⁶ cells/well) were uninfected or infected with MVA, MVAmeso or MVAmesoA35Del. Cells were lysed and samples were loaded onto a pre-cast 4–20% gradient gel. Rabbit anti-mesothelin antibody and anti-rabbit IgG (Fc) AP Conjugate (Promega) detected mesothelin protein (arrow). HEK transected cells, previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193131#pone.0193131.ref007" target="_blank">7</a>], were used as control.</p

MVA meso virus injections are safe.

<p>Female C57BL/6 mice (n = 5) were injected i.m. with PBS, MVA, MVAmeso or MVAmesoA35Del and monitored daily by body score condition (no change) and weight up to 125 days. No adverse effects were noted. Average weights are shown <u>+</u> SEM.</p

MVA replicates in Panc02 cells.

<p>350,000 BHK or Panc02 cells were plated in a 24-well plate and infected with MVA at an MOI of 5. Supernatants and cells were harvested separately at six time points: 0, 12, 16, 24, 36, and 48 h post-infection. These samples were frozen and thawed three times before being titered in BHK cells in 6-well plates.</p

Mesothelin expressing viruses induce IFN-γ responses to Panc02 cells.

<p>Mice (n = 5) were infected with MVA, MVAmeso, MVAmesoA35Del (A35Dmeso), or mock infected with PBS, and splenocytes were harvest 1 month later. 100 live Panc02 cells were added to splenocytes and incubated with Panc02 cells for 40 h at 37°C. Plates were then washed and incubated with biotinylated rat anti-mouse IFN-gamma and streptavidin-AP and developed with BCIP. Average numbers are shown <u>+</u> SEM. * MVAmeso and MVAmesoA35Del vaccinated mice had significantly more responding splenocytes (p<0.05) than MVA and PBS treated mice.</p