# Patent

## Title
Water-in-oil emulsion vaccines

## Abstract

Oil-in-Water emulsion vaccines induce higher biliary IgA responses which decrease mucosal/internal organ invasion and fecal shedding, increase specific activity of serum IgG subpopulations, and increase the relative avidity index of serum IgG subpopulations, resulting in increased protection from disease in animals.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the control of disease in animals through the use of water-in-oil (W/O) emulsion vaccines containing emulsifier and surfactants.
2. Description of the Related Art
Vaccines are used for the prevention of disease in animals. The cornerstone of many infectious disease control programs is the induction of specific immunity by vaccination with either live or inactivated microorganisms or their products. Vaccine efficacy depends on many variables, such as the nature and the amount of antigen administered and the presence of adjuvants to enhance immunogenicity.
Water-in-oil vaccines are proven to be more efficacious than vaccines comprised of oil-in-water or aqueous antigen or oil phase alone (Stone, Avian Dis., Volume 27(3), 688-697, 1993). Stone et al. (Avian Dis., Volume 34, 979-983, 1990) disclose the use of the terpene oils, squalene, and squalane, in a vaccine for Newcastle disease in place of mineral oil. They found that the cumulative HI titers using these oils were similar to mineral oil but the viscosity was up to four times greater than those vaccines using mineral oil. The supply for these terpene oils is limited and more expensive.
Stone (1993, supra) discloses water-in-oil vaccines using animal and vegetable oils. The vaccines contained an aqueous antigen with an oil phase-to-aqueous phase ratio of 4:1. Emulsification was done with both oil-soluble and water-soluble surfactant added to the oil phase. Beeswax was used as an emulsifier for the non-mineral oil vaccines since the known mineral oil surfactants were not suitable for animal and vegetable oil-containing vaccines. It was reported that the non-mineral oil containing emulsion vaccines had a higher viscosity than mineral oil emulsions of the same relative oil and aqueous components. Low viscosity is an important characteristic for oil emulsion vaccines because it eases the vaccination process, lowering fatigue of working, saving time and work when large numbers of birds are involved. Low viscosity of the oil phase also allows emulsification of a greater amount of aqueous phase for increased volume of antigen or multiple antigens before prohibitive viscosity is reached.
U.S. Pat. No. 5,109,026 to Hoskinson et al., discloses water-in-oil vaccines with mineral oils, squalene, and squalene. The water phase includes a polycationic polyelectrolyte. Emulsifiers such as Arlacel A and Arlacel 80 were added as oil-soluble emulsifiers or Tween 80 as a water-soluble emulsifier.
The accepted standard emulsifying agents for mineral oil vaccines are Arlacel A, Arlacel 80, and Tween 80. These easily emulsify water phases in mineral oil but do not function in the same capacity with non-mineral oils. Beeswax is suggested as a surface active agent for use with non-mineral oil vaccines. However, the beeswax-containing emulsion vaccines have viscosities that are much higher than mineral oil vaccines using emulsifying agents such as Arlacel A or Arlacel 80, and Tween 80 (Stone, Avian Diseases, Volume 37, 39-405, 1993).
While various water-in-oil vaccines have been developed, there remains a need in the art for water-in-oil vaccines which induce higher biliary IgA responses thereby increasing immune protection of the intestinal tract, increase specific activity of serum IgG subpopulations, and increase the relative avidity index of serum IgG subpopulations while maintaining a satisfactory low viscosity. The present invention provides water-in-oil emulsion vaccines which are different from prior art compositions and solves some of the problems associated with prior art water-in-oil vaccines.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide water-in-oil vaccines with low viscosity that contain an emulsifier, and a mixture of at least two surfactants.
Another object of the present invention to provide water-in-oil emulsion vaccines with low viscosity having a mixture of at least two nonionic surfactants.
A still further object of the present invention is to provide water-in-oil emulsion vaccines with low viscosity having an emulsifier, and a mixture of polyoxyethylene sorbitan trioleate and sorbitan trioleate.
Another object of the present invention is to provide a method for vaccination that includes administering a water-in-oil emulsion vaccine with low viscosity having an emulsifier, and at least two surfactants.
A further object of the present invention is to provide a method for vaccination that includes administering a water-in-oil emulsion vaccine with low viscosity having an emulsifier, and at least two nonionic surfactants.
A still further object of the present invention is to provide a method for vaccination that includes administering a water-in-oil emulsion vaccine with low viscosity having an emulsifier, and a mixture of polyoxyethylene sorbitan trioleate and sorbitan trioleate.
Another object of the present invention is to provide a priming water-in-oil emulsion composition that contains a fatty acid ester, an emulsifier, and a mixture of at least two surfactants.
A further object of the invention is to provide a priming water-in-oil emulsion composition having a C18-C32 fatty acid ester, an emulsifier, and a mixture of at least two surfactants.
A still further object of the present invention is to provide a priming water-in-oil emulsion composition have a C1-C32 fatty acid ester, sorbitan monooleate, a mixture of polyoxyethylene sorbitan trioleate and sorbitan trioleate, and a C14-C18 straight chain saturated hydrocarbon.
Another object of the present invention is to provide a method that includes injecting an animal with a vaccine including a priming emulsion wherein said vaccine includes a C18-C32 fatty acid ester, sorbitan monooleate, a mixture of polyoxyethylene sorbitan trioleate and sorbitan trioleate, and a C14-C18 straight chain saturated hydrocarbon.
