Journal: bioRxiv

Article Title: A tale of two fusion proteins: understanding the metastability of human respiratory syncytial virus and metapneumovirus and implications for rational design of uncleaved prefusion-closed trimers

doi: 10.1101/2024.03.07.583986

Figure Lengend Snippet: Biopanning of a phage scFv library prepared from PBMCs of ten health adult individuals using the RSF UFC R1 -P2-NQ trimer as a probe was performed to isolate RSV (and potentially hMPV) neutralizing antibodies. ( a ) Phage ELISA was performed to assess randomly picked phage clones for their ability to bind an RSV prefusion F trimer, sc9-10 DS-Cav1, which has a disulfide bond-locked “closed” conformation. The 36 scFv clones that were sequenced are shown in red bars. Of these, 16 scFv clones with complete variable sequences are labeled with solid purple circles and 8 representative scFv clones with red diamond symbols. ( b ) Amino acid sequences of heavy and light chains from 8 representative scFv clones that have complete variable regions from sequencing. A sequencing error was manually corrected for the first amino acid of IXL-F1 HC, which is shown in red. ( c ) Gene family analysis of the phage library-derived antibodies and their yield in transient expression of HEK293 F cells. Key antibody features are summarized in the table. Notably, the scFv gene was cloned into an Fc vector for expression, and as such, the antibodies tested in the initial characterization were in the scFv-Fc form. ( d ) ELISA binding of eight phage library-derived antibodies, in scFv-Fc and some in IgG forms, tested against RSV-F (sc9-10 DS-Cav1) and hMPV-F (UFC M1 -P2-iSS) antigens. The EC 50 values are plotted to facilitate comparison between different clones, while the binding curves are shown below the EC 50 plot. ( f ) Neutralization of eight phage library-derived antibodies, in scFv-Fc and some in IgG forms, tested against live RSV-A2-GFP and hMPV-GFP viruses. The ID 50 values are plotted to facilitate comparison between different clones, while the neutralization curves are shown below. For ( f )-( h ), A total of five libraries, including the pre-panning library and the library after each of the four biopanning steps, were processed into heavy (HC), kappa (KC), and lambda chain (LC) libraries, which were deep sequenced on an Ion GeneStudio S5 sequencer for detailed antibodyomics analysis. ( f ) Pipeline processing of deep sequencing data obtained from the five scFv libraries. ( g ) Quantitative library profiles, including full germline gene usage profiles for the scFv libraries before and after each of the four panning steps (top three panels) and kappa chain-specific profiles for the scFv libraries before and after each of the four panning steps, including germline gene usage (gene families shown), germline divergence (or somatic hypermutation, SHM), and KCDR3 loop length (bottom panel). ( h ) Divergence-identity analysis of seven phage library-derived antibodies (IXL-D1, -E3, -F3, -D12, -E5, -F1, and -H9) within the scFv library before and after each of the four panning steps. To simplify the labeling, the prefix in the clone name “IXL” is not shown for any of the 2D plots. HC and LC/KC sequences are plotted as a function of sequence identity to a given template antibody and sequence divergence from putative germline V genes. Color coding indicates sequence density. On the 2D plots, reference antibodies are shown as black dots, whereas NGS-derived somatic variants that were identified based on the germline V gene match, CDR3 length (with ≤ 1-residue error of margin), CDR3 identity of 95% or greater are shown as orange dots, with the number of sequences and percentage within the germline gene family labeled accordingly.

Article Snippet: Peripheral blood mononuclear cells (PBMC) from 10 healthy donors were purchased from iXCells Biotechnologies (San Diego, CA) and used for scFv library construction following our previously described method ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Labeling, Sequencing, Derivative Assay, Expressing, Plasmid Preparation, Binding Assay, Comparison, Neutralization, Residue

Journal:

Article Title: Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses

doi: 10.1046/j.1365-2567.2003.01632.x

Figure Lengend Snippet: Apoptotic cell death and costimulation induce proliferation of allogeneic lymphocytes. NR and apoptotic SOM 157d cells expressing CD80 or not were cocultured with PBMC from healthy donors in the presence (a) or absence (b) of DCs for 5 days and subsequent thymidine incorporation was determined (c.p.m.). Results show a representative of three independent experiments where bars represent the mean (± SD) of quadruplicates (NR = non-replicating). Percentage apoptotic cells after MMC treatment < 5% and after irradiation treatment > 40%.

Article Snippet: Separation of peripheral blood mononuclear cells from whole blood and DC generation Peripheral blood was obtained from healthy donors or from patients with uveal melanoma into heparin, and peripheral blood mononuclear cells (PBMC) isolated by standard gradient centrifugation in Lymphoprep (Nycomed, Pharmaceuticals Buckinghamshire, UK).

Techniques: Expressing, Irradiation

Journal:

Article Title: Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses

doi: 10.1046/j.1365-2567.2003.01632.x

Figure Lengend Snippet: Apoptotic tumour cells expressing CD80 promotes T cell activation and IFN-γ production. Melanoma cells were cocultured in the presence of DCs (a), or with PBMC alone (not shown) for a period of 5 days and lymphocyte activation was detected by flow cytometry [apoptotic (AP) and non-replicating (NR)]. ELISA-determined expression levels of IFN-γ (b). Lymphocyte activation results are represented as a mean of two donors ± SD. IFN-γ ELISA results represent the mean ± SD from two donors (each of which was assayed in duplicate).

Article Snippet: Separation of peripheral blood mononuclear cells from whole blood and DC generation Peripheral blood was obtained from healthy donors or from patients with uveal melanoma into heparin, and peripheral blood mononuclear cells (PBMC) isolated by standard gradient centrifugation in Lymphoprep (Nycomed, Pharmaceuticals Buckinghamshire, UK).

Techniques: Expressing, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay