Journal: PLoS ONE

Article Title: Improved NYVAC-Based Vaccine Vectors

doi: 10.1371/journal.pone.0025674

Figure Lengend Snippet: A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.

Article Snippet: 3×10 6 BSC-40 (ATCC) cells were infected with an MOI of 0.01 of NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm using Lipofectamine (Invitrogen, San Diego, CA).

Techniques: Plasmid Preparation, Clone Assay, Infection, Transfection, Sonication, Recombinant, Mutagenesis, Purification, Fluorescence, Expressing, SDS Page, Western Blot, Staining

Journal: PLoS ONE

Article Title: Improved NYVAC-Based Vaccine Vectors

doi: 10.1371/journal.pone.0025674

Figure Lengend Snippet: Five groups of mice (n = 6) received an IP inoculation of 2×10 7 PFU/mouse of WR (TK − ), NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At different times post-inoculation (24, 48 and 72 hours) animals were sacrificed and different mouse tissues (peritoneal cells, ovaries, livers, spleens and draining lymph nodes) were harvested and processed for titration as described in . The production of infectious virus was determined by plaque assay in BSC-40 cells. The virus titer was expressed as Plaque Forming Units (pfu) per gram of protein.

Article Snippet: 3×10 6 BSC-40 (ATCC) cells were infected with an MOI of 0.01 of NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm using Lipofectamine (Invitrogen, San Diego, CA).

Techniques: Titration, Plaque Assay

Journal: The Journal of General Virology

Article Title: Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system

doi: 10.1099/jgv.0.001624

Figure Lengend Snippet: Recombinant virus containing hTEE-658. ( a ) Schematic outlining the use of homologous recombination to insert the firefly luciferase gene, either with or without hTEE-658 immediately upstream, into the TK locus of the NYVAC strain of VACV. In this strategy, the coding region for GFP is replaced with the inserted sequence of interest to aid in the detection of proper insertion. ( b ) Luciferase expression following infection of BSC40 cells with recombinant NYVAC. Luciferase assay results were made relative those when hTEE-658 was not included upstream of the luciferase coding region. Expression experiments were conducted in duplicate, with error reported as standard deviation.

Article Snippet: HeLa and BSC40 cells were obtained from American Type Culture Collection (ATCC).

Techniques: Recombinant, Homologous Recombination, Luciferase, Sequencing, Expressing, Infection, Standard Deviation