A still further object of the present invention is to provide a method that includes priming avian embryos for post-hatch vaccination by injecting a priming water-in-oil emulsion containing a C18-C32 fatty acid ester, an emulsifier, and a mixture of at least two surfactants.
Further objects and advantages of the invention will become apparent from the following description.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 is a graph of IDA-Fe3+ chromatograph of non-immunized chicken serum.
FIG. 2 is a photograph showing the immunoeletrophoresis (IEP) of IgG subpopulations of non-immune serum from hens.
FIGS. 3A-3C are graphs showing: (3A) IDA-Fe3+ chromatography of serum from hens receiving a Marcol 52 (mineral oil) emulsion containing polyoxyethylene sorbitan monooleate, and sorbitan monooleate; (3B) ELISA end-point for serum from hens receiving Marcol 52/polyoxyethylene sorbitan monooleate/sorbitan monooleate; (3C) Relative Avidity Index for serum IgG subpopulations from hens receiving Marcol 52/polyoxyethylene sorbitan monooleate/sorbitan monooleate.
FIGS. 4A-4D are graphs showing: (4A) IDA-Fe3+ chromatography of serum from hens receiving Marcol 52 emulsion containing polyoxyethylene sorbitan trioleate and sorbitan trioleate; (4B) ELISA end-point comparisons for serum from hens receiving Marcol 52 emulsion containing polyoxyethylene sorbitan monooleate/sorbitan monooleate vs. polyoxyethylene sorbitan trioleate/sorbitan trioleate; (4C) Comparison of Relative Avidity Indexes (6M Urea) for serum from hens receiving Marcol 52 emulsion containing polyoxyethylene sorbitan monooleate/sorbitan monooleate vs. polyoxyethylene sorbitan trioleate/sorbitan trioleate (4D) Comparison of Relative Avidity Indexes (8M Urea) for serum from hens receiving Marcol 52 emulsion containing polyoxyethylene sorbitan monoloeate/sorbitan monooleate vs. polyoxyethylene sorbitan trioleate/sorbitan trioleate.
FIGS. 5A-5C are graphs showing: (5A) IDA-Fe3+ chromatography of immune serum taken three weeks post-vaccination from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan monooleate and sorbitan monooleate; (5B) ELISA end-point for serum from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan monooleate and sorbitan monooleate; and (5C) Relative Avidity Index for serum from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan monooleate and sorbitan monooleate.
FIGS. 6A-6D are graphs showing: (6A) IDA-Fe3+ chromatography of immune serum taken three weeks post-vaccination from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan trioleate and sorbitan trioleate; (6B) ELISA end-point comparisons for serum from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan trioleate and sorbitan trioleate or n-hexadecane emulsion containing polyoxyethylene sorbitan monooleate and sorbitan monooleate; (6C) Comparison of the Relative Avidity Indexes (6M Urea) for serum from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan trioleate and sorbitan trioleate and n-hexadecane emulsion containing polyoxyethylene sorbitan monooleate and sorbitan monooleate; (6D) Comparison of the Relative Avidity Indexes (8M Urea) for serum from hens receiving n-hexadecane emulsion containing polyoxyethylene sorbitan trioleate and sorbitan trioleate and n-hexadecane emulsion containing polyoxyethylene sorbitan monooleate and sorbitan monooleate.
FIGS. 7A-7C are graphs showing: (7A) Comparison of the ELISA end-points of immune serum taken three weeks post-vaccination from hens receiving Marcol 52 and n-hexadecane emulsions containing polyoxyethylene sorbitan trioleate and sorbitan trioleate; (7B) Comparison of the Relative Avidity Indexes (6M Urea) for serum from hens receiving Marcol 52 and n-hexadecane emulsions containing polyoxyethylene sorbitan trioleate and sorbitan trioleate; and (7C) Comparison of the Relative Avidity Indexes (8M Urea) for serum from hens receiving Marcol 52 and n-hexadecane emulsions containing polyoxyethylene sorbitan trioleate and sorbitan trioleate.
FIGS. 8A-8D are graphs showing comparison of protection against Salmonella enteriditis (SE) infection in hens receiving n-hexadecane inactivated SE emulsions containing polyoxyethylene sorbitan trioleate, sorbitan trioleate, and sorbitan monooleate versus three commercially available vaccines (1) Layermune SE (Biomune Co., Lenexa, Kans.)(BIO), (2) Poulvac SE (Fort Dodge Animal Health, Overland Park, Kans.)(FtD), and (3) Inactivac/SE4 (Maine Biological Laboratories, Waterville, Me.)(MBL); and control (non-immunized hens). FIG. 8A compares the % intestinal Salmonella enteriditis present at 13 days and 19 days post challenge. FIG. 8B is a comparison of the number of Salmonella enteriditis per gram of cecum 13 days and 19 days post challenge. FIG. 8C is a comparison of vaccines for % liver Salmonella enteriditis 13 and 19 days post challenge and FIG. 8D is a comparison of number of Salmonella enteriditis per gram of liver tissue at 13 and 19 days post challenge.
FIGS. 9A-9D are graphs showing comparisons of protection against SE infection in hens receiving n-hexadecane inactivated SE emulsions containing polyoxyethylene sorbitan trioleate, sorbitan trioleate, and sorbitan monooleate versus three commercially available vaccines (1) Layermune SE (Biomune Co., Lenexa, Kans.)(BIO), (2) Poulvac SE (Fort Dodge Animal Health, Overland Park, Kans.)(FtD), and (3) Inactivac/SE4 (Maine Biological Laboratories, Waterville, Me.)(MBL); and control (non-immunized hens). FIG. 9A compares the % intestinal Salmonella enteriditis present at 7 days and 15 days post challenge. FIG. 9B is a comparison of the number of Salmonella enteriditis per gram of cecum 7 days and 15 days post challenge. FIG. 9C is a comparison of vaccines for % liver Salmonella enteriditis 7 and 15 days post challenge and FIG. 9D is a comparison of the number of Salmonella enteriditis per gram of liver tissue at 7 and 15 days post challenge.
FIG. 10a is a graph showing serum HI-titers in hens receiving Avian Influenza Virus n-hexadecane vaccine.
FIG. 10b is a graph showing serum HI-titer in hens receiving NewCastle Disease Virus n-hexadecane vaccine.



