U.S. patent application number 10/246195 was filed with the patent office on 2003-08-07 for adjuvant formulation comprising a submicron oil droplet emulson.
Invention is credited to Barchfeld, Gail, Ott, Gary, Van Nest, Gary.
Application Number | 20030147898 10/246195 |
Document ID | / |
Family ID | 23404033 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030147898 |
Kind Code |
A1 |
Van Nest, Gary ; et
al. |
August 7, 2003 |
Adjuvant formulation comprising a submicron oil droplet emulson
Abstract
An adjuvant composition, comprising a metabolizable oil and an
emulsifying agent, wherein the oil and the detergent are present in
the form of an oil-in-water emulsion having oil droplets
substantially all of which are less than 1 micron in diameter. In
preferred embodiments, the emulsifying agent is also an
immunostimulating agent, such as a lipophilic muramyl peptide.
Alternatively, an immunostimulating agent separate from the
emulsifying agent can be used.
Inventors: |
Van Nest, Gary; (El
Sobrante, CA) ; Ott, Gary; (Livermore, CA) ;
Barchfeld, Gail; (Hayward, CA) |
Correspondence
Address: |
CHIRON CORPORATION
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
23404033 |
Appl. No.: |
10/246195 |
Filed: |
September 17, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10246195 |
Sep 17, 2002 |
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08434512 |
May 4, 1995 |
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6451325 |
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08434512 |
May 4, 1995 |
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08418870 |
Apr 7, 1995 |
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6299884 |
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08418870 |
Apr 7, 1995 |
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08215007 |
Mar 21, 1994 |
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08215007 |
Mar 21, 1994 |
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08041519 |
Apr 1, 1993 |
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08041519 |
Apr 1, 1993 |
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07885905 |
May 18, 1992 |
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07885905 |
May 18, 1992 |
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07528593 |
May 24, 1990 |
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07528593 |
May 24, 1990 |
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07357035 |
May 25, 1989 |
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Current U.S.
Class: |
424/184.1 ;
424/776; 514/21.9; 514/3.2 |
Current CPC
Class: |
Y02A 50/412 20180101;
Y02A 50/30 20180101; Y02A 50/466 20180101; A61K 9/1272 20130101;
A61K 2039/55566 20130101; A61K 2039/55555 20130101; Y10S 977/918
20130101; Y10S 977/802 20130101; A61K 39/39 20130101; A61K 9/1075
20130101; Y10S 514/975 20130101; A61P 37/04 20180101; Y10S 514/97
20130101 |
Class at
Publication: |
424/184.1 ;
424/776; 514/7; 514/8 |
International
Class: |
A61K 039/00; A61K
039/38; A61K 038/14 |
Claims
What is claimed is:
1. An adjuvant composition, comprising: (1) a metabolizable oil and
(2) an emulsifying agent, wherein said oil and said emulsifying
agent are present in the form of an oil-in-water emulsion having
oil droplets substantially all of which are less than 1 micron in
diameter and wherein said composition exists in the absence of any
polyoxypropylene-polyoxyeth- ylene block copolymer.
2. The composition of claim 1, wherein said oil is an animal
oil.
3. The composition of claim 2, wherein said oil is an unsaturated
hydrocarbon.
4. The composition of claim 1, wherein said oil is a terpenoid.
5. The composition of claim 1, wherein said oil is a vegetable
oil.
6. The composition of claim 1, wherein said composition comprises
0.5 to 20% by volume of said oil in an aqueous medium.
7. The composition of claim 1, wherein said emulsifying agent
comprises a non-ionic detergent.
8. The composition of claim 20, wherein said emulsifying agent
comprises a polyoxyethylene sorbitan mono-, di-, or triester or a
sorbitan mono-, di-, or triether.
9. The composition of claim 8, wherein said composition comprises
0.01 to 0.5% by weight of said emulsifying agent.
10. The composition of claim 9, wherein said composition further
comprises a separate immunostimulating agent.
11. The composition of claim 8, wherein said immunostimulating
agent comprises alum or a bacterial cell wall component.
12. The composition of claim 11, wherein said composition comprises
0.0001 to 1.0% by weight of said immunostimulating agent.
13. The composition of claim 11, wherein said immunostimulating
agent comprises a muramyl peptide.
14. The composition of claim 1, wherein said emulsifying agent also
functions as an immunostimulating agent.
15. The composition of claim 14, wherein said composition comprises
0.01 to 0.5% by weight of said immunostimulating agent.
16. The composition of claim 14, wherein said immunostimulating
agent comprises a lipophilic muramyl peptide.
17. The composition of claim 16, wherein said peptide comprises a
muramyl dipeptide or a muramyl tripeptide.
18. The composition of claim 17, wherein said peptide further
comprises a phospholipid.
19. The composition of claim 18, wherein said phospholipid
comprises a phosphoglyceride.
20. The composition of claim 14, wherein said peptide is a compound
of the formula 4wherein R is H or COCH.sub.3; R.sup.1, R.sup.2, and
R.sup.3 independently represent H or a lipid moiety; R.sup.4 is
hydrogen or alkyl; X and Z independently represent an aminoacyl
moiety selected from the group consisting of alanyl, valyl, leucyl,
isoleucyl, .alpha.-aminobutyryl, threonyl, methionyl, cysteinyl,
glutamyl, isoglutamyl, glutaminyl, isoglutaminyl, aspartyl,
phenylalanyl, tyrosyl, tryptophanyl, lysyl, ornithinyl, arginyl,
histidyl, asparaginyl, prolyl, hydroxypropyl, seryl, and glycyl; n
is 0 or 1; Y is --NHCHR.sup.5CH.sub.2CH.sub.2CO--, wherein R.sup.5
represents an optionally esterified or amidated carboxyl group; and
L is OH, NR.sup.6R.sup.7 where R.sup.6 and R.sup.7 independently
represent H or a lower alkyl group, or a lipid moiety.
21. The composition of claim 20, wherein R.sup.4 is methyl, X is
alanyl, and Y is isoglutaminyl.
22. The composition of claim 20, wherein n is 1; Z is alanyl; R is
acetyl; and R.sup.1, R.sup.2, and R.sup.3 are all H.
23. The composition of claim 22, wherein L comprises a phospholipid
moiety.
24. The composition of claim 23, wherein said phospholipid moiety
comprises a diacylphosphoglyceride.
25. The composition of claim 20, wherein said peptide is
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn--
glycero-3-(hydroxy-phosphoryloxy)]ethylamide.
26. The composition of claim 20, wherein at least one of R.sup.1
and R.sup.2 represents an acyl group containing from 1 to 22
carbons.
27. The composition of claim 20, wherein at least one of R.sup.1,
R.sup.2, and R.sup.3 represents an acyl group containing from 14 to
22 carbons.
28. A vaccine composition, comprising: (1) an immunostimulating
amount of an antigenic substance, and (2) an immunostimulating
amount of the adjuvant of claim 1.
29. A method of stimulating an immune response in a host animal,
comprising: administering a protective antigen to said animal in
the presence of an immunostimulating amount of submicron
metabolizable oil droplets in a continuous aqueous phase and in the
absence of any polyoxypropylene-polyoxyethylene block
copolymer.
30. The method of claim 29, wherein said oil droplets further
comprise an emulsifying agent.
31. The method of claim 30, wherein said oil droplets further
comprise an immunostimulating agent separate from said oil and said
emulsifying agent.
32. The method of claim 31, wherein said immuno-stimulating agent
comprises alum or a bacterial cell wall component.
33. The method of claim 31, wherein said immuno-stimulating agent
comprises a muramyl peptide.
34. The method of claim 30, wherein said emulsifying agent is also
effective as an immunostimulating agent.
35. The method of claim 34, wherein said immuno-stimulating agent
comprises a lipophilic muramyl peptide.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 357,035, filed May 25, 1989, which is herein
incorporated by reference.
INTRODUCTION
TECHNICAL FIELD
[0002] This invention relates generally to immunological adjuvants
for use in increasing efficiency of vaccines and is particularly
directed to adjuvants comprising oil-in-water emulsions.
BACKGROUND
[0003] The emergence of new subunit vaccines created by recombinant
DNA technology has intensified the need for safe and effective
adjuvants. Traditional live anti-viral vaccines require no
adjuvants. Killed virus vaccines are generally much more
immunogenic than subunit vaccines and can be effective with no
adjuvant or with adjuvants that have limited ability to stimulate
immune responses. The new, recombinant DNA-derived subunit
vaccines, while offering significant advantages over the
traditional vaccines in terms of safety and cost of production,
generally represent isolated proteins or mixtures of proteins that
have limited immunogenicity compared to whole viruses. Such
materials are referred to generally in this specification as
molecular antigens, to distinguish them from the whole organisms
(and parts thereof) that were previously used in vaccines. These
vaccines will require adjuvants with significant immunostimulatory
capabilities to reach their full potential in preventing
disease.
[0004] Currently, the only adjuvants approved for human use in the
United States are aluminum salts (alum). These adjuvants have been
useful for some vaccines including hepatitis B, diphtheria, polio,
rabies and influenza, but may not be useful for others, especially
if stimulation of cell-mediated immunity is required for
protection. Reports indicate that alum failed to improve the
effectiveness of whooping cough and typhoid vaccines and provided
only a slight effect with adenovirus vaccines. Problems with
aluminum salts include induction of granulomas at the injection
site and lot-to-lot variation of alum preparations.
[0005] Complete Freund's adjuvant (CFA) is a powerful
immunostimulatory agent that has been used successfully with many
antigens on an experimental basis. CFA is comprised of three
components: a mineral oil, an emulsifying agent such as Arlacel A,
and killed mycobacteria such as Mycobacterium tuberculosis. Aqueous
antigen solutions are mixed with these components to create a
water-in-oil emulsion. CFA causes severe side effects, however,
including pain, abscess formation, and fever, which prevent its use
in either human or veterinary vaccines. The side effects are
primarily due to the host's reactions to the mycobacterial
component of CFA. Incomplete Freund's adjuvant (IFA) is similar to
CFA without the bacterial component. While not approved for use in
the United States, IFA has been useful for several types of
vaccines in other countries. IFA has been used successfully in
humans with influenza and polio vaccines and with several animal
vaccines including rabies, canine distemper, and foot-and-mouth
disease. Experiments have shown that both the oil and emulsifier
used in IFA can cause tumors in mice, indicating that an
alternative adjuvant would be a better choice for human use.
[0006] Muramyl dipeptide (MDP) represents the minimal unit of the
mycobacterial cell wall complex that generates the adjuvant
activity observed with CFA; see Ellouz et al. (1974) Biochem.
Biophys. Res. Comm., 59:1317. Many synthetic analogues of MDP have
been generated that exhibit a wide range of adjuvant potency and
side effects (reviewed in Chedid et al. (1978) Prog. Allergy,
25:63). Three analogues that may be especially useful as vaccine
adjuvants are threonyl derivatives of MDP, see Byars et al. (1987)
Vaccine, 5:223; n-butyl derivatives of MDP, see Chedid et al.
(1982) Infect. and Immun., 35:417; and lipophilic derivative of
muramyl tripeptide, see Gisler et al. (1981) in Immunomodulations
of Microbial Products and Related Synthetic Compounds, Y. Yamamura
and S. Kotani, eds., Excerpta Medica, Amsterdam, p. 167. These
compounds effectively stimulate humoral and cell-mediated immunity
and exhibit low levels of toxicity.
[0007] One promising lipophilic derivative of MDP is
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn--
glycero-3-3(hydroxyphosphoryl-oxy)]ethylamide (MTP-PE). This
muramyl tripeptide has phospholipid tails that allow association of
the hydrophobic portion of the molecule with a lipid environment
while the muramyl peptide portion associates with the aqueous
environment. Thus the MTP-PE itself can act as an emulsifying agent
to generate stable oil in water emulsions.
