Journal: Journal of Virology

Article Title: Development of a Primary Human Cell Model for the Study of Human Cytomegalovirus Replication and Spread within Salivary Epithelium

doi: 10.1128/JVI.01608-18

Figure Lengend Snippet: Human salivary cells infected with HCMV-TB40E are able to support all phases of HCMV lytic gene expression and release nascent virus into the culture medium. (A) Human parotid gland-derived (left) or submandibular gland-derived (right) salivary epithelial cells were infected with TB40E at an MOI of 1, and cell extracts were prepared at the indicated time points. Replicate Western blots prepared with these infected cell extracts were then probed with anti-CMV IE1/IE2 (Chemicon), anti-UL44 (12G5), anti-pp65 (Virusys), and β-actin antibody (Bethyl Laboratories). (B) Human parotid gland-derived (left) or submandibular gland-derived (right) salivary epithelial cells were infected with TB40E at an MOI of 0.1, the culture supernatant was isolated at the indicated times postinfection, and the titers were subsequently determined on HS68 fibroblasts. The growth curves depicted in panel B are representative of data from multiple independent experiments, each performed in duplicate.

Article Snippet: Western blots were probed with the following primary antibodies: anti-IE1/IE2 (Chemicon), anti-UL44 (kind gift of John Shanley), anti-pp65 (Virusys Corporation), and cellular β-actin antibody (Bethyl Laboratories).

Techniques: Infection, Gene Expression, Virus, Derivative Assay, Western Blot, Isolation

Journal: PLoS ONE

Article Title: Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

doi: 10.1371/journal.pone.0000415

Figure Lengend Snippet: (A) Schematic outline of Gag/Δpp65 before and after in vivo assembly into chimeric yeast VLPs. (B) SDS-PAGE and anti-pp65 immunoblot of crude extracts from yeast expressing either Δpp65 (lane 1), Gag (lane 2), or Gag/Δpp65 (lane 3). To ensure in vivo translation initiation of N-terminally truncated Δpp65 (24.9 kDa), a methionine residue was added to the N-terminus of Δpp65 [M, prestained PAGE Ruler, Fermentas].

Article Snippet: Blots were probed with monoclonal anti-pp65 (Novocastra), anti-T7 (Novagen), anti-GFP (Roche) or polyclonal antibodies raised in rabbit against native Gag-VLPs (anti-Gag) or chimeric Gag/K28α particles (anti-Gag/K28α) followed by treatment with an alkaline phosphatase-coupled secondary anti-mouse immunoglobulin (Sigma).

Techniques: In Vivo, SDS Page, Western Blot, Expressing, Residue

Journal: PLoS ONE

Article Title: Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

doi: 10.1371/journal.pone.0000415

Figure Lengend Snippet: (A) Western analysis of chimeric Gag/Δpp65 particles and natural L-A virions assembled in yeast and purified by ultracentrifugation through a linear sucrose gradient. Aliquots of each gradient fraction were separated by SDS-PAGE and probed with monoclonal anti-pp65 and polyclonal anti-Gag, respectively [GP, gradient pellet; M, full range rainbow marker, Amersham]. (B) Electron micrograph of sucrose-gradient-purified Gag/Δpp65 after negative staining with uranyl acetate/methyl cellulose [magnification 150,000; arrows indicate Gag/Δpp65 particles].

Article Snippet: Blots were probed with monoclonal anti-pp65 (Novocastra), anti-T7 (Novagen), anti-GFP (Roche) or polyclonal antibodies raised in rabbit against native Gag-VLPs (anti-Gag) or chimeric Gag/K28α particles (anti-Gag/K28α) followed by treatment with an alkaline phosphatase-coupled secondary anti-mouse immunoglobulin (Sigma).

Techniques: Western Blot, Purification, SDS Page, Marker, Negative Staining

Journal: PLoS ONE

Article Title: Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

doi: 10.1371/journal.pone.0000415

Figure Lengend Snippet: (A) Western blot of Gag/Δpp65 (lane 1) and Gag (lane 2) expressed in yeast and partially purified as sucrose cushion pellet after ultracentrifugation. Aliquots (20 µl each) of the indicated VLP preparation were subjected to SDS-PAGE followed by Coomassie-Blue staining and western analysis probed with anti-Gag and/or anti-pp65 [M, full range rainbow marker, Amersham]. (B) Frequencies of antigen-specific CD4 and (C) CD8 T cell activation after stimulation by sucrose cushion-purified yeast Gag and Gag/Δpp65 particles. Activated T cells were identified as CD69/IFN-γ double-positive lymphocytes by flow cytometry. Antigen samples were added to whole blood from HCMV seropositive donor 1. A VLP-free sample containing PBSE buffer was included as negative control (NC), a lysate from HCMV-infected fibroblasts served as positive control (PC). The threshold of significant T cell responses (0.05% of counted lymphocytes ) is indicated as dashed line.

Article Snippet: Blots were probed with monoclonal anti-pp65 (Novocastra), anti-T7 (Novagen), anti-GFP (Roche) or polyclonal antibodies raised in rabbit against native Gag-VLPs (anti-Gag) or chimeric Gag/K28α particles (anti-Gag/K28α) followed by treatment with an alkaline phosphatase-coupled secondary anti-mouse immunoglobulin (Sigma).

Techniques: Western Blot, Purification, SDS Page, Staining, Marker, Activation Assay, Flow Cytometry, Negative Control, Infection, Positive Control

Journal: PLoS ONE

Article Title: Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

doi: 10.1371/journal.pone.0000415

Figure Lengend Snippet: (A) SDS-PAGE and anti-pp65 immunoblot of sucrose gradient purified Gag and Gag/Δpp65 particles expressed and assembled in yeast [Coomassie-Blue staining and BSA (2.7 µg) were used for semi-quantitative signal detection; M, full range rainbow marker, Amersham]. (B and C) Whole blood cells from three HCMV seropositive donors were stimulated by the addition of either Gag or Gag/Δpp65 (5 µg each), and specifically activated CD4 (B) and CD8 (C) T cells were quantified as CD69/IFN-γ double-positive lymphocytes by flow cytometry. A lysate from HCMV-infected fibroblasts served as positive control (PC), whereas a lysate from noninfected fibroblasts, a VLP-free buffer sample as well as blood cells from HCMV seronegative donor 5 were used as negative controls (NC). The threshold of significant T cell responses (0.05% of counted lymphocytes ) is indicated as dashed line.

Article Snippet: Blots were probed with monoclonal anti-pp65 (Novocastra), anti-T7 (Novagen), anti-GFP (Roche) or polyclonal antibodies raised in rabbit against native Gag-VLPs (anti-Gag) or chimeric Gag/K28α particles (anti-Gag/K28α) followed by treatment with an alkaline phosphatase-coupled secondary anti-mouse immunoglobulin (Sigma).

Techniques: SDS Page, Western Blot, Purification, Staining, Marker, Flow Cytometry, Infection, Positive Control