DETAILED DESCRIPTION OF THE INVENTION
The vaccine formulations of the present invention are applicable to any animal and are especially useful in avian species, whether domestic or wild, and particularly to those which are commercially reared for meat or egg production. Without limitation thereto, exemplary avians include chickens, turkeys, geese, ducks, pheasant, emu, ostrich, etc.
One advantage of the present invention is in the prevention of lethal diseases such as those which threaten avians. Avian diseases include any disease or contamination of viral, bacterial, or other microbial origin. Examples of such, without limitation thereto, include Salmonella, Newcastle disease, avian leukosis, infectious bursal disease, adenovirus disease, reovirus, pox, laryngotracheitis, avian influenza, Marek's disease, etc. The present invention water-in-oil emulsion vaccines induce higher biliary IgA (Table 1) responses which decreases mucosal/internal organ invasion and fecal shedding, increase specific activity of serum IgG subpopulations, and increase the relative avidity index of serum IgG subpopulations, causing increased protection. The vaccines of the present invention provide excellent protection against organ invasion and significantly reduce intestinal enteropathogenic bacteria levels when compared to two of the three commercial preparations during challenge studies. Furthermore, the vaccine of the present invention is useful for in ovo vaccination to stimulate early immune protection. In combination with a fatty acid ester priming emulsion, the present invention gives a secondary boost to increase IgG activity. The present invention is also useful for the production of monoclonal antibodies.
The term vaccine is defined to mean all types of biological agents used to produce active immunity or competitive exclusion. More particular, the present invention is drawn to water-in-oil vaccine formulations.
Examples of oils useful in the present invention include C14 to C18 aliphatic straight-chain saturated hydrocarbons, such as for example, hexadecane, isohexadecane, pentadecane, heptadecane, octadecane, tetradecane, etc.
The emulsifier used in the present invention is a hydrophobic nonionic surfactant, sorbitan monooleate (Arlacel 80). The surfactants of the present invention are polyoxyethylene sorbitan trioleate and sorbitan trioleate.
Optionally, C18 to C32 fatty acid esters in a primary emulsion is useful with the present invention. Examples of useful esters include butyl stearate, butyl myristate, tridecyl stearate, octastearate, isopropyl myristate, isocetyl myristate, isopropyl isostearate, etc and mixtures thereof.
Oil vaccines are prepared using an oil to aqueous phase (O:A) of approximately 4 parts oil to approximately 1 part aqueous phase (volume:volume). The surfactant mixture is added to the oil phase or to the aqueous phase for approximately 2-3 minutes on a rotor Stator model Pro400 (ProScientific, Inc., Monroe, Conn.) at maximum speed. The aqueous phase consists of antigen prepared in PBS or normal allantoic fluid. The antigen can be live or inactivated. The oil phase containing the surfactant composition is added to the aqueous phase containing antigen and is either manually shaken for about 15 to about 20 seconds or emulsified for about 20 seconds using a high-shear probe Polytron 10/35 (Brinkman homogenizer, Brinkman Instruments, Westbury, N.Y.). For large volumes of water-in-oil emulsion vaccines, the aqueous antigen is usually added to the oil phase and dispersed during stir. Mineral oil vaccines are prepared as described in Stone et al., Avian Diseases, Volume 22, 666-674, 1978; and Stone et al., Avian Diseases, Volume 34, 979-983, 1990; which are both herein incorporated by reference. Total surfactant volume for vaccines is approximately about 20% of the oil phase.
For industrial preparation of the surfactant composition-containing water-in-oil emulsion vaccines, the oil and surfactant-containing aqueous phases are mixed with a Silverson turbine by mixing for about 5 minutes at about 30° C.
The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. An inactivated Salmonella enterica serovar. enteritidis (SE) is used as a model system for the present invention.
EXAMPLE 1
