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Order Rowavirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Concerning genetic modifications in the hexon protein, Di et al. (2012) proposed a method for the rapid generation of the adenovirus vectors with the chimeric hexons. As a result, the targeting NGR, RGD or Tat PTD peptides were inserted into the hexon HVR5, and the transduction efficiency of the Tat PTD-modified virions was significantly enhanced in the specific cell lines.
Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying a bispecific T cell engager against hepatocellular carcinoma
Published in OncoImmunology, 2023
Xiangfei Yuan, Yang Lu, Yuanyuan Yang, Wencong Tian, Dongmei Fan, Ruoqi Liu, Xiaomin Lei, Yafei Xia, Lei Yang, Shu Yan, Dongsheng Xiong
The tumor tissues of orthotopic hepatocarcinoma models treated with adenovirus-armed HUMSCs for 4 days were cut into paraffin-embedded sections for immunofluorescence to explore the adenovirus delivery by HUMSCs. The sections were stained with mouse anti-Hexon primary antibody (#GTX36896, GeneTex, USA) and AF488-conjugated goat anti-mouse secondary antibody (#ab150113, Abcam, UK) to detect the adenovirus hexon protein. Meanwhile, rabbit anti-human CD90 primary antibody (#ab226123, Abcam, UK) and AF647-conjugated goat anti-rabbit secondary antibody (#ab150079, Abcam, UK) were used to indicate HUMSCs. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; #D9564, Sigma, USA). The images were captured using a two-photon laser scanning confocal microscope (FV1200 MPE, Olympus, Japan). Similarly, the tumor tissues of model mice treated with MSC.Ad and PBMC for 16 days were cut into paraffin-embedded sections and then stained with mouse anti-Hexon primary antibody, AF488-conjugated goat anti-mouse secondary antibody, rabbit anti-His tag primary antibody (#ab213204, Abcam, UK), and AF647-conjugated goat anti-rabbit secondary antibody to indicate hexon and αCD3HAC.
A phase 1 trial extension to assess immunologic efficacy and safety of prime-boost vaccination with VXM01, an oral T cell vaccine against VEGFR2, in patients with advanced pancreatic cancer
Published in OncoImmunology, 2018
Friedrich H. Schmitz-Winnenthal, Nicolas Hohmann, Thomas Schmidt, Lilli Podola, Tobias Friedrich, Heinz Lubenau, Marco Springer, Sébastien Wieckowski, Klaus M. Breiner, Gerd Mikus, Markus W. Büchler, Anne-Valerie Keller, Ruhan Koc, Christoph Springfeld, Phillip Knebel, Mariana Bucur, Lars Grenacher, Walter E. Haefeli, Philipp Beckhove
The quantitative evaluation of antigen-specific T cells secreting IFNγ was conducted as described previously41 ELIspot plates (MAHA S4510; Millipore, Ireland) were coated with 1 µg of anti-IFNγ antibody (clone 1-D1K; Mabtech, Nackastrand, Sweden) per well, incubated overnight at 4 °C, washed and blocked with complete RPMI medium (10% human AB serum). 2 × 104 dendritic cells were plated in 100 µL per well in triplicate, and loaded with 10 µg/mL of antigenic peptides. A pool of 20-mer synthetic peptides #1-135 derived from human VEGFR2 with 10 amino acids overlap (omitting #21+22+76+77) (Proimmune, Oxford, UK) was used as test peptides. A pool of 15-mer synthetic peptides covering the complete sequence of pp65 protein (CMV pp56 PepTivator) with 11 amino acids overlap, mixed with a pool of 15-mer synthetic peptides covering the complete sequence of the hexon protein of human adenovirus 5 with 11 amino acids overlap (AdV5 Hexon PepTivator), as well as SEB (Staphylococcus enterotoxin B), was used as a positive control. Human Normal Immunoglobulin (KIOVIG, Baxter) was used as a negative control. After 14 h of antigen pulsing, 1 × 105 purified T cells (DynaBeads) were added to each well for an additional 40-h co-culture. IFNγ spots were developed using an enzyme-coupled detection antibody system using an anti-IFNγ antibody coupled to biotin (clone 7_B6-1 diluted 1:1000, Mabtech), streptavidin ALP (diluted 1:1000, Mabtech), and NCT/BCIP substrate kit (BioRad, Munich, Germany). Plates were analyzed using an automated ELIspot reader and ImmunoSpot V 5.0.9 Smart Count Software (CTL, Bonn, Germany) using the following spot definition settings: Size: 100%; Size Count: normal; Maximum Spot Size: 9.6250 mm2; Minimum Spot size 0.0051 mm2; Spot Separation: 3; Diffuse Processing: normal; Background Balance: 0–80.
Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery
Published in Journal of Extracellular Vesicles, 2020
Heikki Saari, Tiia Turunen, Andres Lõhmus, Mikko Turunen, Matti Jalasvuori, Sarah J. Butcher, Seppo Ylä-Herttuala, Tapani Viitala, Vincenzo Cerullo, Pia R. M. Siljander, Marjo Yliperttula
Further characterization of the viral cargo in IEVs is provided in Figures 9 and 10. For assessing individual Ad proteins, western blot analysis of the viral proteins in pooled IEV fractions (1–6) and cell lysates was performed with a polyclonal anti-Ad antibody (Figure 9(a,b)). In line with the dot blot analysis of the hexon protein, the amount of other viral proteins increased with time in the IEVs and in the cell lysates when compared to α-tubulin. In contrast to the cell lysates, the IEVs seemed to contain more of the large (>100 kDa) Ad protein bands corresponding to the hexon protein (108 kDa), while the cell lysates were more enriched in Ad proteins under 70 kDa, corresponding to Ad penton proteins (64 kDa) and protein V (42 kDa). The topological arrangement of Ad proteins on IEVs was also examined by a dotblot analysis with proteinase K (Figure 9(c,d)): samples from fractions 3–6 of time points D1, D3 and D5 were treated with proteinase K, cleaving any exposed proteins on the vesicle surface, followed by heat inactivation of the enzyme and dotblot analysis with the polyclonal anti-Ad antibody. Addition of Triton X-100 with proteinase K was used to completely cleave all proteins in the sample to estimate any non-specific binding caused by the presence of inactive proteinase K. Compared to samples without proteinase K treatment, the signal intensity dropped 20–100% in all samples, with the highest drops (approx. 75–100%) occurring in fractions 5 and 6, suggesting that Ad proteins are more exposed in these fractions, although fraction 4 usually had the highest overall intensity. Fractions 1 and 2 were also analysed, but they produced very weak signals that could not be analysed reliably. A control sample with purified free Ad virions resulted in complete eradication of signal as Ad has no lipid membrane to protect itself from the enzyme. PC-3 D1 samples also produced only a weak signal to begin with, as the non-specific binding of antibodies caused by the presence of proteinase K was stronger than the in the samples without treatment themselves (Figure 9(c)). As the proteinase K background signal in the PC-3 D1 samples was nearly identical to the detergent treated replicates, a complete removal of proteins was estimated. Overall, these results demonstrate that all of the IEV samples analysed contain Ad proteins that are exposed outside the vesicles.