U.S. patent application number 10/389025 was filed with the patent office on 2003-09-18 for adjuvants for use in vaccines.
Invention is credited to Dearwester, Don A., Roberts, David S., Swearingin, Leroy A..
Application Number | 20030175308 10/389025 |
Document ID | / |
Family ID | 26815550 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175308 |
Kind Code |
A1 |
Roberts, David S. ; et
al. |
September 18, 2003 |
Adjuvants for use in vaccines
Abstract
The invention relates to adjuvants that contain a lecithin, an
oil and an amphiphilic surfactant and that are capable of forming a
stable oil-in-water emulsion vaccine so as to minimize local
reactions to the vaccine in the injected animal.
Inventors: |
Roberts, David S.;
(Philadelphia, PA) ; Swearingin, Leroy A.;
(Waterford, CT) ; Dearwester, Don A.; (Westerly,
RI) |
Correspondence
Address: |
KOHN & ASSOCIATES, PLLC
SUITE 410
30500 NORTHWESTERN HWY.
FARMINGTON HILLS
MI
48334
US
|
Family ID: |
26815550 |
Appl. No.: |
10/389025 |
Filed: |
March 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10389025 |
Mar 13, 2003 |
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09489713 |
Jan 24, 2000 |
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6572861 |
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60117705 |
Jan 29, 1999 |
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60121760 |
Feb 26, 1999 |
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Current U.S.
Class: |
424/234.1 ;
424/240.1; 424/257.1 |
Current CPC
Class: |
A61K 39/0241 20130101;
A61P 27/16 20180101; A61P 11/00 20180101; A61K 2039/55566 20130101;
Y10S 424/825 20130101; A61K 2039/70 20130101; A61P 31/00 20180101;
A61K 39/39 20130101; A61K 2039/521 20130101; A61K 39/102 20130101;
A61P 37/04 20180101; A61P 11/02 20180101; A61P 31/04 20180101; A61P
37/00 20180101; A61K 2039/552 20130101; A61K 39/099 20130101 |
Class at
Publication: |
424/234.1 ;
424/240.1; 424/257.1 |
International
Class: |
A61K 039/02; A61K
039/10; A61K 039/108 |
Claims
What is claimed is:
1. A vaccine composition comprising from about 0.25% to about 12.5%
v/v of a lecithin, from about 1% to about 23% v/v of an oil, from
about 1.5% to 3.5% v/v of at least one amphiphilic surfactant and
an antigen.
2. The vaccine composition of claim 1 wherein said oil is a mineral
oil and wherein said lecithin is de-oiled.
3. The vaccine composition of claim 1 further comprising an aqueous
carrier.
4. The vaccine composition of claim 1 wherein said antigen is
selected from the group consisting of Erysipelothrix rhusiopathiae
antigens, Bordetella bronchiseptica antigens, Pasteurella multocida
antigens, antigens of Eschericia coli strains that cause neonatal
diarrhea, Actinobacillus pleuropneumoniae antigens, Pasteurella
haemolytica antigens and combinations thereof.
5. The vaccine composition of claim 1 wherein two amphiphilic
surfactants are used.
6. The vaccine composition of claim 5 wherein one hydrophilic
surfactant and one lipophilic surfactant are used.
7. The vaccine composition of claim 5 wherein said lecithin is at a
concentration of about 0.5% v/v, wherein said oil is at a
concentration of about 4.5% v/v and wherein said amphiphilic
surfactants are at a concentration of about 2% v/v.
8. The vaccine composition of claim 5 further comprising an aqueous
carrier.
9. An adjuvant composition comprising from about 0.25% to about
12.5% v/v of a lecithin, from about 1% to about 23% v/v of an oil
and from about 1.5% to 3.5% v/v of at least one amphiphilic
surfactant when said adjuvant is formulated in a vaccine
composition.
10. A method of making a vaccine composition comprising adding an
adjuvant composition of claim 9 to an antigen composition.
11. The method of claim 10 wherein said antigen is selected from
the group consisting of Erysipelothrix rhusiopathiae antigens,
Bordetella bronchiseptica antigens, Pasteurella multocida antigens,
antigens of Eschericia coli strains that cause neonatal diarrhea,
Actinobacillus pleuropneumoniae antigens, Pasteurella haemolytica
antigens and combinations thereof.
12. An antigen composition comprising a Bordetella bronchiseptica
culture that has been inactivated by adding formalin followed by
adding glutaraldehyde.
13. A vaccine composition comprising the antigen composition of
claim 12 and an adjuvant.
14. The vaccine composition of claim 13 wherein said adjuvant is
the adjuvant of claim 1.
15. A method of inactivating a Bordetella bronchiseptica culture
comprising adding formalin to said culture followed by adding
glutaraldehyde to said culture.
16. A Bordetella bronchiseptica composition comprising a Bordetella
bronchiseptica culture, formalin and glutaraldehyde.
17. A vaccine for protecting a mammal against a Bordetella
bronchiseptica infection comprising an amount of Bordetella
brochiseptica cells from a culture inactivated according to the
method of claim 15 effective to protect said mammal against a
Bordetella bronchiseptica infection and a physiologically
acceptable carrier.
18. A vaccine according to claim 17, further comprising an amount
of toxigenic Pasteurella multocida antigens effective to protect
the mammal against a toxigenic Pasteurella multocida infection.
19. A method for protecting a pig against a Bordetella
bronchiseptica infection and/or a toxigenic Pasteuralla multocida
infection comprising vaccinating the pig with an amount of a
vaccine according to claim 18 effective to protect against a
Bordetella bronchiseptica infection and a toxigenic Pasteuralla
multocida infection.
20. A method for protecting a piglet against atrophic rhinitis
which comprises: a) vaccinating a pig prior to furrowing and during
pregnancy with an amount of a vaccine according to claim 18
effective to provide anti-Bordetella bronchiseptica antibodies and
anti-toxigenic Pasteurella multocida antibodies in the colostrum
produced by said female pig; and b) providing colostrum produced by
said female pig to the piglet within about 24 hours subsequent to
the piglet's birth.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/117,705, filed Jan. 29, 1999 and U.S.