Three (3) specific-pathogen free White Leghorn hen birds (3 birds/emulsion/experiment) were immunized subcutaneously, in the neck region, with a water-in-oil (w/o) emulsion containing inactivated Salmonella enterica serovar. enteritidis (SE). Each bird received about a 100 μg dose of acetone-inactivated Salmonella enterica serovar. enteritidis whole cells contained within about a 500 μl injection volume. Serum samples, collected by venous wing vein puncture, were obtained at about three (3) weeks post-primary immunization and were stored at about −20° C. until used. The oils tested included Marcol-52 (mineral oil; Esso Chemicals, Toronto, Canada) and n-hexadecane (Sigma Chemical Co., St. Louis, Mo.). To test emulsion composition, Marcol-52 and n-hexadecane were formulated in the following manner: about 8 ml of n-hexadecane or Marcol-52, about 1 ml sorbitan monooleate (Arlacel 80; ICI United States, Inc., Wilmington, Del.), about 0.7 ml polyoxyethylene sorbitan trioleate (Tween 85, Fisher Scientific, Atlanta, Ga.), about 0.3 ml sorbitan trioleate (Span 85, Sigma Scientific Company) to form formulation 1. The antigen is about 5 mg/ml in distilled water. Approximately 8 mls of formulation 1 is mixed with about 2 mls of the antigen. The antigen is drawn up into a 3 ml syringe containing a 25 g needle. It is slowly added to formulation 1 while mixing at about 7500 rpms. Mixing continues until all the antigen has been added. A second formulation includes about 8 ml n-hexadecane or Marcol-52, about 1 ml sorbitan monooleate (Arlacel 80), about 0.75 ml polyoxyethylene sorbitan monooleate (Tween 80, Polysorbate 80, Fisher Scientific, Atlanta, Ga.), about 0.3 ml sorbitan monooleate (Span 80, Sigma Chemical Company) to form formulation 2. This is mixed with the antigen as described above.
The serum samples collected from the immunized chickens were applied to an Iron Chelate Chromatography column in order to separate the IgG into subpopulations. Iminodiacetic acid (IDA) epoxy-activated Sepharose 6B (Pharmacia Biotech, Piscataway, N.J.) was poured into a G10×150 Moduline medium pressure laboratory column (Amicon, Beverly, Mass.) to obtain a final packing bed volume of about 7.5 ml. Following equilibration with deionized water, the column was converted to the IDA-Fe3+ form by charging with aqueous FeCl3·6H2O. After charging, the column washed with deionized water and then equilibrated with about 50 mM MES (2-[N-Morpholino]ethanesulfonic acid) about pH 6 (Sulkowski, Makromol. Chem. Macromol. Symp., Volume 17, 335, 1988). After completion of each run, the IDA gel is stripped (chelate annihilation) with about 50 mM EDTA+about 500 mM NaCl about pH 7 and regenerated.
About a 350 μl delipidized serum sample is applied to the column after buffer removal from the top of the column bed. The serum sample was allowed to enter the column bed at a flow rate of about 1 ml/min while the fraction collector was operated in the time collection mode at about 3 min/tube and a sensitivity setting of about 0.1 AUFS. The column was subsequently refilled with buffer. The column washed with the equilibration buffer until the unabsorbed peak returned to baseline. Bound proteins and immunoglobulins were eluted using an ascending linear pH gradient of about pH 6 to about pH 8 consisting of about 100 ml equilibration buffer and about 100 ml of about 50 mM HEPES, about pH 8 (Sulkowski, 1988; supra). Following gradient completion, the column washed with about 50 mM HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethane-sulfonic acid]), about pH 8, and subjected to chelate annihilation. Each protein-containing peak was tested for the presence of IgG using the Ouchterlony double diffusion technique and for immunological activity using the enzyme-linked immunosorbent assay (ELISA), while the total IgG content for each peak was determined using the single radial immunodiffusion method (Fahey and McKelvey, Quantitative Determination of Serum Immunoglobulins in Antibody-Agar Plates, J. Immunol., Volume 94, 84-90, 1965).
For the ELISA, purified SE flagella (Ibrahim et al., J. Clin. Micro., Volume 22, 1040, 1985), diluted to about 1 μg/ml in about 100 mM bicarbonate buffer about pH 9.6, was bound to the solid phase by incubation for about two hours at about 37° C. followed by overnight incubation at about 4° C. Following immobilization, the microtiter plate (solid phase) (Immulon 4, Dynatech Laboratories, Chantilly, Va.) was blocked with about 100 mM phosphate buffer (PB)+about 1% (w/v) polyvinylpyrrolidone, about pH 8, for about 1 hour at about 37° C. After blocking, the microtiter plate washed twice with about 20 mM PBS+about 0.05% (v/v) Tween-20, about pH 7.4 (PBST), and then two-fold dilutions, diluted in PBST, of each eluted subpopulation were added to the appropriate wells of the microtiter plate and incubated overnight at about 4° C. The plate washed twice with PBST and alkaline phosphatase labeled affinity purified rabbit anti-chicken IgG Heavy and Light chain antiserum (Jackson ImmunoResearch Laboratories, West Grove Pa.), diluted about 1:5000 in PBST, was added to the plate and incubated for about 60 minutes at about 37° C. After twice washing the plate with PBST, p-nitrophenyl phosphate, about 1 mg/ml diethanolamine buffer at about pH 9.8, was added to the appropriate wells of the microtiter plate. Color development was allowed to proceed for about 30 minutes at about room temperature and was stopped by the addition of about 3N NaOH. Absorbance was measured at about 405 nm using a Multiscan MS microtiter plate reader (Labsystems, Needham, Mass.).
