U.S. patent application number 10/818433 was filed with the patent office on 2004-12-23 for microfluidized oil-in-water emulsions and vaccine compositions.
This patent application is currently assigned to PFIZER INC.. Invention is credited to Dominowski, Paul J., Klose, Pamela, Krebs, Richard L., Mannan, Ramasamy.
Application Number | 20040258701 10/818433 |
Document ID | / |
Family ID | 33131921 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040258701 |
Kind Code |
A1 |
Dominowski, Paul J. ; et
al. |
December 23, 2004 |
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.
Inventors: |
Dominowski, Paul J.;
(Hickory Corners, MI) ; Klose, Pamela; (East Lyme,
CT) ; Krebs, Richard L.; (Ashland, NE) ;
Mannan, Ramasamy; (Kalamazoo, MI) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
GARDEN CITY
NY
11530
|
Assignee: |
PFIZER INC.
NEW YORK
NY
PFIZER PRODUCTS INC.
GROTON
CT
|
Family ID: |
33131921 |
Appl. No.: |
10/818433 |
Filed: |
April 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60460301 |
Apr 4, 2003 |
|
|
|
Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
A61K 2039/55505
20130101; A61K 39/12 20130101; A61K 2039/55511 20130101; A61K
39/092 20130101; Y02A 50/474 20180101; A61P 37/02 20180101; A61K
9/1075 20130101; A61K 2039/521 20130101; A61K 2039/70 20130101;
A61K 39/0225 20130101; Y02A 50/30 20180101; A61K 2039/55577
20130101; A61K 2039/55566 20130101; A61P 37/04 20180101; A61K 39/39
20130101; A61K 39/0258 20130101; A61K 2039/55555 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 039/00; A61K
039/38; A61K 039/02 |
Claims
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
.mu.m.
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 .mu.m.
4. The emulsion of claim 3 wherein said mean droplet size is
between 0.1 to 0.5 .mu.m.
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.RTM.-80, about 0.08% v/v of SPAN.RTM.-80,
and an aqueous phase, wherein said oil is dispersed in said aqueous
phase and the mean oil droplet size is between 0.1 .mu.m to 0.5
.mu.m.
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 .mu.m to 1.1 .mu.m ; 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 .mu.m.
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 .mu.m.
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 .mu.m.
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.RTM. or SPAN.RTM..
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 .mu.m.
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 .mu.m.
27. The vaccine composition of claim 26 wherein said mean droplet
size is between 0.1 to 0.5 .mu.m.
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.RTM. or SPAN.RTM..
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 .mu.m to 1.1 .mu.m; 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 .mu.m.
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 .mu.m to 1.1
.mu.m; 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
.mu.m.
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 .mu.m.
43. The method of claim 42 wherein said mean oil droplet size is
between 0.1 to 0.8 .mu.m.
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 .mu.m.
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 .mu.m.
59. The vaccine composition of claim 58 wherein said mean droplet
size is between 0.1 to 0.5 .mu.m.
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.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application No. 60/460,301, filed on Apr. 4, 2003.
FIELD OF INVENTION
[0002] This invention relates generally to the field of vaccines
and particularly, to adjuvant formulations for enhancing immune
response in veterinary animals. In particular, the invention
relates to the use of a submicron oil-in-water emulsion as a
vaccine adjuvant for enhancing the immunogenicity of antigens.
Submicron oil-in-water emulsion formulations, vaccine compositions
containing an antigen incorporated into such emulsions, as well as
methods of preparing the emulsions and vaccines, are provided by
the present invention.
BACKGROUND OF THE INVENTION
[0003] Bacterial, viral, parasitic and mycoplasma infections are
wide spread in the veterinary animals such as cattle, swine and
companion animal. Diseases caused by these infectious agents are
often resistant to antimicrobial pharmaceutical therapy, leaving no
effective means of treatment. Consequently, a vaccinology approach
is increasingly used to control the infectious disease in the
veterinary animals. A whole infectious pathogen can be made
suitable for use in a vaccine formulation after chemical
inactivation or appropriate genetic manipulation. Alternatively, a
protein subunit of the pathogen can be expressed in a recombinant
expression system and purified for use in a vaccine
formulation.
[0004] Adjuvant generally refers to any material that increases the
humoral and/or cellular immune response to an antigen. The
traditional vaccines are composed of crude preparation of killed
pathogenic microorganisms, and the impurities associated with the
cultures of pathological microorganisms could act as adjuvant to
enhance the immune response. However, when homogeneous preparations
of pathological microorganisms or purified protein subunits are
used as antigens for vaccination, the immunity invoked by such
antigens is poor and the addition of certain exogenous materials as
adjvuant therefore becomes necessary. Further, synthetic and
subunit vaccines are expensive to produce. Therefore, with the aid
of adjuvant, a smaller dose of antigen may be required to stimulate
the immune response, thereby saving the production cost of
vaccines.
[0005] Adjuvants are known to act in a number of different ways to
enhance the immune response. Many adjuvants modify the cytokine
network associated with immune response. These immunomodulatory
adjuvants can exert their effect even when they are not together
with antigens. In general the immunomodulatory adjuvants cause a
general up-regulation of certain cytokines and a concomitant down
regulation of others leading to a cellular Th1 and/or a humoral Th2
response.
[0006] Some adjuvants have the ability to preserve the
conformational integrity of an antigen so that the antigens can be
efficiently presented to appropriate immune effector cells. As a
result of this preservation of antigen conformation by the adjuvant
formulation, the vaccine would have an increased shelf-life such as
that shown for immune stimulating complexes (ISCOMs). Ozel
M.,et.al.; Quarternary Structure of the Immunestimmulating Complex
(Iscom), J.of Ultrastruc. and Molec. Struc. Res. 102, 240-248
(1989).
[0007] Some adjuvants have the property of retaining the antigen as
a depot at the site of injection. As a result of this depot effect
the antigen is not quickly lost by liver clearance. Aluminum salts
and the water-in-oil emulsions act through this depot effect for a
shorter duration. For example, one can obtain a long-term depot by
using Freund's complete adjuvant (FCA) which is an water-in-oil
emulsion. FCA typically remains at the injection site until
biodegradation permits removal of the antigen by antigen-presenting
cells.
[0008] Based on their physical nature, adjuvants can be grouped
under two very broad categories, namely particulate adjvuants and
non-particulate adjvuants. Particulate adjuvants exist as
microparticles. The immunogen is either able to incorporate or
associate with the microparticles. Aluminum salts, water-in-oil
emulsions, oil-in-water emulsions, immune stimulating complexes,
liposomes, and nano- and microparticles are examples of particulate
adjuvants. The non-particulate adjuvants are generally
immunomodulators and they are generally used in conjunction with
particulate adjuvants. Muramyl dipeptide (an adjuvant-active
component of a peptidoglycan extracted from Mycobacteria),
non-ionic block copolymers, Saponins (a complex mixture of
triterpenoids extracted from the bark of the Quillaja saponaria
tree), Lipid A (a disaccharide of glucosamine with two phosphate
groups and five or six fatty acid chains generally C12 to C16 in
length), cytokines, carbohydrate polymers, derivatized
polysaccharides, and bacterial toxins such as cholera toxin and E.
coli labile toxin (LT) are examples of non-particulate
adjuvants.
[0009] Some of the best-known adjuvants are combination of
non-particulate immunomodulators and particulate materials which
could impart depot effect to the adjuvant formulation. For example,
FCA combines the immunomodualtory properties of Mycobacterium
tuberculosis components along with the short-term depot effect of
oil emulsions.
[0010] Oil emulsions have been used as vaccine adjuvant for a long
time. Le Moignic and Pinoy found in 1916 that a suspension of
killed Salmonella typhimurium in mineral oil increased the immune
response. Subsequently in 1925, Ramon described starch oil as one
of the substances augmenting the antitoxic response to diptheria
toxoid. However, the oil emulsions did not become popular until
1937 when Freund came out with his adjuvant formulation now known
as Freund's Complete Adjuvant (FCA). FCA is a water-in-oil emulsion
composed of mineral (paraffin) oil mixed with killed Mycobateria
and Arlacel A. Arlacel A is principally mannide monooleate and is
used as an emulsifying agent. Although FCA is excellent in inducing
an antibody response, it causes severe pain, abscess formation,
fever and granulomatous inflammation. To avoid these undesirable
side reactions, Incomplete Freund's Adjuvant (IFA) was developed.
IFA is similar to FCA in its composition except for the absence of
mycobacterial components. IFA acts through depot formulation at the
site of injection and slow release of the antigen with stimulation
of antibody-producing cells.
[0011] Another approach to improve FCA was based on the notion that
replacing the mineral oil with a biocompatible oil would help
eliminate the reactions associated with FCA at the injection site.
It was also believed that the emulsion should be oil-in-water
rather than water-in-oil, because the latter produces a
long-lasting depot at the injection site. Hilleman et al. described
an oil-based adjuvant "Adjuvant 65", consisting of 86% peanut oil,
10% Arlacel A as emulsifier and 4% aluminum monostearate as
stabilizer. Hilleman, 1966, Prog. Med. Virol. 8:131-182; Hilleman
and Beale, 1983, in New Approaches to Vaccine Development (Eds.
Bell, R. and Torrigiani, G.), Schwabe, Basel. In humans, Adjuvant
65 was safe and potent but exhibited less adjuvanticity than IFA.
Nevertheless, the use of Adjvuant 65 was discontinued due to
reactogenicity for man with certain lots of vaccine and reduction
in adjuvanticity when a purified or synthetic emulsifier was used
in place of Arlacel A. U.S. Pat. Nos. 5,718,904 and 5,690,942 teach
that the mineral oil in the oil-in-water emulsion can be replaced
with metabolizable oil for the purpose of improving the safety
profile.
[0012] Besides the adjuvanticity and safety, the physical
appearance of an emulsion is also an important commercial
consideration. Physical appearance depends on the stability of the
emulsion. Creaming, sedimentation and coalescence are indicators of
the emulsion instability. Creaming occurs when oil and aqueous
phases of the emulsion have different specific gravity. Creaming
also occurs when the initial droplet size of the emulsion is large
and the emulsion droplets are not having any Brownian motion. When
the droplet size is large, there is a tendency for the interfacial
rupture and the droplets coalesce into large particles. The
stability of the emulsion is determined by a number of factors such
as the nature and amount of emulsifier used, the size of the
droplet size in the emulsion, and the difference in the density
between the oil and water phase.
[0013] Emulsifiers promote stabilization of dispersed droplet by
reducing the interfacial free energy and creating physical or
electrostatic barriers to droplet coalescence. Nonionic as well as
ionic detergents have been used as emulsifiers. Nonionic
emulsifiers orient at the interface and produce relatively bulky
structures, which leads to steric avoidance of the dispersed
droplets. Anionic or cationic emulsifiers induce formation of an
electrical double layer by attracting counter ions; the double
layer repulsive forces cause droplets to repel one another when
they approach.
[0014] Besides using the emulsifiers, the stability of the emulsion
can also be achieved through reducing the droplet size of the
emulsion by mechanical means. Typically propeller mixers, turbine
rotors, colloid mills, homogenizers, and sonicators have been used
to manufacture emulsions. Microfluidization is another way to
increase the homogeneity of the droplet size in the emulsion.
Microfluidization can produce an elegant, physically stable
emulsion with consistent particle size in the submicron range.
Besides increasing the stability of the emulsion, the process of
microfluidization allows terminal filtration which is a preferred
way of ensuring the sterility of the final product. Moreover,
submicron oil particles can pass from injection sites into the
lymphatics and then to lymph nodes of the drainage chain, blood and
spleen. This reduces the likelihood of establishing an oily depot
at the injection site which may produce local inflammation and
significant injection site reaction.
[0015] Microfluidizers are now commercially available. Emulsion
formation occurs in a microfluidizer as two fluidized streams
interact at high velocities within an interaction chamber. The
microfluidizer is air or nitrogen driven and can operate at
internal pressures in the excess of 20,000 psi. U.S. Pat. No.
4,908,154 teaches the use of microfluidizer for obtaining emulsions
essentially free of any emulsifying agents.
