Pulmonary Delivery of Virosome-Bound Antigen Enhances Antigen-Specific CD4 + T Cell Proliferation Compared to Liposome-Bound or Soluble Antigen pictureRebecca AM Blom 1,2,3 , pictureMario Amacker 4 , pictureR. Maarten van Dijk 5 , pictureChristian Moser 6 , picturePhilip A. Stumbles 7,8 , pictureFabian Blank 1,2 * † and pictureChristophe von Garnier 1,2 † 1 Department of Pulmonary Medicine, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland 2 Department of Clinical Research, University of Bern, Bern, Switzerland 3 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland 4 Mymetics SA, Epalinges, Switzerland 5 Institute of Anatomy, University of Zürich, Zürich, Switzerland 6 Swiss Federal Institute of Intellectual Property, Bern, Switzerland 7 School of Veterinary and Life Sciences, Medical and Molecular Sciences, Murdoch University, Perth, WA, Australia 8 Telethon Kids Institute, Perth, WA, Australia
Immune modulation in the lung may represent a direct approach for treating respiratory disorders such as allergic asthma ( 1 , 2 ), given that the respiratory tract is readily accessible, making it an ideal target for non-invasive treatments. A dense network of dendritic cells (DCs) ensures mucosal uptake and transport of antigen to lymph nodes for presentation and activation of T cells, as previous studies showed that free antigen is insufficient to induce a strong immune reaction in the respiratory tract ( 3 , 4 ). In recent years, biomedical nanoparticles for targeted delivery of antigen for vaccinations have been developed ( 5 - 7), but to date there is insufficient understanding on how such nanoparticles interact with immune cells in the lung.
Influenza virosomes were prepared as follows. In short: per ml of final formulation, 8 mg of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine; Merck, Darmstadt, Germany
The incorporation of lipids and adjuvant into the virosomes was analyzed by thin-layer chromatography (TLC). The results of the TLC analysis confirmed that DOPC, DOPE and 3D-PHAD® were present in the virosome preparation (Fig. ?(Fig.3c).3c). To roughly quantify the amount of incorporated lipids and adjuvant, the intensities of the spots observed after TLC analysis of the virosomal lipids, shown in Fig. ?Fig.3c,3c, were determined using ImageJ analysis. The relative recoveries of DOPE and DOPC in the virosomes were essentially equal and represented approximately half of the amounts that were initially added, indicating a somewhat lower recovery than that of the viral protein (64%). However, the total quantity of virosome-associated adjuvant 3D-PHAD® was found to be less than 10% of the amount initially added. It therefore appeared that 3D-PHAD® was specifically lost during the production process.
and with synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; Merck & Cie, Switzerland). The concentrated virosome intermediate mixture with the integrated PfCyRPA protein was obtained after removal of OEG on polystryrene beads (Bio-Beads; Bio-Rad, Switzerland) in a batch chromatography.
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