[0008] Original mouse experiments in the laboratories of the
present inventors with MTP-PE showed that this adjuvant was
effective in stimulating anti-HSV gD antibody titers against herpes
simplex virus gD antigen and that effectiveness was vastly improved
if the MTP-PE and gD were delivered in oil (IFA) rather than in
aqueous solution. Since IFA is not approved for human use, other
oil delivery systems were investigated for MTP-PE and antigen. An
emulsion of 4% squalene with 0.008% Tween 80 and HSV gD gave very
good immunity in the guinea pig. This formulation, MTP-PE-LO (low
oil), was emulsified by passing through a hypodermic needle and was
quite unstable. Nevertheless, this formulation gave high antibody
titers in the guinea pig and good protection in a HSV challenge of
immunized guinea pigs. The formulation was most effective when
delivered in the footpad but also gave reasonable antibody titers
and protection when delivered intramuscularly. These data have
appeared in 2 publications (Sanchez-Pescador et al., J. Immunology
141, 1720-1727, 1988 and Technological Advances in Vaccine
Development, Lasky et al., ed., Alan R. Liss, Inc., p. 445-469,
1988). The MTP-PE-LO formulation was also effective in stimulating
the immune response to the yeast-produced HIV envelope protein in
guinea pigs. Both ELISA antibody titers and virus neutralizing
antibody titers were stimulated to a high level with the MTP-PE
formulation. However, when the same formulation was tested in large
animals, such as goats and baboons, the compositions were not as
effective. The desirability of additional adjuvant formulations for
use with molecular antigens in humans and other large animals is
evident.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is an object of the present invention to
provide an adjuvant formulation suitable for stimulating immune
responses to molecular antigens in large mammals.
[0010] Surprisingly, it has been found that a satisfactory adjuvant
formulation is provided by a composition comprising a metabolizable
oil and an emulsifying agent, wherein the oil and the emulsifying
agent are present in the form of an oil-in-water emulsion having
oil droplets substantially all of which are less than 1 micron in
diameter and wherein the composition exists in the absence of any
polyoxy-proplyene-polyoxyeth- ylene block copolymer. Such block
copolymers were previously thought to be essential for the
preparation of submicron oil-in-water emulsions. The composition
can also contain an immunostimulating agent (which can be the same
as the emulsifying agent, if an amphipathic immunostimulating agent
is selected).
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0011] The present invention provides an adjuvant composition
comprising a metabolizable oil and an emulsifying agent, wherein
the oil and the emulsifying agent are present in the form of an
oil-in-water emulsion having oil droplets substantially all of
which are less than 1 micron in diameter. Investigations in the
laboratories of the present inventors, reported in detail in the
examples that follow, show a surprising superiority over adjuvant
compositions containing oil and emulsifying agents in which the oil
droplets are significantly larger than those provided by the
present invention.
[0012] The individual components of the adjuvant compositions of
the present invention are known, although such compositions have
not been combined in the same manner and provided in a droplet size
of such small diameter. Accordingly, the individual components,
although described below both generally and in some detail for
preferred embodiments, are well known in the art, and the terms
used herein, such as metabolizable oil, emulsifying agent,
immunostimulating agent, muramyl peptide, and lipophilic muramyl
peptide, are sufficiently well known to describe these compounds to
one skilled in the art without further description.
[0013] One component of these formulations is a metabolizable,
non-toxic oil, preferably one of 6 to 30 carbon atoms including,
but not limited to, alkanes, alkenes, alkynes, and their
corresponding acids and alcohols, the ethers and esters thereof,
and mixtures thereof. The oil may be any vegetable oil, fish oil,
animal oil or synthetically prepared oil which can be metabolized
by the body of the subject to which the adjuvant will be
administered and which is not toxic to the subject. The subject is
an animal, typically a mammal, and preferably a human. Mineral oil
and similar toxic petroleum distillate oils are expressly excluded
from this invention.
[0014] The oil component of this invention may be any long chain
alkane, alkene or alkyne, or an acid or alcohol derivative thereof
either as the free acid, its salt or an ester such as a mono-, or
di- or triester, such as the triglycerides and esters of
1,2-propanediol or similar poly-hydroxy alcohols. Alcohols may be
acylated employing a mono- or poly-functional acid, for example
acetic acid, propanoic acid, citric acid or the like. Ethers
derived from long chain alcohols which are oils and meet the other
criteria set forth herein may also be used.
[0015] The individual alkane, alkene or alkyne moiety and its acid
or alcohol derivatives will have 6-30 carbon atoms. The moiety may
have a straight or branched chain structure. It may be fully
saturated or have one or more double or triple bonds. Where mono or
poly ester- or ether-based oils are employed, the limitation of
6-30 carbons applies to the individual fatty acid or fatty alcohol
moieties, not the total carbon count.
[0016] Any metabolizable oil, particularly from an animal, fish or
vegetable source, may be used herein. It is essential that the oil
be metabolized by the host to which it is administered, otherwise
the oil component may cause abscesses, granulomas or even
carcinomas, or (when used in veterinary practice) may make the meat
of vaccinated birds and animals unacceptable for human consumption
due to the deleterious effect the unmetabolized oil may have on the
consumer.
[0017] Sources for vegetable oils include nuts, seeds and grains.
Peanut oil, soybean oil, coconut oil, and olive oil, the most
commonly available, exemplify the nut oils. Seed oils include
safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil
and the like. In the grain group, corn oil is the most readily
available, but the oil of other cereal grains such as wheat, oats,
rye, rice, teff, triticale and the like may also be used.
[0018] The technology for obtaining vegetable oils is well
developed and well known. The compositions of these and other
similar oils may be found in, for example, the Merck Index, and
source materials on foods, nutrition and food technology.
[0019] The 6-10 carbon fatty acid esters of glycerol and
1,2-propanediol, while not occurring naturally in seed oils, may be
prepared by hydrolysis, separation and esterification of the
appropriate materials starting from the nut and seed oils. These
products are commercially available under the name NEOBEE.RTM. from
PVO International, Inc., Chemical Specialties Division, 416
Division Street, Boongon, N.J. and others.
[0020] Oils from any animal source, may be employed in the
adjuvants and vaccines of this invention. Animal oils and fats are
usually solids at physiological temperatures due to the fact that
they exist as triglycerides and have a higher degree of saturation
than oils from fish or vegetables. However, fatty acids are
obtainable from animal fats by partial or complete triglyceride
saponification which provides the free fatty acids. Fats and oils
from mammalian milk are metabolizable and may therefore be used in
the practice of this invention. The procedures for separation,
purification, saponification and other means necessary for
obtaining pure oils from animal sources are well known in the
art.
[0021] Most fish contain metabolizable oils which may be readily
recovered. For example, cod liver oil, shark liver oils, and whale
oil such as spermaceti exemplify several of the fish oils which may
be used herein. A number of branched chain oils are synthesized
biochemically in 5-carbon isoprene units and are generally referred
to as terpenoids. Shark liver oil contains a branched, unsaturated
terpenoids known as squalene,
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene which
is particularly preferred herein. Squalane, the saturated analog to
squalene, is also a particularly preferred oil. Fish oils,
including squalene and squalane, are readily available from
commercial sources or may be obtained by methods known in the
art.
[0022] The oil component of these adjuvants and vaccine
formulations will be present in an amount from 0.5% to 20% by
volume but preferably no more than 15%, especially in an amount of
1% to 12%. It is most preferred to use from 1% to 4% oil.
[0023] The aqueous portion of these adjuvant compositions is
buffered saline or, in preferred embodiments, unadulterated water.
Because these compositions are intended for parenteral
administration, it is preferable to make up final buffered
solutions used as vaccines so that the tonicity, i.e., osmolality,
is essentially the same as normal physiological fluids in order to
prevent post-administration swelling or rapid absorption of the
composition because of differential ion concentrations between the
composition and physiological fluids. It is also preferable to
buffer the saline in order to maintain a pH compatible with normal
physiological conditions. Also, in certain instances, it may be
necessary to maintain the pH at a particular level in order to
insure the stability of certain composition components such as the
glycopeptides.
[0024] Any physiologically acceptable buffer may be used herein,
but phosphate buffers are preferred. Other acceptable buffers such
as acetate, tris, bicarbonate, carbonate, or the like may be used
as substitutes for phosphate buffers. The pH of the aqueous
component will preferably be between 6.0-8.0.
[0025] However, when the adjuvant is initially prepared,
unadulterated water is preferred as the aqueous component of the
emulsion. Increasing the salt concentration makes it more difficult
to achieve the desired small droplet size. When the final vaccine
formulation is prepared from the adjuvant, the antigenic material
can be added in a buffer at an appropriate osmolality to provide
the desired vaccine composition.
[0026] The quantity of the aqueous component employed in these
compositions will be that amount necessary to bring the value of
the composition to unity. That is, a quantity of aqueous component
sufficient to make 100% will be mixed, with the other components
listed above in order to bring the compositions to volume.
[0027] A substantial number of emulsifying and suspending agents
are generally used in the pharmaceutical sciences. These include
naturally derived materials such as gums from trees, vegetable
protein, sugar-based polymers such as alginates and cellulose, and
the like. Certain oxypolymers or polymers having a hydroxide or
other hydrophilic substituent on the carbon backbone have
surfactant activity, for example, povidone, polyvinyl alcohol, and
glycol ether-based mono- and poly-functional compounds. Long chain
fatty-acid-derived compounds form a third substantial group of
emulsifying and suspending agents which could be used in this
invention. Any of the foregoing surfactants are useful so long as
they are non-toxic.
[0028] Specific examples of suitable emulsifying agents (also
referred to as surfactants or detergents) which can be used in
accordance with the present invention include the following:
[0029] 1. Water-soluble soaps, such as the sodium, potassium,
ammonium and alkanol-ammonium salts of higher fatty acids
(C.sub.10-C.sub.22), and, particularly sodium and potassium tallow
and coconut soaps.
[0030] 2. Anionic synthetic non-soap detergents, which can be
represented by the water-soluble salts of organic sulfuric acid
reaction products having in their molecular structure an alkyl
radical containing from about 8 to 22 carbon atoms and a radical
selected from the group consisting of sulfonic acid and sulfuric
acid ester radicals. Examples of these are the sodium or potassium
alkyl sulfates, derived from tallow or coconut oil; sodium or
potassium alkyl benzene sulfonates; sodium alkyl glyceryl ether
sulfonates; sodium coconut oil fatty acid monoglyceride sulfonates
and sulfates; sodium or potassium salts of sulfuric acid esters of
the reaction product of one mole of a higher fatty alcohol and
about 1 to 6 moles of ethylene oxide; sodium or potassium alkyl
phenol ethylene oxide ether sulfonates, with 1 to 10 units of
ethylene oxide per molecule and in which the alkyl radicals contain
from 8 to 12 carbon atoms; the reaction product of fatty acids
esterified with isethionic acid and neutralized with sodium
hydroxide; sodium or potassium salts of fatty acid amide of a
methyl tauride; and sodium and potassium salts of
SO.sub.3-sulfonated C.sub.10-C.sub.24 .alpha.-olefins.
[0031] 3. Nonionic synthetic detergents made by the condensation of
alkylene oxide groups with an organic hydrophobic compound. Typical
hydrophobic groups include condensation products of propylene oxide
with propylene glycol, alkyl phenols, condensation product of
propylene oxide and ethylene diamine, aliphatic alcohols having 8
to 22 carbon atoms, and amides of fatty acids.
[0032] 4. Nonionic detergents, such as amine oxides, phosphine
oxides and sulfoxides, having semipolar characteristics. Specific
examples of long chain tertiary amine oxides include
dimethyldodecylamine oxide and bis-(2-hydroxyethyl) dodecylamine.
Specific examples of phosphine oxides are found in U.S. Pat. No.
3,304,263 which issued Feb. 14, 1967, and include
dimethyldodecyl-phosphine oxide and dimethyl-(2hydroxydodecyl)
phosphine oxide.
[0033] 5. Long chain sulfoxides, including those corresponding to
the formula R.sup.1--SO--R.sup.2 wherein R.sup.1 and R.sup.2 are
substituted or unsubstituted alkyl radicals, the former containing
from about 10 to about 28 carbon atoms, whereas R.sup.2 contains
from 1 to 3 carbon atoms. Specific examples of these sulfoxides
include dodecyl methyl sulfoxide and 3-hydroxy tridecyl methyl
sulfoxide.
