Non-specific immunostimulating agents
A use, virus envelope technology, applied in the direction of vaccines, antifungal agents, antiviral agents, etc.
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Embodiment 1
[0112] Example 1 (preparative example): Reagents used in the preparation and working examples
[0113] Reagent: octaethylene glycol-mono-(n-dodecyl) ether (OEG, C 12 E. 8 ) was purchased from Fluka Chemie GmbH - (Buchs, Switzerland). Sucrose (Eur. Phar.) was purchased from Merck (Dietikon, Switzerland). Egg phosphatidylcholine (PC) was obtained from Lipoid (Cham, Switzerland). 1-Oleoyl-3-palmitoyl-rac-glycero-2-phosphoethanolamine was obtained from Bachem (Bubendorf, Switzerland). Bio-Beads SM2 were purchased from Bio-Rad Laboratories (Glattbrugg, Switzerland). Cholesteryl N(trimethylammoniumethyl)carbamate chloride (TC-chol) was purchased from Merck Eprova (Schaffhausen, Switzerland).
[0114] Influenza virus A / Singapore / 6 / 86 (A / Sing) strain and other influenza A strains proliferate in the allantoic cavity of embryonated eggs (Gerhard, W. (1976), Journal of Experimental Medicine 144:985 -995), obtained from Berna Biotech AG (Bern, Switzerland), purified as described b...
Embodiment 2
[0115] Example 2 (preparation example): Preparation of standard virus particles as influenza virus particles
[0116] For a final volume of 4 ml, 32 mg of egg PC and 8 mg of OPPE were dissolved in 3 ml of PBS, 100 mM OEG (PBS / OEG). 2 mg of HA of inactivated influenza A / Singapore / 6 / 86 virus or another influenza A strain was centrifuged at 100,000 xg for 1 hour at 4°C and the pellet was dissolved in 1 ml of PBS / OEG. Detergent-dissolved phospholipids and virus were mixed and sonicated for 1 min. This mixture was centrifuged at 100,000 xg for 1 hour at 18°C. Virus particles were formed by removing the detergent using two 1.5 g moistened SM2 Bio-Beads shaking each for 1 hour at room temperature. Newly formed virus particles were then sterile filtered (0.22 μm).
Embodiment 3
[0117] Example 3 (Preparation Example): Preparation of Virus Particles Containing TC-Chol
[0118] Virus particles containing TC-Chol were prepared by a detergent removal method. For a final volume of 4 ml, 32 mg of egg PC and 8 mg of OPPE and 5 mg of cholestenyl N-(trimethylammoniumethyl)carbamate chloride (TC-Chol) were dissolved in 2.6 ml of PBS, 100 mM OEG (PBS / OEG). 2 mg of HA of inactivated influenza A / Singapore / 6 / 86 virus or another influenza A strain was centrifuged at 100,000 xg for 1 hour at 4°C and the pellet was dissolved in 1 ml of PBS / OEG. Detergent-dissolved phospholipids and virus were mixed with 0.4 ml of 50% (w / v) sucrose and sonicated for 1 min. The mixture was centrifuged at 100,000 xg for 1 hour at 18°C. Virus particles were formed by removing the detergent using two 1.5 g moistened SM2 Bio-Beads shaking each for 1 hour at room temperature. Newly formed virus particles were then sterile filtered (0.22 μm) and aliquoted in sterile glass vials. The c...
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