[0016] A number of submicron oil-in-water adjuvant formulations
have been described in the literature. U.S. Pat. No. 5,376,369
teaches a submicron oil-in-water emulsion adjuvant formulation
known as Syntax Adjuvant Formulation (SAF). SAF contains squalene
or squalane as the oil component, an emulsion-forming amount of
Pluronic L121 (polyoxy-proplyene-polyoxyethylene) block polymer and
an immunopotentiating amount of muramyldipeptide. Squalene is a
linear hydrocarbon precursor of cholesterol found in many tissues,
notably in the livers of sharks and other fishes. Squalane is
prepared by hydrogenation of squalene and is fully saturated. Both
squalene and squalane can be metabolized and have a good record of
toxicological studies. Squalene or squalane emulsions have been
used in human cancer vaccines with mild side effects and a
desirable efficacy. See, e.g., Anthony C. Allison, 1999, Squalene
and Squalane emulsions as adjuvants, Methods 19:87-93.
[0017] U.S. Pat. No. 6,299,884 and International Patent Publication
WO 90/14837 teach that the polyoxy-proplyene-polyoxyethylene block
copolymers are not essential for the formation of submicron
oil-in-water emulsion. Moreover, these references teach the use of
non-toxic, metabolizable oil and expressly exclude the use of
mineral oil and toxic petroleum distillate oils in their emulsion
formulations.
[0018] U.S. Pat. No. 5,961,970 teaches yet another submicron
oil-in-water emulsion to be used as a vaccine adjuvant. In the
emulsion described in this patent, the hydrophobic component is
selected from the group consisting of a medium chain triglyceride
oil, a vegetable oil and a mixture thereof. The surfactant included
in this emulsion can be a natural biologically compatible
surfactant such as phospholipid (e.g., lecithin) or a
pharmaceutically acceptable non-natural surfactant such as
TWEEN-80. This patent also teaches incorporating the antigen into
the emulsion at the time the emulsion is formed, in contrast to
mixing the antigen with the emulsion after the emulsion has been
independently and extrinsically formed.
[0019] U.S. Pat. No. 5,084,269 teaches that an adjuvant formulation
containing lecithin in combination with mineral oil causes a
decrease in irritation within the host animal and simultaneously
induces increased systemic immunity. The adjuvant formulation
resulting from U.S. Pat. 5,084,269 is commercially used in
veterinary vaccines under the trade name AMPHIGEN.RTM.. The
AMPHIGEN.RTM. formulation is made up of micelles--oil droplets
surrounded by lecithin. These micelles allow more whole cell
antigens to attach than traditional oil-based adjuvants. Moreover,
the AMPHIGEN.RTM.-based vaccine formulations contain a low oil
content of 2.5 to 5% mineral oil, compared to other vaccine
formulations containing oil adjuvants, which typically contain from
10% to 20% oil. Its low oil content makes this adjuvant-based
vaccine formulation less irritating to tissues at the injection
site, resulting in fewer lesions and less trim at slaughter. In
addition, the lecithin coating surrounding the oil droplets further
reduces injection site reactions resulting in a vaccine that is
both safe and efficacious.
[0020] The AMPHIGEN.RTM. formulation is used as an adjuvant in a
number of veterinary vaccines and there is need to maintain the
physical appearance of the vaccine product during short and long
storage periods as well as at the time of reconstitution. In
addition, a lyophilized antigen is mixed with the pre-made adjuvant
formulation just before the injection. This practice does not
always ensure that there is a uniform distribution of the antigen
within the oil-in-water emulsion and the appearance of the emulsion
may not be desirable. Moreover, upon standing, the homogenized
emulsion can show phase separation. Therefore, there exists a need
for a stable adjuvant formulation which does not show phase
separation upon long shelf-life. One way to prevent the phase
separation is to reduce the droplet size and increase the particle
homogeneity of the emulsion. While the process of microfluidization
of metabolizable oil-based emulsion formulations has been
documented, microfluidization of oil-in-water emulsions such as the
AMPHIGEN.RTM. formulation has not yet been carried out.
[0021] In the present invention, microfluidization has been used to
bring the size of lecithin-surrounded mineral oil droplets to
submicron size. Unexpectedly, it has been discovered by the present
inventors that microfluidization of vaccine formulations adjuvanted
with an oil-in-water emulsion comprised of a mixture of lecithin
and oil not only improves the physical appearance of the
formulations, but also enhances the immunizing effects of the
formulations. Microfluidized formulations are also characterized by
an improved safety profile.
SUMMARY OF THE INVENTION
[0022] It has been unexpectedly discovered by the present inventors
that the adjuvant activity and the safety profile of
non-metabolizable oil based oil-in-water emulsions can be improved
through microfluidization. Antigens incorporated in microfluidized
emulsions are stable even when the antigens are intrinsically
incorporated into the emulsions prior to microfluidization.
[0023] Accordingly, in one embodiment, the present invention
provides submicron oil-in-water emulsion formulations useful as a
vaccine adjuvant. The submicron oil-in-water emulsions of the
present invention are composed of a non-metabolizable oil, at least
one surfactant, and an aqueous component, where the oil is
dispersed in the aqueous component with an average oil droplet size
in the submicron range. A preferred non-metabolizable oil is light
mineral oil. Preferred surfactants include lecithin, TWEEN.RTM.-80
and SPAN.RTM.-80.
[0024] A preferred oil-in-water emulsion provided by the present
invention is composed of an AMPHIGEN.RTM.formulation.
[0025] The oil-in-water emulsions of the present invention can
include additional components that are appropriate and desirable,
including preservatives, osmotic agents, bioadhesive molecules, and
immunostimulatory molecules. Preferred immunostimulatory molecules
include, e.g., Quil A, cholesterol, GPI-0100,
dimethyldioctadecylammonium bromide (DDA).
[0026] In another embodiment, the present invention provides
methods of preparing a submicron oil-in-water emulsion. According
to the present invention, the various components of the emulsion,
including oil, one or more surfactants, an aqueous component and
any other component appropriate for use in the emulsion, are mixed
together. The mixture is subjected to a primary emulsification
process to form an oil-in-water emulsion, which is then passed
through a microfluidizer to obtain an oil-in-water emulsion with
droplets of less than 1 micron in diameter, preferably with a mean
droplet size of less than 0.5 micron.
[0027] In still another embodiment, the present invention provides
vaccine compositions which contain an antigen and a submicron
oil-in-water emulsion described hereinabove. The antigen is
incorporated into the emulsion either extrinsically or
intrinsically, preferably, intrinsically.
[0028] The antigen which can be included in the vaccine
compositions of the present invention can be a bacterial, fungal,
or viral antigen, or a combination thereof. The antigen can take
the form of an inactivated whole or partial cell or virus
preparation, or the form of antigenic molecules obtained by
conventional protein purification, genetic engineering techniques
or chemical synthesis.
[0029] In a further embodiment, the present invention provides
methods of preparing vaccine compositions containing an antigen or
antigens combined with a submicron oil-in-water emulsion.
[0030] In preparing the vaccine compositions of the present
invention, the antigen(s) can be combined either intrinsically
(e.g., prior to microfluidization) or extrinsically (e.g., after
microfluidization) with the components of the oil-in-water
emulsion. Preferably, the antigen is combined with the components
of the oil-in-water emulsion intrinsically.
[0031] In still another embodiment, the present invention provides
vaccine compositions which contain a microencapsulated antigen and
a submicron oil-in-water emulsion described hereinabove, where the
microencapsulated antigen is combined with the emulsion
extrinsically.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 depicts the process for the batch preparation of
non-microfluidized vaccine compositions. In this process the
various vaccine components are added to the addition vessel on the
left and ultimately pumped into the blend vessel where the
components are mixed together through simple mechanical means.
[0033] FIG. 2 depicts the process for preparation of microfluidized
vaccine compositions containing intrinsically incorporated antigen.
The various vaccine components are added to the addition vessel and
transferred to the pre-emulsion blending unit for mixing through
simple mechanical means. Subsequently, the emulsion is passed
through a microfluidizer and is collected in the
post-microfluidization chamber.
[0034] FIG. 3 depicts the droplet size distribution of the
non-microfluidized AMPHIGEN.RTM. formulation-based vaccine, the
microfluidized AMPHIGEN.RTM. formulation-based vaccine, and the
bench blend vaccine preparation.
[0035] FIG. 4 shows absence of phase separation in the
microfluidized vaccine preparation.
[0036] FIG. 5 depicts a comparison of the stability of antigens
intrinsically incorporated in microfluidized AMPHIGEN.RTM.
formulation-based vaccine preparation (A907505) and three control,
non-microfluidized AMPHIGEN.RTM.) formulation-based vaccine
preparations (A904369, A904370, and A904371). All four vaccine
preparations were stored at 4.degree. C. for two years. At
different points during the storage (0, 6, 12 or 24 months), all
four formulations were used to vaccinate the three months old cows.
Vaccination was done Day 0 and 21 with a 2 ml vaccine dose and the
sera were collected two weeks post second vaccination. Neutralizing
antibody titer for BVD Type II virus was determined in each of the
serum samples. The data are presented as the geometric mean for 5
animals.
[0037] FIG. 6 shows least squares mean rectal temperature of cattle
prior to and following administration of microfluidized and
non-microfluidized vaccines. T01: Placebo group--single dose; T02:
Placebo group--Double dose; T03: Non-microfluidized
formulation--Single Dose; T04: Non-microfluidized
formulation--Double dose; T05: Microfluidized formulation--Single
Dose; T06: Microfluidized formulation--Double dose.
[0038] FIG. 7 depicts least squares mean injection site reaction
volumes observed in cattle following administration of
non-microfluidized and microfluidized vaccine formulations. T03:
Non-microfluidized formulation--Single Dose; T04:
Non-microfluidized formulation--Double dose; T05: Microfluidized
formulation--Single Dose; T06: Microfluidized formulation--Double
dose.
[0039] FIG. 8 depicts geometric mean IgG titers for recombinant
PauA antigen from Streptococcus uberis after vaccination with the
various vaccine formulations containing both recombinant PauA
antigen and E. coli whole cell antigen.
[0040] FIG. 9 depicts geometric mean IgG titers for E. coli whole
cell antigen from Streptococcus uberis after vaccination with the
various vaccine formulations containing both recombinant PauA
antigen and E. coli whole cell antigen.
[0041] FIGS. 10A and 10B depict the particle size distribution of a
Microfluidized Amphigen formulation at initial production (FIG.
10A) and at 22 months post production (FIG. 10B).
DETAILED DESCRIPTION OF THE INVENTION
[0042] It has been unexpectedly discovered by the present inventors
that microfluidization of vaccine formulations adjuvanted with an
oil-in-water emulsion comprised of a mixture of lecithin and
mineral oil not only improves the physical appearance of the
vaccine formulations, but also enhances the immunizing effects of
the vaccine formulations. Microfluidized vaccine formulations are
also characterized by an improved safety profile.
[0043] Based on these discoveries, the present invention provides
submicron oil-in-water emulsions useful as an adjuvant in vaccine
compositions. Methods of making these submicron oil-in-water
emulsions by using a microfluidizer are also provided. Furthermore,
the present invention provides submicron vaccine compositions in
which an antigen is combined with a submicron oil-in-water
emulsion. Methods for making such vaccine compositions are also
provided. The present invention further provides vaccine
compositions containing microencapsulated antigens combined with a
submicron oil-in-water emulsion and methods for making such
vaccines.
[0044] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the following
subsections which describe or illustrate certain features,
embodiments or applications of the invention.
[0045] Submicron Oil-In-Water Emulsions
[0046] In one embodiment, the present invention provides submicron
oil-in-water emulsion formulations useful as a vaccine adjuvant.
The submicron oil-in-water emulsions of the present invention
enhance the immunogenicity of antigens in vaccine compositions, are
safe for administration to animals and stable during storage.
[0047] The submicron oil-in-water emulsions of the present
invention are composed of a non-metabolizable oil, at least one
surfactant, and an aqueous component, where the oil is dispersed in
the aqueous component with an average oil droplet size in the
submicron range.
[0048] By "submicron" is meant that the droplets are of a size of
less than 1 .mu.m (micron) and the average or mean oil droplet size
is less than 1 .mu.m. Preferably, the mean droplet size of the
emulsion is less than 0.8 .mu.m; more preferably, less than 0.5
.mu.m; and even more preferably, less than 0.4 .mu.m, or about
0.1-0.3 .mu.m.
[0049] The "mean droplet size" is defined as the Volume Mean
Diameter (VMD) particle size within a volume distribution of
particle sizes. The VMD is calculated by multiplying each particle
diameter by the volume of all particles of that size and summing.
