Journal: Molecular Therapy. Nucleic Acids

Article Title: Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

doi: 10.1038/mtna.2014.19

Figure Lengend Snippet: General scheme of aptamer-facilitated protection of oncolytic virus from neutralizing antibodies . ( a ) Anti–vesicular stomatitis virus neutralizing antibodies bind to the viruses and cause several effects: (i) aggregation, (ii) blocking attachment of the virus to the cell membrane, and (iii) preventing uncoating of the virus inside the cell. ( b ) Aptamers blocking the Fab fragments of antibodies and shielding aptamers binding to the virus prevent the neutralization of the virus, thus allowing it to infect the cell.

Article Snippet: Selected aptamer pools were then subjected to competitive binding analysis, where 200 nmol/l of aptamers were incubated with 10 ng/μl DyLight 488-conjugated anti-VSV antibodies (Rockland, Immunochemicals, Boyertown, PA) for 1 hour, followed by addition of 6 × 10 8 PFUs of VSV.

Techniques: Virus, Blocking Assay, Membrane, Binding Assay, Neutralization

Journal: Molecular Therapy. Nucleic Acids

Article Title: Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

doi: 10.1038/mtna.2014.19

Figure Lengend Snippet: Selection of DNA aptamers to VSV and VSV-nAbs . ( a ) Selection of aptamers against VSV with 11 rounds of selection: (i) four positive rounds of selection, (ii) three negative rounds of selection against blood cells, and (iii) four rounds of selection using a competitive approach with a 96-well plate. Binding of resulting pools was analyzed by flow cytometry using FAM-labeled aptamer pools. ( b ) Selection of aptamers aginst VSV-nAbs with 15 rounds of selection. Each positive selection was preceded with a negative selection: (i) five negative selections against magnetic beads (MB), (ii) five rounds of negative selection against non-VSV Abs, and (iii) five rounds of selection against MB and non-VSV Abs. Binding of resulting pools was analyzed by flow cytometry using FAM-labeled aptamer pools. nAb, neutralizing antibody; VSV, vesicular stomatitis virus.

Article Snippet: Selected aptamer pools were then subjected to competitive binding analysis, where 200 nmol/l of aptamers were incubated with 10 ng/μl DyLight 488-conjugated anti-VSV antibodies (Rockland, Immunochemicals, Boyertown, PA) for 1 hour, followed by addition of 6 × 10 8 PFUs of VSV.

Techniques: Selection, Binding Assay, Flow Cytometry, Labeling, Magnetic Beads, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

doi: 10.1038/mtna.2014.19

Figure Lengend Snippet: Flow cytometry binding and competitive analysis of aptamer pools . Flow cytometric analysis of competitive binding between FAM-labeled weak, moderate and strong aptamer pools (200 nmol/l) and VSV (1 × 10 7 PFU), followed by introduction of anti-VSV nAbs. Red curve represents virus alone, which was considered as a negative control. Green curve corresponds to the VSV binding with aptamer pools, or ssDNA library without anti-VSV nAbs. Blue and orange curves show the binding after incubation with anti-VSV nAbs (3.75 µg/ml or 2.5 mg/ml, respectively). nAb, neutralizing antibody; VSV, vesicular stomatitis virus.

Article Snippet: Selected aptamer pools were then subjected to competitive binding analysis, where 200 nmol/l of aptamers were incubated with 10 ng/μl DyLight 488-conjugated anti-VSV antibodies (Rockland, Immunochemicals, Boyertown, PA) for 1 hour, followed by addition of 6 × 10 8 PFUs of VSV.

Techniques: Flow Cytometry, Binding Assay, Labeling, Virus, Negative Control, Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

doi: 10.1038/mtna.2014.19

Figure Lengend Snippet: Cell-based viral infectivity assay . Neutralizing antibodies (nAbs) were preincubated with or without 1 µmol/l of anti-nAbs aptamers. The complex was then incubated with vesicular stomatitis virus (VSV) preincubated with anti-VSV aptamers and 100 PFUs were added to Vero cells in a 12-well plate. ( a ) Cells infected with VSV in the presence of nAbs (0% infectivity); ( b ) Cells infected with VSV without nAbs (100% infectivity); ( c ) Cells infected with VSV in the presence of nAbs, and anti-nAbs and anti-VSV aptamer pools; and ( d ) Cells infected with VSV in the presence of nAbs and DNA Library as a control experiment. Results of additional control experiments are presented in the adjusted table. All plaque forming assays were performed in triplicates.

Article Snippet: Selected aptamer pools were then subjected to competitive binding analysis, where 200 nmol/l of aptamers were incubated with 10 ng/μl DyLight 488-conjugated anti-VSV antibodies (Rockland, Immunochemicals, Boyertown, PA) for 1 hour, followed by addition of 6 × 10 8 PFUs of VSV.

Techniques: Infection, Incubation, Virus, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

doi: 10.1038/mtna.2014.19

Figure Lengend Snippet: Dimeric and tetrameric aptamers . ( a ) General scheme of construction of dimers and tetramers using bridge 1, or bridges 1 and 2, respectively. ( b ) Plaque forming assay results showing the infectivity of VSV using unmodified aptamer pools, dimer pools or tetramer pools for VSV, nAbs, or both VSV and nAbs. Plaque forming assays were performed in triplicates. nAb, neutralizing antibody; VSV, vesicular stomatitis virus.

Article Snippet: Selected aptamer pools were then subjected to competitive binding analysis, where 200 nmol/l of aptamers were incubated with 10 ng/μl DyLight 488-conjugated anti-VSV antibodies (Rockland, Immunochemicals, Boyertown, PA) for 1 hour, followed by addition of 6 × 10 8 PFUs of VSV.

Techniques: Infection, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

doi: 10.1038/mtna.2014.19

Figure Lengend Snippet: Fluorescence microscopy aggregation assay . Aptamers in monomeric, dimeric, and tetrameric form were incubated in equal amounts of VSV-YFP and VSV-RFP and used to infect a monolayer of Vero cells. ( a ) Overlay of YFP and RFP expression of cells infected with virus incubated (i) without the aptamers and (ii) with tetrameric aptamers. Cells expressing YFP (green), RFP (red), or both (orange) were counted. ( b ) Cells infected with virus alone, virus with monomeric, dimeric, and tetrameric aptamers showed coexpression of both YFP and RFP in 8, 3, 5, and 4% of all cells, respectively. VSV, vesicular stomatitis virus.

Article Snippet: Selected aptamer pools were then subjected to competitive binding analysis, where 200 nmol/l of aptamers were incubated with 10 ng/μl DyLight 488-conjugated anti-VSV antibodies (Rockland, Immunochemicals, Boyertown, PA) for 1 hour, followed by addition of 6 × 10 8 PFUs of VSV.

Techniques: Fluorescence, Microscopy, Incubation, Expressing, Infection, Virus