Systemic Delivery, Selectivity, and Oncolytic Activity of <i>WR-delB18R</i> Virus

<div><p>(A) Immunocompetent (BALB/c) mice bearing JC tumors were treated via a single tail vein injection with 1 × 10⁸ PFU of vaccinia strains WR or <i>WR-delB18R</i>. Viral titer (PFU/g) in indicated tissues was determined by plaque assay after sacrifice of animals at indicated time points (<i>n</i> = 3 animals/time point).</p> <p>(B) Immunohistochemistry staining for vaccinia coat proteins in tumor tissue sections of animals treated as above and sacrificed 24 h after viral treatment (magnification 40×).</p> <p>(C) Survival of immunocompetent mice (C57/B6) bearing subcutaneous CMT-93 tumors and treated when tumors reached 50–100 mm³ with a single intratumoral (left) or intravenous (right) injection of <i>WR-delB18R</i> or PBS. Doses of 1 × 10⁴ (circles), 1 × 10⁶ (diamonds), or 1 × 10⁸ (triangles) PFU of virus or PBS controls (squares) were used (<i>n</i> = 5 mice/group; <i>p</i> = 0.0047 for intravenous injections).</p></div

Effect of Type-I IFN on Replication of Vaccinia Strains in Tumor and Normal Cell Lines

<div><p>(A) Primary human cell lines (SAECs and NHBEs) were grown to 50% confluence in six-well plates and treated with human IFN-α (50 U/ml) either 24 h prior to or 5 h after infection with vaccinia (or else PBS was used as a control). Vaccinia strains WR (white bars) or <i>WR-delB18R</i> (WR with deletion of the <i>B18R</i> gene; black bars) were used at an MOI of 1.0 PFU/cell. After 72 h, viral titers in the wells were determined by plaque assay (Student <i>t</i>-test for WR versus <i>WR-delB18R</i> with IFN treatment postinfection, <i>p</i> = 0.0055 for SAECs and 0.0012 for NHBEs).</p> <p>(B) This assay was repeated using human tumor cell lines C33A, A2780, and HCT 116.</p> <p>(C) Serial dilutions of vaccinia strains were added to cells (NHBE or A2780) 5 h before addition of human IFN-alpha (or PBS) as before. Cell viability in the different wells was determined 72 h later by MTS assay, and EC50 values (viral PFU/ml required to reduce cell viability to 50% of untreated well) were determined.</p> <p>An asterisk (*) indicates significant difference (<i>p</i> = 0.0055 for SAECs and 0.0012 for NHBEs).</p></div

In Vitro Testing of <i>JX-795</i> (Western Reserve Vaccinia Virus Containing Deletions of <i>B18R</i> and <i>Thymidine Kinase</i> Genes and Expressing Luciferase and mIFN-β)

<div><p>(A) mIFN-β production following infection of human A2780 cells with <i>JX-795</i> at an MOI of 5.0. IFN-β secretion into the medium during each hour postinfection (up to 14 h) was determined by ELISA.</p> <p>(B) Replication of <i>JX-795</i> (solid symbols and lines; ΔB18RΔTK-IFNb) and an equivalent virus (<i>B18R</i>- and <i>TK</i>-deleted, expressing luciferase only; Δ18ΔTK: open symbols; dashed lines) following infection of murine tumor (CMT-64 [squares] and JC [diamonds]) or nontransformed (NIH 3T3; triangles) cells at an MOI of 1.0.</p> <p>(C) Viral gene expression (luciferase) as measured by bioluminescence 24 h after infection of indicated cell lines with indicated viruses at an MOI of 1.0, and quantified (average of three experiments).</p></div