Provisional Application No. 60/121,760, filed Feb. 26, 1999.
FIELD OF THE INVENTION
[0002] The invention relates to immunological adjuvants. In
particular, the invention relates to adjuvants which comprise an
oil-in-water emulsion and a surfactant. Adjuvants of the invention
are useful in a variety of vaccine formulations, including vaccines
comprising bacterial or viral components.
BACKGROUND OF THE INVENTION
[0003] The generation of immunity to infectious organisms is a
powerful tool in disease control. Those antigens that induce
immunity to infection are known as immunogens. The protective
antibody they induce may collaborate with other natural defenses to
inhibit the infective process, or they may neutralize harmful
products of the infective organism such as toxins.
[0004] An effective means of enhancing the antibody response is the
use of an adjuvant. Thus, an adjuvant is included in a vaccine as
an additive or vehicle to enhance the response to the antigen. An
adjuvant may function by different mechanisms, including (1)
trapping the antigen in the body to cause a slow release, (2)
attracting cells of the immune system to the injection site, (3)
stimulating cells of the immune system to proliferate and to become
activated, and (4) improving antigen dispersion in the recipient's
body.
[0005] A number of agents with diverse chemical properties have
been used as adjuvants, including water-in-oil and oil-in-water
emulsions, mineral salts, polynucleotides and natural substances.
One adjuvant, known under the trademark AMPHIGEN.TM., is described
in U.S. Pat. No. 5,084,269. AMPHIGEN.TM. adjuvant consists of
de-oiled lecithin dissolved in an oil, usually light liquid
paraffin. In vaccine preparations AMPHIGEN.TM. is dispersed in an
aqueous solution or suspension of the immunizing antigen as an
oil-in-water emulsion.
[0006] Problems were observed when using an AMPHIGEN.TM. adjuvant
according to U.S. Pat. No. 5,084,269, above. For example, the
lecithin in the AMPHIGEN.TM. does not suffice to produce a stable
emulsion of the oil, thus leading to a pool or depot of oil in the
injected tissues. Mineral oil can not be metabolized or removed by
the animal. As a result, the oil becomes a source of severe chronic
inflammation and scarring. Emulsifying the AMPHIGEN.TM. directly in
the antigenic preparation carries the risk of damaging the antigen.
Also, if the desired emulsion fails to form, the valuable antigen
must be discarded.
[0007] An adjuvant useful in vaccines for animals, including
humans, that is effective and solves the above problems would
therefore be highly desirable.
SUMMARY OF THE INVENTION
[0008] The invention relates to an adjuvant useful for the
enhancement of the immune response of an animal to an antigen. In
particular, the invention relates to an adjuvant that is capable of
forming an oil-in-water emulsion in a vaccine composition. The
invention also relates to an adjuvant that, when used in a vaccine
formulation, causes minimal inflammation and scarring at the
vaccination site. The invention further relates to a vaccine
formulation that contains an adjuvant of the invention. Finally,
the invention relates to a method of using an adjuvant of the
invention in a vaccination.
[0009] In one embodiment, the adjuvant of the invention comprises a
lecithin, an oil and an amphiphilic surfactant capable of
emulsifying the adjuvant, for example, a Tween or a Span
surfactant. In another preferred aspect, the surfactant is Tween
80, Tween 85, Span 80 or Span 85.
[0010] In another embodiment, the adjuvant of the invention
comprises a lecithin, an oil and two amphiphilic surfactants
capable of emulsifying the adjuvant or a vaccine composition that
contains the adjuvant. In a preferred aspect, one of the two
surfactants is predominantly found in the aqueous phase, for
example, Tween 80, and the other surfactant is predominantly found
in the oil phase, for example, Span 80.
[0011] A lecithin is a phosphatide. Crude preparations of lecithin
may include triglycerides. For purposes of the present invention,
"lecithin" encompasses both purified and crude preparations. In a
preferred aspect, the lecithin is de-oiled.
[0012] Suitable oils include a mineral oil, for example,
DRAKEOL.TM. light mineral oil.
[0013] In a further embodiment, the adjuvant of the invention
contains an aqueous carrier solution, for example, a
physiologically acceptable buffer, water or a saline solution.
[0014] In a preferred embodiment, the adjuvant of the invention
contains a lecithin, a mineral oil, two amphiphilic surfactants and
an aqueous carrier solution (e.g., saline).
[0015] In another embodiment of the invention, a method to
inactivate a culture of Bordetella bronchiseptica ("B.
bronchiseptica") using formalin and glutaraldehyde is described. In
another aspect, a culture of B. bronchiseptica is provided that was
inactivated using formalin and glutaraldehyde. In yet another
aspect, an antigen composition from a B. bronchiseptica culture is
provided that was inactivated using formalin and glutaraldehyde. In
yet another aspect, a vaccine composition is provided that contains
an antigen composition from a B. bronchiseptica culture that was
inactivated using formalin and glutaraldehyde.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 presents a graph depicting the distribution of
droplet sizes of an emulsion prepared as described below. Lines (a)
and (b) depict that about 94% of the droplets have a diameter of 1
.mu.m or less.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The invention relates to an adjuvant useful for the
enhancement of the immune response to an antigen. In particular,
the invention relates to an oily adjuvant that is capable of
emulsifying a vaccine formulation. Further, the invention relates
to an adjuvant that, when used in a vaccine formulation, is capable
of substantially avoiding the inflammation or scarring at the
injection site, typical of vaccines containing mineral oil.
Adjuvants of the invention comprise a lecithin, an oil and an
amphiphilic surfactant capable of emulsifying the adjuvant or a
vaccine composition that contains the adjuvant.
[0018] The invention is based, in part, on the discovery that
adding from about 1.5% v/v (i.e., 1.5% volume per volume
concentration obtained by, e.g., mixing 98.5 volumes of the vaccine
comprising the adjuvant with 1.5 volumes of the amphiphilic
surfactant) to 3.5% v/v of an amphiphilic surfactant to a vaccine
containing an adjuvant as described in U.S. Pat. No. 5,084,269 is
effective to sufficiently emulsify a vaccine composition formulated
with such an adjuvant and to minimize irritation in the injection
site of the vaccinated animal.