This is then divided by the total volume of all particles.
[0050] The term "non-metabolizable oil" as used herein refers to
oils that cannot be metabolized by the body of the animal subject
to which the emulsion is administered.
[0051] The terms "animal" and "animal subject" as used herein refer
to all non-human animals, including cattle, sheep, and pigs, for
example.
[0052] Non-metabolizable oils suitable for use in the emulsions of
the present invention include alkanes, alkenes, alkynes, and their
corresponding acids and alcohols, the ethers and esters thereof,
and mixtures thereof. Preferably, the individual compounds of the
oil are light hydrocarbon compounds, i.e., such components have 6
to 30 carbon atoms. The oil can be synthetically prepared or
purified from petroleum products. Preferred non-metabolizable oils
for use in the emulsions of the present invention include mineral
oil, paraffin oil, and cycloparaffins, for example.
[0053] The term "mineral oil" refers to a mixture of liquid
hydrocarbons obtained from petrolatum via a distillation technique.
The term is synonymous with "liquefied paraffin", "liquid
petrolatum" and "white mineral oil." The term is also intended to
include "light mineral oil," i.e., oil which is similarly obtained
by distillation of petrolatum, but which has a slightly lower
specific gravity than white mineral oil. See, e.g., Remington's
Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing
Company, 1990, at pages 788 and 1323). Mineral oil can be obtained
from various commercial sources, for example, J. T. Baker
(Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred
mineral oil is light mineral oil commercially available under the
name DRAKEOL.RTM..
[0054] Typically, the oil component of the submicron emulsions of
the present invention is present in an amount from 1% to 50% by
volume; preferably, in an amount of 10% to 45; more preferably, in
an amount from 20% to 40%.
[0055] The oil-in-water emulsions of the present invention
typically include at least one (i.e., one or more) surfactant.
Surfactants and emulsifiers, which terms are used interchangeably
herein, are agents which stabilize the surface of the oil droplets
and maintain the oil droplets within the desired size.
[0056] Surfactants suitable for use in the present emulsions
include natural biologically compatible surfactants and non-natural
synthetic surfactants. Biologically compatible surfactants include
phospholipid compounds or a mixture of phospholipids. Preferred
phospholipids are phosphatidylcholines (lecithin), such as soy or
egg lecithin. Lecithin can be obtained as a mixture of phosphatides
and triglycerides by water-washing crude vegetable oils, and
separating and drying the resulting hydrated gums. A refined
product can be obtained by fractionating the mixture for acetone
insoluble phospholipids and glycolipids remaining after removal of
the triglycerides and vegetable oil by acetone washing.
Alternatively, lecithin can be obtained from various commercial
sources. Other suitable phospholipids include phosphatidylglycerol,
phosphatidylinositol, phosphatidylserine, phosphatidic acid,
cardiolipin, and phosphatidylethanolamine. The phospholipids may be
isolated from natural sources or conventionally synthesized.
[0057] Non-natural, synthetic surfactants suitable for use in the
submicron emulsions of the present invention include sorbitan-based
non-ionic surfactants, e.g. fatty-acid-substituted sorbitan
surfactants (commercially available under the name SPAN.RTM. or
ARLACEL.RTM.), fatty acid esters of polyethoxylated sorbitol
(TWEEN.RTM.), polyethylene glycol esters of fatty acids from
sources such as castor oil (EMULFOR); polyethoxylated fatty acid
(e.g., stearic acid available under the name SIMULSOL M-53),
polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL),
polyoxyethylene fatty alcohol ethers (BRIJ.RTM.); polyoxyethylene
nonphenyl ethers (TRITON.RTM. N), polyoxyethylene isooctylphenyl
ethers (TRITON.RTM. X). Preferred synthetic surfactants are the
surfactants available under the name SPAN.RTM. and TWEEN.RTM..
[0058] Preferred surfactants for use in the oil-in-water emulsions
of the present invention include lecithin, Tween-80 and
SPAN-80.
[0059] Generally speaking, the surfactant, or the combination of
surfactants, if two or more surfactants are used, is present in the
emulsion in an amount of 0.01% to 10% by volume, preferably, 0.1%
to 6.0%, more preferably 0.2% to 5.0%.
[0060] The aqueous component constitutes the continuous phase of
the emulsion and can be water, buffered-saline or any other
suitable aqueous solution.
[0061] The oil-in-water emulsions of the present invention can
include additional components that are appropriate and desirable,
including preservatives, osmotic agents, bioadhesive molecules, and
immunostimulatory molecules.
[0062] It is believed that bioadhesive molecules can enhance the
delivery and attachment of antigens on or through the target mucous
surface conferring mucosal immunity. Examples of suitable
bioadhesive molecules include acidic non-naturally occurring
polymers such as polyacrylic acid and polymethacrylic acid (e.g.,
CARBOPOL.RTM.), CARBOMER); acidic synthetically modified natural
polymers such as carboxymethylcellulose; neutral synthetically
modified natural polymers such as (hydroxypropyl) methylcellulose;
basic amine-bearing polymers such as chitosan; acidic polymers
obtainable from natural sources such as alginic acid, hyaluronic
acid, pectin, gum tragacanth, and karaya gum; and neutral
non-naturally occurring polymers, such as polyvinylalcohol; or
combinations thereof.
[0063] The phrase "immunostimulatory molecules", as used herein,
refers to those molecules that enhance the protective immune
response induced by an antigenic component in vaccine compositions.
Suitable immunostimulatory materials include bacterial cell wall
components, e.g., derivatives of N-acetyl
muramyl-L-alanyl-D-isoglutamine such as murabutide, threonyl-MDP
and muramyl tripeptide; saponin glycosides and derivatives thereof,
e.g., Quil A, QS 21 and GPI-0100; cholesterol; and quaternary
ammonium compounds, e.g., dimethyldioctadecylammonium bromide (DDA)
and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine
("avridine").
[0064] Saponis are glycosidic compounds that are produced as
secondary metabolites in a wide variety of plant species. The
chemical structure of saponins imparts a wide range of
pharmacological and biological activities, including some potent
and efficacious immunological activity.
[0065] Structurally, saponins consist of any aglycone attached to
one or more sugar chains. Saponins can be classified according to
their aglycone composition: Triterpene glycosides, Steroid
glycosides, and Steroid alkaloid glycosides.
[0066] Saponin can be isolated from the bark of Quillaja saponaria.
Saponin has long been known as an immunostimulator. Dalsgaard, K.,
"Evaluation of its adjuvant activity with a special reference to
the application in the vaccination of cattle against foot-and-mouth
disease", Acta. Vet. Scand. 69: 1-40 1978. Crude extracts of plants
containing saponin enhanced potency of foot and mouth disease
vaccines. However, the crude extracts were associated with adverse
side effects when used in vaccines. Subsequently, Dalsgaard
partially purified the adjuvant active component from saponin by
dialysis, ion exchange and gel filtration chromatography.
Dalsgaard, K. et al., "Saponin adjuvants III. Isolation of a
substance from Quillaja saponaria Morina with adjuvant activity in
foot-and-mouth disease vaccines", Arch. Gesamte. Virusforsch. 44:
243-254 1974. An adjuvant active component purified in this way is
known as "Quil A." On a weight basis Quil A showed increased
potency and exhibited reduced local reactions when compared to
crude saponin. Quil A is widely used in veterinary vaccines.
[0067] Further analysis of Quil A by high pressure liquid
chromatography (HPLC) revealed a heterogenous mixture of closely
related saponins and led to discovery of QS21 which was a potent
adjuvant with reduced or minimal toxicity. Kensil C. R. et al.,
"Separation and characterization of saponins with adjuvant activity
from Quillaja saponaria Molina cortex," J. Immunol. 146: 431-437,
1991. Unlike most other immunostimulators, QS 21 is water-soluble
and can be used in vaccines with or without emulsion type
formulations. QS21 has been shown to elicit a Th1 type response in
mice stimulating the production of IgG2a and IgG2b antibodies and
induced antigen-specific CD8+CTL (MHC class I) in response to
subunit antigens. Clinical studies in humans have proved its
adjuvanticity with an acceptable toxicological profile. Kensil, C.
R. et al., "Structural and imunological charaterization of the
vaccine adjuvant QS-21. In Vaccine Design: the subunit and Adjvuant
Approach," Eds. Powell, M. F. and Newman, M. J. Plenum Publishing
Corporation, New York. 1995, pp. 525-541.
[0068] U.S. Pat. No. 6,080,725 teaches the methods of making and
using saponin-lilpophile conjugate. In this saponin-lipophile
conjugate, a lipophile moiety such as lipid, fatty acid,
polyethylene glycol or terpene is covalently attached to a
non-acylated or desacylated triterpene saponin via a carboxy group
present on the 3-O-glucuronic acid of the triterpene saponin. The
attachment of a lipophilic moiety to the 3-O-glucuronic acid of a
saponin such as Quillaja desacylsaponin, lucyoside P, or saponin
from Gypsophila, saponaria and Acanthophyllum enhances their
adjuvant effects on humoral and cell-mediated immunity.
Additionally, the attachment of a lipophile moiety to the
3-O-glucuronic acid residue of non- or desacylsaponin yields a
saponin analog that is easier to purify, less toxic, chemically
more stable, and possesses equal or better adjuvant properties than
the original saponin.
[0069] GPI-0100 is a saponin-lipophile conjugate described in the
U.S. Pat. No. 6,080,725. GPI-0100 is produced by the addition of
aliphatic amine to desacylsaponin via the carboxyl group of
glucuronic acid.
[0070] Quaternary ammonium compounds--A number of aliphatic
nitrogenous bases have been proposed for use as immunological
adjuvants, including amines, quaternary ammonium compounds,
guanidines, benzamidines and thiouroniums. Specific such compounds
include dimethyldioctadecylammonium bromide (DDA) and
N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine
("avridine").
[0071] U.S. Pat. No. 5,951,988 teaches adjuvant formulation
containing quarternary ammonium salts such as DDA in conjunction
with an oil component. This formulation is useful in conjunction
with known immunological substances, e.g., viral or bacterial
antigens in a vaccine composition, in order to enhance the
immunogenic response. The composition is also useful without an
incorporated antigen as nonspecific immunostimulatory
formulation.
[0072] U.S. Pat. No. 4,310,550 describes the use of N,N-higher
alkyl-N,N'-bis(2-hydroxyethyl)-propanediamine and N,N-higher
alkyl-xylylenediamines formulated with fat or lipid emulsion as a
vaccine adjuvant. A method of inducing or enhancing the immunogenic
response of an antigen in man or an animal through parenteral
administration of the adjuvant formulation is described in the U.S.
Pat. No. 4,310,550.
[0073] In a preferred embodiment, the present invention provides a
submicron oil-in-water emulsion useful as vaccine adjuvant, which
is composed of an AMPHIGEN.RTM. formulation, with droplets of a
size less than 1 .mu.m and a mean droplet size of about 0.25
.mu.m.
[0074] The term "AMPHIGEN.RTM. formulation" as used herein refers
to a solution formed by mixing a DRAKEOL.RTM. lecithin oil solution
(Hydronics, Lincoln, NE) with saline solution in the presence of
TWEEN.RTM. 80 and SPAN.RTM. 80. A typical AMPHIGEN.RTM. formulation
contains 40% light mineral oil by volume (v/v), about 25% w/v
lecithin, about 0.18% TWEEN 80 by volume (v/v) and about 0.08% Span
80 by volume (v/v).
[0075] Methods of Preparing Submicron Oil-In-Water Emulsions
[0076] In another embodiment, the present invention provides
methods of preparing the submicron oil-in-water emulsions described
hereinabove.
[0077] According to the present invention, the various components
of the emulsion, including oil, one or more surfactants, an aqueous
component and any other component appropriate for use in the
emulsion, are combined and mixed together.
[0078] The mixture formed is subjected to an emulsification
process, typically by passage one or more times through one or more
homogenizers or emulsifiers to form an oil-in-water emulsion which
has a uniform appearance and an average droplet size of about 0.5
.mu.m. Any commercially available homogenizer or emulsifier can be
used for this purpose, e.g., Ross emulsifier (Hauppauge, N.Y.),
Gaulin homogenizer (Everett, Mass.).
[0079] The emulsion so formed is then subjected to
microfluidization to bring the droplet size in the submicron range.