The relative avidity index (RAI) for each subpopulation of IgG was determined using about 6M and 8M urea as previously described by Chargelegue et al. (Clin. Exp. Immunol., Volume 93, 331, 1993). Statistical differences for the relative avidity index were determined by Students' paired t-test or the Rank Sum Test using SigmaStat software version 1.0 (Jandel Scientific Com., San Rafael, Calif.).
Immunoelectrophoresis (IEP) was performed in about 1% (w/v) agarose (Type II, Medium EEO, Sigma Chemical Co.) in Tris-Tricine buffer, about pH 8.6. Samples of 5 μl each were electrophoresed in an EC 360 flatbed apparatus (E-C Apparatus Corp., St. Petersburg, Fla.) at about 200 constant volts for about 45 minutes. After electrophoresis, the IEP films were incubated overnight in a humidity box and then processed and stained with about 0.1% (w/v) coomassie brilliant blue R-250 (Sigma Chemical Co.).
Normal chicken serum (non-immune serum) from non-immunized birds, was chromatographed on a previously equilibrated IDA-Fe3+ column (FIG. 1). Using an ascending linear pH gradient, seven IgG containing peaks, as confirmed by double diffusion analysis, were resolved. Peak 1 comprises the unabsorbed fraction, while peaks 2-5 elute during gradient formation. Peak 6 is eluted during the about pH 8 washing step, while peak 7 is collected during the chelate annihilation phase. Analysis of the elution pH indicates that peak 2 elutes from approximately pH 6.15-6.52; peak 3 from approximately pH 6.57-6.90; peak 4 from approximately pH 6.90-7.53; while peak 5 elutes from approximately pH 7.53-7.75. Examination of each IgG subpopulation by immunoelectrophoresis demonstrates an electrophoretic mobility difference (FIG. 2). This difference is clearly demonstratable between peaks 1 and 7, while minor differences exist between peaks 2-6.
Serum from hens immunized with the mineral oil water-in-oil emulsion vaccine containing mineral oil (Marcol-52), sorbitan monooleate (Arlacel-80), polyoxyethylene sorbitan (Tween-80) and sorbitan monooleate (Span-80) shows a profile very similar to normal chicken serum but with some notable exceptions: peaks 3 and 4 are more resolved, while peak 4 is enhanced, and the almost complete absence of peak 5 (FIG. 3A). Immunological activity (tested by ELISA) was detected in all peaks with peaks 4 and 7 exhibiting the highest activity (FIG. 3B). The relative avidity index was determined for each IgG subpopulation. This emulsion primarily elicits low avidity antibodies and the distribution of high avidity antibodies is skewered towards those subpopulations that do not bind to the column or else elute very early in the gradient formation; that is, the relative percentages of high avidity antibodies decrease with increasing net positive charge (FIG. 3C).
The mineral oil (Marcol-52), sorbitan monooleate (Arlacel-80), polyoxyethylene sorbitan trioleate (Tween 85), and sorbitan trioleate (Span 85) emulsion vaccine was used to test the effect of the emulsion composition. The serum chromatography profile for this emulsion is quite different from that previously seen: peaks 2-6 have been greatly enhanced (FIG. 4A), while the immunological activity for peaks 5 and 6, and especially 7 have been highly upregulated (FIG. 4B). By substituting polyoxyethylene sorbitan monooleate (Tween 80) and sorbitan oleate (Span 80) with polyoxyethylene sorbitan trioleate (Tween 85) and sorbitan trioleate (Span 85), the relative avidity index for both high and low avidity antibodies have been upregulated for subpopulations 1-4 and is statistically significant (6M, P=0.0029; 8M, P=0.0326) (FIGS. 4C and 4D).