[0019] In one embodiment, the adjuvant of the invention contains a
lecithin and an oil and an amphiphilic surfactant. In one
embodiment, the adjuvant of the invention contains a lecithin and
an oil and an amphiphilic surfactant capable of emulsifying a
vaccine composition formulated with an adjuvant of the invention.
In another preferred embodiment, two amphiphilic surfactants are
used in an adjuvant of the invention, for example a Tween and a
Span surfactant.
[0020] A preferred adjuvant, herein referred to as "No. 1
Adjuvant", comprises about 2% v/v lecithin, about 18% v/v mineral
oil, and about 8% v/v surfactant (e.g., about 5.6% v/v Tween 80 and
about 2.4% v/v Span 80), with the remaining volume being a saline
solution. In a preferred aspect, a vaccine composition is
formulated comprising an antigen at a concentration of about 75%
v/v and an adjuvant, preferably No. 1 Adjuvant, at a concentration
of about 25% v/v of the vaccine composition. All concentrations
provided herein in percentage are indicated in volume per volume
unless the context indicates otherwise.
Surfactants Useful in the Adjuvant of the Invention
[0021] Surfactants useful for the adjuvant of the invention are
amphiphilic and acceptable for veterinary or medical use. Whether
or not a particular surfactant is acceptable for medical or
veterinary use can be determined by those of ordinary skill in the
art. A surfactant is amphiphilic if a part of the surfactant
molecule is hydrophobic and a part is hydrophilic. See U.S. Pat.
Nos. 5,690,942; 5,376,369; 4,933,179 and 4,606,918, which describe
surfactants than can be used in the adjuvant of the invention.
Examples of surfactants useful in the adjuvant of the invention
include, but are not limited to, a Tween surfactant and a Span
surfactant. Tween and Span surfactants include, but are not limited
to, monolaureate (Tween 20, Tween 21, Span 20), monopalmitate
(Tween 40, Span 40), monostearate (Tween 60, Tween 61, Span 60),
tristearate (Tween 65, Span 65), monooleate (Tween 80, Tween 81,
Span 80) and trioleate (Tween 85, Span 85). In a preferred
embodiment, Tween 80, Tween 85, Span 80 or Span 85 is used.
[0022] It is preferred that a surfactant useful in the adjuvant of
the invention is amphiphilic and has a hydrophilic-lipophilic
balance ("HLB") value that is preferably at least about half the
sum of the HLB values of all other components of the adjuvant. More
preferably, the surfactant has an HLB value that is from about half
to about twice the sum of the HLB values of all other components of
the adjuvant. More preferably, the surfactant has an HLB value that
is about the same as the HLB value of all other components of the
adjuvant. HLB values are readily available for surfactants,
lecithins, oils and carrier solutions or, if necessary, can be
determined through routine experimentation. For example, see U.S.
Pat. Nos. 4,504,275 and 4,261,925 and references provided
therein.
[0023] Amphiphilic surfactants useful in the adjuvant of the
invention have HLB values from about 2 to about 20, preferably from
about 3 to about 17. Methods for determining the HLB value of
particular surfactants are known in the art. See for example U.S.
Pat. Nos. 5,603,951; 4,933,179 and 4,606,918, which describe
surfactants having particular HLB values.
[0024] The concentration of a surfactant in a vaccine composition
formulated with the adjuvant of the invention is from about 1.5% to
3.5% v/v, more preferably from about 1.5% to about 3% v/v, more
preferably from about 1.5% to about 2.5%, and most preferably about
2% v/v. When more than one surfactant is used, the sum of the
concentrations of all surfactants used in a vaccine composition
formulated with the adjuvant of the invention is also from about
1.5% to 3.5%, more preferably from about 1.5% to about 3%, more
preferably from about 1.5% to about 2.5%, and most preferably about
2% v/v.
[0025] The concentration of a surfactant in the adjuvant of the
invention also depends on the concentration at which the adjuvant
is used in a vaccine composition. For example, a vaccine
composition may be formulated with the adjuvant of the invention so
that about 25% of the volume of the vaccine composition is the
adjuvant ("25% adjuvant") and the remaining about 75% is made up of
other components, for example the antigen composition. In one
aspect, the concentration of the surfactant in a 25% adjuvant is
from about 6% to 14% v/v. More preferably, the surfactant
concentration in a 25% adjuvant is from about 6% to about 12%, more
preferably from about 6% to about 10%, and most preferably about 8%
v/v.
[0026] The concentration of the surfactant in the adjuvant of the
invention is dependent on different factors. For example, the
higher the concentration of oil in the adjuvant the more surfactant
is required to emulsify a vaccine composition formulated with the
adjuvant of the invention. Another factor that is useful to
determine the concentration of a surfactant is the concentration of
a lecithin. The higher the concentration of a lecithin in the
adjuvant, the less surfactant may be required for
emulsification.
[0027] When the adjuvant of the invention is used in a vaccine
composition at a concentration of less than 25% v/v, the
concentration of the adjuvant components in the adjuvant has to be
increased accordingly. The aqueous carrier is an exception as the
carrier always comprises the volume that remains unoccupied by all
other components; thus if the concentration of all components
except the carrier increases, the concentration of the carrier in
the adjuvant will decrease and vice versa. For example, when the
adjuvant is used at a concentration of about 12.5% v/v in a vaccine
composition, the concentration of the components in the adjuvant is
about twice the concentration of the components in a 25% adjuvant.
Similarly, when the adjuvant of the invention is used in a vaccine
composition at a concentration that is above 25% v/v, the
concentration of the components in the adjuvant has to be decreased
accordingly, for example when the adjuvant is used at a
concentration of about 50% v/v in a vaccine composition, the
concentration of the components in the adjuvant is about half the
concentration of the components in a 25% adjuvant.