Microfluidization can be achieved by use of a commercial
mirofluidizer, such as model number 11 OY available from
Microfluidics, Newton, Mass.; Gaulin Model 30CD (Gaulin, Inc.,
Everett, Mass.); and Rainnie Minilab Type 8.30H (Miro Atomizer Food
and Dairy, Inc., Hudson, Wis.). These microfluidizers operate by
forcing fluids through small apertures under high pressure, such
that two fluid streams interact at high velocities in an
interaction chamber to form emulsions with droplets of a submicron
size.
[0080] Droplet size can be determined by a variety of methods known
in the art, e.g., laser diffraction, by use of commercially
available sizing instruments. The size may vary depending on the
type of surfactant used, the ratio of surfactant to oil, operating
pressure, temperature, and the like. The skilled artisan can
determine the desired combination of these parameters to obtain
emulsions with desired droplet size without undue experimentation.
The droplets of the emulsions of the present invention are less
than 1 .mu.m in diameter, preferably with a mean droplet size of
less than 0.8 .mu.m, and more preferably with a mean droplet size
less than 0.5 .mu.m, and even more preferably with a mean droplet
size of less than 0.3 .mu.m.
[0081] In a preferred embodiment of the present invention, the
DRAKEOL lecithin oil solution, which is commercially available from
Hydronics (Lincoln, Nebr.) and contains 25% lecithin in light
mineral oil, is combined and mixed with saline as well as
surfactants TWEEN.RTM. 80 and SPAN.RTM. 80 to form an "AMPHGEN.RTM.
solution" or "AMPHIGEN.RTM. formulation". The AMPHGEN.RTM. solution
is then emulsified with a Ross.RTM. (Hauppauge, N.Y. 11788)
emulsifier at approximately 3400 rpm to form an oil-in-water
emulsion. Subsequently the emulsion is passed once through a
Microfluidizer operating at about 4500.+-.500 psi. The
microfluidized oil-in-water emulsion has droplets of a size less
than 1 .mu.m, with a mean droplet size of about 0.25 .mu.m.
[0082] Vaccine Compositions Containing Antigens Incorporated in
Submicron Oil-In-Water Emulsions
[0083] In another embodiment, the present invention provides
vaccine compositions which contain an antigen(s) and a submicron
oil-in-water emulsion described hereinabove. These vaccine
compositions are characterized by having an enhanced immunogenic
effect and an improved physical appearance (e.g., no phase
separation is observed after an extended period of storage). In
addition, the vaccine compositions of the present invention are
safe for administration to animals.
[0084] According to the present invention, the antigen can be
combined with the emulsion extrinsically, or preferably,
intrinsically. The term "intrinsically" refers to the process
wherein the antigen is combined with the emulsion components prior
to the microfluidization step. The term "extrinsically" refers to
the process where the antigen is added to the emulsion after the
emulsion has been microfluidized. The extrinsically added antigen
can be free antigen or it can be encapsulated in microparticles as
further described herein below.
[0085] The term "antigen" as used herein refers to any molecule,
compound or composition that is immunogenic in an animal and is
included in the vaccine composition to elicit a protective immune
response in the animal to which the vaccine composition is
administered.
[0086] The term "immunogenic" as used in connection with an antigen
refers to the capacity of the antigen to provoke an immune response
in an animal against the antigen. The immune response can be a
cellular immune response mediated primarily by cytotoxic T-cells,
or a humoral immune response mediated primarily by helper T-cells,
which in turn activates B-cells leading to antibody production.
[0087] A "protective immune response" is defined as any immune
response, either antibody or cell mediated immune response, or
both, occurring in the animal that either prevents or detectably
reduces the occurrence, or eliminates or detectably reduces the
severity, or detectably slows the rate of progression, of the
disorder or disease caused by the antigen or a pathogen containing
the antigen.
[0088] Antigens which can be included in the vaccine composition of
the present invention include antigens prepared from pathogenic
bacteria such as Mycoplasma hyopneumoniae, Haemophilus somnus,
Haemophilus parasuis, Bordetella bronchiseptica, Actinobacillus
pleuropneumonie, Pasteurella multocida, Manheimia hemolytica,
Mycoplasma bovis, Mycoplasma galanacieum, Mycobacterium bovis,
Mycobacterium paratuberculosis, Clostridial spp., Streptococcus
uberis, Streptococcus suis, Staphylococcus aureus, Erysipelothrix
rhusopathiae, Campylobacter spp., Fusobacterium necrophorum,
Escherichia coli, Salmonella enterica serovars, Leptospira spp.;
pathogenic fungi such as Candida; protozoa such as Cryptosporidium
parvum, Neospora canium, Toxoplasma gondii, Eimeria spp.; helminths
such as Ostertagia, Cooperia, Haemonchus, Fasciola, either in the
form of an inactivated whole or partial cell preparation, or in the
form of antigenic molecules obtained by conventional protein
purification, genetic engineering techniques or chemical synthesis.
Additional antigens include pathogenic viruses such as Bovine
herpesviruses-1,3,6, Bovine viral diarrhea virus (BVDV) types 1 and
2, Bovine parainfluenza virus, Bovine respiratory syncytial virus,
bovine leukosis virus, rinderpest virus, foot and mouth disease
virus, rabies, swine fever virus, African swine fever virus,
Porcine parvovirus, PRRS virus, Porcine circovirus, influenza
virus, swine vesicular disease virus, Techen fever virus,
Pseudorabies virus, either in the form of an inactivated whole or
partial virus preparation, or in the form of antigenic molecules
obtained by conventional protein purification, genetic engineering
techniques or chemical synthesis.
[0089] The amount of the antigen should be such that the antigen
which, in combination with the oil-in-water emulsion, is effective
to induce a protective immune response in an animal. The precise
amount of an antigen to be effective depends on the nature,
activity and purity of the antigen, and can be determined by one
skilled in the art.
[0090] The amount of the oil-in-water emulsion present in the
vaccine compositions should be sufficient for potentiating the
immunogenicity of the antigen(s) in the vaccine compositions. When
desirable and appropriate, additional amounts of surfactant(s) or
additional surfactant(s) can be added in the vaccine composition in
addition to the surfactant(s) provided by the oil-in-water
emulsion. Generally speaking, the oil component is present in the
final volume of a vaccine composition in an amount from 1.0% to 20%
by volume; preferably, in an amount of 1.0% to 10%; more
preferably, in an amount from 2.0% to 5.0%. The surfactant, or the
combination of surfactants if two or more surfactants are used, is
present in the final volume of a vaccine composition in an amount
of 0.1% to 20% by volume, preferably, 0.15% to 10%, more preferably
0.2% to 6.0%.
[0091] In addition to the antigen(s) and the oil-in-water emulsion,
the vaccine composition can include other components which are
appropriate and desirable, such as preservatives, osmotic agents,
bioadhesive molecules, and immunostimulatory molecules (e.g., Quil
A, cholesterol, GPI-0100, dimethyldioctadecylammonium bromide
(DDA)), as described hereinabove in connection with the
oil-in-water emulsion.
[0092] The vaccine compositions of the present invention can also
include a veterinarily-acceptable carrier. The term "a
veterinarily-acceptable carrier" includes any and all solvents,
dispersion media, coatings, adjuvants, stabilizing agents,
diluents, preservatives, antibacterial and antifungal agents,
isotonic agents, adsorption delaying agents, and the like. Diluents
can include water, saline, dextrose, ethanol, glycerol, and the
like. Isotonic agents can include sodium chloride, dextrose,
mannitol, sorbitol, and lactose, among others. Stabilizers include
albumin, among others.
[0093] In a preferred embodiment, the present invention provides a
vaccine composition which includes at least one of a BVDV type I or
BVDV type II antigen, incorporated intrinsically in an oil-in-water
emulsion which has droplets of a size of less than 1 .mu.m,
preferably with a mean droplet size of less than 0.8 .mu.m, more
preferably less than 0.5 .mu.m, and even more preferably with a
mean droplet size of about 0.5 .mu.m. The BVDV type I and/or II
antigen is preferably in the form of an inactivated viral
preparation. The submicron oil-in-water emulsion preferably is
composed of an AMPHIGEN.RTM. formulation (i.e., a formulation which
contains light mineral oil, lecithin, TWEEN.RTM. 80, and SPAN.RTM.
80). The vaccine composition preferably also includes Quil-A,
cholesterol, and thimerosol.
[0094] In another preferred embodiment, the present invention
provides a vaccine composition which includes a Leptospira antigen
and at least one of a BVDV type I or BVDV type II antigen in an
oil-in-water emulsion. The antigens, preferably in the form of
inactivated cell or viral preparation, are incorporated
intrinsically in the oil-in-water emulsion having droplets of a
size of less than 1 .mu.m, preferably with a mean droplet size of
less than 0.8 .mu.m, more preferably less than 0.5 .mu.m, and even
more preferably with a mean droplet size of about 0.5 .mu.m. The
submicron oil-in-water emulsion preferably is composed of an
AMPHIGEN formulation (i.e., a formulation which contains light
mineral oil, lecithin, TWEEN.RTM. 80, and SPAN.RTM. 80). The
vaccine composition preferably also includes one or more
immunostimulatory molecules selected from Quil-A, cholesterol, DDA,
GPI-100 and aluminum hydroxide (AIOH).
[0095] In still another preferred embodiment, the present invention
provides a vaccine composition which includes at least one
bacterial antigen, e.g., the recombinant Streptococcus uberis PauA
protein or a cell preparation of E. coli or a combination of both,
in an oil-in-water emulsion. The antigen(s) is combined
intrinsically with the oil-in-water emulsion which has droplets of
a size of less than 1 .mu.m, preferably with a mean droplet size of
less than 0.8 .mu.m, more preferably less than 0.5 .mu.m, and even
more preferably with a mean droplet size of about 0.25 .mu.m. The
submicron oil-in-water emulsion preferably is composed of an
AMPHIGEN.RTM. formulation (i.e., a formulation which contains light
mineral oil, lecithin, TWEEN.RTM. 80, and SPAN.RTM. 80). The
vaccine composition preferably also includes one or more
immunostimulatory molecules selected from Quil A, DDA and
GPI-100.
[0096] The vaccine compositions of the present invention can be
administered to an animal by known routes, including the oral,
intranasal, mucosal, topical, transdermal, and parenteral (e.g.,
intravenous, intraperitoneal, intradermal, subcutaneous or
intramuscular) route. Administration can be achieved using a
combination of routes, e.g., first administration using a parental
route and subsequent administration using a mucosal route.
[0097] Methods of Preparing Vaccine Compositions
[0098] In a further embodiment, the present invention provides
methods of preparing vaccine compositions containing an antigen or
antigens and a submicron oil-in-water emulsion.
[0099] In preparing the vaccine compositions of the present
invention, the antigen(s) can be combined either intrinsically or
extrinsically with the components of the oil-in-water emulsion.
Preferably, the antigen is combined with the components of the
oil-in-water emulsion intrinsically.
[0100] The antigen can be combined with the various components of
the emulsion, including oil, one or more surfactants, an aqueous
component and any other appropriate component, to form a mixture.
The mixture is subjected to a primary blending process, typically
by passage one or more times through one or more homogenizers or
emulsifiers, to form an oil-in-water emulsion containing the
antigen. Any commercially available homogenizer or emulsifier can
be used for this purpose, e.g., Ross emulsifier (Hauppauge, N.Y.),
Gaulin homogenizer (Everett, Mass.), or Microfluidics (Newton,
Mass.). Alternatively, the various components of the emulsion
adjuvant, including oil, one or more surfactants, and an aqueous
component can be combined first to form an oil-in-water emulsion by
using a homogenizer or emulsifier; and the antigen is then added to
this emulsion. The mean droplet size of the oil-in-water emulsion
after the primary blending is approximately 1.0-1.2 micron.
[0101] The emulsion containing the antigen is then subjected to
microfluidization to bring the droplet size in the submicron range.
Microfluidization can be achieved by use of a commercial
mirofluidizer, such as model number 11OY available from
Microfluidics, Newton, Mass.; Gaulin Model 30CD (Gaulin, Inc.,
Everett, Mass.); and Rainnie Minilab Type 8.30H (Miro Atomizer Food
and Dairy, Inc., Hudson, Wis.).