In the n-hexadecane emulsion vaccine containing polyoxyethylene sorbitan monooleate (Tween 80) and sorbitan oleate (Span 80) (FIGS. 5A-5D), immunological activity was detected in peaks 2-7, while peak 1 demonstrated no activity (FIG. 5B). The relative avidity index was comparable to that obtained for the mineral oil emulsion containing Tween 80 and Span 80 (FIG. 5C). When n-hexadecane was formulated with polyoxyethylene sorbitan trioleate (Tween 85) and sorbitan trioleate (Span 85) (FIGS. 6A-6D), both the immunological activity (FIG. 6B) and the relative avidity index for all subpopulations were significantly upregulated (FIGS. 6C and 6D). The relative avidity index and the immunological activity for the n-hexadecane emulsion containing Tween 85 and Span 85 was statistically significant (P<0.001) over the n-hexadecane emulsion containing Tween 80 and Span 80, and the mineral oil emulsion containing Tween 85 and Span 85 (P=0.000122) (FIGS. 7A-7C).
EXAMPLE 2
Hens were vaccinated and boosted about 4 weeks post primary vaccination with an inactivated Salmonella enterica serovar. enteriditis water-in-oil vaccine containing hexadecane and Arlacel 80, polyoxyethylene sorbitan trioleate and sorbitan trioleate (SEPRL) as described above in Example 1. Another group of hens were vaccinated and boosted about 4 weeks post primary vaccination with three commercially available Salmonella enterica serovar. enteriditis water-in-oil vaccines from Maine Biological Laboratories, Fort Dodge, and Biomune. The vaccines were 1) Layermune SE (Biomune Co., Lenexa, Kans.)(BIO), (2) Poulvac SE (Fort Dodge Animal Health, Overland Park, Kans.)(FtD), and (3) Inactivac/SE4 (Maine Biological Laboratories, Waterville, Me.) (MBL). Non-immunized birds were used as the control. Three weeks following the boost, all hens were challenged with about 1×108 S. enteriditis. Tissues were taken and cultured on the days 13 and 19 post boost (FIGS. 7A-7D). Hens were sacrificed by cervical dislocation and a portion of the liver, the whole spleen, a portion of the ovaries, and a portion of the right cecum were aseptically removed and placed into pre-tared sterile stomacher bags. For each bird, the liver and spleen samples were combined while the ovaries and the cecum were placed in individual bags. The samples were diluted about 1:10 in tetrathionate brilliant green (Difco Laboratories, Detroit, Mich.) and stomached for about 60 seconds. The stomached samples (about 0.1 ml) were plated onto Brilliant Green agar containing about 20 μg/ml novobiocin (Sigma Chemical Co., St. Louis, Mo.) and about 20 μg/ml nalidixic acid (BGNN)(Sigma Chemical Co., St. Louis, Mo.). The cecum samples were serially diluted (about 10 fold) in phosphate-buffered saline, and about 0.1 ml samples were incubated overnight at about 37° C., and SE colony-forming units (CFU) was determined by plate count. For any negatives, about 0.1 ml of the respective tetrathionate enrichment was plated onto BGNN, which were then incubated overnight at about 37° C. As the enumeration method had a minimum detection threshold of about 1×102 CFU/g, samples that were negative for direct enumeration but positive after tetrathionate enrichment were arbitrarily assigned a value of about 50 CFU/g. The numbers of SE CFU/g in each treatment group were transformed to log10 then means were calculated. Statistical differences were determined by one-way analysis of variance using GraphPad Software (San Diego, Calif.) at P<0.05. Groups with different letters are significantly different on that particular day. Two trials were conducted.
In Trial 1 (FIGS. 8A-8D), the % of intestinal SE+ hens was lower in the hens receiving the vaccine of the present invention (SEPRL) compared to the 3 commercial vaccines at day 13 post challenge and lower levels were observed at day 19 post challenge compared with hens receiving the commercial vaccines. (FIGS. 8A and 8B). Internal extraintestinal organ levels were less affected by the SEPRL vaccine (FIGS. 8C and 8D).
In trial 2 (FIGS. 9A-9D), significantly fewer hens receiving the SEPRL vaccine were intestinally SE+ compared with two of the commercial vaccines (FIGS. 9A and 9B). At day 15 post challenge, fewer hens were intestinally SE+, though not significantly, in the SEPRL vaccinated group compared to the commercial groups. Internal organ levels were numerically less in the SEPRL vaccinated group compared to the commercial groups (FIGS. 9C and 9D).
Blood was drawn weekly from the vaccinated birds for serum. Bile was collected from sacrificed birds at the termination of the experiment on weeks 8 and 9. Microagglutination titers against SE stained antigen were determined on sera and bile according to methods previously described using serial two-fold dilutions of an original dilution of each sample (Gast et al, Avian Dis., Volume 37, 6, 992-999, 1992; herein incorporated by reference). See Table 1 for results. Serum and bile anti-SE titers were elevated in hens vaccinated with the vaccine of the present invention compared with the three commercial vaccines.







