Method of preparing adjuvanted live attenuated vaccines and adjuvanted live attenuated vaccines thus obtained
The invention relates to a method of preparing live vaccines and to the live vaccines thus obtained. 
     It is known that live attenuated viruses generally are less immunogenic than the original virulent virus from which they are derived. The attenuation is meant to remove the virulence or at any rate to reduce it so strongly that the virus thus attenuated can safely be administered. However, this attenuation usually also results in a reduced immunogenic activity. 
     Live virus vaccines are as a rule freeze-dried in order to stabilise the infectiosity, as a result of which such a live vaccine is longer stable. A live vaccine is dissolved prior to administration, for example, in a physiological saline solution or sometimes in an inactivated vaccine. Such an inactivated vaccine is nearly always an Al(OH) 3  adsorbed vaccine. Only for practical reasons is such a live virus vaccine combined with an inactivated vaccine: as a matter of fact both can now be administered simultaneously. As a rule, such a combination does not result in a better potency of the live component. 
     It has so far been taken for granted that a higher immune response could be achieved with live vaccines, for example, by increasing the virus content, or by using a more immunogenic strain. 
     It has surprisingly been found that the use of an oil-in-water (o/w) emulsion as the &#34;solvent&#34; for live vaccines has a positive effect on the serological and immune response in the vaccinated animals. In this oil-in-water emulsion, the aqueous phase is present on the outside and the freezedried live vaccine can easily be dissolved in it. 
     A further aspect of the invention is that it has been found that by using the o/w-emulsion as solvent for live vaccines a very high serological response is obtained in young animals still having maternal immunity. This surprising effect may be caused by a protective action of the o/w-emulsion on the live virus against neutralisation by the antibodies which are present in the animal. 
     A still further embodiment of the invention is that instead of taking up the live vaccine(s) in the o/w-emulsion as such, said live vaccine may be taken up in an inactivated vaccine or combination of vaccines containing the o/w-emulsion. 
     The oily phase of the emulsion to be used according to the invention is a mineral oil, for example Marcol® or Drakeol®. The quantity of oil in the emulsion is usually between 15 and 50% by volume, preferably 20-30% by volume. 
     The emulsion to be used can be obtained in the conventional manner while using emulsifying agents suitable for the preparation of o/w-emulsions. 
     The invention will now be described in greater detail with reference to a few experiments with a live attenuated Aujeszky vaccine based on the Bartha strain, with a live attenuated Infectious Bovine Rhinotracheitisvirus (IBRV) vaccine, and with a live attenuated Respiratory Syncytial-Virus vaccine (RSV). 
     It is known that the Bartha strain is not very immunologically active, in particular in piglets which still have maternally obtained antibodies. Nor did an increased virus content of this vaccine prove capable of inducing a sufficiently high serum titer. 
     By using an oil-in-water emulsion according to the invention, a surprisingly higher serological response was obtained with the same virus material. 
    
    
     EXAMPLE I 
     Since an important field of application of live Aujeszky vaccines is the vaccination of young fattening pigs which still have a maternal antibody titer with respect to Aujeszky&#39;s disease, a comparative serological experiment has been set up using the said animals. The results hereof are recorded in Table A. 
     
                                           TABLE A                                 
__________________________________________________________________________
Average serum neutralisation (SN)-titers prior to and after vaccination   
with the Bartha vaccine, dissolved in physiological saline or in a 25%    
o/w emulsion.                                                             
Type     age on                                                           
             Number                                                       
                  2 weeks                                                 
                       Day                                                
                          Number of weeks                                 
of       vaccin-                                                          
             of   before                                                  
                       of after vaccination                               
vaccine                                                                   
     Group                                                                
         ation                                                            
             animals                                                      
                  vaccin.                                                 
                       vac.                                               
                          2  4  6  8  10                                  
__________________________________________________________________________
Physiol.                                                                  
     I   18 w                                                             
             6    3.8  1.6                                                
                           7.3                                            
                             10.8                                         
                                -- -- --                                  
saline                                                                    
     II  16 w                                                             
             6    9.1  1.6                                                
                          11.5                                            
                              5.8                                         
                                -- -- --                                  
     III 14 w                                                             
             8    4.3  1.3                                                
                           5.3                                            
                              6.1                                         
                                 4.5                                      
                                   --  6.8                                
o/w  IV  14 w                                                             
             8    4.5  2.3                                                
                          16.2                                            
                             18.9                                         
                                22.9                                      
                                   -- 47.3                                
emuls.                                                                    
     V   11 w                                                             
             8    23.8 6.6                                                
                          11.7                                            
                             13.4                                         
                                19.4                                      
                                   29.4                                   
                                      --                                  
__________________________________________________________________________
 -- = not carried out                                                     
 