[0102] Droplet size can be determined by a variety of methods known
in the art, e.g., laser diffraction, by use of commercially
available sizing instruments. The size may vary depending on the
type of surfactant used, the ratio of surfactant to oil, operating
pressure, temperature, and the like. One can determine a desired
combination of these parameters to obtain emulsions with a desired
droplet size. The oil droplets of the emulsions of the present
invention are less than 1 .mu.m in diameter. Preferably the mean
droplet size is less than 0.8 .mu.m. More preferably, the mean
droplet size is less than 0.5 .mu.m. Even more preferably, the mean
droplet size is about 0.1 to 0.3 .mu.m.
[0103] In a preferred embodiment of the present invention, the
DRAKEOL.RTM. lecithin oil solution, which contains 25% lecithin in
light mineral oil, is combined and mixed with surfactants
TWEEN.RTM. 80 and SPAN.RTM. 80 and saline solution to form a
mixture that contains 40% light mineral oil, lecithin, 0.18%
TWEEN.RTM. 80, and 0.08% SPAN.RTM. 80. The mixture is then
emulsified with a Ross.RTM. (Hauppauge, N.Y. 11788) emulsifier at
approximately 3400 rpm to form an emulsion product, which is also
referred to as an "AMPHIGEN.RTM. formulation" or "AMPHIGEN.RTM.
solution". Subsequently, the desired antigen(s) are combined with
the AMPHIGEN.RTM. solution and any other appropriate components
(e.g., immunostimulatory molecules) with the aid of an emulsifier,
e.g., a Ross homogenizer, to form an oil-in-water emulsion
containing the antigen(s). Such emulsion is passed once through a
Microfluidizer operating at about 10000.+-.500 psi. The
microfluidized oil-in-water emulsion has droplets of a size of less
than 1 .mu.m, with the mean droplet size of about 0.25 .mu.m.
[0104] In another preferred embodiment, prior to combining an
oil-in-water emulsion (e.g., an AMPHIGEN.RTM. formulation) with a
desired antigen(s), the antigen(s) is combined with a saponin
glycoside, e.g., Quil A, to form a mixture. This antigen(s)-saponin
mixture is subjected to homogenization, e.g., in a homogenization
vessel. A sterol, e.g., cholesterol, is then added to the
homogenized antigen(s)-saponin mixture. The mixture containing the
antigen(s), saponin and sterol is then subjected to further
homogenization. The homogenized antigen(s)-saponin-sterol mixture
is then combined with an oil-in-water emulsion (e.g., an AMPHIGEN)
formulation) with the aid of a homogenizer, for example. The
homogenized oil-in-water emulsion containing the antigen(s),
saponin and sterol is then subjected to high pressure
homogenization, such as microfluidization.
[0105] Vaccine Compositions Containing Microencapsulated Antigens
in a Submicron Oil-in-Water Emulsion and Methods of Preparation
[0106] In still another embodiment, the present invention provides
vaccine compositions which contain an antigen encapsulated in
microparticles (or "microencapsulated antigen"), where the
microencapsulated antigen is extrinsically incorporated into a
submicron oil-in-water emulsion described hereinabove.
[0107] Methods for absorbing or entrapping antigens in particulate
carriers are known in the art. See, e.g., Pharmaceutical
Particulate Carriers: Therapeutic Applications (Justin Hanes,
Masatoshi Chiba and Robert Langer. Polymer microspheres for vaccine
delivery. In: Vaccine design. The subunit and adjuvant approach.
Eds. Michael F. Powell and Mark J. Newman, 1995 Plenum Press, New
York and London ). Particulate carriers can present multiple copies
of a selected antigen to the immune system in an animal subject and
promote trapping and retention of antigens in local lymph nodes.
The particles can be phagocytosed by macrophages and can enhance
antigen presentation through cytokine release. Particulate carriers
have also been described in the art and include, e.g., those
derived from polymethyl methacrylate polymers, as well as those
derived from poly(lactides) and poly(lactide-co-glycolides)- ,
known as PLG. Polymethyl methacrylate polymers are
non-biodegradable while PLG particles can be biodegrade by random
non-enzymatic hydrolysis of ester bonds to lactic and glycolic
acids which are excreted along normal metabolic pathways.
[0108] Biodegradable microspheres have also used to achieve
controlled release of vaccines. For example, a continuous release
of antigen over a prolonged period can be achieved. Depending upon
the molecular weight of the polymer and the ratio of lactic to
glycolic acid in the polymer, a PLGA polymer can have a hydrolysis
rate from a few days or weeks to several months or a year. A slow,
controlled release may result in the formation of high levels of
antibodies similar to those observed after multiple injections.
Alternatively, a pulsatile release of vaccine antigens an be
achieved by selecting polymers with different rates of hydrolysis.
The rate of hydrolysis of a polymer typically depends upon the
molecular weight of the polymer and the ratio of lactic to glycolic
acid in the polymer. Microparticles made from two or more different
polymers with varying rates of antigen release provide pulsatile
releases of antigens and mimics multiple-dose regimes of
vaccination.
[0109] According to the present invention, an antigen, including
any of those described hereinabove, can be absorbed to a
particulate polymer carrier, preferably a PLG polymer, by using any
procedure known in the art (such as one exemplified in Example 17),
to form a microencapsulated antigen preparation. The
microencapsulated antigen preparation is then mixed with and
dispersed in a submicron oil-in-water emulsion, which emulsion has
been described hereinabove, to form the vaccine composition.
[0110] In a preferred embodiment, the present invention provides a
vaccine composition which contains an antigen encapsulated in a PLG
polymer, wherein the microencapsulated antigen is dispersed
extrinsically in a microfluidized oil-in-water emulsion which is
composed of light mineral oil, lecithin, TWEEN80, SPAN80 and
saline, and has a mean droplet size of less than 1.0 .mu.m.
[0111] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
EXAMPLE 1
Preparation of an AMPHIGEN.RTM. Formulation
[0112] An AMPHIGEN.RTM. formulation was prepared in a two-step
process. In the first step, 80 liters of Drakeol Lecithin oil
solution, 116 liters of Tetanus Toxoid saline, 1.2 liters of SPAN
80, and 2.8 liters of Tween 80 were mixed together and emulsified
using a Ross emulsifier. The Drakeol Lecithin oil solution
contained 25% soya lecithin and 75% mineral oil. Emulsified product
was recirculated through Ross emulsifier for a minimum of 5 volumes
or a minimum of 10 minutes. The emulsified product was stored at
2-7.degree. C. for a maximum of 24 hours for further processing.
The emulsion from the Ross emulsifier tank was transferred to a
Gaulin homogenizer and was homogenized for 20 minutes under a
pressure of 4500 psi. The resulting 40% Drakeol Lecithin oil
solution (hereinafter the "AMPHIGEN.RTM.) formulation" or
"AMPHIGEN.RTM. solution") was then dispensed into sterile
polypropylene carboxy containers. The dispensing was performed
inside a class 100 dispensing hood located in a class 10,000
controlled environment. The containers were stored at 2-7.degree.
C. This AMPHIGEN.RTM. formulation was used in the experiments
described hereinbelow unless indicated otherwise.
EXAMPLE 2
Primary Blending by Flashblend Homogenization of the BVD
Vaccine
[0113] The apparatus used for this homogenization process is shown
in FIG. 1. Using aseptic technique or steam cross valves, a bottle
containing an BVD Type I antigen (an inactivated BVD Type I viral
preparation) was attached to the bottom side port on the blend
vessel. After the transfer of required volume of the BVD Type I
antigen was completed, the BVD Type I bottle was replaced with the
bottle containing an inactivated BVD Type II viral preparation (an
inactivated BVD type II viral preparation). After the required
amount of a BVD Type II antigen transfer was completed, the Ross
homogenizer was attached to the portable vessel and the
recirculation was initiated at maximum RPM (3300 rpm). Vessel
agitation was maintained at medium speed.
[0114] Using aseptic technique or stream cross valve, a bottle
containing Quil-A at 50 mg/ml concentration was attached to the
homogenizer in-line port on the blend vessel. A required amount of
the Quil-A solution was passed into the vessel through line
suction. After the transfer of the Quil-A solution was completed,
the bottle was removed. In the same way, a required amount of
cholesterol in ethanol solution (18 mg/ml) was transferred to the
blend vessel. Subsequently, a required amount of the AMPHIGEN.RTM.
formulation, 10% thimerosol solution, and Basic Modified Eagles
media ("BME") extender solutions were added to the blend
vessel.
[0115] Once all the additions were complete, the mixing was
continued for an additional 15 minutes. The resulting formulation
was aliquoted into 2 ml doses and represented a non-microfluidized
AMPHIGEN.RTM. formulation-based BVD vaccine. Each dose of the
vaccine contained 500 .mu.g Quil-A, 500 .mu.g Cholesterol, 2.5%
AMPHIGEN.RTM. formulation and 0.009% thimerosol. The antigen
concentration for the two different BVD strains was determined in
terms of the ELISA titer for gp53.
EXAMPLE 3
Secondary Blending by Microfluidization
[0116] FIG. 2 illustrates the process used for the secondary
blending through microfluidization. The microfluidizer was steam
sterilized. First the auxiliary processing module chamber was
installed in the unit and the blank chamber was installed on the
second chamber position. The vessel containing the fully adjuvanted
BVD vaccine prepared as described in the Example 2 was connected to
the microfluidizer by attaching a transfer line from the supply
vessel drain valve to the microfluidizer inlet. Nitrogen gas was
connected to the supply vessel air filter inlet and the vessel
pressure setting was adjusted to 20+/-5 PSI. Collection vessel
drain valve was connected to the transfer line from the
microfluidizer outlet. After making all the necessary connections,
the valves were opened and microfluidization was initiated at an
operating pressure of 10,000+/-500 PSI. The entire content of the
vaccine was passed through the microfluidizer one time and was
collected in the post-microfluidization chamber. This preparation
was aliquoted into 2 mL doses and represents the microfluidized
AMPHIGEN.RTM. formulation-based BVD vaccine.
EXAMPLE 4
[0117] Preparation of a Vaccine Composition through Bench
Blend.
[0118] The AMPHIGEN.RTM. formulation prepared as described in
Example 1 was diluted to the 2.5% with the addition of BVD antigens
and the extender. The resulting solution was blended at the bench
using a stir bar instead of using a homogenizer. The final
preparation contained the following composition: BVD Type 1 and
Type 2 antigens, 2.5% AMPHIGEN.RTM. formulation (which contains
oil, lecithin, SPAN.RTM. and TWEEN.RTM., as described in Example
1), and saline. TWEEN 80 and SPAN 80 are present in the final
vaccine preparation at 0.18% and 0.08% by volume, respectively.
EXAMPLE 5
Comparison of Droplet Size Distribution between the
Non-Microfluidized and Microfluidized AMPHIGEN.RTM.
Formulation-Based Vaccine Preparations
[0119] The non-microfluidized AMPHIGEN.RTM. formulation-based
vaccine prepared as described in the Example 2, the microfluidized
AMPHIGEN.RTM. formulation-based vaccine prepared as described in
the Example 3, and the preparation made through bench blend as
described in Example 4, were used to compare the droplet size of
the vaccine preparations. Two mililiters of the sample from each of
the preparations were added to a Malvern 2000 Laser Diffraction
meter and the droplet size distribution was determined. As shown in
FIG. 3, the results indicate that the microfluidized AMPHIGEN.RTM.
formulation-based vaccine preparation had the maximum particle
volume around 0.1 micron while the non-microfluidized AMPHIGEN.RTM.
formulation-based vaccine preparation had the maximum particle
distribution volume around 1 micron.
EXAMPLE 6
Reduction in Vaccine Phase Separation
[0120] Three different vaccine preparations: the non-microfluidized
AMPHIGEN.RTM. formulation-based vaccine prepared as described in
the Example 2, the microfluidized AMPHIGEN.RTM. formulation-based
vaccine prepared as described in the Example 3, and the vaccine
prepared through bench blend as described in Example 4, were
compared side by side to determine their phase separation
properties upon long storage. All these preparations were allowed
to stand at 4.degree. C. for about one month and the phase
separation was monitored in terms of appearance of a creamy layer
at the top of the vaccine preparations. As shown in FIG. 4, there
was no phase separation in the microfluidized AMPHIGEN.RTM.
formulation-based preparation when compared to the other two
preparations.