TABLE 1







Comparison of serum and biliary microagglutination (# of


positive wells) for Hexadecane, MBL, Biomune, and Ft. Dodge.









WEEK
















se-
se-
se-
se-
se-
se-





rum
rum
rum
rum
rum
rum
bile
bile


Vaccine
2
3
4
5
6
7
8
9


















Hexa-
11
9.91

9.32
9.45
9.04
7.6
6.6


decane


MBL
9.64
8.92

8.44
8.66
8.33
5.8
5.11


Biomune
9.79
8.68

8.29
7.46
6.6
4.8
4.33


Ft.
8.16
7.92

6.92
7.92
8.0
4.8
4.8


Dodge









EXAMPLE 3
Water-in-oil vaccines for viruses were prepared using the same adjuvant base and procedures as for the Salmonella bacterin described above in Example 1. Vaccine antigen for Turkey Wisconsin 68 and La Sota NDV was prepared from virus propagated in 9-day-old chicken embryos and harvested in the allantoic fluids (Beard et al., Avian Dis., Volume 19, 6920699, 1975; herein incorporated by reference). Inactivation of the fluids was with beta-proprio-lactone (BPL) as described by Beard et al. (supra). One milliliter of vaccine emulsion containing about 0.25 ml of AI allantoic antigen or about 1 mg of NDV antigen was given subcutaneously in the mid-dorsal neck region. Sera, in groups of 6 chickens, vaccinated at 4-weeks-old, were tested for hemagglutination-inhibition titers (Beard et al, supra) at 3, 4, and 5 weeks post vaccination. The antiviral titers were exceptionally high (FIGS. 10a and 10b). Chickens receiving n-hexadecane emulsions of the present invention containing avian influenza virus (AIV) and Newcastle Disease Virus (NDV) exhibited exceptionally elevated anti-viral titers (FIGS. 10a and 10b).
EXAMPLE 4
A series of fatty acid esters were formulated to test their ability to enhance secondary immune responses in chickens. The fatty acid esters tested included butyl stearate, butyl myristate, tridecyl stearate, octastearate, isopropyl myristate, isocetyl myristate, isocetyl stearate, and isopropyl isostearate. These esters were formulated as water-in-oil emulsion vaccines using about 8 ml fatty acid ester, about 1 ml Imwitor 780K (isostearyl diglyceryl succinate; Condea, Piscataway, N.J.), plus about 0.7 ml Tween-85 and about 0.3 ml Span-85 surfactants (Stone H. D., Avian Dis., July-September, Volume 41(3), 591-597, 1997; herein incorporated by reference) and about 1 mg of antigen. Birds were vaccinated by the subcutaneous route in the mid-dorsal region of the neck. Each bird received about a 1 mg dose of acetone inactivated SE upon primary immunization and at the secondary boost about 6 weeks later. The time frame between the primary immunization and the secondary boost was about 6 weeks (Davis and Glick, Poult. Sci., May, Volume 67 (5), 855-857, 1988; herein incorporated by reference). At about three weeks post-secondary boost, the immunological activity and the relative avidity index for each IgG subpopulation was determined (Tables 2a-2c). These results indicated that fatty acid ester priming emulsions initiate a stronger secondary immune response than do those vaccines which have a mineral oil base.







TABLE 2a







ELISA Activity for Secondary Immune Response








Fatty Acid
IgG Subpopulations













Ester
2
3
4
5
6
7
















Butyl Stearate
819200

819200

204800
409600


Butyl Myristate
204800

409600

102400
409600


Tridecyl
204800
204800
204800
102400
204800
819200


Stearate


Octastearate
204800

204800
102400
102400
204800


Isopropyl
102400
102400
204800

51200
204800


Myristate


Isocetyl
204800
204800
204800

204800
204800


Myristate


Isocetyl
204800

409600

102400
204800


Stearate


Isopropyl
204800
204800
204800
204800
204800
204800


Isostearate





*NOTE:


Not all IgG subpopulations were well resolved/present.













TABLE 2b







Relative Avidity for Secondary Immune Responses (6M Urea)









IgG Subpopulations













Fatty Acid Ester
2
3
4
5
6
7
















Butyl Stearate
84.17

78.07

72
66.91


Butyl Myristate
77.49

75.72

62.62
54.26


Tridecyl Stearate
79.82
72.13
65.27
68.95
69.47
65.08


Octastearate
70.16

75.47
70.56
69.64
67.66


Isopropyl Myristate
76.4 
71.06
60.71

54.24
58.55


Isocetyl Myristate
79.77
77.24
67.83

69.03
66.56


Isocetyl Stearate
76.22

70.78

65.6
62.58


Isopropyl Isostearate
80.95
78.78
82.76
82.18
78.99
78.86





*NOTE:


Relative Avidity Indexes are expressed as % optical density remaining after treatment with 6M Urea.













TABLE 2c







Relative Avidity Indexes for Secondary Immune Responses (8M Urea)









IgG Subpopulations













Fatty Acid Ester
2
3
4
5
6
7
















Butyl Stearate
66.79

58.50

54.40
52.66


Butyl Myristate
67.73

59.85

49.09
39.3


Tridecyl Stearate
65.67
56.51
49.63
54.41
51.09
46.32


Octastearate
62.78

58.01
56.26
54.75
41.82


Isopropyl Myristate
60.06
51.65
42.50

35.89
38.14


Isocetyl Myristate
71.38
63.44
55.37

53.88
49.57


Isocetyl Stearate
61.3

51.12

49.37
43.57


Isopropyl Isostearate
68.78
67.25
65.25
64.94
61.92
59.21





*NOTE:


Relative Avidity Indexes are expressed as % optical density remaining after treatment with 8M Urea.