[0028] In one embodiment, two amphiphilic surfactants may be used
in the adjuvant of the invention. Preferably, the two surfactants
would include one surfactant that would be more concentrated in an
aqueous phase than in an oil phase of the adjuvant ("hydrophilic
surfactant") and one surfactant that would be more concentrated in
an oil phase of the adjuvant ("lipophilic surfactant"). For
example, Tween 80 would concentrate more in an aqueous phase and
Span 80 would concentrate more in an oil phase. A preferred
hydrophilic surfactant has an HLB value from about 9 to about 20
and a preferred lipophilic surfactant has an HLB value from about 2
to about 9. See U.S. Pat. Nos. 5,603,951; 4,933,179 and 4,606,918,
which describe surfactants with HLB values in both ranges useful
for the adjuvant of the invention.
[0029] When two surfactants are used in the adjuvant of the
invention, the total concentration of both surfactants combined in
a vaccine composition formulated with the adjuvant of the invention
is from about 1.5% to 3.5%, more preferably from about 1.5% to
about 3%, more preferably from about 1.5% to about 2.5%, and most
preferably about 2% v/v. The concentration of each of two
surfactants used in the adjuvant of the invention may differ from
each other. For example, when a hydrophilic surfactant and a
lipophilic surfactant are used, for example Tween 80 and Span 80,
the concentration of Tween 80 may be from about 1.2.times. to about
5.times., more preferably from about 1.5.times. to about 4.times.,
more preferably from about 1.8.times. to about 3.times., more
preferably from about 2.times. to about 2.5.times. and more
preferably about 2.3.times. as high as the concentration of Span
80, preferably when used in an adjuvant with a lecithin and an oil
concentration as in No. 1 Adjuvant.
[0030] The concentration of the hydrophilic surfactant used in the
adjuvant of the invention depends, in part, on the size of the
aqueous phase, and the concentration of the lipophilic surfactant
depends, in part, on the size of the oil phase. In one embodiment,
the adjuvant of the invention that consists of an aqueous phase at
80% v/v and of an oil phase at 20% v/v, may contain a hydrophilic
surfactant at a concentration of up to about 4 times (i.e., 80/20)
the concentration of a lipophilic surfactant, or for example up to
about 2 times.
Non-Surfactant Components of the Adjuvant of the Invention
[0031] In addition to an amphiphilic surfactant, the adjuvant of
the invention contains a lecithin and an oil. In another aspect,
the adjuvant of the invention contains an aqueous carrier
solution.
[0032] Any lecithin known in the art is useful for the adjuvant of
the invention. Lecithin refers to a mixture of phosphatides. When
provided as a crude extract, a lecithin may also contain
triglycerides. Lecithins may be of plant or animal origin. In
addition, lecithins may be synthetically derived. Examples of
lecithins are described in U.S. Pat. Nos. 5,690,942; 5,597,602 and
5,084,269. In a preferred embodiment, the contents of triglycerides
in a lecithin used in the adjuvant of the invention is lowered
compared to its natural source, ie., the lecithin is de-oiled. A
number of ways are known in the art to de-oil a lecithin, for
example as described in U.S. Pat. No. 5,597,602.
[0033] The concentration of a lecithin in a vaccine composition
formulated with the adjuvant of the invention is from about 0.25%
to about 12.5% v/v, more preferably from about 0.5% to about 10%
v/v, more preferably from about 0.5% to about 7.5%, more preferably
from about 0.5% to about 5%, more preferably from about 0.5% to
about 2.5%, and most preferably from about 0.5% to about 1.25%
v/v.
[0034] The concentration of a lecithin in a 25% adjuvant is at
least about 1% v/v, preferably at least about 2% v/v. In another
aspect, the lecithin concentration in a 25% adjuvant is from about
1 % to about 50% v/v, more preferably from about 2% to about 40%
v/v, more preferably from about 2% to about 30% v/v, more
preferably from about 2% to about 20% v/v, more preferably from
about 2% to about 10% v/v and most preferably from about 2% to
about 5% v/v. The concentration of a lecithin in the adjuvant of
the invention with a higher or lower concentration is determined as
exemplified above.
[0035] The adjuvant of the invention contains an oil, for example
an oil described in U.S. Pat. Nos. 5,814,321; 5,084,269. In a
preferred aspect, the adjuvant of the invention contains a mineral
oil, for example DRAKEOL.TM.. In another aspect, a mixture of oils
is used. The oil may be provided for preparation of the adjuvant of
the invention as pure oil or as a mixture that contains the oil and
another component, for example the lecithin.
[0036] The concentration of an oil in a vaccine composition
formulated with the adjuvant of the invention is from about 1% to
about 23% v/v, more preferably from about 1.5% to about 20% v/v,
more preferably from about 2.5% to about 15%, more preferably from
about 3.5% to about 10%, more preferably from about 3.5% to about
7.5%, more preferably from about 4% to about 6% v/v, and most
preferably about 4.5%.
[0037] The concentration of an oil in a 25% adjuvant is at least
about 5% v/v, preferably at least about 8% v/v and more preferably
at least about 12% v/v. In another aspect, the oil concentration in
a 25% adjuvant is from about 4% to about 92% v/v, more preferably
from about 6% to about 80% v/v, more preferably from about 10% to
about 60% v/v, more preferably from about 14% to about 40% v/v,
more preferably from about 14% to about 30% v/v, more preferably
from about 16% to about 24% and most preferably about 18%. The
concentration of an oil in the adjuvant of the invention with a
higher or lower concentration is determined as exemplified
above.
[0038] In another embodiment, an aqueous carrier is used in the
adjuvant of the invention, for example saline (e.g.,
phosphate-buffered saline), tris-HCl, citrate-phosphate buffer,
Hepes buffers, other pharmaceutically acceptable buffers known in
the art or water. The pH of the carrier preferably is
physiologically acceptable, for example between 6 and 8, most
preferably around 7.
[0039] The aqueous carrier used in the adjuvant of the invention
preferably takes up the volume that is not needed for any of the
other components.
[0040] The adjuvant of the invention is preferably provided at a
concentration that is from about 2.times. to about 10.times. the
concentration after formulation of the adjuvant in a vaccine
composition, more preferably from about 2.times. to about 8.times.,
more preferably from about 3.times. to about 6.times. and most
preferably about 4.times..