EXAMPLE 7
Preparation of Microfluidized and Non-Microfluidized Cattle Vaccine
against Bovine Viral Diarrhea Virus
[0121] Bovine Virus Diarrhea viral antigen was intrinsically
incorporated into the AMPHIGEN.RTM. formulation through
microfluidization. The term "intrinsically incorporated" refers to
the process whereby the antigen was added to the AMPHIGEN.RTM.
formulation prior to the microfluidization. The antigen was
subjected to the physical forces of the microfluidization process
along with the components of the adjuvant formulation. In the
control non-microfluidized group, the antigen preparation was
dispersed in the AMPHIGEN.RTM. formulation through blending.
[0122] The final composition of both the control and microfluidized
preparations was as follow: BVD type I with a post-inactivation
ELISA titer of 2535 RU/ dose for gp53, BVD Type II with a
post-inactivation ELISA titer of 3290 RU/dose for gp53, Quil-A at
the concentration of 1.25 mg/dose, cholesterol at the concentration
of 1.25 mg/dose, the AMPHIGEN.RTM. formulation at the final
concentration of 2.5%, and thimerosol at the final concentration of
0.009%. The vaccine dose was 5 ml.
EXAMPLE 8
Long Term Stability of Intrinsically Incorporated BVD Viral
Antigens in the Microfluidized AMPHIGEN.RTM. Formulation-Based
Vaccine Preparation
[0123] This experiment was carried out to determine the stability
of the intrinsically incorporated antigen during the long storage.
Killed BVD Type II viral antigen was intrinsically incorporated
into the AMPHIGEN.RTM. formulation during microfluidization process
to obtain microfluidized vaccine preparation (A907505). Three other
vaccine preparations containing the same antigen in
non-microfluidized AMPHIGEN.RTM. formulation (A904369, A904370, and
A904371) served as the control. In the non-microfluidized
preparations, the antigen was mixed with AMPHIGEN.RTM. formulation
and mixed through blending using a Ross homogenizer. All four
vaccine preparations were stored at 4.degree. C. for two years. At
different points during the storage (0, 6, 12 or 24 months), all
four formulations were used to vaccinate three months old cows.
[0124] On days 0 and 21, three-month old cows were vaccinated
through subcutaneous route with a 2 ml vaccine formulation. The
serum from the vaccinated animals was collected on day 35, and
serological response to the vaccine was measured in terms of the
antibody titer through BVDV-E2 ELISA. As shown in FIG. 5, the
microfluidized vaccine preparation showed a higher antibody titer
at all the time points tested (0, 6, 12, and 24 months), suggesting
the stability of the antigen preparation is not lost during the
intrinsic incorporation of the antigen during the microfuidization
process. Moreover, it was also surprisingly found that the
microfluidized vaccine preparation induced an enhanced immune
response at all time points.
EXAMPLE 9
Reduction in the Vaccine-Induced Increase in Rectal Temperature
after Microfluidization
[0125] The microfluidized and non-microfluidized vaccine
preparations made as described in Example 7 were used to vaccinate
the cattle on day zero and the rectal temperature was monitored
during the period from one day prior to vaccination till four days
post vaccination. The vaccine dose was 2 ml. The groups were
vaccinated either with a single or double dose of the vaccine.
Rectal temperatures were measured and recorded daily on Day -1
through Day 4, inclusive. Rectal temperatures on day 0 were
measured prior to administration of test article.
[0126] As shown in FIG. 6, the results indicate that there was a
steep rise in the rectal temperature in about 24 hours following
vaccination in those animals vaccinated with either a single or
double dose of the non-microfluidized vaccine formulation. However,
in the animals vaccinated with microfluidized forms of vaccine, the
rise in rectal temperature following the vaccination was only
minimal and significantly lower than in the animals vaccinated with
the non-microfluidized formulation (FIG. 6).
EXAMPLE 10
The Injection Site Reaction Volume was Resolved Faster when
Vaccinated with Microfluidized Vaccine Formulations
[0127] The microfluidized and non-microfluidized vaccine
preparations made as described in the Example 7 were used to
vaccinate the cattle on day zero. The animals included in this
study were cross-bred beef cattle. There were three animals in each
of the placebo treatment groups (T01 and T02). There were six
animals in each of the groups T03 through T06. The vaccine dose was
2 ml and the groups were vaccinated either with one or two doses of
the vaccine on day zero. On day 0, test article was administered in
the right neck. Animals receiving the double dose (4 ml) of the
test article (T02, T04, and T06) received the entire double dose as
a single injection at one side. Observation of injection sites,
including estimation of reaction size at the injection site were
made on the right side of the neck on Day 0 through Day 4,
inclusive, and Days 6, 9, and 14. On Day 0 injection sites were
observed prior to administration of test articles. The groups
vaccinated with one or two doses of the placebo did not show any
significant increase in the injection site reaction volume and
therefore those data are not shown in the FIG. 7. In the case of
the non-microfluidized vaccine formulation, there was a
proportional increase in the injection site reaction volume between
the one dose and two dose vaccination. On the other hand, in the
case of the microfluidized vaccine formulation, although the single
dose induced a larger injection site reaction volume, the injection
with second dose did not cause any further increase. Moreover, in
the case of the animals injected with microfluidized vaccine
formulation, the injection site reaction site volume was resolved
at a faster rate when compared to that in the animals injected with
a non-microfluidized vaccine formulation. These results are shown
in FIG. 7.
EXAMPLE 11
Preparation of Microfluidized AMPHIGEN.RTM. Formulation-Based
Vaccine Preparations with Intrinsically Incorporated BVD Viral and
Leptospira Antigens and Immunostimulatory Molecules Such as Quil A
and DDA
[0128] Formalin-inactivated Leptospira hardjo-bovis strain CSL was
formulated in the appropriate adjuvant at direct counts of about
1.4.times.10.sup.9 organisms/5 ml dose. Formalin-inactivated
Leptospira Pomona strain T262 was formulated at about 2400
Nephalomeric Units/5 ml dose. Nephalomeric units were calculated
based on nephalometric measurement of preprocessed fermentation
fluid. BVD virus Type 1 was formulated at E2 Elisa titer of about
3000 Relative Units/5 ml dose. BVD virus Type 2 was formulated at
E2 Elisa titer of about 3500 Relative Units/5 ml dose. The Relative
Unit was calculated based on the E2 ELISA titer of pre-assembly
post-inactivation bulk fluid. Both Quil-A and cholesterol were used
at the concentration of 0.5 mg per dose. Thimerosol and the
AMPHIGEN.RTM. formulation were used at the final concentration of
0.009% and 2.5%, respectively. Aluminum hydroxide (Rehydragel LV)
was used at the final concentration of 2.0%. When DDA was used as
an immunomodulator, DDA was included within the AMPHIGEN.RTM.
formulation. The AMPHIGEN.RTM. formulation (i.e., the 40%
Drakeol-lecithin stock solution) contained 1.6 mg/ml of DDA and,
when appropriately diluted, the final vaccine preparation contained
2.5% AMPHIGEN.RTM. formulation and 0.1 mg/ml of DDA.
[0129] In the preparation of different vaccine formulations, BVD
fractions, Leptos, Quil-A, chloestrol, thimerosol, the
AMPHIGEN.RTM. formulation, and saline as an extender were added to
a Silverson homogenizer and mixed for 15 minutes at 10,000.+-.500
RPM. Components were then microfluidized through a 200 micron
screen at 10,000 psi.
[0130] When the vaccine formulation contained aluminum hydroxide,
the microfluidization was carried out without aluminum hydroxide.
After the microfluidization was completed, aluminum hydroxide was
added and mixed with a stir bar overnight at 4.degree. C.
EXAMPLE 12
Preparation of BVD Viral Vaccine for Challenge Studies
[0131] The vaccine preparation used in this experiment contained
antigens from both BVD virus Type 1 and BVD Virus Type 2. BVD1
-5960 antigen was used at the post-inactivation ELISA titer of 2535
RU/dose for gp53. BVD2-890 antigen was used at the
post-inactivation ELISA titer of 3290 RU/dose for gp53. Quil A and
cholesterol were used at the concentration of 0.5 mg/ml. Thimersol
and the AMPHIGEN.RTM. formulation were used at the final
concentration of 0.009% and 2.5%, respectively. When DDA was used
as an immune modulator, DDA was included within the the
AMPHIGEN.RTM. formulation. The AMPHIGEN.RTM. stock solution (40%
Drakeol-lecithin solution) contained varying amounts of DDA and
when appropriately diluted, the final vaccine preparation contained
2.5% AMPHIGEN.RTM.) formulation and DDA concentration ranging from
0.5 mg/dose to 2.0 mg/dose. Aluminum gel (Rehydragel-LV) was used
at the final concentration of 2%. GPI-0100 was used in the range of
2, 3, and 5 mg/dose.
[0132] All the components were added to a Silverson homogenizer and
blended for 15 minutes at 10,500 rpm and then microfluidized by
passing through a 200 micron chamber with 10,000 psi. When the
vaccine preparation contained aluminum hydroxide, the
microfluidization was carried out without aluminum hydroxide. After
the microfluidization was completed, aluminum hydroxide was added
and mixed with a stir bar overnight at 4.degree. C.
EXAMPLE 13
Protection against Leptospira Challenge after Vaccination with a
Microfluidized Amphigen Vaccine Formulation with Leptospira
Antigens
[0133]
1TABLE 1 Treatment Groups Treatment group Composition of adjuvant
T01 Salilne T02 Quil-A, Cholesterol, and the AMPHIGEN .RTM.
formulation (QAC) T03 Quil-A, Cholesterol, the AMPHIGEN .RTM.
formulation and AIOH (QAC-AIOH) T04 DDA, Cholesterol, and the
AMPHIGEN .RTM. formulation (DDA) T05 DDA, Cholesterol, the AMPHIGEN
.RTM. formulation, and AIOH (DDA-AIOH)
[0134] Table 1 shows the composition of the adjuvant formulations
in the vaccine preparations tested in this study. The vaccine
preparations were prepared as described in the Example 11. There
were six animals in each group. About seven-month old beef
cross-bred heifers were used in this study. Vaccination was done on
Day 0 and Day 21 through subcutaneous route with 5 ml vaccine
volume. Challenge was done with L. hardjo-bovis strain 203 from
NADC (National agricultural Disease Center). Challenge was done
during Days 57-59 with a 1-ml innoculum. Challenge was administered
conjunctively in the eye and vaginally. The challenge material
contained 5.0.times.10.sup.6 leptospires/ml. Urine was collected
weekly for lepto culture, FA and PCR. Kidney collection was made
during Days 112 and 113.
2TABLE 2 Results of the Leptospira Challenge Study Percent Percent
of calves ever Percent of Percent of Calves ever positive for
calves ever calves ever positive for Leptospira positive for
positive for Leptospira in urine Leptospira Leptospira in Urine and
Kidney in urine in urine and Kidneys through and Kidney and Kidney
across Treatment Culture through FA through PCR all assays Saline
100 83.3 83.3 100 QAC 0 0 0 0 QAC/AIOH 0 50.0 0 50.0 DDA 0 0 0 0
DDA/AIOH 0 33.3 16.7 50.0
[0135] Table 2 shows the data from the Leptospira challenge study.
In determining the percentage of Leptospira infection in the
challenged animal, the following criteria were used. If the kidney
culture was positive for only one sample, the animal is considered
to be positive for Leptospira. If an animal is positive in only one
sample for either FA or PCR, the animal is considered to be
negative. If the sample is positive for both FA and PCR in only one
sample, it was considered positive for Leptospira.
[0136] The results shown in Table 2 indicate that there was a
significant shorter duration of urinary shedding in all vaccine
groups based on all the three assays. As far as urinary and kidney
colonization are concerned, the efficacies of the QAC- and
DDA-containing formulations without AIOH were comparable. AIOH did
not improve and even reduced the efficacies of the QAC- or
DDA-containing vaccines in this challenge study.
3TABLE 3 Microscopic Agglutination Titer Range On Day Of Peak
Geometric Mean Titer Prior To Challenge (Day 35) Treatment L.
Hardjo L. pomona Saline <20 <20 QAC 160-640 1280-10240
QAC/AIOH 160-2560 8-10240 DDA 40-1280 320-2560 DDA/AIOH 320-640
1280-5120
[0137] Serological responses against both of the Leptospira
antigens in the vaccine formulation ere detected in the vaccinated
animal and the peak response was noted on Day 35. There was no
correlation between the serological response and the protection
against the challenge. The presence of aluminum gel in the vaccine
formulation reduced the level of protection although the
serological response was enhanced by the presence of aluminum gel
in the vaccine.