[0034] 6. Ampholytic synthetic detergents, such as sodium
3-dodecylaminopropionate and sodium 3-dodecylaminopropane
sulfonate.
[0035] 7. Zwitterionic synthetic detergents, such as
3-(N,N-dimethyl-N-hexadecylammonio) propane-1-sulfonate and
3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy
propane-1-sulfonate.
[0036] Additionally, all of the following types of emulsifying
agents can be used in a composition of the present invention: (a)
soaps (i.e., alkali salts) of fatty acids, rosin acids, and tall
oil; (b) alkyl arene sulfonates; (c) alkyl sulfates, including
surfactants with both branched-chain and straight-chain hydrophobic
groups, as well as primary and secondary sulfate groups; (d)
sulfates and sulfonates containing an intermediate linkage between
the hydrophobic and hydrophilic groups, such as the fatty acylated
methyl taurides and the sulfated fatty monoglycerides; (e)
long-chain acid esters of polyethylene glycol, especially the tall
oil esters; (f) polyethylene glycol ethers of alkylphenols; (g)
polyethylene glycol ethers of long-chain alcohols and mercaptans;
and (h) fatty acyl diethanol amides. Since surfactants can be
classified in more than one manner, a number of classes of
surfactants set forth in this paragraph overlap with previously
described surfactant classes.
[0037] There are a number of emulsifying agents specifically
designed for and commonly used in biological situations. For
example, a number of biological detergents (surfactants) are listed
as such by Sigma Chemical Company on pages 310-316 of its 1987
Catalog of Biochemical and Organic Compounds. Such surfactants are
divided into four basic types: anionic, cationic, zwitterionic, and
nonionic. Examples of anionic detergents include alginic acid,
caprylic acid, cholic acid, 1-decanesulfonic acid, deoxycholic
acid, 1-dodecanesulfonic acid, N-lauroylsarcosine, and taurocholic
acid. Cationic detergents include dodecyltrimethylammonium bromide,
benzalkonium chloride, benzyldimethylhexadecyl ammonium chloride,
cetylpyridinium chloride, methylbenzethonium chloride, and
4-picoline dodecyl sulfate. Examples of zwitterionic detergents
include 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate
(commonly abbreviated CHAPS),
3-[(cholamidopropyl)-dimethylammonio]-2-hydroxy-1-pro-
panesulfonate (generally abbreviated CHAPSO),
N-dodecyl-N,N-dimethyl-3-amm- onio-1-propanesulfonate, and
lyso-.alpha.-phosphatidylcholine. Examples of nonionic detergents
include decanoyl-N-methylglucamide, diethylene glycol monopentyl
ether, n-dodecyl .beta.-D-glucopyranoside, ethylene oxide
condensates of fatty alcohols (e.g., sold under the trade name
Lubrol), polyoxyethylene ethers of fatty acids (particularly
C.sub.12-C.sub.20 fatty acids), polyoxyethylene sorbitan fatty acid
ethers (e.g., sold under the trade name Tween), and sorbitan fatty
acid ethers (e.g., sold under the trade name Span).
[0038] A particularly useful group of surfactants are the
sorbitan-based non-ionic surfactants. These surfactants are
prepared by dehydration of sorbitol to give 1,4-sorbitan which is
then reacted with one or more equivalents of a fatty acid. The
fatty-acid--substituted moiety may be further reacted with ethylene
oxide to give a second group of surfactants.
[0039] The fatty-acid-substituted sorbitan surfactants are made by
reacting 1,4-sorbitan with a fatty acid such as lauric acid,
palmitic acid, stearic acid, oleic acid, or a similar long chain
fatty acid to give the 1,4-sorbitan mono-ester, 1,g-sorbitan
sesquiester or 1,4-sorbitan triester. The common names for these
surfactants include, for example, sorbitan monolaurate, sorbitan
monopalmitate, sorbitan monoestearate, sorbitan monooleate,
sorbitan sesquioleate, and sorbitan trioleate. These surfactants
are commercially available under the name SPAN.RTM. or
ARLACEL.RTM., usually with a letter or number designation which
distinguishes between the various mono, di- and triester
substituted sorbitans.
[0040] SPAN.RTM. and ARLACEL.RTM. surfactants are hydrophilic and
are generally soluble or dispersible in oil. They are also soluble
in most organic solvents. In water they are generally insoluble but
dispersible. Generally these surfactants will have a
hydrophilic-lipophilic balance (HLB) number between 1.8 to 8.6.
Such surfactants can be readily made by means known in the art or
are commercially available from, for example, ICI America's Inc.,
Wilmington, Del. under the registered mark ATLAS.RTM..
[0041] A related group of surfactants comprises polyoxyethylene
sorbitan monoesters and polyoxyethylene sorbitan triesters. These
materials are prepared by addition of ethylene oxide to a
1,4-sorbitan monester or triester. The addition of polyoxyethylene
converts the lipophilic sorbitan mono- or triester surfactant to a
hydrophilic surfactant generally soluble or dispersible in water
and soluble to varying degrees in organic liquids.
[0042] These materials, commercially available under the mark
TWEEN.RTM., are useful for preparing oil-in-water emulsions and
dispersions, or for the solubilization of oils and making anhydrous
ointments water-soluble or washable. The TWEEN.RTM. surfactants may
be combined with a related sorbitan monester or triester
surfactants to promote emulsion stability. TWEEN.RTM. surfactants
generally have a HLB value falling between 9.6 to 16.7. TWEEN.RTM.
surfactants are commercially available from a number of
manufacturers, for example ICI America's Inc., Wilmington, Del.
under the registered mark ATLAS.RTM. surfactants.
[0043] A third group of non-ionic surfactants which could be used
alone or in conjunction with SPAN.RTM., ARLACEL.RTM. and TWEEN.RTM.
surfactants are the polyoxyethylene fatty acids made by the
reaction of ethylene oxide with a long-chain fatty acid. The most
commonly available surfactant of this type is solid under the name
MYRJ.RTM. and is a polyoxyethylene derivative of stearic acid.
MYRJ.RTM. surfactants are hydrophilic and soluble or dispersible in
water like TWEEN.RTM. surfactants. The MYRJ.RTM. surfactants may be
blended with TWEEN.RTM. surfactants or with TWEEN.RTM./SPAN.RTM. or
ARLACEL.RTM. surfactant mixtures for use in forming emulsions.
MYRJ.RTM. surfactants can be made by methods known in the art or
are available commercially from ICI America's Inc.
[0044] A fourth group of polyoxyethylene based non-ionic
surfactants are the polyoxyethylene fatty acid ethers derived from
lauryl, acetyl, stearyl and oleyl alcohols. These materials are
prepared as above by addition of ethylene oxide to a fatty alcohol.
The commercial name for these surfactants is BRIJ.RTM.. BRIJ.RTM.
surfactants may be hydrophilic.TM.or lipophilic depending on the
size of the polyoxyethylene moiety in the surfactant. While the
preparation of these compounds is available from the art, they are
also readily available from such commercial sources as ICI
America's Inc.
[0045] Other non-ionic surfactants which could potentially be used
in the practice of this invention are for example: polyoxyethylene,
polyol fatty acid esters, polyoxyethylene ether, polyoxypropylene
fatty ethers, bee's wax derivatives containing polyoxyethylene,
polyoxyethylene lanolin derivative, polyoxyethylen fatty
glycerides, glycerol fatty acid esters or other polyoxyethylene
acid alcohol or ether derivatives of long-chain fatty acids of
12-22 carbon atoms.
[0046] As the adjuvant and the vaccine formulations of this
invention are intended to be multi-phase systems, it is preferable
to choose an emulsion-forming non-ionic surfactant which has an HLB
value in the range of about 7 to 16. This value may be obtained
through the use of a single non-ionic surfactant such as a
TWEEN.RTM. surfactant or may be achieved by the use of a blend of
surfactants such as with a sorbitan mono, di- or triester based
surfactant; a sorbitan ester polyoxyethylene fatty acid; a sorbitan
ester in combination with a polyoxyethylene lanolin derived
surfactant; a sorbitan ester surfactant in combination with a high
HLB polyoxyethylene fatty ether surfactant, or a polyethylene fatty
ether surfactant or polyoxyethylene sorbitan fatty acid.
[0047] It is more preferred to use a single non-ionic surfactant,
most particularly a TWEEN.RTM. surfactant, as the emulsion
stabilizing non-ionic surfactant in the practice of this invention.
The surfactant named TWEEN.RTM. 80, otherwise known as polysorbate
80 for polyoxyethlyene 20 sorbitan monooleate, is the most
preferred of the foregoing surfactants.
[0048] Sufficient droplet size reduction can usually be effected by
having the surfactant present in an amount of 0.02% to 2.5% by
weight (w/w). An amount of 0.05% to 1% is preferred with 0.01 to
0.5% being especially preferred.
[0049] The manner in which the droplet size of the invention is
reached is not important to the practice of the present invention.
One manner in which submicron oil droplets can be obtained is by
use of a commercial emulsifiers, such as model number 11OY
available from Microfluidics, Newton, Mass. Examples of other
commercial emulsifiers include Gaulin Model 30CD (Gaulin, Inc.,
Everett, Mass.) and Rainnie Minilab Type 8.30H (Miro Atomizer Food
and Dairy, Inc., Hudson, Wis.). These emulsifiers operate by the
principle of high shear forces developed by forcing fluids through
small apertures under high-pressure. When the model 11OY is
operated at 5,000-30,000 psi, oil droplets having diameters of
100-750 nm are provided.
[0050] The size of the oil droplets can be varied by changing the
ratio of detergent to oil (increasing the ratio decreases droplet
size), operating pressure (increasing operating pressure reduces
droplet size), temperature (increasing temperature decreases
droplet size), and adding an amphipathic immunostimulating agent
(adding such agents decreases droplet size). Actual droplet size
will vary with the particular detergent, oil, and immunostimulating
agent (if any) and with the particular operating conditions
selected. Droplet size can be verified by use of sizing
instruments, such as the commercial Sub-Micron Particle Analyzer
(Model N4MD) manufactured by the Coulter Corporation, and the
parameters can be varied using the guidelines set forth above until
substantially all droplets are less than 1 micron in diameter,
preferably less than 0.8 microns in diameter, and most preferably
less than 0.5 microns in diameter. By substantially all is meant at
least 80% (by number), preferably at least 90%, more preferably at
least 95%, and most preferably at least 98%. The particle size
distribution is typically Gaussian, so that the average diameter is
smaller than the stated limits.
[0051] The present invention is practiced by preparing an oil
emulsion in the absence of other components previously taught in
the prior art to be used with submicron emulsions for satisfactory
immunogenicity, namely polyoxypropylene-polyoxyethlyne block
polymers such as those described for use with adjuvants in U.S.
Pat. No. 4,772,466 and 4,770,874 and in European Patent Application
0 315 153 A2.
[0052] An adjuvant composition of the invention consists
essentially of a metabolizable oil in water and an emulsifying
agent other than than a POP-POE copolymer. The emulsifying agent
need not have any specific immunostimulating activity, since the
oil composition by itself can function as an adjuvant when the oil
droplets are in the submicron range. However, increased
immunostimulating activity can be provided by including any of the
known immunostimulating agents in the composition. These
immunostimulating agents can either be separate from the
emulsifying agent and the oil or the immunostimulating agent and
the emulsifying agent can be one and the same molecule. Examples of
the former situation include metabolizable oils mixed with killed
mycobacteria, such as Mycobacterium tuberculosis, and subcellular
components thereof. Additional immunostimulating substances include
the muramyl peptides that are components of the cell walls of such
bacteria. A number of preferred muramyl peptides are listed below.
