Journal: bioRxiv

Article Title: Bioactive Enhanced Adjuvant Chemokine Oligonucleotide Nanoparticles (BEACONs) for Mucosal Vaccination Against Genital Herpes

doi: 10.1101/2025.07.31.667899

Figure Lengend Snippet: (A) Schematic of preparation of DNA-chemokine nanoparticles (BEACONs). (B) Hydrodynamic diameter of BEACONs (mouse) as measured by dynamic light scattering. (C) Representative negative stain TEM image showing spherical morphology of BEACONs (mouse). Scale bar, 200 nm. (D) Measurement of CXCL9 functional activity via chemotaxis assay, quantifying the number of T cells migrated across the Transwell by measuring calcein AM fluorescence. (E) TLR9 signaling assessed by measuring fold change (relative to saline) in reporter activation in HEK-mouse-TLR9 reporter cells 48 hours after treatment with free CpG-ODNs, CXCL9, or BEACONs. (F) Frequency of CD86 + BMDCs after stimulation with BEACONs compared to controls (free CpG-ODNs, CXCL9, or medium-only).

Article Snippet: Mouse TL9 reporter assay: HEK-BlueTM mTLR9 cells (InvivoGen) were seeded at 3 × 10 4 cells per well in flat-bottom 96-well plates (Corning) for 24 hours and then treated with water (vehicle control), CXCL9, CpG-ODN, or BEACONs for 48 hours.

Techniques: Staining, Functional Assay, Activity Assay, Chemotaxis Assay, Fluorescence, Saline, Activation Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Nanofilament immunotherapy induces potent antitumor vaccine responses

doi: 10.1136/jitc-2024-011331

Figure Lengend Snippet: Key properties and validation of the TAT003 nanofilament. ( a ) Activation of tumor-associated macrophages (TAMs) after exposure to M13. Flow cytometric analysis of activation markers CD80 and CD86, and antigen presentation markers H2Kb and H2Db (representative data). ( b ) Left side: Schematic representation of TAT003’s key features. Right side: Mechanisms of immunogenicity, a TAT003 nanofilament activating a TAM, and, in the inset, a TAT003 nanofilament interacting simultaneously with a cancer cell, through the binding to PD-L1 via a displayed anti-PD-L1 scFv, and with a T cell, through the binding to IL-2 receptor (IL-2R) via a displayed mIL-2; resulting in the blockade of PD-1/PD-L1 interactions and stimulation of IL-2R. ( c ) Competitive binding flow cytometry assay on A20 mouse cells between an anti-PD-L1-PE and M13, TAT003ΔIL2, TAT003 or atezolizumab (representative data). The dotted line serves as a reference point for the evaluation of PD-L1 signal status. ( d ) Single confocal slices of A20 cells exposed to M13 or TAT003 for 15 min then incubated in Hoechst dye and anti-M13-FITC antibody conjugate. ( e ) Phagocytosis of TAT003 and TAT003-coated A20 cancer cells by bone-marrow-derived macrophages. ( d ) M13 and TAT003 were detected using an anti-M13-FITC antibody (green), and cell nuclei were stained using Hoechst dye (cyan) (representative images). Scale bars: 10 µm. ( e ) PD-1 blockade bioassay using engineered cells that output luminescence on PD-1/PD-L1 axis blockade. Dose response of cells was obtained with various concentrations of either TAT003∆IL2, TAT003 or atezolizumab as a positive control (technical duplicate). ( f ), Dose response of the HEK Blue-IL-2 cell line to various concentrations of human and mouse IL-2 compared with the signal obtained with a TAT003 dose equivalent to 0.1 ng/mL of mIL-2 molecules displayed on the bacteriophage (n=3). ( g ), Indirect ELISA to assess the presence of both anti-PD-L1 scFV and mIL-2 on the same bacteriophage particle (n=3). Measurement of TLR9 dose response activation using HEK Blue TLR9 cell lines for the murine ( h ) and human ( i ) versions of the receptor using commercial ODN as positive controls (n=2). ( j ), Cancer cell phagocytosis frequency evaluation. Bone marrow-derived macrophages were generated and put in contact with CFSE-stained A20 cancer cells and treated with either PBS or TAT003. The proportion of macrophages having phagocytized A20 cells corresponds to the number of CFSE+macrophages (n=4). APC, Allophycocyanin; CTL, cytotoxic T lymphocytes; FITC, Fluorescein isothiocyanate; hIL-2, human IL-2; IL-2, interleukin-2; mIL-2, mouse IL-2; ODN, oligodesoxynucleotides; PBS, phosphate-buffered saline; PD-L1, Programmed Death Ligand 1; PD1, Programmed Death protein 1; PE, phycoetrythrin; scFv, single-chain antibody fragment; TLR, toll-like receptor.

Article Snippet: HEK-Blue STAT5-inducible SEAP reporter cells, which are specifically designed to detect IL-2 (InvivoGen; hkb-il2) or TLR9 (InvivoGen; hkb-hTLR9 and hkb-mTLR9), were cultured under optimal conditions according to the manufacturer’s guidelines.

Techniques: Biomarker Discovery, Activation Assay, Immunopeptidomics, Binding Assay, Flow Cytometry, Incubation, Derivative Assay, Staining, Bioassay, Positive Control, Indirect ELISA, Generated, Saline