    
     Blood samples for the determination of the antibody titer were taken 2 weeks before vaccination, on the day of vaccination and on several weeks after vaccination as recorded in Table A. 
     From the SN-titers stated in Table A the following appears: 
     The Bartha vaccine in physiological saline is not capable of producing a satisfactory serological response, even in animals which have only a very low maternal immunity (1.3-1.6). At the moment of vaccination, these animals were 14-18 weeks of age. 
     The Bartha vaccine in o/w emulsion on the contrary gives good serum titers upon administration to piglets having a maternal immunity of 2.3 or 6.6. Upon vaccination these animals were 11 to 14 weeks of age. 
     EXAMPLE II 
     Another field of application is the vaccination of young guilts and sows. The usefulness in this case is determined substantially by the titer height reached in the mother animal, because only then can a sufficiently long containing maternal immunity with respect to Aujeszky&#39;s disease in the offspring be ensured. 
     Starting from maternally immune young guilts, a comparison has been made between the Bartha vaccine dissolved in physiological saline solution and the same vaccine dissolved in 25% o/w emulsion. Both vaccines were administered twice. 
     
                                           TABLE B                                 
__________________________________________________________________________
Average SN-titers before and after 1st and 2nd vaccination with the       
Bartha vaccine dissolved in a 25% o/w emulsion and the same vaccine       
dissolved in physiological saline solution.                               
Type     Numb.                                                            
             Age on                                                       
                  SN-titer                                                
                        Number of weeks after                             
of       of  1st vac.                                                     
                  before 1st                                              
                        1st vaccination                                   
vaccine                                                                   
     Group                                                                
         animals                                                          
             (weeks)                                                      
                  vaccin.                                                 
                        2  4  8  10 12                                    
__________________________________________________________________________
Physiol.                                                                  
     VI  6   12   6.3    6.3                                              
                           6.3*                                           
                              63 -- --                                    
saline                                                                    
o/w  VII 4   11   5.8   11.6                                              
                           20.3                                           
                              54*                                         
                                 7080                                     
                                    3928                                  
emul VIII                                                                 
         4   11   4.9   10.3                                              
                           22.5                                           
                              23*                                         
                                 4623                                     
                                    3012                                  
sion                                                                      
__________________________________________________________________________
 -- = not carried out                                                     
 *= moment of 2nd vaccination                                             
 
    
     From Table B it again appears that the Bartha vaccine in o/w formulation in the first vaccination breaks through the maternal immunity considerably better and that the same vaccine dissolved in physiological saline solution does not produce any serological response in these maternally immune animals after one vaccination. After the second vaccination there also is a clear difference between the two formulations: only a 10-fold rise in titer is reached with the Bartha vaccine in physiological saline solution; a 130 to 200-fold rise was reached with the Bartha vaccine in o/w emulsion. 
     EXAMPLE III 
     In this example, a combined Aujeszky-influenza vaccine has been used. The influenza vaccine for pigs is an inactivated vaccine based on a o/w emulsion. The freeze-dried Barth vaccine is dissolved in it immediately before use. For this experiment piglets were used which have no maternal antibodies any longer with respect to Aujeszky&#39;s disease, so as to be better able to study a possible negative influence of the influenza component on the Bartha vaccine. The quantity of influenza virus per dose was varied and is expressed as &#34;high&#34;, &#34;medium&#34; and &#34;low&#34;. 
     
