Microfluidized oil-in-water emulsions and vaccine compositions

Abstract

This invention provides submicron oil-in-water emulsions useful as a vaccine adjuvant for enhancing the immunogenicity of antigens. The present invention also provides vaccine compositions containing an antigen combined with such emulsions intrinsically or extrinsically. Methods of preparing the emulsions and vaccines are also provided by the present invention.

What is claimed is:  
     
         1 . A submicron oil-in-water emulsion useful as a vaccine adjuvant comprising a light hydrocarbon non-metabolizable oil, a surfactant, and an aqueous component, wherein said oil is dispersed in said aqueous component and the mean oil droplet size is less than 1 μm.  
     
     
         2 . The emulsion of  claim 1 , wherein said oil is in an amount of 1% to 50% v/v, and said surfactant is in an amount of 0.01% to 10% v/v.  
     
     
         3 . The emulsion of  claim 1  wherein said mean droplet size is less than 0.8 μm.  
     
     
         4 . The emulsion of  claim 3  wherein said mean droplet size is between 0.1 to 0.5 μm.  
     
     
         5 . The emulsion of  claim 1  wherein said light hydrocarbon non-metabolizable oil is light mineral oil.  
     
     
         6 . The emulsion of  claim 1  wherein said surfactant comprises a phospholipid compound or a mixture of phospholipid compounds.  
     
     
         7 . The emulsion of  claim 6  wherein said phospholipid is selected from the group consisting of phosphstidylchloine, phosphatidylethanolmine, phosphatidylserine, phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.  
     
     
         8 . The emulsion of  claim 6  wherein said mixture of phospholipid compounds is lecithin.  
     
     
         9 . The emulsion of  claim 1 , wherein said surfactant comprises at least one of TWEEN or SPAN.  
     
     
         10 . A submicron oil-in-water emulsion useful as a vaccine adjuvant comprising about 40% v/v of mineral oil, about 10% w/v of lecithin, about 0.18% v/v of TWEEN®-80, about 0.08% v/v of SPAN®-80, and an aqueous phase, wherein said oil is dispersed in said aqueous phase and the mean oil droplet size is between 0.1 μm to 0.5 μm.  
     
     
         11 . The emulsion of  claim 1  or 10, further comprising an immunostimulatory molecule selected from Quil-A, GP-100, cholesterol or DDA.  
     
     
         12 . A method of preparing a submicron oil-in-water emulsion, comprising: 
 (a) preparing a mixture by combining a light hydrocarbon non-metabolizable oil, a surfactant, and an aqueous component;    (b) subjecting said mixture to a primary emulsification process to produce an oil-in-water emulsion which has a mean oil droplet size of 1.0 μm to 1.1 μm ; and    (c) subjecting the oil-in-water emulsion prepared in (b) to microfluidization to produce said submicron oil-in-water emulsion, wherein the submicron emulsion has a mean oil droplet size of less than 1 μm.    
     
     
         13 . The method of  claim 12 , wherein said oil is in an amount of 1% to 50% v/v, and said surfactant is in an amount of 0.01% to 10% v/v.  
     
     
         14 . The method of  claim 13  wherein said mean oil droplet size in said submicron oil-in-water emulsion is less than 0.8 μm.  
     
     
         15 . The method of  claim 14  wherein said mean oil droplet size in said submicron oil-in-water emulsion is between 0.1 -0.5 μm.  
     
     
         16 . The method of  claim 12  wherein said light hydrocarbon non-metabolizable oil is mineral oil.  
     
     
         17 . The method of  claim 12  wherein said surfactant comprises a phospholipid compound or a mixture of phospholipid compounds.  
     
     
         18 . The method of  claim 17  wherein said phospholipid is selected from the group consisting of phosphstidylchloine, phosphatidylethanolmine, phosphatidylserine, phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.  
     
     
         19 . The method of  claim 17  wherein said mixture of phospholipid compounds is lecithin.  
     
     
         20 . The method of  claim 12  wherein said surfactant comprises at least one of TWEEN® or SPAN®.  
     
     
         21 . The method of  claim 12  wherein said microfluidization is conducted in a microfluidizer at an operating pressure in the range of about 1,000 to 15,000 psi.  
     