Uses of Adjuvants of the Invention
[0041] Adjuvants of the invention may be used to enhance the immune
response to an antigen of a vaccine formulation. Adjuvants of the
invention can be used with antigens derived from any bacteria or
from any virus, provided the antigen does not get destroyed or
denatured. Examples of antigens, and not by way of limitation, are
Erysipelothrix rhusiopathiae antigens, Bordetella bronchiseptica
antigens, antigens of toxigenic strains of Pasteurella multocida,
antigens of Eschericia coli strains that cause neonatal diarrhea,
Actinobacillus pleuropneumoniae antigens, Pasteurella haemolytica
antigens, or any combination of the above. Adjuvants of the
invention are also useful in vaccine compositions that contain an
antigen described in U.S. Pat. Nos. 5,616,328 and 5,084,269.
[0042] In a preferred embodiment, the adjuvant of the invention is
used in a vaccine formulation containing an antigen obtained from
the liquid phase of an Erysipelothrix rhusiopathiae ("E.
rhusiopathiae") culture. In a preferred aspect, a culture of E.
rhusiopathiae is inactivated by adding formalin (about 0.5% v/v
final concentration) and, after incubation for 24 hours at
37.degree. C., the cells were removed, for example by
centrifugation or filtration. The culture supernatant, in a
preferred embodiment, is concentrated about 10 fold and aluminum
hydroxide gel (preferably REHYDRAGEL.TM.) is added to the
concentrated supernatant at a final concentration of about 30% v/v
to stabilize the antigen. In another preferred embodiment,
thimerosal (about 0.01% v/v final concentration) (Dimportex, Spain,
imported through Flavine Inc., Klosters, N.J.) with EDTA (about
0.07% v/v final concentration) are added to the antigens as
preservatives. In another preferred embodiment, a vaccine
composition is formulated comprising the antigen and the adjuvant
of the invention (e.g. No. 1 Adjuvant) with the adjuvant
comprising, for example, about 25% v/v of the vaccine composition.
This preferred E. rhusiopathiae antigen is described in U.S. patent
application Ser. No. 60/117,704, filed Jan. 29, 1999, entitled
"Erysipelothrix rhusiopathiae Antigens and Vaccine Compositions",
which is incorporated herein by reference.
[0043] In another preferred embodiment, the adjuvant of the
invention is used in a vaccine composition containing antigens from
a B. bronchiseptica culture that has been inactivated by adding
formalin thereto in log phase, preferably late log phase, followed
by the addition of glutaraldehyde. In addition to killing the
bacterial cells, the purpose of this novel and unique inactivation
is to make nontoxic the endotoxin and exotoxin B. bronchiseptica,
while leaving the antigens of B. bronchiseptica cells effective in
eliciting the desired immune response. Formalin is added to a
concentration in the B. bronchiseptica culture of from about 0.2%
v/v to about 1% v/v, more preferably from about 0.4% v/v to about
0.8% v/v and most preferably about 0.6% v/v. Glutaraldehyde is
added from about 10 minutes to about 40 minutes following the
addition of formalin to the culture, more preferably from about 15
minutes to about 30 minutes and most preferably about 20 minutes.
Glutaraldehyde is added to a concentration in the B. bronchiseptica
culture of from about 0.2% v/v to about 1% v/v, more preferably
from about 0.4% v/v to about 0.8% v/v and most preferably about
0.6% v/v. Prior to adding the glutaraldehyde to the culture, it has
a concentration of from about 10% v/v to about 50% v/v, more
preferably from about 15% v/v to about 35% v/v and most preferably
about 25% v/v. Following the addition of formalin and
glutaraldehyde to the B. bronchiseptica culture, the resulting mix
is incubated under stirring at from about 32.degree. C. to about
42.degree. C., more preferably at from about 35.degree. C. to about
39.degree. C. and most preferably at about 37.degree. C. The mix is
incubated from about 12 hours to about 60 hours, more preferably
from about 24 hours to about 48 hours. All other processing steps
in preparing an antigen composition of the invention from B.
bronchiseptica culture are described in Example 7, infra, and in
U.S. Pat. Nos. 5,019,388 and 4,888,169.
Vaccine Compositions Comprising Adjuvants of the Invention and
their Administration
[0044] The adjuvant of the invention may be used in a vaccine
formulation to immunize an animal. In one embodiment, the vaccine
formulation contains the adjuvant of the invention and an antigen.
The optimal ratios of each component in the vaccine formulation may
be determined by techniques well known to those skilled in the
art.
[0045] A vaccine formulation may be administered to a subject per
se or in the form of a pharmaceutical or therapeutic composition.
Pharmaceutical compositions comprising the adjuvant of the
invention and an antigen may be manufactured by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Pharmaceutical compositions may be formulated in
conventional manner using one or more physiologically acceptable
carriers, diluents, excipients or auxiliaries which facilitate
processing of the antigens of the invention into preparations which
can be used pharmaceutically. Proper formulation is dependent upon
the route of administration chosen. For purposes of this
application, "physiologically acceptable carrier" encompasses
carriers that are acceptable for human or animal use without
relatively harmful side effects (relative to the condition being
treated), as well as diluents, excipients or auxiliaries that are
likewise acceptable.
[0046] Systemic formulations include those designed for
administration by injection, e.g. subcutaneous, intradermal,
intramuscular or intraperitoneal injection.
[0047] For injection, the vaccine preparations may be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hanks's solution, Ringer's solution, phosphate buffered
saline, or any other physiological saline buffer. The solution may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the proteins may be in powder
form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0048] Determination of an effective amount of the vaccine
formulation for administration is well within the capabilities of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0049] An effective dose can be estimated initially from in vitro
assays. For example, a dose can be formulated in animal models to
achieve an induction of an immune response using techniques that
are well known in the art. One having ordinary skill in the art
could readily optimize administration to all animal species based
on results described herein. Dosage amount and interval may be
adjusted individually. For example, when used as a vaccine, the
vaccine formulations of the invention may be administered in about
1 to 3 doses for a 1-36 week period. Preferably, 1 or 2 doses are
administered, at intervals of about 3 weeks to about 4 months, and
booster vaccinations may be given periodically thereafter.
