Journal: Nature medicine

Article Title: Broad Antigenic Coverage Induced by Viral cDNA Library-based Vaccination Cures Established Tumors

doi: 10.1038/nm.2390

Figure Lengend Snippet: a. The ASEL VSV-expressed cDNA library contains cDNA from normal human prostate cloned into VSV in direct, or reverse, orientation. b. Human prostate specific genes – , but not the melanocyte-specific TRP-2 , detected by PCR in the original human prostate plasmid library (lane 1) and in the VSV-cDNA plasmid library (2). rtPCR from cDNA of HT1080 cells infected with ASEL (MOI 0.1) (3) compared to uninfected cells (4) (predicted size for hPSCA shown with an arrow). c. BHK cells infected with ASEL Direct (1) or Reverse (2), or with control viruses (lanes 3&4) (MOI ~10) assayed for human PSA by Western Blot. Lane 5, uninfected BHK cells; Lane 6, 10 4 human prostate LnCap cells. d. BHK cells infected with 10 fold dilutions of ASEL virus (1–6) assayed by rtPCR for PSA or human GAPDH. No PSA-specific signal was detected at dilutions lower than 1:100 of the original virus stock. (Expression of GFP from 100 pfu of VSV-GFP could be detected by this assay). +Positive/*negative for PCR upon nested PCR. e . Splenocytes infected with VSV-GFP or VSV-cDNA libraries from cells which did, or did not, express OVA (MOI 0.1), co-cultured with naive OT-I T cells and assayed for IFN-γ . Lane 1, splenocytes alone; (2–4), splenocytes infected with VSV-GFP, without OT-I (2), with OT-I (3) or with irrelevant T cells ; (5&6), splenocytes infected with VSV-cDNA library from B16ova cells in Direct (5) or Reverse (6) orientation with OT-I; (7&8), splenocytes infected with VSV-cDNA library from B16 cells (no OVA) in Direct (7) or Reverse (8) orientations with OT-I. (9), OT-I activated by SIINFEKL peptide. (10–13), splenocytes infected with VSV-ova at MOI 0.01 (10), 0.1 (11), 1.0 (12) and 10 (13). (14), OT-I (no splenocytes, no VSV).

Article Snippet: Murine B16ova melanoma cells (H2-K b ) were derived from B16 cells by transduction with a cDNA encoding the chicken ovalbumin gene . cDNA from normal human prostate (Biochain, Hayward, CA) was amplified from the BioExpress shuttle vector by PCR and cloned into the VSV genomic plasmid pVSV-XN2 between the G and L genes.

Techniques: cDNA Library Assay, Clone Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Infection, Control, Western Blot, Virus, Expressing, Nested PCR, Cell Culture

Journal: Nature medicine

Article Title: Broad Antigenic Coverage Induced by Viral cDNA Library-based Vaccination Cures Established Tumors

doi: 10.1038/nm.2390

Figure Lengend Snippet: a. Prostate weights 60d following 10 7 pfu i.v. of ASEL (n=5) or VSV-GFP (n=7). b. Mean levels of IL-17 secreted by splenocytes from these mice co-cultured with lysates of B16 melanoma, TC2 prostate, normal mouse prostate or pancreas. c. Survival of mice bearing 7d TC2 tumors (n=7–8) injected intra-tumorally or intravenously with 10 7 pfu of VSV-GFP, ASEL, or heat inactivated VSV-GFP (days 7,9,11). d,e . Survival of mice bearing 7d TC2 ( d ) or B16 ( e ) tumors injected intravenously with ASEL, or with a VSV-cDNA library from human melanoma cells (Altered Self Melanoma Epitope Library, ASMEL) (days 7,9,11). f. Cumulative percentages of mice cured of 7d TC2 tumors when administered 3, 6 or 9 injections of ASEL or VSV-GFP i.t. or i.v. every other day. g. IL-17 secreted by splenocytes from the three mice cured of TC2 tumors by 9 intra-tumoral injections of ASEL in f. , as well as from 3 mice treated similarly with VSV-GFP, co-cultured with lysates of B16, TC2, normal mouse prostate or pancreas. h. Survival of mice bearing 7d TC2 tumors (n=7/8), mock depleted, or depleted of CD4+, CD8+ or NK cells, injected intravenously with ASEL on days 7,9,11,14,16,18,21,23,25.

Article Snippet: Murine B16ova melanoma cells (H2-K b ) were derived from B16 cells by transduction with a cDNA encoding the chicken ovalbumin gene . cDNA from normal human prostate (Biochain, Hayward, CA) was amplified from the BioExpress shuttle vector by PCR and cloned into the VSV genomic plasmid pVSV-XN2 between the G and L genes.

Techniques: Cell Culture, Injection, cDNA Library Assay

Journal: Nature medicine

Article Title: Broad Antigenic Coverage Induced by Viral cDNA Library-based Vaccination Cures Established Tumors

doi: 10.1038/nm.2390

Figure Lengend Snippet: a. The IEEL contained cDNA from three TCR2 tumors cloned into VSV. b. Western blot for murine N-Cadherin from BHK cells infected with VSV (1), IEEL Reverse (2) or Direct (3) (MOI ~10). Equal loading confirmed by ß-actin probing. c. Survival of mock, or VSV-GFP,-vaccinated mice bearing 7d TC2 tumors treated with three i.v. injections of VSV-GFP or ASEL, either as viral supernatant (10 7 pfu) or as virus pre-loaded onto CD8+ T cells [T(ASEL)]. d. Mice bearing 7d TC2 tumors were treated i.v. with VSV-GFP or ASEL (days 7,9,11). On days 25,27,29 mice initially treated with ASEL received i.v. IEEL virus pre-loaded onto CD8+ T cells [T(IEEL)]. e. VSV-vaccinated mice bearing 7d TC2 tumors were injected intravenously with VSV-GFP, ASEL or IEEL (10 7 pfu) or with virus pre-loaded onto CD8+ T cells [T(ASEL)/T(IEEL)] (d 7,9,11). On days 20,22,24, surviving mice were treated i.v. with T cells loaded with IEEL [T(ASEL)/T(IEEL)] or ASEL [T(ASEL)/T(ASEL)]. f-i. Splenocytes from mice which either did ( f,g ) or did not ( h,i ) reject TC2/TC2R tumors following i.v. ASEL + T(IEEL) ( d ) or T(ASEL) + T(IEEL) ( e ) were co-cultured with lysates of TC2, TC2R, B16, normal mouse prostate or pancreas and assayed for ( f,g ) IL-17 or ( h,i ) IFN-γ. Results are from three survivor mice ( f,g ) or two mice which succumbed to TC2R tumors ( h,i ).

Article Snippet: Murine B16ova melanoma cells (H2-K b ) were derived from B16 cells by transduction with a cDNA encoding the chicken ovalbumin gene . cDNA from normal human prostate (Biochain, Hayward, CA) was amplified from the BioExpress shuttle vector by PCR and cloned into the VSV genomic plasmid pVSV-XN2 between the G and L genes.

Techniques: Clone Assay, Western Blot, Infection, Virus, Injection, Cell Culture