EXAMPLE 5
A series of fatty acid esters were formulated into vaccine preparations to prime 18 day old White Plymouth Rock embryos to respond more fully to the SE hexadecane vaccine administered at about 3 weeks post hatch. The fatty acid esters tested included butyl stearate, butyl myristate, ethyl oleate, isopropyl isostearate, octyl palmitate, isopropyl palmitate, isopropyl oleate, and ethyl oleate. These esters were formulated as water-in-oil emulsions using Imwitor 780K (isostearyl diglyceryl succinate; Condea, Piscataway, N.J.) plus Tween-85 and Span-85 surfactants. Embryos were immunized as described previously (Stone et al., Avian Dis., Volume 41, 856-863, 1997; herein incorporated by reference) using about 20 μg acetone inactivated SE in about 200 μl of the respective emulsion. About three weeks post hatch, the birds were vaccinated by the subcutaneous route in the mid-dorsal region of the neck using about 1 ml of hexadecane vaccine containing about 1 mg acetone inactivated SE described above (Example 1). All birds were bled weekly for the next 3 weeks post vaccination and microagglutination titers determined on serum from each group using methods previously described. Results are presented in Table 3 below and indicate that individuals receiving in ovo the vaccine containing the butyl stearate emulsion were primed to respond to secondary vaccination after hatch, exhibiting a decreased delay in responding to vaccination.







TABLE 3







Serum microagglutination titers for In Ovo immuni-


zations utilizing fatty acid ester priming emulsions.


WEEKS POST HATCH













Fatty Acid Ester
3
4
5
6

















Ethyl Oleate


7.3
8.6



Isopropyl

6
7.5
9.7



Isostearate



Butyl
4.17
4.1
9.6
9.9



Stearate



Octyl


7.6
9.4



Palmitate



Isopropyl


11.2
9.9



Palmitate



Isopropyl


9.3
10.5



Oleate



Isopropyl


9.6
10.9



Myristate



Butyl


10.2
10



Myristate



Control


9.3
9.8










For isopropyl isostearate, 4 out of 10 birds responded at 4 weeks post hatch or 1 week post-revaccination.
For butyl stearate, 6 out of 10 birds responded at 3 weeks post hatch while 10 of 10 birds responded at 4 weeks (or 1 week post revaccination) post hatch.
The foregoing detailed description is for the purpose of illustration. Such detail is solely for that purpose and those skilled in the art can make variations without departing from the spirit and scope of the invention.

## Claims

1. A water-in-oil emulsion vaccine comprising:
(a) an antigen in an aqueous solution,
(b) an emulsifier, sorbitan monooleate added to an oil phase,
(c) an oil phase having a C14 to C18 aliphatic straight-chain saturated hydrocarbon, and
(d) an adjuvant mixture consisting essentially of polyoxyethylene sorbitan trioleate and sorbitan trioleate; wherein said antigen is emulsified into said oil phase and said emulsifier and said adjuvant mixture is in a concentration of about 20% by volume of an oil phase.

2. A method for conferring immunity in an animal comprising: injecting an animal with an water-in-oil emulsion vaccine consisting essentially of an antigen wherein said antigen is emulsified into an oil phase, the emulsifier, sorbitan monooleate, and an adjuvant mixture consisting essentially of polyethoxyethylene sorbitan trioleate and sorbitan trioleate wherein said emulsifier and said adjuvant is in a concentration of about 20% of an oil phase.

3. The method of claim 2 wherein said oil phase comprises a C14 to C18 aliphatic straight-chain saturated hydrocarbon.

4. A water-in-oil emulsion vaccine comprising:
(a) an antigen in an aqueous solution,
(b) an oil phase,
(c) an emulsifier, sorbitan monooleate, and
(d) an adjuvant mixture consisting essentially of two nonionic surfactants, wherein said emulsifier and said adjuvant mixture is in a concentration of about 20% by volume if an oil phase, said antigen is emulsified into said oil phase to form a water-in-oil vaccine and said vaccine confers immunity with increases in levels of biliary IgA responses, and increases the relative avidity index of serum IgG subpopulations.

5. The water-in-oil vaccine of claim 4 wherein said adjuvant mixture includes polyoxyethylene sorbitan trioleate and sorbitan trioleate.

6. The water-in-oil emulsion vaccine of claim 4 wherein said oil phase comprises a C14 to C18 aliphatic straight-chain saturated hydrocarbon.

7. A method for conferring immunity in an animal comprising injecting an animal with a water-in-oil emulsion vaccine comprising an aqueous antigen, an emulsifier wherein said emulsifier is added to an oil phase wherein said oil phase comprises a C14 to C18 aliphatic straight-chain saturated hydrocarbon, and adjuvant mixture consisting essentially of two nonionic surfactants, said aqueous antigen is emulsified into said oil phase and said emulsifier and said adjuvant mixture is in a concentration of about 20% by volume of an oil phase to form a water-in-oil vaccine, wherein said method of conferring immunity increases levels of biliary IgA responses, and increases relative avidity index of serum IgG subpopulations.

8. The method of claim 7 wherein said emulsifier is sorbitan monooleate.

9. The method of claim 7 wherein said adjuvant consists essentially of polyoxyethylene sorbitan trioleate and sorbitan trioleate.