Alternative protocols may be appropriate for individual animals. A
suitable dose is an amount of the vaccine formulation that, when
administered as described above, is capable of raising an immune
response in an immunized animal sufficient to protect the animal
from an infection for at least 4 to 12 months. In general, the
amount of the antigen present in a dose ranges from about 1 pg to
about 100 mg per kg of host, typically from about 10 pg to about 1
mg, and preferably from about 100 pg to about 1 .mu.g. Suitable
dose range will vary with the route of injection and the size of
the patient, but will typically range from about 0.1 mL to about 5
mL.
[0050] The invention having been described, the following examples
are offered by way of illustration and not limitation.
EXAMPLE 1
The Use of an Adjuvant that Contains Oil and Lecithin
[0051] The following example describes the use of an adjuvant that
contains de-oiled lecithin dissolved in an oil ("oil-lecithin
adjuvant"), usually mineral oil (light liquid paraffin) in
veterinary vaccines. See U.S. Pat. No. 5,084,269, which describes
an oil-lecithin adjuvant. A vaccine preparation using an
oil-lecithin adjuvant is an oil-in-water emulsion.
[0052] All percentage concentrations herein are provided in volume
per volume unless indicated otherwise. Percentage values, unless
otherwise indicated, of an oil-lecithin adjuvant refer to the
concentration of a mixture of lecithin (10% of the mixture) and a
carrier oil (DRAKEOL.TM.) (90% of the mixture) in an aqueous
carrier (continuous phase). For example, a 20% oil-lecithin
adjuvant contains 2% v/v lecithin (Central Soya, Fort Wayne, Ind.),
18% v/v DRAKEOL.TM. 5 (Penreco, Karns City, Pa.) and 80% v/v saline
solution (with the saline content being reduced if other
components, for example surfactants, are added). The percentage
values of an oil-lecithin adjuvant in a vaccine composition, i.e.,
following dilution of the adjuvant solution with the antigen
solution, refer to the concentration of a mixture of lecithin (10%
of mixture) and a carrier oil (DRAKEOL.TM.) (90% of mixture) in the
vaccine preparation which comprises the adjuvant and a solution
containing an antigen, unless the context indicates otherwise. In
all cases where a surfactant was added to an adjuvant composition,
the percentage values for a surfactant concentration refer to the
total concentration of all added surfactants in the adjuvant or the
vaccine preparation, unless the context indicates otherwise.
[0053] When an oil-lecithin adjuvant was used as an adjuvant in
vaccine formulations, it was found that it does not emulsify
aqueous preparations without the addition of extra surfactants as
the lecithin in the oil-lecithin adjuvant did not suffice for
emulsification. Therefore, vaccines made using inadequately
dispersed oil-lecithin adjuvant formed a pool or depot of mostly
mineral oil in the tissues at the injection site. This oil can not
be metabolized or removed by the injected animal and so it remains
as a source of severe chronic inflammation and scarring.
[0054] It was also determined that adding surfactants to a vaccine
formulation comprising an oil-lecithin adjuvant and an antigen in
order to emulsify the formulation was not an adequate solution.
Problems encountered when adding oil and surfactants to the vaccine
formulation before emulsifying were that the antigen could get
damaged and, it a suitable emulsion was not achieved, that the
formulation would have to be discarded including the valuable
antigen.
[0055] Different adjuvant compositions were tested comprising an
oil-lecithin adjuvant in combination with surfactants to emulsify
the vaccine formulations.
EXAMPLE 2
The Use of an Adjuvant Containing a Surfactant at a Low
Concentration
[0056] The following example describes the use of an emulsion
containing 40% oil-lecithin and 2% of synthetic surfactants, i.e.,.
Tween 80 and Span 80 (Van Water & Rogers, Omaha, Nebr.) in
phosphate buffered saline. This adjuvant was prepared aseptically
and separate from the antigen. The emulsion was added to the
antigen preparation without further emulsification. The synthetic
surfactants helped the oil-lecithin adjuvant to disperse as a
coarse, relatively stable emulsion. The adjuvant emulsion was added
to the aqueous antigenic preparation at the rate of one in eight,
decreasing the oil-lecithin adjuvant content from 40% to 5%, and
the surfactants from a combined 2% to 0.25%.
[0057] The adjuvant was used in several vaccines. It was found that
because the emulsion is coarse and not very stable, the oil
droplets tend to coalesce and to separate as a permanent,
irritating depot of oil in the injected tissues. Another problem
observed with this adjuvant was that it aggregates with Al gel. A
number of vaccines contain Al gel for a number of purposes like,
for example, as an adjuvant or to stabilize an antigen or to bind
endotoxin. The oil-lecithin adjuvant carries a negative charge
which causes it to bind to the positively charged Al gel to form
coarse aggregates. These aggregates are unsightly, difficult to
pass through a hypodermic needle, and very irritating to the
injected tissues.
EXAMPLE 3
The Use of an Adjuvant Containing a Surfactant at a High
Concentration
[0058] An oil-lecithin adjuvant (5% v/v) was emulsified in the
antigenic preparation with the help of Tween 80 and Span 80
surfactants, as above, but at a total surfactant concentration of
8% in the vaccine composition. The emulsion was very fine and
stable. It had almost the clarity of a solution and it did not
cream on standing. Under the microscope, with maximum magnification
(resolution 0.2 micron), most droplets were too small to be
visible. Thus, it was a microemulsion. This adjuvant, when used in
a vaccine formulation, was found to be virtually free of
injection-site reactivity and, when Al gel was added, there was no
detectable aggregation of oil and gel. As a result of its high
surfactant content, this adjuvant is easy to emulsify, attractive
in appearance, stable, unreactive with Al gel, and virtually free
of irritating effects at the site of vaccination. Despite these
advantages, however, this emulsion had slightly lower adjuvant
potency compared to the coarse version made with surfactants at a
low concentration.