Examples of the joint emulsifying agent/immunostimulating agent are
the lipophilic muramyl peptides described in the two
Sanchez-Pescador et al. publications cited above. These materials
comprise the basic N-acetylmuramyl peptide (a hydrophilic moiety)
that acts as an immunostimulating group, but also include a
lipophilic moiety that provides surface-active characteristics to
the resulting compound. Such compounds, as well as other types of
amphipathic immunostimulating substances, act as both
immunostimulating agents and emulsifying agents and are preferred
in the practice of the present invention. In addition, it is also
possible to practice the present invention by using a amphiphatic
immunostimulating substance in combination with a second
immunostimulating substance that is not amphipathic. An example
would be use of a lipophilic muramyl peptide in combination with an
essentially unsubstituted (i.e., essentially hydrophilic) muramyl
dipeptide.
[0053] The preferred immune-response-stimulating muramyl peptides
(or more accurately glycopeptides) of this invention are a group of
compounds related to and generally derived from
N-acetylmuramyl-L-alanyl-D-isogluta- mine, which was determined by
Ellouz et al. (1974) Biochem. & Biophys. Res. Comm., 59(4):
1317, to be the smallest effective unit possessing immunological
adjuvant activity in M. tuberculosis, the mycobacterial component
of Freund's complete adjuvant. A number of dipeptide- and
polypeptide-substituted muramic acid derivatives were subsequently
developed and found to have immunostimulating activity.
[0054] Though these glycopeptides are a diverse group of compounds,
they can be generally represented by Formula I below: 1
[0055] wherein the pyran ring oxygens are substituted by hydrogen,
alkyl, or acyl or the like, or may be replaced by nitrogen-based
substituents, particularly the 6-position oxygen; the 2-amino group
is an acyl group or some other amide; the lactyl side chain is
modified, e.g., is ethyl or another two-position alkyl moiety; and
the peptide function is a dipeptide or polypeptide, which may be
further derivatized. Furanosyl analogues of the pyranosyl compounds
also have immunopotentiating activity and are useful in this
invention.
[0056] Among the glycopeptides of this invention are those
disaccharides and tetrasaccharides linked by
meso-.alpha.,.epsilon.-diaminopimelic acid such as described in
U.S. Pat. Nos. 4,235,771 and 4,186,194.
[0057] Immune response stimulating glycopeptides which may be used
in the practice of this invention are disclosed in U.S. Pat. Nos.
4,094,971; 4,101,536; 4,153,684; 4,235,771; 4,323,559; 4,327,085;
4,185,089; 4,082,736; 4,369,178; 4,314,998 and 4,082,735; and
4,186,194. The glycopeptides disclosed in these patents are
incorporated herein by reference and made a part hereof as if set
out in full herein. The compounds of Japanese patent application
Nos. JP 40792227, JP 4079228, and JP 41206696 would also be useful
in the practice of this invention.
[0058] Methods for preparing these compounds are disclosed and
well-known in the art. Preparative process exemplification can be
found in U.S. Pat. Nos. 4,082,736 and 4,082,735. Additionally,
similar preparative processes may be found in the U.S. patents
referenced in the preceding paragraph.
[0059] Preferred glycopeptides are those having the Formula II
2
[0060] wherein
[0061] R is an unsubstituted or substituted alkyl radical
containing from 1 to 22 carbon atoms, or an unsubstituted or
substituted aryl radical containing from 6 to 10 carbon atoms;
[0062] R.sup.1and R.sup.2 are the same or different and are
hydrogen or an acyl radical containing from 1 to 22 carbon
atoms;
[0063] R.sup.3 is hydrogen, alkyl of 1 to 22 carbons, or aryl of 7
to 10 carbon atoms;
[0064] R.sup.3 is hydrogen or alkyl;
[0065] n is 0 or 1;
[0066] X and Z are independently alanyl, valyl, leucyl, isoleucyl,
.alpha.-aminobutyryl, threonyl, methionyl, cysteinyl, glutamyl,
glutaminyl, isoglutamyl, isoglutaminyl, aspartyl, phenylalanyl,
tyrosyl, lysyl, ornithinyl, arginyl, histidyl, asparaginyl, prolyl,
hydroxyprolyl, seryl, or glycyl;
[0067] R.sup.5 is an optionally esterified or amidated carboxyl
group of the terminal amino acid; and
[0068] Y is --NHCHR.sup.6CH.sub.2CH.sub.2CO--, wherein R.sup.6 is
an optionally esterified or amidated carboxyl group.
[0069] Alkyl is a straight or branched radical comprised of 1 to 7
carbon atoms unless otherwise specified, exemplified by methyl,
ethyl, propyl, butyl, pentyl, hexyl or heptyl or an isomer. Lower
alkyl is a radical of 1 to 4 carbon atoms.
[0070] An optionally esterified or amidated carboxyl group is the
carboxyl group itself or a carboxyl group esterified with a lower
alkanol, such as methanol, ethanol, propanol, butanol, or the
carbamoyl group, which, on the nitrogen atom, is unsubstituted or
monosubstituted or di-substituted by alkyl, especially lower alkyl,
aryl, particularly phenyl, or arylalkyl, particularly benzyl. The
carbamoyl group may also be substituted with an alkylidene radical
such as butylidene or pentylidene radical. In addition, the
carbamoyl group R.sup.5 may also be substituted with a
carbamoylmethyl group on the nitrogen atom.
[0071] Particularly preferred compounds are those of Formula II
wherein R and R.sup.1 are the same or different and are hydrogen or
an acyl radical containing from 1 to 22 carbon atoms; R.sup.2 is
methyl; R.sup.3 is hydrogen; X is L-alanyl, Y is D-isoglutaminyl,
and n is 0.
[0072] A different preferred group of glycopeptides are the
compounds of Formula II wherein R and R.sup.1 are hydrogen or acyl
of 1 to 22 carbon atoms, R.sup.2 is methyl, R.sup.2 is hydrogen,
R.sup.4 is methyl or butyl, and X is L-valyl, L-seryl, L-alanyl,
L-threonyl or L-.alpha.--aminobutyryl.
[0073] Specific examples include the following compounds:
[0074] N-acetylmuramyl-L-.alpha.-aminobutyryl-D-isoglutamine;
[0075]
6-0-stearoyl-N-acetylmuramyl-L-.alpha.-aminobutyryl-D-isoglutamine;
[0076] N-acetylmuramyl-L-threonyl-D-isoglutamine;
[0077] N-acetylmuramyl-L-valyl-D-isoglutamine;
[0078] N-acetylmuramyl-L-alanyl-D-glutamine n-butyl ester;
[0079] N-acetyl-desmethyl-D-muramyl-L-alanyl-D-isoglutamine;
[0080] N-acetylmuramyl-L-alanyl-D-glutamine;
[0081] N-acetylmuramyl-L-seryl-D-isoglutamine;
[0082] N-acetyl(butylmuramyl)-L-.alpha.-aminobutyl-D-isoglutamine;
and
[0083] N-acetyl(butylmuramyl)-L-alanyl-D-isoglutamine.
[0084] An effective amount of immunostimulating glycopeptide is
that amount which effects an increase in antibody titer level when
administered in conjunction with an antigen over that titer level
observed when the glycopeptide has not been co-administered
(typically in the range of 0.0001 to 10% of the total composition).
As can be appreciated, each glycopeptide may have an effective dose
range that may differ from the other glycopeptides. Therefore, a
single dose range cannot be prescribed which will have a precise
fit for each possible glycopeptide within the scope of this
invention. However, as a general rule, the glycopeptide will
preferably be present in the vaccine in an amount of between 0.001
and 5% (w/v). A more preferred amount is 0.01 to 3% (w/v).
[0085] Most of the immunostimulating glycopeptides discussed above
are essentially hydrophilic compounds. Accordingly, they are
intended for use with a separate emulsifying agent (which can be,
as discussed above, also an immunostimulating agent). In some case,
the above-described compounds have a lipophilic character, such as
the compounds comprising fatty acid substituents and/or aryl
substituents on the sugar moiety, particularly those containing one
or more acyl radicals containing from 14 to 22 carbon atoms,
particularly those containing more than 1 such acyl substituent.
However, it is also possible to achieve lipophilic character in a
muramyl peptide by providing a lipid moiety linked through the
carboxylate group or side chains of the peptide moiety. In
particular, lipid groups joined to the peptide moiety through the
terminal carboxylate group represent a preferred grouping of
compounds. This linkage can readily be provided either directly,
such as by forming an ester linkage between the terminal
carboxylate and a fatty alcohol containing from 14 to 22 carbon
atoms, or by using a bifunctional linking group, such as
ethanolamine, to link the carboxylate through either a ester or
amide linkage to a lipid. Particularly preferred in this embodiment
of the invention are phospholipids, as the phosphate groups provide
a readily linkable functional group. Diacylphospho-glycerides
provide one such readily linkable phospho-lipid. Phosphatidyl
ethanolamine, a readily available, naturally occurring compound,
can be easily linked to the terminal carboxylate of the peptide
moiety through an amide bond. Other lipids to the terminal carboxyl
include acylglycerols, phosphatidyl choline, phosphatidyl serine,
phosphatidyl inositol, phosphatidylglycerol, cardiolipin, and
sphingomyelin.
[0086] A number of preferred amphipathic immunostimulating peptides
are those having Formula III below: 3
[0087] wherein R, R.sup.1-R.sup.4, X, Y, Z and n have the
previously described meanings. L represents a lipid moiety, such as
the lipid moieties described above.
[0088] In summary, the muramic acid moiety and the peptide moiety
of the molecule together provide a hydrophilic moiety. A lipophilic
moiety is also present in the molecule, lipophilicity generally
being provided by a long-chain hydrocarbon group, typically present
in the form of a fatty acid. The fatty acid or other
hydrocarbon-containing radical can be attached to a hydroxyl group
of the sugar or can be linked to the peptide portion of the
molecule either directly, such as by reacting a fatty acid with a
free amino group present in the peptide moiety, or through a
linking group, such as a hydroxyalkylamine that forms a link
between a carboxylic acid group of the peptide through amide bond
formation and a functional group in a lipid, such as a phosphate
group. Phospholipid moieties are particularly preferred for use in
forming lipophilic muramyl peptides. A group of preferred compounds
include muramyl dipeptides and tripeptides linked to a phospholipid
moiety through a hydroxyalkylamine moiety. An example, and a
particularly preferred compound, is
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn--
glycero-3-(hydroxyphosphoryloxy)]ethylamide (abbreviated
MTP-PE).
[0089] The adjuvant formulations are generally prepared from the
ingredients described above prior to combining the adjuvant with
the antigen that will be used in the vaccine. The word antigen
refers to any substance, including a protein or
protein-poly-saccharide, protein-lipopolysaccharide,
poly-saccharide, lipopolysaccharide, viral subunit, whole virus or
whole bacteria which, when foreign to the blood stream of an
animal, on gaining access to the tissue of such an animal
stimulates the formation of specific antibodies and reacts
specifically in vivo or in vitro with a homologous antibody.
Moreover, it stimulates the proliferation of T-lymphocytes with
receptors for the antigen and can react with the lymphocytes to
initiate the series of responses designated cell-mediated
immunity.
[0090] A hapten is within the scope of this definition. A hapten is
that portion of an antigenic molecule or antigenic complex that
determines it immunological specificity. Commonly, a hapten is a
peptide or polysaccharide in naturally occurring antigens. In
artificial antigens it may be a low molecular weight substance such
as an arsanilic acid derivative. A hapten will react specifically
in vivo or in vitro with homologous antibodies or T-lymphocytes.
Alternative descriptors are antigenic determinant, antigenic
structural grouping and haptenic grouping.
[0091] The formulation of a vaccine of the invention will employ an
effective amount of an antigen. That is, there will be included an
amount of antigen which, in combination with the adjuvant, will
cause the subject to produce a specific and sufficient
immunological response so as to impart protection to the subject
from the subsequent exposure to virus, bacterium, fungus,
mycoplasma, or parasite immunized against.
[0092] Antigens may be produced by methods known in the art or may
be purchased from commercial sources. For example, U.S. Pat. Nos.