     
         22 . The method of  claim 12 , wherein the mixture formed in step (a) further includes an immunostimulatory molecule selected from Quil-A, GP-100, cholesterol or DDA.  
     
     
         23 . A submicron oil-in-water emulsion prepared according to any one of the methods of claims 12-22.  
     
     
         24 . A vaccine composition comprising an oil-in-water emulsion and an antigen, wherein said antigen is dispersed in said emulsion, said emulsion comprises a light hydrocarbon non-metabolizable oil, a surfactant and an aqueous component, and wherein the mean oil droplet size of said emulsion is less than 1 μm.  
     
     
         25 . The vaccine composition of  claim 24  wherein said oil is present in said vaccine composition in an amount of 1% to 20% v/v, and said surfactant is present in said vaccine composition in an amount of 0.01% to 10% v/v.  
     
     
         26 . The vaccine composition of  claim 24  wherein said mean droplet size is in the range of less than 0.8 μm.  
     
     
         27 . The vaccine composition of  claim 26  wherein said mean droplet size is between 0.1 to 0.5 μm.  
     
     
         28 . The vaccine composition of 24 wherein said light hydrocarbon non-metabolizable oil is light mineral oil.  
     
     
         29 . The vaccine composition of  claim 24  wherein said surfactant comprises a phospholipid compound or a mixture of phospholipid compounds.  
     
     
         30 . The vaccine composition of  claim 29  wherein said phospholipid is selected from the group consisting of phosphstidylchloine, phosphatidylethanolmine, phosphatidylserine, phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.  
     
     
         31 . The vaccine composition of  claim 29  wherein said mixture of phospholipid compounds is lecithin.  
     
     
         32 . The vaccine composition of  claim 24 , wherein said surfactant comprises at least one of TWEEN® or SPAN®.  
     
     
         33 . The vaccine composition of  claim 24 , further comprising an immunostimulatory molecule selected from Quil-A, GP-100, cholesterol or DDA.  
     
     
         34 . The vaccine composition of  claim 24 , wherein said antigen comprises a viral antigen.  
     
     
         35 . The vaccine composition of  claim 34 , wherein said viral antigen comprises killed Bovine Viral Diarrhea virus Type 1 or Type 2.  
     
     
         36 . The vaccine composition of  claim 24 , wherein said antigen comprises a bacterial antigen.  
     
     
         37 . The vaccine composition of  claim 36 , wherein said bacterial antigen comprises at least one of an inactivated  Leptospira  bacterin, the recombinant  Streptococcus uberis  PauA protein, or an  E. coli  cell preparation.  
     
     
         38 . A method of preparing a vaccine composition, comprising: 
 (a) preparing a mixture by combining a light hydrocarbon non-metabolizable oil, a surfactant, and an aqueous component;    (b) combining an antigen with the mixture formed in (a);    (c) subjecting the mixture containing said antigen, which is formed in (b), to a primary emulsification process to produce an oil-in-water emulsion which has a mean oil droplet size of 1.0 μm to 1.1 μm; and    (d) subjecting the emulsion formed in (c) to high pressrure homogenization to produce said vaccine composition, wherein the composition has a mean oil droplet size of less than 1 μm.    
     
     
         39 . The method of  claim 38 , wherein the antigen to be combined with the mixture formed in (a) is provided in a mixture comprising a saponin and a sterol that is formed by: 
 (i) combining said antigen with said saponin to form a mixture;    (ii) subjecting the mixture formed in (i) to homogenization;    (iii) adding said sterol to the homogenized mixture formed in (ii); and    (iv) subjecting the mixture formed in (iii) to homogenization.    
     
     
         40 . A method of preparing a vaccine composition, comprising: 
 (a) combining an antigen with a saponin to form a mixture;    (b) subjecting the mixture formed in (a) to homogenization;    (c) adding a sterol to the homogenized mixture formed in (b);    (d) subjecting the mixture formed in (c) to homogenization;    (e) preparing a mixture of a light hydrocarbon non-metabolizable oil, a surfactant, and an aqueous component;    (f) adding the mixture of (e) to the homogenized mixture formed in (d);    (g) subjecting the mixture formed in (f) to further homogenization to produce an oil-in-water emulsion which has a mean oil droplet size of 1.0 μm to 1.1 μm; and    (h) subjecting the emulsion formed in (c) to high pressure homogenization to produce said vaccine composition, wherein the composition has a mean oil droplet size of less than 1 μm.    
     