EXAMPLE 14
Elicitation of Immune Response to the BVD Viral Antigen and
Protection against the BVD Type 2 Virus Challenge after
Immunization with a Microfluidized Vaccine Preparation Containing
AMPHIGEN.RTM. Formulation and DDA
[0138] Four to seven month-old seronegative calves were used in
this experiment. There were six different groups and each group had
ten animals (Table 4). On Day 0 and Day 21 each animal received one
2 ml subcutaneous dose of the vaccine or placebo in the lateral
neck approximately midway between the scapula and poll.
4TABLE 4 Treatment Groups Treatment Adjuvant composition T01 Saline
T02 Quil-A, AMPHIGEN .RTM. formulation, and Chloesterol T03
AMPHIGEN .RTM. formulation, Choloesterol, DDA (0.5 mg/dose) and
AIOH T04 AMPHIGEN .RTM. formulation, Cholesterol, and DDA (0.5
mg/dose) T05 AMPHIGEN .RTM. formulation, Cholesterol, and DDA (1.0
mg/dose) T06 AMPHIGEN .RTM. formulation, Cholesterol, and DDA (2.0
mg/dose)
[0139] A 5 ml dose of the challenge virus preparation
(approximately 2.5 ml per nostril) was administered intranasally on
Day 44 of the study. Noncytopathic BVD virus Type 2, isolate #
24515 (Ellis Strain), lot # 46325-70 was used in this study as the
challenge strain. Retained samples of challenge material were
tittered (two replicates per titration) at the time challenge was
initiated and immediately upon its completion. The mean live virus
titer per 5 ml dose was 5.3 log.sub.10 FAID.sub.50/5 ml prior to
challenge and 5.4 log.sub.50 FAID.sub.50/5 ml post challenge (FAID
is equivalent to TCID.sub.50).
[0140] Animals were monitored daily from Day -3 through Day 58.
Clinical disease scores of 0, 1, 2, or 3, based on clinical signs
attributable to BVD 2 infection were made for each animal on Days
42 through 58. The scores on Day 44 were recorded prior to
challenge. Blood samples (two 13 ml Serum Separation Tubes, SST)
were collected from each animal on Days 0, 21, 35, 44, and 58 for
determination of serum titers of BVD Type 1 and BVD Type 2 virus
neutralization antibodies.
[0141] Blood samples were collected from each animal on Days 42
through Day 58, inclusive, and the presence of BVD virus in buffy
coat cell was determined. On Day 44, samples were obtained prior to
challenge.
[0142] For determining white blood cell counts, blood samples (one
4 ml EDTA tube) were collected from each animal on Day 42 through
Day 58, inclusive. On Day 44, samples were obtained prior to
challenge.
[0143] Leukopenia was defined as a 40% or greater decrease in the
WBC count from baseline (average of pre-challenge WBC counts from
two days prior to, and the day of challenge).
[0144] Clinical disease scores were used to define disease status
as follows; if the score is .ltoreq.1, then disease=no; if the
score is >2, then disease=yes.
[0145] As shown in the Tables 5 and 6, the groups vaccinated with
vaccines containing BVD viral antigens along with the
AMPHIGEN.RTM.) formulation, Quil A or DDA and microfluidized,
seroconverted with significant serum virus neutralization titers
for both BVD Type 1 and BVD Type 2 viruses. In those groups there
was also a significant reduction in the percentage of animals
showing viremia following challenge, while in the control group
100% of the animals were viremic (Table 7). In addition, in those
vaccinated groups the frequency of the disease was also
significantly reduced (Table 8). Similarly, the percentage of
animals showing leukopenia was also reduced in the vaccine groups
and the reduction of leukopenia was more significant in the group
containing DDA than in the group containing Quil A (Table 9). In
the control group there was a significant drop in the weight gain
when compared to the vaccinated groups. (Table 10)
[0146] Serology
[0147] Prior to vaccination on Day 0, all animals in the study were
seronegative (SVN<1:2) for antibodies to BVD virus Types 1 and 2
(data not shown). Fourteen days after the second vaccination (Day
35), all animals that were administered the placebo (T01) remained
seronegative for antibodies to BVD virus Types 1 and 2; and all of
the animals vaccinated with the ITAs (Investigational Test Antigen)
(T02, T03, T04, T05 and T06) were seropositive (SVN.gtoreq.1:8) for
antibodies to BVD virus, Types 1 and 2. One animal which was
administered with the vaccine adjuvanted with the AMPHIGEN.RTM.
formulation at 2 mg/dose of DDA had an SVN titer of 3 for
antibodies to BVD virus Type 2 on Day 35 (Table 11 and 12).
[0148] Prior to challenge on Day 44, all controls (T01), except
one, were seronegative (SVN<1:2) for antibodies to BVD virus
Types 1 and 2 (data now shown). The one control (#2497) was
seropositive (SVN=10) for antibodies to BVD virus Type 1 and
seronegative for antibodies to BVD virus Type 2. Fourteen days
following challenge, all animals in the study were seropositive for
antibodies to BVD virus Types 1 and 2.
5TABLE 5 BVD Virus Type 1 Geometric Mean Serum Virus Neutralization
Titers BVDv Type 1 Geometric Mean SVN Titers on Study Day Treatment
0 21 35 44 58 T01 Saline <2 <2 <2 <2 23.9 T02 Amphigen,
<2 39.1 19824.5 14018.2 27554.5 Quil A T03 Amphigen, <2 51.8
32204.8 22381.1 23170.4 0.5 mg DDA, Al T04 Amphigen, <2 27.0
14512.4 8932.0 21996.2 0.5 mg DDA T05 Amphigen, <2 26.7 11585.2
8194.6 20882.0 1.0 mg DDA T06 Amphigen, <2 23.5 8778.7 6769.3
16961.1 2.0 mg DDA
[0149]
6TABLE 6 BVD Virus Type 2 Geometric Mean Serum Virus Neutralization
Titers BVDv Type 1 Geometric Mean SVN Titers on Study Day Treatment
0 21 35 44 58 T01 Saline <2 <2 <2 <2 522.0 T02
Amphigen, <2 8.9 2272.4 2048.2 24833.6 Quil A T03 Amphigen,
<2 9.5 3565.7 2702.2 20881.8 0.5 mg DDA, Al T04 Amphigen, 0.5 mg
<2 4.1 1260.7 989.1 18496.2 DDA T05 Amphigen, <2 6.4 1398.8
1453.9 30047.8 1.0 mg DDA T06 Amphigen, 2.0 mg <2 7.7 1673.2
1428.9 16384.0 DDA
[0150]
7TABLE 7 BVD Virus Isolation Following Challenge BVD Virus
Isolation Frequency LSMean (%) of Viremic Days with Treatment On
Study Days Animals Viremia T01 Saline 47 through 58 10/10 (100.0)
10.4 T02 Amphigen, 50 through 53 1/10 (10.0) 0.4 Quil A T03
Amphigen, 0.5 mg -- 0/10 (0.0) 0.0 DDA, Al T04 Amphigen, 48, 50
through 3/10 (30.0) 0.5 0.5 mg DDA 52, 57 T05 Amphigen, 1.0 mg 49
through 51 2/10 (20.0) 0.4 DDA T06 Amphigen, 2.0 mg 48 through 52
2/10 (20.0) 0.5 DDA
[0151]
8TABLE 8 Clinical Signs Of BVD Disease Following Challenge
Frequency Frequency (%) Observations with (%) with Clinical Sign of
BVD Disease Total Treatment Disease 0 1 2 3 Obs. T01 Saline 9/10
(90.0) 75 (46) 63 (37.5) 29 (17.3) 1 (0.6) 168 T02 Amphigen, 1/10
(10.0) 105 (61.8) 63 (37.1) 2 (1.2) 0 (0) 170 Quil A T03 Amphigen,
2/10 (20.0) 99 (58.2) 67 (39.4) 4 (2.4) 0 (0) 170 0.5 mg DDA, Al
T04 Amphigen, 0/10 (0.0) 118 (69.4) 52 (30.6) 0 (0) 0 (0) 170 0.5
mg DDA T05 Amphigen, 0/10 (0.0) 101 (59.4) 69 (40.6) 0 (0) 0 (0)
170 1.0 mg DDA T06 Amphigen, 0/10 (0.0) 104 (61.2) 66 (38.8) 0 (0)
0 (0) 170 2.0 mg DDA
[0152]
9TABLE 9 Leukopenia Following Challenge Leukopenia Frequency (%) of
LSMean Days with Treatment Leukemic Animals Leukemia T01 Saline
10/10 (100.0) 7.8 T02 Amphigen, Quil A 6/10 (60.0) 1.2 T03
Amphigen, 0.5 mg 2/10 (20.0) 0.2 DDA, Al T04 Amphigen, 0.5 mg 4/10
(40.0) 0.8 DDA T05 Amphigen, 3/10 (30.0) 0.9 1.0 mg DDA T06
Amphigen, 2.0 mg 2/10 (30.0) 0.5 DDA
[0153]
10TABLE 10 Body Weight and Body Weight Gain During the Study Mean
Body Weight (lb.) on Study Day Weight Treatment -1 43 50 58 Gain
(lb) T01 Saline 378.0 484.9 491.0 476.9 98.9 T02 Amphigen, 428.0
526.5 546.7 579.0 151.0 Quil A T03 Amphigen, 410.5 514.4 534.2
579.0 168.5 0.5 mg DDA, AlOH T04 Amphigen, 373.7 472.3 492.6 538.1
164.4 0.5 mg DDA T05 Amphigen, 358.9 451.4 478.9 507.1 148.2 1.0 mg
DDA T06 Amphigen, 408. 513.3 533.9 560.3 151.6 2.0 mg DDA
[0154] Virus Isolation
[0155] As the data shown in Table 13, during the challenge period
(Days 44 through 58), all ten animals in the control (T01) were
viremic (BVD virus was isolated on one or more days). In the groups
administered with the ITAs, the frequency of viremic animals was
one, zero, three, two and two in each group of ten (T02, T03, T04,
T05 and T06, respectively). The difference between the control and
the groups administered with the ITAs was statistically significant
(P.ltoreq.0.05). The least squares mean number of days of viremia
was also significantly greater (10.4 days) for the control as
compared to the groups administered with the ITAs (0.0 to 0.5
days).
[0156] Clinical Disease
[0157] Animals with clinical sign scores of 2 or 3 were considered
demonstrating signs of BVD disease. As shown in the Table 14, the
frequency of animals with clinical signs of BVD virus disease was
nine of ten in the control (T01) and one, two, zero, zero and zero
of ten in each of the groups administered the ITAs (T02, T03, T04,
T05 and T06, respectively). The difference between the control and
groups that were administered the ITAs was statistically
significant (P.ltoreq.0.05).
[0158] Leukopenia
[0159] As shown in Table 15, during the challenge period (Days 44
through 58), all ten animals in the control (T01) were leukemic (a
40% reduction in white blood cell count from pre-challenge
baseline, Days 42-44). The frequency of animals with leukemia was
six, two, four, three and two of the ten animals in each of the
groups administered with the ITAs (T02, T03, T04, T05 and T06,
respectively). The difference between the control and the group
administered with vaccine which was adjuvanted with the
AMPHIGNEN.RTM.) formulation at 0.5 mg/dose and aluminum hydroxide
(T03) was statistically significant (P.ltoreq.0.05). The least
squares mean number of days of leukemia was significantly greater
(7.8 days) for the control as compared to the groups administered
with the ITAs (0.2 to 1.2 days).
EXAMPLE 15
Elicitation of Immune Response to the BVD Viral Antigen and
Protection against the BVD Type 2 Virus Challenge after
Immunization with Microfluidized Vaccine Formulation Containing
GPI-0100
[0160] A set of experimental conditions as described in the Example
14 was followed and a direct comparison between Quil A and GPI-0100
was made. As shown in the Tables 11 and 12, the animals vaccinated
with BVD antigens in the microfluidized AMPHIGEN.RTM.)
formulation-based preparation containing either Quil A or GPI-0100
had a significant antibody titer both for BVD Type 1 and BVD Type 2
viruses. The antibody titer for BVD Type 1 virus was much more
higher than that for BVD Type 2 virus. However, subsequent
challenge with BVD Type 2 virus showed a strong protection and the
disease incidence was significantly reduced in the calves
vaccinated with the microfluidized AMPHIGEN.RTM. formulation-based
vaccine preparation containing GPI-0100.