4,434,157, 4,406,885, 4,264,587, 4,117,112, 4,034,081, and
3,996,907, incorporated herein by reference, describe methods for
preparing antigens for feline leukemia virus vaccines. Other
antigens may similarly be prepared. Antigens within the scope of
this invention include whole inactivated virus particles, isolated
virus proteins and protein subunits, whole cells and bacteria, cell
membrane and cell wall proteins, and the like. Vaccines of the
invention may be used to immunize birds and mammals against
diseases and infection, including without limitation cholera,
diphtheria, tetanus, pertussis, influenza, measles, meningitis,
mumps, plague, poliomyelitis, rabies, Rocky Mountain spotted fever,
rubella, smallpox, typhoid, typhus, feline leukemia virus, and
yellow fever.
[0093] No single dose designation can be assigned which will
provide specific guidance for each and every antigen which may be
employed in this invention. The effective amount of antigen will be
a function of its inherent activity and purity. It is contemplated
that the adjuvant compositions of this invention may be used in
conjunction with whole cell or virus vaccines as well as with
purified antigens or protein subunit or peptide vaccines prepared
by recombinant DNA techniques or synthesis.
[0094] Since the adjuvant compositions of the invention are stable,
the antigen and emulsion can mixed by simple shaking. Other
techniques, such as passing a mixture of the adjuvant and solution
or suspension of the antigen rapidly through a small opening (such
as a hypodermic needle) readily provides a useful vaccine
composition.
[0095] The invention now being generally described, the same will
be better understood by reference to the following detailed
examples which are provided by way of illustration and are not
intended to be limiting of the invention unless so specified.
EXAMPLE 1
General Techniques
[0096] The following general techniques were used throughout the
examples that follow, except where noted:
[0097] Materials
[0098] MTP-PE was provided by CIBA-GEIGY (Basel, Switzerland).
Squalene and Tween 80 were obtained from Sigma Chemical Co. (St.
Louis, Mo.). CFA and IFA were obtained from Gibco (Grand Island,
N.Y.). Aluminum hydroxide (Rehsorptar) was obtained from Reheis
Chemical Co. (Berkeley Heights, N.J.).
[0099] Preparation of Emulsions
[0100] Method 1--Syringe and needle. A mixture consisting of 4%
squalene, 0.008% Tween 80, 250 .mu.g/ml MTP-PE and antigen in
phosphate buffered saline (PBS) was passed through a 23 gauge
needle 6 times. This emulsion consisted of oil droplet sizes in the
range of 10 microns and is termed MTP-PE-LO.
[0101] Method 2--Kirkland Emulsifier. The above mixture was passed
through a Kirkland emulsifier five times. This emulsion consists of
oil droplets primarily of 1-2 microns and is termed MTP-PE-LO-KE.
The Kirkland emulsifier (Kirkland Products, Walnut Creek, Calif.)
is a small-scale version of the commercial knife-edged homogenizer
(e.g., Gaulin Model 30CD and Rainnie Minilab Type 8.30H) generating
about 1000 psi in the working chamber.
[0102] Method 3--Microfluidizer. Mixtures containing 0.3-18%
squalene and 0.2-1.0 mg/ml MTP-PE with or without Tween 80 were
passed through the Microfluidizer (Model No. 11OY, Microfluidics
Newton, Mass.) at 5,000-30,000 PSI. Typically, 50 ml of emulsion
was mixed for 5 minutes or 100 ml for 10 minutes in the
microfluidizer. The resulting emulsions consisted of oil droplets
of 100-750 nm depending on squalene, MTP-PE, and detergent
concentration and microfluidizer operating pressure and
temperature. This formulation is termed MTP-PE-LO-MF.
[0103] Antigen was added to the adjuvant formulations above after
preparation. The antigen and emulsion were mixed by shaking. When
using CFA and IFA, antigen in PBS was mixed with an equal volume of
either CFA or IFA The mixture was emulsified by passing through a
hypodermic needle until a thick, emulsion was achieved.
[0104] Antigens
[0105] Herpes simplex virus (HSV) rgD2 is a recombinant protein
produced genetically engineered Chinese hamster ovary cells. This
protein has the normal anchor region truncated, resulting in a
glycosylated protein secreted into tissue culture medium. The gD2
was purified in the CHO medium to greater than 90% purity. Human
immunodeficiency virus (HIV) env-2-3 is a recombinant form of the
HIV enveloped protein produced in genetically engineered
Saccharomyces cerevisae. This protein represents the entire protein
region of HIV gp120 but is non-glycosylated and denatured as
purified from the yeast. HIV gp120 is a fully glycosylated,
secreted form of gp120 produced in CHO cells in a fashion similar
to the gD2 above.
[0106] Immunization of Animals
[0107] Mice were injected with the various adjuvant/antigen
formulations by intraperitoneal, intramuscular, or subcutaneous
routes. Guinea pigs were immunized by footpad or intramuscular
routes. Rabbits, goats, and baboons were immunized by the
intramuscular routes.
[0108] Analysis of Immune Response
[0109] Antibody titers against the immunizing antigen were
determined by enzyme linked immunosorbent assay (ELISA).
EXAMPLE 2
MTP-PE-LO Formulation in Large Animals
Comparative Example
[0110] A number of experiments were carried out, first with the HIV
env 2-3 antigen and later with the HSV gD protein, using the
MTP-PE-LO formulation to stimulate immunity in large animals. These
experiments are outlined below.
[0111] 1. HIV env 2-3
[0112] a. Guinea pigs. Guinea pigs were immunized monthly with 50
pg/dose of env 2-3 by either the footpad or intramuscular route.
The vaccine was administered with either the MTP-PE-LO formulation
(4% Squalene, 0/008% Tween 80, 50 pg/dose MTP-PE) or absorbed to
alum (0.7% aluminum hydroxide). Sera were collected one week after
each immunization and analyzed for anti-env 2-3 antibody by ELISA.
The results are shown in Table 1. The MTP-PE-LO formulation gave
high anti-env 2-3 titers when delivered both intramuscularly and in
the footpad. In contrast, alum gave much lower antibody titers by
both routes. This experiment illustrates the effectiveness of the
MTP-PE-LO formulation in guinea pigs.
1TABLE 1 Comparison of Different Adjuvants, As a Function of
Injection Route, In Eliciting Env 2-3 Specific Antibodies.sup.a Env
2-3 ELISA Titers djuvant Animal Immunization Number Group # Route
Zero Two Three MTP-PE 839 FP <<100.sup.c 135,500 382,100 4%
840 FP <<100 331,700 588,700 Squalene 0.008% 841 FP
<<100 247,800 330,900 Tween 842 FP <<100 108,100
570,300 843 FP <<100 65,00 -- 844 FP <<100 25,000 --
(average) (FP) (<<100) (152,000) (468,000) MTP-PE 845 IM
<<100 12,300 19,600 4% 846 IM <<100 10,400 20,500
Squalene 0.008% 847 IM <<100 29,700 80,000 Tween 848 IM
<<100 447,000 640,000 849 IM 350 10,600 78,700 850 IM
<<100 340,000 -- (average) (IM) (<<100) (142,000)
(168,000) Alum 863 FP <<100 <<100 nt 864 FP <<100
2,500 4,100 865 FP <<100 2,400 26,400 866 FP <<100
15,100 103,900 867 FP <<100 2,200 8,800 868 FP <<100
6,500 44,500 (average) (FP) (<<100) (5,700) (38,000) Alum 869
IM <<100 <<100 300 870 IM <<100 <<100 130
871 IM <<100 <<100 1,200 872 IM <<100 <<100
300 873 IM <<100 <<100 990 874 IM <<100
<<100 940 (average) (IM) (<<100) (<<100) (640)
Env 2-3 ELISA Titers djuvant Immunization Number Group Four Five
Six Seven MTP-PE 343,100 401,800 338,000 382,700 4% 542,300 392,900
359,000 292,100 Squalene 0.008% 301,100 285,800 334,400 383,700
Tween 694,300 344,400 289,800 220,300 -- -- -- -- -- -- -- --
(470,000) (356,000) (330,000) (295,000) MTP-PE 23,800 15,100 20,000
27,300 4% 43,600 44,800 121,100 42,000 Squalene 0.008% 136,800
156,000 144,500 164,400 Tween 400,000 71,000 674,000 533,000
311,000 533,000 nt 200,000 -- -- -- -- (183,000) (164,000)
(240,000) (193,000) Alum nt nt nt nt 86,000 47,700 21,000 16,000
80,400 83,500 39,200 4,500 124,100 107,100 56,700 16,800 14,500
11,900 11,400 12,300 34,000 18,800 12,800 (68,000) (54,000)
(28,000) (12,000) Alum 2,600 2,000 1,600 2,300 220 330 270 300
4,300 4,900 3,000 1,600 900 920 770 1,700 41,100 79,800 27,900
15,500 17,300 13,200 10,600 8,600 (11,000) (17,000) (7,000) (5,000)
.sup.aGuinea pigs were immunized monthly with 50 `"m"` g/dose of
env 2-3 with the different adjuvants by either the footpad (FP) or
intramuscular (IM) route. Sera were collected one week following
each immunization. .sup.b" "; no data obtained due to death of the
animal. .sup.c<<100; no detectable ELISA signal at 1:100
serum dilution. .sup.dnt = not tested
[0113] b. Goats. Pairs of goats received 1 mg of env 2-3 on primary
immunizations and 500 .mu.g on secondary immunization with the
MTP-PE-LO formulation containing various amounts of MTP-PE from 0
to 500 .mu.g. Positive control animals received the primary
immunization with CFA and the secondary immunization with IFA. One
group also received 100 .mu.g env 2-3 in the primary immunization
followed by 50 .mu.g in the secondary immunization with the
MTP-PE-LO formulation containing 100 .mu.g MTP-PE. As shown in
Table 2, both goats receiving Freund's adjuvant showed high
antibody titers ranging from 2700 to 62,800. In contrast, most of
the goats receiving the MTP-PE-LO formulation were negative for
anti-env 2-3 antibody. Animals that did respond only developed
titers in the 100-600 range. These results are in stark contrast to
the guinea pig data above.
2TABLE 2 Antibody Responses of Goats Immunized With Env 2-3 and
Various Doses of MTP-PE Adjuvant Env 2-3 ELISA Titer Formu- Animal
Immunization lation Number None One Two Freund's 2295
.sup.b<<100 43,200 62,800 2296 <<100 2,700 7,500
.sup.aST + 0 .mu.g 2297 <<100 .sup.c<100 <100 MTP-PE
2298 <<100 100 300 ST + 20 .mu.g 2290 <<100 <100
<100 MTP-PE 2302 <<100 100 200 ST + 50 .mu.g 2301
<<100 <<100 <100 MTP-PE 2302 <<100 <<100
<100 ST + 100 .mu.g 2303 <<100 <<100 100 MTP-PE 2304
<<100 <<100 <100 ST + 250 .mu.g 2305 <<100
<100 600 MTP-PE 2306 <<100 <<100 <100 ST + 500
.mu.g 2307 <<100 <100 <100 MTP-PE 2308 <<100
<<100 <<100 ST + 100 .mu.g 2309 200 500 200 MTP-PE 2310
<<100 <100 <<100 .sup.aST is the low oil
formulation; 4% Squalene, 0.008% Tween 80. .sup.b<<100
indicates an env 2-3 ELISA titer that was not above background at a
1/100 serum dilution. .sup.c<100 indicates an env 2-3 ELISA
value at a 1/100 serum dilution that was above background but less
than the half maximal signal in the assay.
[0114] c. Dogs. Beagle dogs were immunized with either 250 .mu.g of
env 2-3 in MTP-PE-LO (100 .mu.g MTP-PE) or with the MTP-PE-LO
formulation alone at three week intervals. Ten days after each
immunization the animals were bled and anti-env 2-3 antibody titers
were determined by ELISA. Table 3 shows that the two dogs receiving
env 2-3 plus adjuvant did develop anti-env 23 titers, but these
titers failed to reach the levels seen in guinea pigs (maximum
titers 1700 and 6300 for the two immunized animals). In addition,
these animals failed to develop virus neutralizing antibodies to
either the homologous (SF2) or heterologous (BRU or Zr6) HIV
strains.