EXAMPLE 4
The Use of an Adjuvant Containing a Surfactant at a Medium
Concentration
[0059] An attempt was made to find an adjuvant emulsion that is
acceptably smooth and fully potent as an adjuvant. A 20%
oil-lecithin adjuvant was used in these experiments as it was found
that a 20% oil-lecithin adjuvant emulsion is easier to make than a
40% oil-lecithin adjuvant emulsion. Its addition to vaccines at a
rate of one in four, to make a final oil concentration of 5%, would
leave 75% of the dose volume for antigens. Preliminary experiments
showed that a smooth submicron emulsion (most droplets had a
diameter of less than one micron, see FIG. 1) could be prepared
with 20% oil and 16% of Tween 80 and Span 80 surfactants.
[0060] Two emulsions were prepared for the assays. One contained a
20% oil-lecithin adjuvant and 16% of Tween 80 and Span 80
surfactants. Diluting it one in four resulted in an emulsion
comprising 5% oil-lecithin adjuvant and 4% surfactants in the
vaccine preparation. The other emulsion was prepared with a 40%
oil-lecithin adjuvant and 2% of Tween 80 and Span 80 surfactants.
Diluting it one in eight gave an emulsion with 5% oil-lecithin
adjuvant and 0.25% surfactants.
[0061] Al gel (REHYDRAGEL.TM. obtained from Reheis, Berkeley
Heights, N.J.) was added to a concentration of 10% to samples of
each emulsion. In the emulsion with 0.25% surfactants the oil and
Al gel aggregated and separated to form a thick layer at the top of
the liquid column (creaming). In the emulsion with 4% surfactants,
by contrast, there was no aggregation or creaming. With 4%
surfactants, the Al gel sedimented at the bottom of the tube
leaving the oil droplets dispersed in the supernatant fluid.
EXAMPLE 5
Swelling of Injection Sites when Using an Adjuvant Containing a
Surfactant at a Medium Concentration
[0062] Vaccine preparations were tested in pigs to determine
whether swelling of the injection site occurred when an adjuvant
with a medium concentration of surfactant was used. Vaccine
preparations that contain a 5% oil-lecithin adjuvant and either
0.25% or 4% surfactants caused no swelling in pigs at the injection
site. When Al gel was added to the vaccine preparation at a
concentration of 10%, the preparation with 0.25% surfactants caused
severe injection site swellings whereas the one with 4% surfactants
resulted in almost no swelling.
[0063] Experiments were carried out to determine the range of
surfactant concentrations that are effective in preventing
aggregation with Al gel and swelling of the injection site. When
using a 1.5% surfactant concentration in the vaccine, slight
aggregation of oil and Al gel was observed. The aggregation was
much heavier at lower surfactant concentrations. At 2% and 4%
surfactant concentrations, there was no aggregation. The swelling
induced in pigs by vaccine preparations containing 0.5% or less
surfactants were larger at 2 and 4 weeks after vaccination than
those induced by preparations with 1% or more surfactants. By 6
weeks after vaccination, it was evident that 1.5% surfactants was
the minimum needed to avoid chronic swellings.
EXAMPLE 6
Adjuvants with Useful In Vitro and In Vivo Properties
[0064] Assays were carried out to find an adjuvant that does not
react with Al gel and does not lead to reactivity in the animal
following vaccination. A 20% oil-lecithin adjuvant that contains 8%
surfactants, resulting in a vaccination preparation with 5%
oil-lecithin adjuvant and 2% surfactant, was determined to be
sufficient to avoid both in vitro reactivity with Al gel and
irritation of the tissues at the vaccination site. Evidence of a
relationship between surfactant concentration and adjuvant power
was much less clear. There were occasional indications that 4%
surfactants in the vaccine was excessive, e.g. in the induction of
agglutinin to E. coli K99, and neutralizing antitoxin to the toxin
of P. multocida Type D.
[0065] Thus, it was determined that the optimal concentration of
surfactants was 8% in a 20% oil-lecithin adjuvant, resulting in 2%
surfactants in the vaccine composition. This provided for
reasonably easy emulsification and for good stability in cold
storage. In vaccines with 5% oil-lecithin adjuvant, 2% surfactants
was ideal for both adjuvant power and freedom from irritancy in the
injected tissues.
[0066] The droplet size in the submicron emulsion of a 20%
oil-lecithin adjuvant with 8% surfactants was determined. The 8%
surfactant consisted of 5.6% Tween 80 in the aqueous phase and 2.4%
Span 80 in the oil phase. About 94% of all droplets were less than
1 micron in diameter, see FIG. 1.
[0067] A stock of 1000 mL of a 20% oil-lecithin adjuvant with 8%
surfactants was made from 200 mL filter-sterilized lecithin-oil
solution (10% lecithin in DRAKEOL.TM. mineral oil), autoclaved
Tween 80 (56 mL) and Span 80 (24 mL), and phosphate buffered saline
(720 mL) (Dulbecco PBS). The lecithin-oil solution and Span 80 were
combined and mixed in a sterile tank for at least 1 hour at room
temperature until emulsification was complete. The saline and Tween
80 were combined and mixed in a sterile tank for at least 1 hour at
room temperature. The oil mixture was emulsified in the aqueous
mixture using an emulsifier. Emulsification was continued by
recirculation until all of the adjuvant was added into the saline.
The emulsion was then passed twice through a homogenizer at room
temperature. The adjuvant was stored at 2 to 8.degree. C.
EXAMPLE 7
Atrophic Rhinitis Vaccine Using an Adjuvant Containing a Surfactant
at a Medium Concentration
[0068] The adjuvant as described in Example 4 with a medium
concentration of surfactants was used in an Atrophic Rhinitis
Vaccine which contained antigens of Bordetella bronchiseptica and
toxigenic Pasteurella multocida. A Bordetella
bronchiseptica-Pasteurella multocida Bacterin-Toxoid vaccine was
made from B. bronchiseptica cells and the toxoid of P.
multocida.
[0069] B. bronchiseptica cells, strain 2-9 NADL, were prepared as
described in U.S. Pat. Nos. 5,019,388 and 4,888,169 except that at
the end of the growth cycle, cultures were continuously mixed and
formalin solution was added to a final concentration of 0.6%.