11TABLE 11 BVD virus Type 1 Geometric Mean Serum Virus
Neutralization Titers Geometric mean SVN titer Treatment 0 21 35 43
57 T01 Saline <2 <2 <2 <2 35.5 T02 Amphigen, Quil A
<2 98.7 20171.0 12203.4 44762.4 T03 Amphigen, 2 mg <2 84.6
10998.5 7383.2 25709.2 GPI-0100, AlOH T04 Amphigen, 2 mg <2
106.0 18179.2 8933.2 28526.2 GPI-0100 T05 Amphigen, 3 mg <2 62.9
15024.3 8780.1 19824.4 GPI-0100 T06 Am,phigen, 5 mg <2 71.1
12203.3 7512.0 16670.2 GPI-0100
[0161]
12TABLE 12 BVD virus Type 2 Geometric Mean Serum Virus
Neutralization Titers BVDv Type 1 Geometric Mean SVN Titers on
Study Day Treatment 0 21 35 44 58 T01 Saline <2 <2 <2
<2 14.7 T02 Amphigen, Quil A <2 12.9 2312.0 1692.5 1663.4 T03
Amphigen, 2 mg <2 13.2 1663.5 1116.8 1562.3 GPI-0100, AlOH T04
Amphigen, 2 mg <2 20.5 2610.2 1978.2 2478.7 GPI-0100 T05
Amphigen, 3 mg <2 11.4 1752.8 1305.2 2435.4 GPI-0100 T06
Amphigen, 5 mg <2 12.0 3158.4 2120.2 1845.6 GPI-0100
[0162]
13TABLE 13 BVD Virus Isolation Following Challenge BVD Virus
Isolation Frequency (%) of LSMean Days Treatment Viremic Animals
with Viremia T01 Saline 10/10 (100.0) 8.4 T02 Amphigen, Quil A 3/10
(30.0) 0.3 T03 Amphigen, 2 mg GPI-0100, 0/10 (0.0) 0.0 AIOH T04
Amphigen, 2 mg GPI-0100 1/10 (10.0) 0.1 T05 Amphigen, 3 mg GPI-0100
3/10 (30.0) 0.3 T06 Amphigen, 5 mg GPI-0100 2/10 (20.0) 0.2
[0163]
14TABLE 14 Clinical Signs of BVD Disease Following Challenge
Frequency Frequency (%) Observations with (%) with Clinical Disease
Score of Total Treatment Disease 0 1 2 Obs. T01 Saline 5/10 (50.0)
103 (60.6) 55 (32.4) 12 (7.1) 170 T02 Amphigen, Quil A 5/10 (50.0)
115 (67.6) 48 (28.2) 7 (4.1) 170 T03 Amphigen, 2 mg 0/10 (0.0) 128
(75.3) 42 (24.7) 0 (0) 170 GPI-0100, AlOH T04 Amphigen, 2 mg 0/10
(0.0) 124 (72.9) 46 (27.1) 0 (0) 170 GPI-0100 T05 Amphigen, 3 mg
0/10 (0.0) 104 (61.2) 66 (38.8) 0 (0) 170 GPI-0100 T06 Amphigen, 5
mg 0/10 (0.0) 128 (75.3) 42 (24.7) 0 (0) 170 GPI-0100
[0164]
15TABLE 15 Leukopenia Following Challenge Leukopenia Frequency (%)
of LSMean Days with Treatment Leukopenic Animals Leukopenia T01
Saline 9/10 (90.0) 8.7 T02 Quil A 6/10 (60.0) 1.6 T03 2 mg
GPI-0100, AIOH 7/10 (70.0) 2.6 T04 2 mg GPI-0100 4/10 (40.0) 1.5
T05 3 mg GPI-0100 7/10 (70.0) 2.6 T06 5 mg GPI-0100 8/10 (80.0)
2.9
[0165] In conclusion, safety of each vaccine was demonstrated by
the absence of adverse reactions or mortality in the vaccinated
animals. Potency of each vaccine was demonstrated by seroconversion
(SVN antibody titers to BVD-1 and BVD-2>1:8) in 100% of the
vaccinated animals. Satisfactory resistance to challenge was
demonstrated by the vaccine adjuvanted with 2 mg GPI-0100 only.
EXAMPLE 16
Vaccine Preparation Containing Microencapsulated Antigen in
Microfluidized Oil-In-Water Emulsion
[0166] Three grams of Trehalose (Fluka) was added to water to get a
stock of 333mg/ml of Trehalose solution. Recombinant PauA antigen
solubililzed in 0.8% SDS solution (SDS/rPauA) was added to
Trehalose solution to get a final concentration of 494 .mu.g
rPauA/ml. In the next step 10 grams of polylactide glycolic acid
(PLG- Resomer RE 503H, Boeringher Ingelheim) was dissolved in 200
ml Methylene Chloride (MeCl2). The resulting PLG/MeCl2 solution was
combined with the SDS-rPauA/trehalose solution prepared in the
first step. The combined solution was subjected to
microfluidization using (Microfluidizer from Microfluidics Model
M110EH) and the microfluidized preparation was spray dried using
(Temco Spray Dryer Model SD-05). The spray dried material was
collected using a 500 micron screen.
[0167] The concentration of rPauA in this spray dried material was
quantified using a Western blot analysis. 1.04 mg of spray-dried
material was dissolved in 50 .mu.l of acetone and centrifuged at
13,200 rpm at room temperature for 10 minutes. The supernatant was
removed. The supernatantand the pellet fractions were dried in a
biological safety hood for 2.5 hours. The pellet was resuspended in
47.43 .mu.L of sample solution (25 .mu.l of sample buffer +10 .mu.l
of reducing agent+65 .mu.l of water). The dried supernatant
fraction was resuspended with 20 .mu.l of sample solution. In the
western analysis purified PauA was used as a standard to quantify
the rPauA content of the spray dried material.
[0168] A 20% Manitol stock solution was prepared by dissolving 100
grams of mannitol (Sigma) in 500 ml of Water for Injection (WFI).
Solution was heated to 40.degree. C. with hot plate/stirrer and
cooled to 30.degree. C. Solution was sterile filtered through a
0.22 micron sterile filter (Millipore). 2.5% Carboxymethylcellulose
solution was prepared by dissolving 12.5 grams of
carboxymethyulcellulose (Sigma) in 500 ml of WFI and mixed
overnight at 4.degree. C. Solution was autoclaved at 121.degree.
C.
[0169] The powder resulting from spray drying was reconstituted in
a solution containing 5% mannitol, 0.3% carboxymethyl cellulose,
and 1:5000 of thimerosol. The final solution was aliquoted in to 3
ml vials and lyophilized using a Lyophilizer (USIFROID). The
lyophilized powder represents the microencapsulated rPauA. The
microencapsulated subunit protein antigen is resuspended in 2 ml of
microfluidized oil-in-water emulsion containing an AMPHIGEN.RTM.
formulation (such as the microfluidized emulsion described in
Example 20) and used as a vaccine.
EXAMPLE 17
Preparation of Microfluidized Vaccine Formulation Containing Both
Bacterial Whole Cell Antigen and Recombinant Protein Antigen in
Oil-In-Water Emulsion
[0170] Two vaccine preparations were made which contained both
recombinant Streptococcus uberis PauA protein and Escherichia coli
bacterial cells, added intrinsically to oil-in-water emulsions as
described in Examples 2 and 3. The recombinant PauA antigen was at
the concentration of 100 .mu.g per dose and the E. coli cells were
at the final count of 4.times.10 per dose. The emulsion adjuvant
compositions of the two vaccine formulations are shown in the Table
16.
16TABLE 16 Vaccine formulations containing both the recombinant
protein and whole E. coli cells. Treatment Antigen Adjuvant T01
Placebo Saline T02 Pau A/E. coli SEAM-14 T03 Pau A/E. coli 2.5%
Amphigen, 0.5 mg GPI-0100, 0.5 mg cholesterol T04 Pau A/E. coli
2.5% Amphigen, 0.5 mg dimethyldioctadecylammonium bromide (DDA),
0.5 mg cholesterol
EXAMPLE 18
Immune Response to Microfluidized Vaccine Containing the rPauA and
Whole Cell Bacterial Agents in Oil-In-Water Emulsion
[0171] Mature dairy cows were used in this experiment. Animals were
at the end of their first or second lactation at the time of
enrollment. Two ml of each vaccine formulation was administered
subcutaneously three times, once at the time of drying off (D-0),
28 days later (D=28), and again 4 to 10 days following calving (C+4
- C+10). The first and third dose was administered on the left side
of the neck and the second dose was administered on the right side
of the neck. Blood was collected prior to each vaccination and
approximately 14 days and 32 days following third vaccination. The
antibody titer for E. coli and the rPauA antigen were determined
through ELISA. As shown in FIG. 8, the results indicate that the
antibody titer for rPauA was higher in the group vaccinated with
vaccine formulation containing GPI-0100 as an immunostimulant and
peaked on day 70 post initial vaccination. The antibody titer for
E. coli antigen is shown in FIG. 9. The antibody titer for E. coli
antigen was comparable in both vaccine formulations, although the
presence of GPI-0100 as an immunostimulant induced a relatively
higher antibody titer when compared to the formulation with DDA as
an immunostimulant.
EXAMPLE 19
Analysis of Virucidal Activity of the Microfluidized AMPHIGEN.RTM.
Formulation Based Vaccine Preparations
[0172] In order to determine whether microfluidization inactivates
the virus, the viricidal activity of three microfluidized
AMPHIGEN.RTM. formulation based vaccine preparations were
determined. The three preparations contained three different bovine
infectious viruses, namely bovine herpes virus (BHV), parainfluenza
virus 3 (PI3), and bovine respiratory synctial virus (BRSV).
[0173] Detection of the viricidal activity in the three vaccine
preparations was conducted in accordance with the USDA 9CFR.113.35
requirements.
[0174] The results shown in Table 16 indicate that
microfluidization of AMPHIGEN.RTM. formulation-based vaccine
preparations does not cause any significant inactivation of the
vaccine preparation.
17TABLE 16 Analysis Of Viricidal Activities Of Microfluidized
Vaccines Serial BRSV BHV PI3 A 0 0.2 0 AM200 -0.2 0 -0.2 AM75 0
-0.3 -0.3 AM75@37 C. 0.1 -0.3 -0.2 B 0 -0.1 -0.2 BM200 0 0 -0.2
BM75 -0.2 -0.5 0 BM75@37 C. 0.5 -0.5 0 C 0.1 -0.1 -0.2 CM200 -0.2
-0.1 -0.2 CM75 0.1 0.5 -0.2 CM75@37 C. 0.5 0.5 -0.2 A =
Choloesterol added at 650 ml/min B = Cholesterol added at 28 ml/mim
C = Cholesterol added at 5 ml/min M200 = Microfluidized with 200
micron screen M75 = Microfluidized with 75 micron screen M75@37 C.
= Fluids heated to 37.degree. C. prior to microfluidization
[0175] A value above 0.7 is an indication of viricidal effect.
EXAMPLE 20
Preparation of a Microfluidized AMPHIGEN.RTM. Formulation
[0176] An AMPHIGEN.RTM. formulation was prepared by combining the
DRAKEOL lecithin oil solution (light mineral oil with 25% lecithin)
and TWEEN 80 (with the final concentration of 0.18%) and Span 80
(with the final concentration of 0.08%) with mixing for 8-22 hours
at 36.+-.1.degree. C. The oil mixture was then added to saline with
the aide of a Ross.RTM. (Hauppauge, N.Y. 11788) emulsifier at
approximately 3400 rpm. Subsequently the mixture was passed once
through a microfluidizer with a 200 .mu.m interaction chamber at
4500.+-.500 psi. FIGS. 10A and 10B show the stability of the
microfluidized AMPHIGEN.RTM. formulation. Particle size
distribution, as measured by laser diffraction, at the starting,
initial time point (FIG. 10A) was nearly identical to the particle
size distribution after 22 months of 4.degree. C. storage (FIG.
10B).
* * * * *