In Vivo Testing, Biodistribution, and Efficacy of <i>JX-795</i>

<div><p>(A) mIFN-β levels were determined by ELISA in the serum, and the tumors of animals (BALB/c bearing subcutaneous JC tumors) treated with (1 × 10⁸ PFU) <i>JX-795</i> (ΔB18RΔTK-IFNb), ΔB18RΔTK virus, or PBS 72 h earlier (<i>n</i> = 3/group).</p> <p>(B) Biodistribution of viral gene expression (as determined by bioluminescence imaging of mice treated with viruses expressing luciferase). Mice (BALB/c) bearing subcutaneous JC tumors (arrows) were treated with a single tail vein injection of 1 × 10⁸ PFU of viruses, <i>TK</i>-deleted (ΔTK-luc; squares), ΔB18RΔTK-luc (triangles), or ΔB18RΔTK-luc-IFNb (<i>JX-795</i>; circles), and imaging performed after luciferin addition. Representative images were taken at 72 h post-treatment. Bioluminescence was quantified over the tumor and the torso of the treated animals (<i>n</i> = 3/group) at times post-treatment (middle and bottom right-hand panels; symbols are for both graphs).</p> <p>(C) Survival curves of BALB/c mice bearing subcutaneous JC tumors and treated with a single tail vein injection of 1 × 10⁸ PFU of ΔB18RΔTK virus (diamonds), ΔB18RΔTK-IFNb (<i>JX-795</i>; triangles) or PBS (squares) (<i>p</i> = 0.028 for ΔB18RΔTK relative to ΔB18RΔTK-IFNb).</p></div

Infection of Primary Tumor and Normal Explant Tissues with <i>B18R-</i> and <i>TK</i>- Deleted Virus Expressing GFP

<p>Three tissue samples (top: rectal tumor with normal rectal tissue; middle: colon tumor metastases in liver; and bottom: endometrial tumor [no normal tissue available]:) are represented. Tumor and normal tissue were mixed with virus, washed, and images taken 48 h later.</p

Potential Mechanisms of Tumor Cell Killing Employed by <i>WR-delB18R</i>

<div><p>(A) Immunocompetent (C57/B6) mice bearing subcutaneous CMT-93 tumors and treated with a single tail vein injection of 1 × 10⁸ PFU of either psoralen-UV–inactivated WR (IAVV), WR, or <i>WR-delB18R</i> were sacrificed 7 d post-treatment, and CD4-positive cells in tumor sections were scored blind by a pathologist. Average numbers of positive cells per high-powered field (40×) from an average of ten randomly chosen fields from each of three mice treated under each condition are represented (<i>p</i> = 0.035 for IAVV compared to <i>WR-delB18R</i> treated). An asterisk (*) indicates significantly different counts.</p> <p>(B) Mice treated as in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040353#pmed-0040353-g003" target="_blank">Figure 3</a>C and displaying complete responses were rechallenged with a subcutaneous injection of 5 × 10⁵ CMT-93 cells. Tumor burden was measured by calipers (<i>n</i> = 8 mice).</p> <p>(C) Mice (C57/B6 bearing subcutaneous CMT-93 tumors) were treated with a single tail vein injection of 1 × 10⁸ PFU of <i>WR-delB18R</i> virus and sacrificed after 24 h. Tumor sections stained for viral coat proteins indicated initial infection of tumor vascular endothelial cells (top), whereas tumors from mice treated as above, but with <i>WR-delB18R</i> expressing GFP (WR-ΔB18RΔTK-GFP), were examined by immunofluorescence following staining with PE-conjugated antibody targeting CD31 (endothelial cells) (bottom; green = viral GFP expression; red = endothelial cell CD31 staining).</p> <p>(D) Vascular collapse in tumors of mice treated with <i>WR-delB18R</i>. Subcutaneous CMT-93 tumors implanted into C57/B6 mice were examined by ultrasound immediately prior to (left) and 48 h after (right) intravenous treatment with 1 × 10⁸ PFU <i>WR-delB18R</i>. A nonspecific contrast agent was delivered intravenously during ultrasound data acquisition in order to detect tumor vasculature (green); the border of the tumor was manually delineated (red).</p></div