Within 20 minutes after the addition of formalin, a 25%
glutaraldehyde solution was added to a final concentration of 0.6%.
The culture was stirred for 24 to 48 hours at 37.+-.2.degree. C. to
complete inactivation and detoxification. (See Table 1). Then, the
culture fluids were cooled to 15.degree. C. or less for processing.
Inactivated cultures not processed immediately were stored at 2 to
8.degree. C. for up to 14 days. Following inactivation, the
bacteria were separated from the culture fluid by centrifugation.
The supernatant was discarded and the cells were resuspended in
phosphate-buffered saline at approximately one tenth of the
original volume. The concentrated suspension was stored at 2 to
8.degree. C. The treatment of B. bronchiseptica with two aldehydes
inactivates both the endotoxin and the exotoxin, obviating other
treatments for safety.
[0070] The toxoid of P. multocida was prepared in two different
forms as described in U.S. Pat. Nos. 5,536,496 and 5,695,769. In
one form, the toxin is toxoided within the bacterial cells by the
addition of formaldehyde to the culture; the toxoid stays inside
the cells. In the other form, the live cells are mechanically
disrupted and the toxin extracted. The toxin is toxoided by
exposure to a high pH, as described in U.S. Pat. No. 5,536,496.
Both forms of the toxoid are treated with Al gel to control free
endotoxin by a patented process, as described in U.S. Pat. No.
5,616,328. (See Table 1). A synergy between the two forms of
pasteurella toxoid results in antitoxin responses far exceeding the
sum of the responses to each form when used alone as described in
U.S. Pat. No. 5,695,769.
1TABLE 1 Treatment of cells during preparation of the Bordetella
Bronchiseptica-Pasteurella Multocida Bacterin-Toxoid vaccine Target
Temp. Time Antigen Inactivant Concentration Range (Min/Max) B.
bronchiseptica Formalin 0.6% 37 .+-. 2.degree. C. 24-48 hours
Glutaraldehyde 0.6% P. multocida cell-bound Formalin 0.4% 37 .+-.
2.degree. C. 5 days toxoid P. multocida cell-free 5N NaOH pH 10
.+-. 0.2 15 .+-. 2.degree. C. 1 to 6 hours toxoid
[0071] The adjuvant described in Example 4 was added to result in
concentrations of 5% oil-lecithin adjuvant and 2% surfactants in
the vaccine formulation.
[0072] A trial to determine the minimum immunizing dose of the
atrophic rhinitis vaccine demonstrated the adjuvant properties of
the oil-lecithin adjuvant with a medium concentration of
surfactants. Pregnant sows were vaccinated with two 2 mL doses at
an interval of 4 weeks. They farrowed about 2 weeks after the
second dose. At one month of age, their pigs were subjected to a
severe challenge, consisting of virulent B. Bronchiseptica and P.
Multocida cultures given intranasally in sequence. The pigs born to
7 sows vaccinated only with a placebo developed severe atrophic
rhinitis. The litters of all 7 sows given vaccine containing a full
dose of the antigens were strongly protected by the maternal
antibody still in circulation. Sows given vaccines containing 1/2
or 1/8 dose of antigens did not provide their litters with
satisfactory protection.
EXAMPLE 8
Erysipelas Vaccine Using an Adjuvant Containing a Surfactant at a
Medium Concentration
[0073] The adjuvant described in Example 4 with a medium
concentration of surfactants was used in an erysipelas vaccine
which contained E. rhusiopathiae antigens. The antigens for use in
a vaccine were made from E. rhusiopathiae cultures inactivated with
0.5% formalin for at least 24 hours. The inactivated cultures were
clarified by centrifuging, and concentrated roughly tenfold by
molecular filtration. The concentrates were stabilized by adding Al
gel, i.e., REHYDRAGEL.TM., to a concentration of 30%. The
preadsorbed concentrates were included in the vaccine in- an amount
such that each 2 mL dose contained at least 3.2 opacity units (OU)
as calculated from the optical density (OD) of the culture at
inactivation. (The OD is multiplied by the final concentration
factor to give a value in OU per mL.)
[0074] An Erysipelas vaccination was carried out to determine the
efficacy of a vaccine containing the oil-lecithin adjuvant with a
medium concentration of surfactants. The adjuvant as described in
Example 4 was added at a final concentration of 25% v/v, giving a
final lecithin -oil concentration of 5%. Thimerosal (0.01% w/v),
with EDTA (0.07% w/v), was added as preservative.
[0075] Vaccines made according to this formula were tested twice
for efficacy in pigs. In each case pigs were vaccinated with two 2
mL doses given intramuscularly (IM) one dose at about 3 weeks
(weaning) and the second dose 3 weeks later. Controls received
phosphate-buffered saline as a placebo. Immunity was challenged by
the IM injection of virulent E. Rhusiopathiae at about 9 weeks of
age in one study and 6 months of age in the other. As shown in
Table 2, protection due to vaccination was 100% at 9 weeks and 75%
at 6 months (i.e., slaughter age). These results indicated that the
vaccine provides satisfactory protection against Erysipelas
throughout the normal feeding period.
[0076] The vaccine used in the group challenged at 9 weeks was
already 12 months old. The result confirms that the protective
antigen was successfully stabilized.
2TABLE 2 Protection of pigs against Erysipelas Controls Vaccinates
Age at Challenge (Protected/Challenged) (Protected/Challenged) 9
weeks 0/10 19/19 6 months 0/10 15/20 Note: In the vaccinated group
challenged at 9 weeks, the 20th pig was excluded. A very fractious
animal, it struggled so violently when handled that its temperature
at rest could not be determined. Following challenge this pig
remained completely healthy.
[0077] Note: In the vaccinated group challenged at 9 weeks, the
20th pig was excluded. A very fractious animal, it struggled so
violently when handled that its temperature at rest could not be
determined. Following challenge this pig remained completely
healthy.
[0078] The invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention, and any adjuvants which are
functionally equivalent are within the scope of the invention.
Indeed, various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended
claims.
[0079] All publications cited herein are incorporated by reference
in their entirety.
* * * * *