     
         41 . The method of  claim 38  or  40 , wherein said oil is present in the vaccine composition in an amount of 1% to 20% v/v, and said surfactant is present in said vaccine composition in an amount of 0.01% to 10% v/v.  
     
     
         42 . The method of  claim 38  or  40  wherein said mean oil droplet size in said vaccine is less than 0.8 μm.  
     
     
         43 . The method of  claim 42  wherein said mean oil droplet size is between 0.1 to 0.8 μm.  
     
     
         44 . The method of  claim 38  or  40  wherein said light hydrocarbon non-metabolizable oil is light mineral oil.  
     
     
         45 . The method of  claim 38  or  40  wherein said surfactant comprises a phospholipid compound or a mixture of phospholipid compounds.  
     
     
         46 . The method of  claim 45  wherein said phospholipid is selected from the group consisting of phosphstidylchloine, phosphatidylethanolmine, phosphatidylserine, phosphatidylethanolmine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, spingomyelin and cardiolipin.  
     
     
         47 . The method of  claim 45  wherein said mixture of phospholipid compounds is lecithin.  
     
     
         48 . The method of  claim 38  or  40  wherein said surfactant comprises at least one of TWEEN or SPAN.  
     
     
         49 . The method of  claim 39  or  40 , wherein said saponin is Quil A and said sterol is cholesterol.  
     
     
         50 . The method of  claim 38  or  40  wherein said high pressure homogenization is conducted in a microfluidizer at an operating pressure in the range of about 1,000 to 15,000 psi.  
     
     
         51 . The method of  claim 38  or  40 , wherein said antigen comprises a viral antigen.  
     
     
         52 . The method of  claim 51 , wherein said viral antigen comprises killed Bovine Viral Diarrhea virus Type 1 or Type 2.  
     
     
         53 . The method of  claim 38  or  40 , wherein said antigen comprises a bacterial antigen.  
     
     
         54 . The method of  claim 53 , wherein said bacterial antigen comprises at least one of an inactivated  Leptospira  bacterin, the recombinant  Streptococcus uberis  PauA protein, or an  E. coli  cell preparation.  
     
     
         55 . A vaccine prepared according to any one of the methods of claims  38 - 40 .  
     
     
         56 . A vaccine composition comprising an microencapsulated antigen and an oil-in-water emulsion, wherein said microencapsulated antigen is dispersed in said emulsion, and said emulsion comprises a light hydrocarbon non-metabolizable oil, a surfactant and an aqueous component, and wherein the mean oil droplet size of said emulsion is less than 1 μm.  
     
     
         57 . The vaccine composition of  claim 56  wherein said oil is present in said vaccine composition in an amount of 1.0% to 20% v/v, and said surfactant is present in said vaccine composition in an amount of 0.01% to 10% v/v.  
     
     
         58 . The vaccine composition of  claim 56  wherein said mean droplet size is in the range of less than 0.8 μm.  
     
     
         59 . The vaccine composition of  claim 58  wherein said mean droplet size is between 0.1 to 0.5 μm.  
     
     
         60 . The vaccine composition of 56 wherein said light hydrocarbon non-metabolizable oil is light mineral oil.  
     
     
         61 . The vaccine composition of  claim 56  wherein said surfactant comprises a phospholipid compound or a mixture of phospholipid compounds.  
     
     
         62 . The vaccine composition of  claim 61  wherein said mixture of phospholipid compounds is lecithin.  
     
     
         63 . The vaccine composition of  claim 56 , wherein said surfactant comprises at least one of TWEEN or SPAN.  
     
     
         64 . The vaccine composition of  claim 56 , further comprising an immunostimulatory molecule selected from Quil-A, GP-100, cholesterol or DDA.  
     
     
         65 . The vaccine composition of  claim 56 , wherein said antigen is a viral antigen or a bacterial antigen.  
     
     
         66 . The vaccine composition of  claim 56 , wherein said antigen is encapsulated in a particulate carrier, and wherein said carrier comprises polylactide glycolic acid.