3TABLE 3 ELISA and Neutralizing Antibody Titers of Sera From Beagle
Dogs Immunized With Env 2-3 In MTP-PE-LO Adjuvant.sup.a Env 2-3
Neutralization titers Animal Immunized Immunization ELISA HIV- HIV-
# with # titer HIV-SF2 BRU Zr6 1375 env 2-3 pre- .sup.b<<100
.sup.c<20 <20 <20 bleed MTP-PE-LO 2 1,300 <20 <20
<20 100 .mu.g MTP-PE 3 1,700 <20 <20 <20 4 900 <20
<20 <20 5 400 <20 <20 <20 6 300 <20 <20 <20
7 300 <20 <20 <20 1376 env 2-3 pre- <<100 <20
<20 <20 bleed MTP-PE-LO 2 3,500 <20 <20 <20 100
.mu.g MTP-PE 3 6,300 <20 <20 <20 4 5,100 <20 <20
<20 5 2,100 <20 <20 <20 6 2,200 <20 <20 <20 7
2,000 <20 <20 <20 1377 MTP-PE-LO pre- <<100 <20
<20 <20 bleed O-MTP-PE 2 <<100 <20 <20 <20
control 3 <<100 <20 <20 <20 5 <<100 <20
<20 <20 6 <<100 <20 <20 <20 7 <<100
<20 <20 <20 1378 MTP-PE-LO pre- <<100 <20 <20
<20 bleed O-MTP-PE 2 <<100 <20 <20 <20 control 3
<<100 <20 <20 <20 4 <<100 <20 <20 <20
5 <<100 <20 <20 <20 6 <<100 <20 <20
<20 7 <<100 <20 <20 <20 .sup.aDogs received 250
.mu.g of env 2-3 in Biocine adjuvant (100 .mu.g MTP-PE)
intramuscularly every 21 days. Blood samples were collected 10 days
following each injection. .sup.bELISA titers of <<100 are
listed when no signal was detected at a 1/100 serum dilution.
.sup.cNeutralization titers of <20 indicate that no
neutralization was observed at the most concentrated serum dilution
tested (1/20).
[0115] d. Pigs. Pigs were immunized with 1 mg env 2-3 with
MTP-PE-LO (100 .mu.g MTP-PE) every 21 days. Control animals
received the adjuvant alone. Ten days after each immunization the
animals were bled, and anti-env 2-3 antibody titers were determined
by ELISA. The results in Table 4 show that the two immunized
animals developed only low anti-env 2 titers (140 and 100,
respectively) and no detectable virus neutralizing titers against
either the homologous strain (SF2) or heterologous strains (BRU or
Zr6).
4TABLE 4 ELISA and neutralizing antibody titers of swine immunized
with env 2-3 MTP-PE-LO adjuvant..sup.a env Neutralizing Immuni- 2-3
titer on: Animal zation ELISA HIV- HIV- HIV- Number Antigen Number
titer SF2 BRU Zr6 1371 Env 2-3 pre- .sup.b<<50 .sup.d<20
<20 <20 bleed 2 .sup.c<50 <20 <20 <20 3 70 <20
<20 <20 4 70 <20 <20 <20 5 80 <20 <20 <20 6
70 <20 <20 <20 7 140 <20 <20 <20 1372 Env 2-3
pre- <<50 <20 <20 <20 bleed 2 100 <20 <20
<20 3 70 <20 <20 <20 4 70 <20 <20 <20 5 60
<20 <20 <20 6 90 <20 <20 <20 7 90 <20 <20
<20 1373 Adjuvant pre- <<50 <20 <20 <20 Control
bleed 2 <<50 <20 <20 <20 3 <<50 <20 <20
<20 4 <<50 <20 <20 <20 5 <<50 <20 <20
<20 6 <<50 <20 <20 <20 7 <<50 <20 <20
<20 1374 Adjuvant pre- <<50 <20 <20 <20 Control
bleed 2 <<50 <20 <20 <20 3 <<50 <20 <20
<20 4 <<50 <20 <20 <20 5 <<50 <20 <20
<20 6 <<50 <20 <20 <20 7 <<50 <20 <20
<20 .sup.aSwine received 1 mg of env 2-3 in Biocine adjuvant
(100 .mu.g MTP-PE) intramuscularly every 21 days. Sera were
collected 10 days following each immunization. .sup.bShowing no
signal at 1/50 serum dilution are listed as having titers of
<<50. .sup.cLow but detectable signal at 1/50 serum dilution.
.sup.dNo neutralization seen at a 1/20 serum dilution, the most
concentrated dilution tested.
[0116] e. Monkeys. Rhesus macaques were immunized every 30 days
with 250 .mu.g of env 2-3 with MTP-PE-LO (100 .mu.g MTP-PE).
Control animals received the adjuvant formulation alone. One week
after each immunization, the animals were bled and anti-env 2-3
antibody titers were determined by ELISA. Table 5 shows that,
similar to the dogs, all animals developed antibody titers to env
2-3, but these titers only ranged from 300-3100, far lower than
seen previously with guinea pigs.
5TABLE 5 Titers of env 2-3 specific antibodies in sera from Rhesus
macaques immunized with env 2-3 in MTP-PE-LO adjuvant..sup.a Animal
Immunization Antigen Number Prebleed 1 2 3 4 5 6 Env 2-3 1189
<<100 <<100 300 700 400 400 300 1190 <<100
<<100 1,200 800 800 900 500 1191 <<100 <<100 500
2,000 1,300 1,900 3,100 1192 <<100 <<100 1,100 900 400
400 500 (average) <<100 <<100 780 1,100 700 900 1,100
Adjuvant Control 1197 <<100 <<100 <<100
<<100 <<100 <<100 <<100 1198 <<100
<<100 <<100 <<100 <<100 <<100
<<100 1199 <<100 <<100 <<100 <<100
<<100 <<100 <<100 1978 <<100 <<100
<<100 <<100 <<100 <<100 <<100
(average) <<100 <<100 <<100 <<100
<<100 <<100 <<100 .sup.aAnimals received 250 mg
of antigen in Biocine adjuvant (100 mg MTP-PE) intramuscularly
every 30 days. Sera were collected one week following each
immunization.
[0117] 2. HSv gD
[0118] a. Goats. A series of adjuvant formulations were tested with
gD2 in goats. Animals were immunized with 100 .mu.g of gD2 with the
various adjuvants every 21 days. Ten days after the second and
third immunizations the animals were bled and anti-gD2 titers were
determined by ELISA. The following adjuvant formulations were used.
CFA (1.degree.) followed by IFA (2.degree. & 3.degree.), IFA
containing 100 .mu.g MTP-PE), 0.8 mg/ml aluminum hydroxide (alum),
MTP-PE-LO (100 .mu.g MTP-PE), MTP-PE-LO-KE (100 .mu.g MTP-PE), and
MTP-PE-LO-KE (12% squalene, 5.0 mg MTP-PE). The ELISA results are
shown in Table 6. One CFA/IFA animal, both MTP-PE/IFA animals, and
one MTP-PE-LO-KE (5 mg MTP-PE) animal developed high antibody
titers (2187-13 172). One CFA/IFA animal, both alum animals, and
one MTP-PE-LO-KE (5 mg MTP-PE) animals developed moderate antibody
titers (5691489). The MTP-PE-LO animals and the MTP-PE-LO-KE
animals developed low anti-gD2 titers (46-323). Thus, as with env 2
noted above, the MTP-PE-LO formulation fails to elicit high
antibody titers in goats. Modifying the emulsion by using the
Kirkland emulsifier (1-2 mm oil droplet sizes) did not improve the
adjuvant performance. Vast increases in MTP-PE (to 5.0 mg) dose
appeared to improve the adjuvant performance.
6TABLE 6 Adjuvant effectiveness with gD2 in the goats. ELISA Titer
After 2 Immuni- 3 Immuni- Group Animal Adjuvant zations zations 1
3606 CFA/IFA 2187 13172 3609 738 770 2 3610 Alum 1489 781 3611 921
522 3 3612 MTP-PE-LO 77 194 3613 (100 .mu.g MTP-PE) 145 323 4 3614
MTP-PE-LO-KE 123 227 3615 (100 .mu.g MTP-PE) 56 46 5 3624
MTP-PE-LO-KE 142 569 (12% squalene, 615 2291 5.0 mg MTP-PE
[0119] b. Baboons. Juvenile baboons were immunized with gD2
formulated with alum, MTP-PE-LO-KE, MTP/IFA and IFA alone. In
addition a dose ranging study for gD2 combined with alum and
MTP-PE-LO-KE was done. Baboons of 2-3 yr (3.4 to 12 kg) were
immunized intramuscularly in the thigh three times at three-week
intervals. Sera were collected 3 weeks after the first two
immunizations and 2 weeks after the final vaccine dose for
determination of gD-specific antibody by ELISA. Whole blood was
drawn at each of these time points for complete blood cell analyses
(CBC). Baboons immunized with 100 .mu.g of gD2 bound to alum
developed anti-gD2 mean antibody titers of 3349.+-.550. There was
no significant difference in titers for the three antigen doses
tested, 10, 25, 100
7TABLE 7 HSV vaccine trial in baboons: antibody titers.sup.a
Adjuvant Composition gD2 ELISA Titers.sup.b % of Group (mg) Dose
(mg) Dose (mg) 1.degree. Bleed 2.degree. Bleed 3.degree. Bleed
MTP-PE/IFA.sup.c 1 Alum 400 10 287 (+ 123) 1002 (+ 366) 1566 (+
350) 0.6 2 Alum 400 25 1075 (+ 785) 880 (+ 343) 1993 (+ 1156) 0.8 3
Alum 400 100 720 (+ 184) 1882 (+ 489) 3349 (+ 550) 1.3 4 MTP-PE/LO
50 25 140 (+ 63) 788 (+ 331) 1320 (+ 430) 0.5 5 MTP-PE/LO 250 10
217 (+ 103) 2490 (+ 995) 3244 (+ 1582) 1.3 6 MTP-PE/LO 250 100 57
(+ 34) 925 (+ 254) 2439 (+ 510) 1.0 7 MTP-PE/LO 1000 25 91 (+ 70)
1097 (+ 565) 3883 (+ 2401) 1.6 8 MTP-PE/IFA 250 25 24,101 (+ 5423)
62,775 (+ 28,634) 250,382 (+ 64,771) 100 9 IFA 25 2591 (+ 2280)
7631 (+ 6563) 66,132 (+ 75,095) 26.4 .sup.aAll animals immunized
with gD2 by IM delivery in the thigh; 4 animals/group .sup.b50%
endpoint antibody titer, geometric mean + SE .sup.cFraction of
animals with a positive gD2-specific lymphoproliferative response
defined as a stimulation index >3.0. No adverse reactions to the
vaccines were noted in any of the animals, and the CBC profiles
were normal.
EXAMPLE 3
MTP-PE-LO Formulation Effective In Stimulating Immunity in Large
Animals
[0120] As demonstrated in Example 2, MTP-PE-LO formulations that
were prepared with a syringe and needle (.about.10 micron droplet
size) and the Kirkland emulsifier (1-2 micron droplet size) failed
to give good immunostimulation to vaccine antigens in large animals
and humans (human data not shown). The microfluidizer model 110Y
was used to generate small-droplet-size, stable emulsions. This
machine is a high pressure (5000-30,000 PSI) submerged jet type
emulsifier. A series of emulsions were prepared varying in size and
stability based on the concentrations of squalene, Tween 80, and
MTP-PE and the physical parameters of temperature and operating
pressure. Examples of different emulsions made with the
microfluidizer are given in Table 8. By changing the physical
parameters and emulsion composition, oil droplet sizes from 1
micron to less than 0.2 microns can be achieved. As demonstrated in
Table 8, parameters that decrease emulsion droplet size are
increased detergent, increased MTP-PE to squalene ratio, increased
operating pressure, and increased operating temperature. These
small droplet size emulsions were then tested as adjuvants for
vaccine antigens in goats and baboons.
