Liposome particle containing viral or bacterial antigenic subunit

#Attempts to reduce the side effects often encountered in administration of a vaccine have led to the use of a subunit antigen. Such subunit, however, is usually much less immunogenic than the whole intact antigenin starting material.

#It is an object of the present invention to increase the antigenicity of a subunit antigenic preparation. A further object is to provide a particle wherein the subunit is attached to a liposome. Still another object is to provide a method for preparing such a particle. These and other objects of the present invention will be apparent from the following description.

#A subunit viral or bacterial antigen is incorporated into a lipid which contains a positively charged amino-containing surfactant, or the subunit antigen is reacted with a positively charged amino-containing surfactant to form a complex which is incorporated into a lipid.

#The following examples illustrate the present invention without, however, limiting the same thereto. All temperatures are expressed in degrees Celsius.

#Dioleoylphosphatidylcholine (20 mg in 0.8 ml chloroform) and stearylamine (3 mg dry powder dissolved in 1-2 ml dry chloroform) are mixed together at room temperature for 1-2 minutes in a 50 ml round bottom or pear-shaped flask and then dried down rapidly in vacuo at 37.degree.. Immediately after drying, pure dry nitrogen gas is blown into the flask for 2-3 minutes. The influenza A Victoria subunit antigen at 750 .mu.g hemagglutinin (HA) units/ml in 3.5 ml of deionized distilled water or phosphate buffered saline (pH 7.2) is added slowly. Nitrogen gas is continuously, slowly bubbled through the solution which is placed in the center of a small (13.5 cm.times.13.5 cm) ultrasonic cleaner bath (Cole-Parmer) filled 6 cm 8 cm deep with water which contains 1 ml of Triton X-100 as detergent to lower the surface tension. The cleaner bath operates at 50-55 KHz and is maintained at 20.degree.-25.degree. by addition of ice. The solution is sonicated, with occasional swirling of the contents, for 2-8 minutes, typically for 7 minutes. After sonication the contents of the flask are transferred to a 6 ml serum bottle and capped with a rubber stopper. The sonicated material is stored at 2.degree.-4.degree..

#Stearylamine (3 mg) plus ethanol (0.4 ml) are heated to 55.degree.-60.degree. for 1-2 minutes until the stearylamine is completely soluble. Then deionized, distilled water (2.6 ml) which has been heated to 55.degree.-60.degree. is slowly added with swirling. At this stage the solution may be opalescent, but there should be no precipitate. Pure, dry nitrogen gas is bubbled through the solution for 5-10 minutes, typically 8 minutes, until the solution is cooled to 30.degree.-40.degree.. Then the influenza A Victoria subunit antigen at 750 .mu.g hemagglutinin (HA) units per ml at 25.degree.-30.degree. in 0.9 ml deionized, distilled water is slowly added and nitrogen gas bubbled through for a further 2-6 minutes, typically 4 minutes. No obvious precipitate should be present. Next, the antigenstearylamine preparation is slowly added to a dried down preparation of dioleoylphosphatidylcholine (20 mg) in a 50 ml round bottom glass flask. The solution is sonicated for 2-8 minutes, typically for 7 minutes, in a Cole-Parmer ultrasonic cleaner bath at 50-55 KHz while continuously bubbling nitrogen gas therethrough. The temperature of the cleaner bath is maintained at 20.degree.-25.degree. by the addition of ice. During sonication the contents of the flask are occasionally swirled. After sonication the contents of the flask are transferred to a 6 ml serum bottle, capped with a rubber stopper, and stored at 2.degree.-4.degree..