Journal: iScience

Article Title: Novel adenovirus vaccine vectors lacking thrombosis-associated interactions with platelet factor 4

doi: 10.1016/j.isci.2025.114329

Figure Lengend Snippet: Identification of adenovirus and adeno-associated virus vectors lacking binding to PF4 by ELISA-qPCR and aggregate pull-down screening (A) Principle of the ELISA-qPCR technique. Adenovirus (Ad) vector particles (VPs) are allowed to interact with proteins, e.g., PF4, coated on an ELISA plate. After washing, the genomes of VPs that specifically interact with the proteins are released by heating and alkaline treatment and quantified by qPCR. Figure created with BioRender. (B) PF4 binding of vaccine-equivalent vectors. Ad5 was obtained from the Ad-GLN collection. N ≥ 6, two independent repeats. Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (C and D) Screening of human and gorilla Ad (C) and AAV (D) collections for PF4 binding by ELISA-qPCR. For each experiment repeat, the PF4 binding index is computed as described in (Statistics), with positive values indicating significant binding to PF4 and negative values corresponding to overlap in the number of bound VPs in PF4-coated versus control samples. Averages and minimum/maximum range of the PF4 binding index from two to four (Ad5, Ad11, Ad34, and Ad80) independent repeats are displayed. Data are represented as mean ± standard deviation. (E) ELISA-qPCR of Ad5 hexon genetic and chemical variants for PF4 binding. Studied variants include: D151C and T273C point mutations; a 5 kDa polyethylene-glycol (PEG) polymer covalently linked to a cysteine residue, which prevents binding on part of the hexon surface by steric competition; deletion of the HVR1 loop; and the T425A substitution known to ablate the binding of factor X. The E1-deleted, GFP-expressing Ad5 vector was used as control (Ad5). HVR: hyper-variable region. PEG: polyethylene glycol. Schematics created with BioRender. N = 6, two independent repeats. Data are represented as mean ± standard deviation. (F) ELISA-qPCR of selected Ads for binding to mouse PF4. The binding index is computed as described in (Statistics). Measurements were performed once with technical triplicates. Data are represented as mean. (G) Principle of the aggregate pull-down technique. Aggregates forming upon interaction with PF4 are separated from free VPs by low speed centrifugation and titrated by qPCR. Figure created with BioRender. (H) Aggregate pull-down of selected Ads in the absence or presence of PF4. N = 8, two independent repeats. The aggregation rate was calculated based on the titration of Ad genomes both in the pellet (aggregates) and in the supernatant (free VPs). Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001.

Article Snippet: Proteins of interest were diluted in coating buffer (0.1 M NaHCO 3 , pH set between 9.2 and 9.6 using 1 M Na 2 CO 3 ) to a concentration of 20 μg/mL and 75 μL were added per well of ELISA plate (Sarstedt #82.1581.200), which was sealed with a transparent film and incubated overnight at 4°C.

Techniques: Virus, Binding Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Standard Deviation, MANN-WHITNEY, Control, Polymer, Residue, Expressing, Centrifugation, Titration