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: Journal of Translational Medicine

Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients

doi: 10.1186/1479-5876-11-152

Figure Lengend Snippet: Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

Article Snippet: Healthy donor derived peripheral blood mononuclear cells (PBMC) were isolated from HLA-A2 positive buffycoats (Sanquin, Amsterdam, The Netherlands) by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway).

Techniques: Ex Vivo, Staining, Derivative Assay, Modification, Flow Cytometry, Concentration Assay, Labeling, Adjuvant, Enzyme-linked Immunospot, Binding Assay, Negative Control