8TABLE 8 Composition and Physical Parameters of MTP-PE-Squalene
Emulsions made with the Microfluidizer Formu- MTP-PE Squalene Tween
80 Mannitol Aqueous Temp Pressure Size lation (mg/ml) % % % Phase
(.degree. C.) (KPSI) (m) A .01 2 .004 0 H.sub.2O 40.degree. 5 .23 B
0.2 2 .004 0 H.sub.2O 40 5 .17 C 1.0 2 0.16 5 H.sub.2O 0 10 .19 D
0.5 2 0 5 H.sub.2O 40 10 .16 E 0.5 2 0 0 H.sub.2O 40 10 .17 F 1.0 4
0 0 H.sub.2O 30 10 .19 G 1.0 4 0 0 H.sub.2O 20 10 .20 H 1.0 4 0 0
H.sub.2O 0 15 .20 I 1.0 4 0 0 H.sub.2O 0 10 .29 J 1.0 4 0 0
H.sub.2O 0 5 .39 K 1.0 4 .16 0 H.sub.2O 0 10 .22 L 1.0 4 .016 0
H.sub.2O 0 10 .27 M 1.0 6 0 0 H.sub.2O 0 10 .29
[0121] 1. HSV gD2 in Goats
[0122] The first microfluidizer used with the gD2 antigen was a 4%
squalene, 100 .mu.g/ml MTP-PE emulsion without Tween 80
(MTP-PE-LO-MF #13; number designations of MTP-PE-LO-MF formulations
are arbitrary and are intended only for use as reference numbers).
This material was made at low pressure in the microfluidizer and
had an oil droplet size of about 0.8 microns. Goats were immunized
intramuscularly with 100 .mu.g of gD2 in this formulation three
times at 21 day intervals. Goats immunized with 100 .mu.g gD2, in
CFA for primary and IFA for secondary and tertiary immunizations
served as controls. Ten days after the second and third
immunization the animals were bled and anti-gD2 antibody titers
were determined by ELISA. The results are shown in Table 9. Both
animals receiving the MTP-PE-LO-MF showed significant anti-gD2
titers. These titers 1661-2966 were intermediate compared to the
titers of the two CFA/IFA control goats (140-24,269). The
MTP-PE-LO-MF animals showed titers that were significantly higher
than goats that had received MTP-PE-LO formulations prepared in a
syringe and needle or in the Kirkland emulsifier (see Table 6). In
a second experiment in goats, 100 .mu.g gD2 was administered every
21 days with MTP-PE-LO-MF #16. This formulation consisted of 4%
squalene, 500 .mu.g/ml MTP-PE and 0 Tween 80. The oil droplet size
of this emulsion was 0.5-0.6 microns. As seen in Table 10, this
formulation appeared to give even higher antibody titers than the
previous formulation. Thus, reducing the oil droplet size and/or
increasing the MTP-PE improves the adjuvant performance of this
emulsion.
9TABLE 9 Test of MTP-PE-LO-MF #13 as an adjuvant for gD2 in Goats
ELISA titer after: Animal 2 Immuni- 3 Immuni- Group Number Adjuvant
Antigen zations zations 1 4519 CFA/IFA gD2 9868 24269 (100 .mu.g)
4520 " gD2 140 980 (100 .mu.g) 2 4598 MTP-PE- gD2 2966 2207
LO-MF.sup.a (100 .mu.g) 4599 " gD2 1661 N.T..sup.b (100 .mu.g)
.sup.a4% squalene, 100 .mu.g/ml MTP-PE, O Tween 80, H.sub.2O, about
0.8 micron oil droplet size. .sup.bN.T.--Not tested. Animal died of
causes unrelated to immunization.
[0123]
10TABLE 10 Test of MTP-PE-LO-MF #13 as an adjuvant for gD2 in Goats
ELISA titer after: Animal 2 Immuni- 3 Number Adjuvant Antigen
zations zations 5013 MTP-PE-LO-MF gD2 (100 .mu.g) 1299 386 #16 5014
MTP-PE-LO-MF gD2 (100 .mu.g) 6657 2806 #16 5015 MTP-PE-LO-MF gD2
(100 .mu.g) 8206 1943 #16 5016 MTP-PE-LO-MF gD2 (100 .mu.g) 7886
1514 #16 .sup.aMTP-PE-LO-MF #16 - 4% squalene, 500 .mu.g/ml MTP-PE,
0 Tween 80, H.sub.2O. Oil droplet size of 0.5-0.6 microns.
[0124] 2. HIV env 2-3 and gp120 in Goats.
[0125] Microfluidizer preparations were compared to CFA/IFA and the
MTP-PE-LO-KE as adjuvants using the HIV antigen env 2-3 and gp120.
Animals were immunized three times at 21-day intervals with 100
.mu.g of the gp120 antigen in CFA(1.degree.)/IFA(2.degree. &
3.degree.), MTP-PE-LO-MF #14 (4% squalene, 500 .mu.g/ml MTP-PE, O
Tween, phosphate buffered saline) MTP-PE-LO-KE (4% squalene, 100
.mu.g MTP-PE, 0.008% Tween 80, phosphate buffered saline emulsified
in the Kirkland emulsifier) and MTP-PE-LO-MF #15 (4% squalene, 100
.mu.g MTP-PE, 0.008% Tween 80, phosphate buffered saline). Animals
were also immunized with 100 .mu.g of the HIV antigen env 2-3 in
CFA/IFA and in MTP-PE-LO-MF #14. The animals were bled 10 days
after the second and third immunization and anti-env 2-3 antibody
titers were determined by ELISA. The results are shown in Table 11.
With env 2-3, the animals immunized with the MTP-PE-LO-MF #14
formulation showed equivalent titer to CFA/IFA animals after two
immunizations and higher titers than the CFA/IFA animals after
three immunizations. With gp120 the results were not quite as
clear. The MTP-PE-LO-MF #14 animals show much more variation than
the CFA/IFA animals. Thus the mean titers for the microfluidizer
group is lower than the CFA group, but individual animals receiving
MTP-PE-LO-MF #14 did show titers as high as any animals in the
CFA/IFA group. A direct comparison with gp120 of identical adjuvant
components (4% squalene, 100 .mu.g/ml MTP-PE, 0.008% Tween 80,
phosphate buffered saline) emulsified by two different methods
(Kirkland emulsifier vs. microfluidizer) illustrates the importance
of the small droplet size in the emulsion. The Kirkland emulsifier
group showed mean titer of 632 after these immunizations while the
microfluidizer group showed mean titer of 3277.
11TABLE 11 Test of MTP-PE-LO-MF as an adjuvant with HIV antigens
env 2 and gp120 ELISA Titer after: Animal 2 immuni- Genometric 3
immuni- Genometric Group Number Adjuvant Antigen zation Mean + SE
zation Mean + SE 1 5018 CFA/IFA gp120 (100 mg) 900 1861 + 539 7300
6630 + 996 5019 " gp120 (100 mg) 3700 5700 5020 " gp120 (100 mg)
2000 7100 5021 " gp120 (100 mg) 1800 3400 2 5022 CFA/IFA env 2 (100
mg) 2400 3000 5023 " env 2 (100 mg) 4600 2235 + 680 3400 5074 +
1378 5024 " env 2 (100 mg) 2400 8900 3 5026 MTP-PE-LO-MF gp120 (100
mg) 0 800 #14.sup.a 5027 " gp120 (100 mg) 300 101 + 1089 500 1324 +
994 5029 " gp120 (100 mg) 3407 5800 4 5030 MTP-PE-LO-MF env 2 (100
mg) 7900 19,500 #14.sup.a 5031 " env 2 (100 mg) 4600 2351 + 1688
6600 9896 + 2493 5032 " env 2 (100 mg) 300 6900 5033 " env 2 (100
mg) 2800 10,800 5 5034 MTP-PE-LO-KE.sup.b gp120 (100 mg) 0 600 5035
" gp120 (100 mg) 1400 721 + 416 600 632 + 32 5037 " gp120 (100 mg)
400 700 6 5038 MTP-PE-LO-MF gp120 (100 mg) 1000 5100 #15.sup.c 5040
" gp120 (100 mg) 0 10 + 333 2300 3277 + 767 5041 " gp120 (100 mg) 0
3000 .sup.aMTP-PE-LO-MF #14 - 4% squalene, 500 mg/ml MTP, 0 Tween,
phosphate buffered saline. .sup.bMTP-PE-LO-KE - 4% squalene, 100
mg/ml MTP-PE, 0.008% Tween 80 phosphate buffered saline emulsified
in the Kirkland emulsifier. .sup.cMTP-PE-LO-MF #15 - 4% squalene,
100 mg/ml MTP-PE, 0.008% Tween 80, phosphate buffered saline.
[0126] 3. HIV env 2-3 and gp120 in baboons.
[0127] MTP-PE-LO-MF #1 (2% squalene, 500 .mu.g/ml MTP-PE, O Tween
80, H20, oil droplet size .about.0.17 microns) was tested as an
adjuvant with the HIV antigens env 2-3 and gp120 in baboons. MTP-PE
in IFA and alum were used as controls. Animals were immunized at
one month intervals. Two weeks after the second immunization, the
animals were bled and anti-env 2-3 antibody virus neutralizing
titers were determined. The results are shown in Table 12. Antibody
titers against gp120 were higher with MTP-PE-LO-MF #1 than with
MTP-PE-IFA. Anti-env 2-3 titers were similar in the MTP-PE-IFA and
MTP-PE-LO-MF #1 groups. Anti-gp120 titers achieved with alum were
in the same range as with MTP-PE-LO-MF. #1 but anti env 2-3 titers
achieved with alum appear lower than with the MTP-PE adjuvants.
12TABLE 12 Test of MTP-PE-LO-MF #1 as an Adjuvant for HIV Protein
env2 and gp120 in Baboons Virus ELISA Animal Titer After 2
Neutralizing Group Number Adjuvant Antigen Immunizations Antibody
Titer 1 2947 MTP/IFA gp120 (55 mg) <100 <10 2948 (350
mgMTP-PE) gp120 (55 mg) <100 <10 2949 " gp120 (55 mg) 3000
<10 2 2550 MTP-PE/IFA env2 (25 mg) 400 <10 2451 (250
mgMTP-PE) env2 (25 mg) 34,500 30 2952 " env2 (25 mg) 142,300 200 3
2953 MTP-PE-LO-MF #1.sup.a gp120 (55 mg) 51,000 200 2957 " gp120
(55 mg) 43,000 35 2595 " gp120 (55 mg) 800 50 4 2956 MTP-PE-LO-MF
#1 env2 (25 mg) 600 <10 2957 " env2 (25 mg) 14,400 35 2958 "
env2 (25 mg) 87,400 >250 5 2964 Alum.sup.b gp120 (55 mg) 56,000
150 2965 " gp120 (55 mg) 100 <10 6 2966 Alum env2 (25 mg) 4900
80 " env2 (25 mg) 700 <10 .sup.aMTP-PE-LO-MF #1 - 2% squalene,
500 mg/ml MTP-PE, 0 Tween 80, H.sub.2O. Oil droplet size -0.17
microns. .sup.bAlum antigen bound to 0.8 mg/ml aluminum
hydroxide.
EXAMPLE 4
Additional Adjuvant/Antigen Formulations
[0128] In addition to the detailed examples set forth above, a
number of other antigens have been prepared in vaccine formulations
containing adjuvant compositions of the invention. These include
antigens from pathogens responsible for influenza and malaria, as
well as antigens associated with HIV and HSV other than those
described in previous examples. Antigens from cytomegalovirus (CMV)
and hepatitis C virus (HCV) are also described, as these antigens
can be used in the same adjuvant formulations described for the
other indicated antigens.