                       TABLE C                                                     
______________________________________                                    
Average Aujeszky SN-titers after 1 or 2 vaccinations with the             
Aujeszky-influenza vaccine on o/w basis.                                  
                    Number   SN-titer                                     
                                     SN-titer                             
Influenza                                                                 
         Number of  of       8 wks after                                  
                                     2 wks after                          
dose     vaccinations                                                     
                    animals  1st vaccin.                                  
                                     2nd vaccin.                          
______________________________________                                    
&#34;high&#34;   2x         4        195*    3016                                 
         1x         3        159      132                                 
&#34;medium&#34; 2x         4        116*    3887                                 
         1x         3        112      58                                  
&#34;low&#34;    2x         4        107*    3887                                 
         1x         3        113      103                                 
______________________________________                                    
 *the second vaccination was administered to these animals 8 weeks after  
 the first vaccination                                                    
 
    
     It will be clear from Table C that the presence of inactivated influenza virus in the o/w emulsion has no detrimental effect on the results of the live Aujeszky component. The serological response both after 1 and after 2 vaccinations is extremely good. 
     In various experiments the body temperature of the animals was measured from 1 day before up to and including 3 days after vaccination. At no instant whatsoever after vaccination was an increased temperature observed in the animals vaccinated with the o/w formulation. No local vaccination reactions were found either. The live vaccine in an o/w formulation therefore is to be considered as being safe. 
     EXAMPLE IV 
     The effect of the o/w-emulsion of the invention was also examined with live attenuated infectious bovine rhinotracheitis-virus (IBRV) by immunizing cows with the usual amount of vaccine-virus either dissolved in a 25% o/w-emulsion or in a physiological saline solution. 
     The serological responses are indicated in Table D: 
     
                       TABLE D                                                     
______________________________________                                    
Average SN-titers before and after 1 and 2 vaccinations with              
IBRV-vaccine in 25% o/w or physiological saline.                          
             SN-titer                                                     
Type   Number  Number  before                                             
                             3 wks after                                  
                                     6 wks after 1st                      
of     of      of      1st   1st     resp. 3 wks                          
vaccine                                                                   
       animals vaccin. vaccin.                                            
                             vaccination                                  
                                     after 2nd vac.                       
______________________________________                                    
physiol.                                                                  
       4       1       0      1.4 (1)*                                    
                                     1.6 (1)                              
saline 4       2       0     1.3 (2) 3.2 (3)                              
o/w-   4       1       0     3.8 (4) 3.8 (4)                              
emul.  4       2       0     2.9 (4) 8.5 (4)                              
not vacc.                                                                 
       4       0       0     0       0                                    
______________________________________                                    
 *The numbers in parentheses indicate the number of cows having a positive
 SNtiter after vaccination.                                               
 
    
     The following can be concluded from the SN-titers as indicated in Table D: 
     1. 100% of the animals vaccinated with the vaccine containing o/w-emulsion according to the invention gives a serological response, whereas only 50% of the animals of the physiological saline group gives a positive response. 
     2. The SN-titers after vaccination in the o/w-group are 2-3 times as high as in the other group. 
     EXAMPLE V 
     In this example the influence of the o/w-emulsion according to the invention on the serological response of live vaccine was examined by vaccinating cows with a live attenuated respiratory syncytial virus-(RSV) vaccine, either in 25% o/w-emulsion or in a physiological saline solution. 
     The serological responses after vaccination with both types of vaccine are indicated in Table E. With both types of vaccine equal amounts of virus were administered to the cows. 
     
                       TABLE E                                                     
______________________________________                                    
Average indirect immunofluorescence (i.I.F)-RSV-titers after              
vaccination with RSV-vaccine dissolved in physiological saline            
solution or in a 25% o/w-emulsion.                                        
Type    Number                                                            
of      of        i-IF-titer                                              
vaccine animals   before vaccination                                      
                               3 wks after vacc.                          
______________________________________                                    
physiol.                                                                  
        8         380          269                                        
saline                                                                    
o/w-emuls.                                                                
        8         453          3044                                       
not vacc.                                                                 
        3         172          50                                         
______________________________________                                    
 
    
     It appears from the results of Table E that: 
     Animals vaccinated with RSV-vaccine in a o/w-emulsion show a very high titer, even when a considerable titer is present before vaccination (due to maternal immunity). However, the vaccine dissolved in physiological saline solution causes no increase of the titer in animals with maternal immunity.