Abstract:
Influenza virus is extracted with a mixture of ether and ethanol, the aqueous and organic phases are separated and residual ether removed from the virus suspension under reduced pressure. The resulting product is useful as an influenza vaccine.

Description:
BACKGROUND OF THE INVENTION 
     Attempts have been made to detoxify influenza vaccines by disrupting the influenza virus with detergents, organic solvents or a combination of both. These attempts, however, were not entirely successful because of loss of immunogenic potency. 
     OBJECTS OF THE INVENTION 
     It is an object of the present invention to provide an improved method for detoxifying influenza virus without lowering immunogenic potency and, more particularly, to detoxify influenza virus for use in a vaccine by treating the virus with a combination of ether and ethanol. These and other objects of the present invention will be apparent from the following description. 
     SUMMARY OF THE INVENTION 
     Influenza virus is extracted with a mixture of ether and ethanol, the aqueous and organic phases are separated and residual ether removed from the virus suspension under reduced pressure. The resulting product is useful as an influenza vaccine. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to the detoxification of influenza virus, and, more particularly, to a method for detoxifying influenza virus without lowering its immunogenic potency. 
     It has now been found that influenza virus may be detoxified without lowering immunogenic potency by treatment with a combination of ether and ethanol. 
     According to the present invention, influenza virus is delipidated by treatment with a mixture comprising from about 0.5 to about 5, preferably about 1, volumes of diethyl ether plus from about 0.02 to about 0.1 volume of ethanol per volume of aqueous influenza virus-containing fluid. 
     According to the present invention live influenza virus is concentrated by sedimentation from the egg allantoic fluid in which it was grown and dialyzed against phosphate buffered saline. To the dialyzed concentrate there is added from about 2% to about 10% ethanol. This solution is then mixed with diethyl ether, preferably at a temperature of from about 5° to about 25° C. The mixture separates into two layers, an ether layer and an aqueous layer. The ether layer is discarded and the aqueous layer is stripped of ether by any suitable means, for example by passing an inert gas such as nitrogen through the aqueous layer or by the use of a rotary evaporator under vacuum for a period of from about 1 to about 6 hours, typically for about 2 hours. The resulting influenza virus is inactivated but retains its immunogenic efficacy. 
     The following example illustrates the present invention without, however, limiting the same thereto. All temperatures are expressed in degrees Celsius. 
     EXAMPLE 1 
     Type A/Victoria influenza virus was grown in the allantoic fluid of embryonated eggs, harvested and concentrated by density gradient centrifugation. The sedimentation band containing the influenza virus was isolated and approximately 200 ml of the A/Victoria type were added to each of two 750 ml fluted flasks. Each flask contained a 5 cm stirring bar. 200 Ml of diethyl ether containing 2% ethanol, pre-chilled to 5° were than added to one flask, and 200 ml of diethyl ether containing 10% ethanol, pre-chilled to 5° were then added to the other flask. The resulting mixtures were stirred for 10 minutes and then each was transferred to a 500 ml separatory funnel. After 5 minutes to allow for phase separation, each aqueous phase was removed and transferred to a 500 ml round bottom flask and rotary evaporated in the cold using a 15° water bath and a water aspirator. The evaporation was continued until the ether concentration was below 100 μg/ml (approximately 120 minutes). The virus preparations were then transferred to suitable vessels and stored at 5°. 
     Hemagglutinating activity was determined by rocket immunoelectrophoresis. Influenza A/Victoria anti-serum was used at a concentration of 5 μl/ml in the gel. 
     Protein was determined by the method of Lowry using bovine serum albumin as a protein standard. Ethanol and ether were determined by gas chromatography. 
     Lipid material in the organic phase was measured by the phospho-vanillin method. 
     EXAMPLE 2 
     Type B/Hong Kong influenza virus was treated similarly to the A/Victoria in Example 1 except that for determining hemagglutinating activity, influenza type B/Hong Kong anti-serum was prepared in guinea pigs from Bromelain purified hemagglutinin and was used at a concentration of 3 μl/ml per gel. 
     The following results were obtained: 
     
         ______________________________________Ethanol-Ether Extraction - Summary          A/Vic     B/HK          2%    10%     2%      10%______________________________________μg HA   Start     510     510   350   350ml         End       520     490   240   200      % Yield   100     96    68    58μg/protein      Start     1900    1900  1400  1400ml         End       2200    1600  1200  1100      % Yield   100     84    85    82Residual   Vaccine   14      55    14    64ethanol(μg/ml)Residual   Vaccine   4       17    4     5Ether(μg/ml)Lipid      Vaccine   13      17    6     11Removed(μg/ml)______________________________________ 
    
     EXAMPLE 3 
     The following tables show in vivo activity of influenza vaccine treated according to the process of the present invention. 
     
                       TABLE 1______________________________________Geometric Mean Titers of Mice 21 Days Postinjectionwith A/Victoria Influenza Virus Vaccine         HA Units/0.25 mlVaccine         20      6.25    2    0.625                                     0.2______________________________________Whole Non-Inactivated           103.7   113.1   61.7 43.6 19.5Whole HCHO Inactivated           67.3    36.7    25.9  8.7 2.8Whole Non-Inactivated           146.7   80.0    47.6 13.8 8.7Treated with 2%Alcohol-EtherWhole Non-Inactivated           87.2    67.3    47.6 21.8 5.0Treated with 10%Alcohol-Ether______________________________________ 
    
     
                       TABLE 2______________________________________Number of Positive Responses of Mice 21 Days Postinjectionwith A/Victoria Influenza Virus Vaccine           HA Units/0.25 mlVaccine           20     6.25   2    0.625                                     0.2______________________________________Whole Non-Inactivated             8/8    8/8    8/8  8/8  7/8Whole HCHO Inactivated             8/8    8/8    8/8  6/8  3/8Whole Non-Inactivated Treated             8/8    8/8    8/8  7/8  6/8with 2% Alcohol-EtherWhole Non-Inactivated Treated             8/8    8/8    8/8  8/8  5/8with 10% Alcohol-Ether______________________________________ 
    
     
                       TABLE 3______________________________________Relative Potencies of A/Victoria Influenza Virus Vaccines            Reference VaccineVaccine            A      B       C    D______________________________________Whole Non-Inactivated (A)              --     8.07    2.17 2.86Whole HCHO Inactivated (B)              0.12   --      0.27 0.35Whole Non-Inactivated Treated              0.46   3.71    --   1.31with 2% Alcohol-Ether (C)Whole Non-Inactivated Treated              0.35   2.83    0.76 --with 10% Alcohol-Ether (D)______________________________________ 
    
     
                       TABLE 4______________________________________Geometric Mean Titers of Mice 21 Days Postinjectionwith B/Hong Kong Influenza Virus Vaccine           HA Units/0.25 mlVaccine           20     6.25   2    0.625                                     0.2______________________________________Whole Non-Inactivated B/HK             190    147    113  57   34Whole HCHO Inactivated B/HK              87     48    34   28   22B/HK treated with 103    113    48   13    82% Alcohol-EtherB/HK treated with  73     36    13   25   1310% Alcohol-Ether______________________________________