[0129] Antigens
[0130] Influenza antigens suitable for use in vaccine preparations
are commercially available. Antigens used in the following examples
are Fluogen.RTM., manufactured by Parke-Davis; Duphar, manufactured
by Duphar B. V.; and influenza vaccine batch A41, manufactured by
Instituto Vaccinogeno Pozzi.
[0131] Malaria antigens suitable for use in vaccine preparations
are described in U.S. patent application Ser. No. 336,288, filed
Apr. 11, 1989, and in U.S. Pat. No. 4,826,957, issued May 2,
1989.
[0132] Additional HIV antigens suitable for use in vaccine
preparations are described in U.S. application Ser. No. 490,858,
filed Mar. 9, 1990. Also see published European application number
181150 (May14, 1986) for additional HIV antigens.
[0133] Additional HSV antigens suitable for use in vaccine
preparations are described in PCT WO85/04587, published Oct. 24,
1985, and PCT WO88/02634, published Apr. 21, 1988. Mixtures of gB
and gD antigens, which are truncated surface antigens lacking the
anchor regions, are particularly preferred.
[0134] Cytomegalovirus antigens suitable for use in vaccine
preparations are described in U.S. Pat. No. 4,689,225, issued Aug.
25, 1987, and in PCT application PCT/US89/00323, published Aug. 10,
1989 under International Publication Number WO 89/07143. Also see
U.S. application Ser. NO. 367,363, filed Jun. 16, 1989.
[0135] Hepatitis C antigens suitable for use in vaccine
preparations are described in PCT/US88/04125, published European
application number 318216 (May 31, 1989), published Japanese
application number 1-500565 (filed Nov. 18, 1988), and Canadian
application 583,561. A different set of HCV antigens is described
in European patent application 90/302866.0, filed Mar. 16, 1990.
Also see U.S. application Ser. No. 456,637, filed Dec. 21, 1989,
and PCT/US90/01348.
[0136] It should be noted that published versions of the various
unpublished application numbers listed above can be obtained from
an indexing service such as World Patent Index, as well as a
listing of corresponding applications in other countries.
[0137] Adjuvant Formulations and Preparation Techniques
[0138] The following summaries describe adjuvant formulations and
how they are prepared as well as vaccine compositions prepared
using the adjuvants and various antigenic substances. In some cases
summaries of vaccination studies are provided, but without the
detail of the examples above, since the vaccination studies set
forth above already provide sufficient guidance for use of the
vaccine compositions.
[0139] Influenza
[0140] In a series of experiments, hamsters were immunized with a
commercial influenza vaccine from Instituto Vaccinogeno Pozzi. This
vaccine consists of purified HA from two A strains
(A/Leningrad/360/86 and A/Singapore/6/86) and one B strain (B/Ann
Arbor/1/86). The vaccine was tested alone, with an MTP-PE/LO
emulsion made with a Kirkland emulsifier (Fluoromed Pharmaceutical,
Inc., La Mesa, Calif.) and with an MTP-PE/MF emulsion made in a
microfluidizer (model 110Y, Microfluidics, Newton, Mass.). The
first two are comparative compositions, while the "MF" composition
is a composition of the invention. MTP-PE/MF stands for "MTP-PE
Microfluidizer" emulsion and contains 4% squalene and 1.0 mg/ml
MTP-PE emulsified with the Microfluidizer. The MTP-PE Kirkland
emulsion contained 4% squalene, 0.5 mg/ml MTP-PE, and 0.008% Tween
80 emulsified with the Kirkland emulsifier. Animals received three
immunizations containing 8.3 .mu.g of each HA antigen. MTP-PE was
used at 50 .mu.g per dose in both formulations. ELISA titers were
determined against the immunizing antigens after each immunization
and HAI titers were determined after the second immunization. ELISA
titers were increased substantially by both of the adjuvant
formulations tested.
[0141] In other experiments, hamsters were immunized with either
the commercially available Parke-Davis Fluogen vaccine (HA
A/Shanghai/11/87, A/Taiwan/1/86 and B/Yamagata/16/88) or the
commercially available Duphar influenza vaccine (HA A/Sechuan/2/87,
A/Singapore/6/86 and B/Beijing/1/87) alone or with the MF69
adjuvant formulation (MF69 is 5% squalene, 0.2% Tween 80, 0.8%,
Span 85, and 400 .mu.g/ml MTP-PE, emulsified in the
Microfluidizer). Equal volumes of vaccine were mixed with the MF69
adjuvant. Animals received three immunizations of 11.25 .mu.g of
the Parke-Davis vaccine or 7.5 .mu.g of the Duphar vaccine at three
week intervals. Animals receiving the MF69 adjuvant received 50
.mu.g doses of MTP-PE. The animals receiving Duphar plus MF69
showed significantly higher anti-HA titers than Duphar alone after
one and two immunizations (mean titers 80-fold higher than vaccine
alone after one immunization and 170-fold higher than after two
immunizations). The MF69 adjuvant showed good stimulation of
antibody response to the Parke-Davis vaccine, generating mean
titers of 2951, 14,927 and 12,878 after one, two or three
immunizations. This represents titers 82, 29 and 10-fold higher
than vaccine alone after one, two or three immunizations,
respectively. For both vaccines, peak antibody titers were seen
after two immunizations with MF 69.
[0142] In further experiments, the immunogenicity of two commercial
influenza vaccines, Parke-Davis Fluogen and Duphar subunit
influenza, were compared with no adjuvant and with several MTP-PE
containing adjuvant formulations in goats. The animals were
immunized intramuscularly with 0.5 ml of each vaccine mixed with
either 0.5 ml of PBS or 0.5 ml of MTP-PE adjuvant formulations.
Three adjuvant formulations were compared: 200 .mu.g of MTP-PE
dissolved in PBS, and 200 .mu.g of MTP-PE in two different
microfluidized emulsions, referred to as Gaulin 1/4 and MF40/4
emulsions. Gaulin 1/4 consists of 1.6% squalene and 400 .mu.g/ml
MTP-PE emulsified in the Gaulin homogenizer (APV Gaulin, Everett,
Mass.). MTP-PE/MF-40/4 consists of 1.6% squalene, 400 .mu.g/ml
MTP-PE, 0.154% Tween 85, and 0.166% Span 85 emulsified in the
Microfluidizer (Model 110Y, Microfluidics, Newton, Mass.). Animals
received 0.5 ml of vaccine mixed with either 0.5 ml of PBS or 0.5
ml of the indicated adjuvant formulation to generate a 1.0 ml
injection volume. As with the hamsters, the goats receiving the
influenza vaccines combined with the adjuvant emulsions showed much
higher antibody titers than goats receiving vaccine alone. This is
especially pronounced early in the immunization schedule. After one
immunization the Gaulin 1/4 emulsion generated anti-HA titers
greater than 30-fold higher than the Parke-Davis vaccine alone. The
MTP-PE/MF-40 emulsion generated anti-HA titers that were greater
than 130-fold higher than Parke-Davis vaccine alone and 60-fold
higher than Duphar vaccine alone. MTP-PE in PBS showed no
stimulation of antibody titer after one immunization. After two
immunizations, similar increases in antibody titers with the
emulsions were seen. The early stimulation of anti-HA titers seen
with the adjuvant emulsions is especially significant since
influenza vaccines are generally given as one dose vaccines to
adults and two dose vaccines to infants. Thus, as in hamsters, the
MTP-PE-emulsions show large increases in the immune response to
influenza vaccines.
[0143] In another experiment, the Duphar vaccine was compared alone
and with adjuvant formulation MF69. The Parke-Davis vaccine was
compared alone and with MF101, MF69, MF-68+MTP-PE, and the Ribi
Adjuvant system made in the Gaulen homogenizer (micro-fluidizer).
MF-101 consists of 1.6% squalene and 400 ug/ml MTP-PE, emulsified
in the Microfluidizer. MF-68 consists of 5% squalene, 0.8% Span 85,
and 0.2% Tween 80, emulsified in the Microfluidizer. MF-68+MTP
consists of MF-68 to which was added 400 ug/ml MTP-PE per ml post
emulsification. Ribi-MF consists of 2% squalene, 0.4% Tween 20, 250
ug/ml monophosphoryl lipid A, 250 ug/ml Trehalose dimycolate, and
250 ug/ml cell wall skeleton (Ribi Immunochem, Hamilton Mont.),
emulsified in the Gaulin homogenizer. All adjuvants were used at a
dose of 0.5 ml per injection with equal volumes of vaccine
(antigen). MF69 significantly increased the ELISA titer to the
Duphar vaccine. All of the adjuvants tested also significantly
increased the immunogenicity of the Parke-Davis vaccine as measured
by both ELISA titer and hemagglutination titer.
[0144] In a further experiment, MF69 and MF59 formulations
(differing only in the Tween 80:Span 85 ratio; see descriptions
above) were compared as adjuvants with the Parke-Davis influenza
vaccine in goats. The animals were immunized once with one-half of
the human vaccine dose (7.5 .mu.g each of the three HA components)
combined with the adjuvant formulations. MTP-PE was used at a dose
of 100 .mu.g in the formulations. As expected, the two formulations
give very similar titers with the MF69 showing a mean titer of 926
and the MF59 showing a mean titer of 821.
[0145] Malaria
[0146] A vaccination study has been initiated using MF59 (described
above) as adjuvant. A mixture of commercially available antigens
from the sporozoite, merozoite, and erythrocytic stages of the
disease was used: Falc. 2.3 circumsporozoite antigen, HP 195
merozoite antigen, and SERA 1 red blood stage antigen. Vaccine
compositions are prepared as described above, namely mixing equal
volumes of the previously prepared MF59 adjuvant and the antigen
composition.
[0147] HIV
[0148] An immunization experiment was carried out to compare
production of neutralizing antibodies by a number of different
gp120 antigens. Details of preparation of the antigens are set
forth in U.S. application Ser. No. 490,858, filed Mar. 9, 1990. One
antigen was a gp120 analog (env 2-3) prepared in yeast, which is
denatured and non-glycosylated. Another antigen was glycosylated
gp120 retaining its natural configuration. Both gp120 materials
were derived from the same gene source, HIV-1 SF-2 isolate.
Antibody production was measured in baboons. Initial studies using
oil-containing adjuvants with particle sizes larger than 1 micron
produced titers less than those produced using conventional alum
adjuvants. However, later studies with submicron particle adjuvants
produced antibody titers at least 10-fold higher than with alum.
The initial submicron composition contained 2% squalene and 0.500
mg/ml MTP-PE in water and had oil droplets averaging about 0.17
microns in diameter. Vaccine compositions using MF59 (described
above) or MF58 (MF59 but with MTP-PE added exogenously) as an
adjuvant in baboons have proven even more effective in stimulating
antibody production than the initial submicron composition used.
MF59 was used at a 1:2 dilution at a rate of 0.100 mg MTP-PE.
[0149] Herpes Simplex Virus
[0150] In addition to the gD2 experiments described above,
additional experiments have been carried out using MF59 and various
amounts of MTP-PE and antigens. Satisfactory antibody tiers have
been obtained using from 0.003 to 0.250 mg gD2 with MF59 adjuvant
and 0.050 mg MTP-PE in guinea pigs (intramuscular administration)
and using from 0.010 to 0.100 mg gD2 with MF59 and 0.100 mg
MTP-PE.
[0151] Cytomegalovirus
[0152] Vaccine formulations can be prepared by mixing from 0.001 to
0.250 mg of CMV antigens in 0.5 ml physiological saline with 0.5 ml
MF59 adjuvant containing 0.050 mg MTP-PE. MF69, MF101, and other
submicron particle adjuvants can be used in the same manner.
[0153] Hepatitis C Virus
[0154] Vaccine formulations can be prepared by mixing from 0.001 to
0.250 mg of HCV antigens in 0.5 ml physiological saline with 0.5 ml
MF59 adjuvant containing 0.050 mg MTP-PE. MF69, MF101, and other
submicron particle adjuvants can be used in the same manner.
[0155] All publications and patent applications cited herein are
incorporated by reference in the location where cited to the same
extent as if each individual publication or patent application had
been individually indicated to be incorporated by reference.
[0156] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *