Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 334 1 65 TLC-172
_
AFFINITY ASSOCIATED VACCINE
FIELD OF THE INVENTION
This invention concerns a vaccine against an infective agent, the
vaccine comprising a liposome having an exterior and an interior and
having externally disposed affinity (noncovalently) associated
antigen material of at least one, preferably nonpartitioning,
antigen representative of said infective agent. Also disclosed is a
method of preparation and use of this vaccine.
BACKGROUND OF THE INVENTION
In the vaccine art antigens are introduced into an organism in a
manner so as to stimulate an immune response in the host organism.
The induction of an immune response depends on many factors among
which are believed to be the chemical composition and configuration
of the antigen, the potential of the immune system of the challenged
organism, and the manner and period of administration of the
antigen. An immune response has many aspects some of which are
exhibited by the cells of the immune system, (e.g., B-lymphocytes,
T-lymphocytes, macrophages, and plasma cells). Immune system cells
may participate in the immune response through interaction with
antigen, interaction with other cells of the immune system, the
release of cytokines and reactivity to those cytokines. Immune
response is conveniently (but arbitrarily) divided into two main
categories -- humoral and cell-mediated. The humoral component of
the immune response includes production of immunoglobulins specific
for the antigen. The cell-mediated component includes the
generation of delayed-type hypersensitivity and cytotoxic effector
cells against the antigen.
l ~
,..
1 334 1 65
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In some instances immune response is the result of an initial or
priming dose of an antigen that is followed by one or more booster
exposures to the antigen. Priming with relatively strong immuno-
gens and liposomes is discussed in "Liposomal Enhancement of the
Immunogenicity of Adenovirus Type 5 Hexon and Fiber Vaccines,
Kramp, W.J. et al., Infection and Immunitv, 25:771-773 (1979) and
"Liposomes as Adjuvants with Immunopurified Tetanus Toxoid: the
Immune Response", Davis, D. et al., Immunoloav Letters, 14:341-8
(1986/1987).
Ideally, an antigen will exhibit two properties, the capacity to
stimulate the formation of the corresponding antibodies and the
propensity to react specifically with these antibodies. Antigens
bear one or more epitopes which are the smallest part of an antigen
recognizable by the combining site of an antibody.
In particular instances antigens or fractions of antigens or with
particular presenting conditions the immune response precipitated by
the desired antigen is inadequate or nonexistent and insufficient
immunity is produced. This is particularly the case with peptide or
other small molecules used as antigens.
In such cases the vaccine art recognizes the use of substances called
adjuvants to potentiate an immune response when used in conjunction
with an antigen or immunogen. Adjuvants are further used to elicit
immune response sooner, or a greater response, or with less antigen
or immunogen or to increase production of certain antibody subclasses
that afford immunological protection, or to enhance components of the
immune response (e.g., humoral, cellular). Liposomal vaccines and
adjuvancy are further discussed in Canadian Patent Application Ser.
No. 609,464 to Popescu et al., filed on August 25, 1989.
Known adjuvants are Freund's Adjuvants (and other oil emulsions),
Bordetella pertussis, Lipid A (the glycophospholipid moiety of
lipopolysaccharide found in Gram-negative bacteria), aluminum salts
(and other metal salts), Mycobacterial products (including muramyl
_ _ 3 _ l 334 1 65
dipeptides), and liposomes. As used herein the term "adjuvant" will
be understood to mean a substance or material administered together
or in conjunction with an antigen which increases the immune
response to that antigen. Adjuvants may be in a number of forms
including emulsion (e.g., Freund's adjuvant) gels (aluminum
hydroxide gel) and particles (liposomes) or as a solid material.
It is believed that adjuvant activity can be affected by a number of
factors. Among such factors are (a) carrier effect, (b) depot
formation, (c) altered lymphocyte recirculation, (d) stimulation of
T-lymphocytes, (e) direct stimulation of B-lymphocytes and (f)
stimulation of macrophages.
With many adjuvants adverse reactions are seen. In some instances
adverse reactions include granuloma formation at the site of
injection, severe inflammation at the site of injection,
pyrogenicity, adjuvant induced arthritis or other autoimmune
response, or oncogenic response. Such reactions have hampered the
use of adjuvants such as Freund's adjuvant.
In particular embodiments liposome adjuvants are utilized. U.S.
Patent No. 4,053,585 issued October 17, 1977 to Allison et al.
states that liposomes of a particular charge are adjuvants.
Other substances such as immunomodulators (e.g., cytokines such as
the interleukins) may be combined in adjuvants as well.
Humoral immune response may be measured by many well known methods.
Single Radial Immunodiffusion Assay (SRID), Enzyme Immunoassay (EIA)
and Hemagglutination Inhibition Assay (HAI) are but a few of the
commonly used assays.
EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to
determine total antibodies in a sample. The antigen is adsorbed to
the surface of a microtiter plate. The test serum is exposed to the
plate followed by an enzyme linked immunoglobulin, such as IgG. The
enzyme activity adherent to the plate is quantified by any
convenient means such as spectrophotometry and is proportional to
the concentration of antibody directed against the antigen present
in the test sample.
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Tests to measure cellular immune response include determination of
delayed-type hypersensitivity or measuring the proliferative
response of lymphocytes to target antigen.
Liposomes are completely closed lipid bilayer membranes containing
an entrapped aqueous volume. Liposomes may be unilamellar vesicles
(possessing a single bilayer membrane ) or multilamellar vesicles
(onion-like structures characterized by multiple membrane bilayers,
each separated from the next by an aqueous layer). The bilayer is
composed of two lipid monolayers having a hydrophobic "tail" region
and a hydrophilic "head" region. The structure of the membrane
bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid
monolayers orient toward the center of the bilayer while the
hydrophilic "head" orient towards the aqueous phase.
The original liposome preparation of Bangham, et al. ~. Mol. Biol.,
1965, 13:238-252) involves suspending phospholipids in an organic
solvent which is then evaporated to dryness leaving a phospholipid
film on the reaction vessel. Next, an appropriate amount of aqueous
phase is added, the mixture is allowed to "swell," and the resulting
liposomes which consist of multilamellar vesicles (MLVs) are
dispersed by mechanical means. This technique provides the basis
for the development of the small sonicated unilamellar vesicles
described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968,
135:624-638), and large unilamellar vesicles.
Unilamellar vesicles may be produced using an extrusion apparatus by
a method described in Cullis et al., PCT Application No. WO
86/00238, published January 16, 1986, entitled "Extrusion Technique
for Producing Unilamellar Vesicles". Vesicles made by this
technique, called L W ETs, are extruded under pressure once or a
number of times through a membrane filter. L W ETs will be
understood to be included in the term "unilamellar vesicle".
- 5 - l 33 4 1 65
Another class of liposomes are those characterized as having
substantially equal lamellar solute distribution. This class of
llposomes is denominated as stable plurilamellar vesicles (SPLV) as
defined in U.S. Patent No. 4,522,803 to Lenk, et al., monophasic
vesicles (MPVs) as described in U.S. Patent No. 4,588,578 to
Fountain, et al. and frozen and thawed multilamellar vesicles
(FATMLV) wherein the vesicles are exposed to at least one freeze and
thaw cycle, as described in Bally et al., PCT Publication No.
87/00043, January 15, 1987, entitled /'Multilamellar Liposomes Having
0 Improved Trapping Efficiencies/', corresponding to U.S. Patent No.
4,975,282. U.S. Patent No. 4,721,612 to Janoff et al. describes
steroidal liposomes for a variety of uses.
Lipids of net negative charge are well known in the art and include
for example, phosphatidylserine, phosphatidic acid, and phosphatidyl-
glycerol. Lipids of net positive charge are well known in the art
and include for example, aminodiglycerides, glyceridecholine, stearyl-
amine, trimethylstearylamine, and dioctadecyl trimethylammonio pro-
pane. In general any bilayer forming amphiphile which has a charged
hydrophilic moiety may be used.
In addition, lipid charge may be manipulated by a number of methods
well known in the art, such as by linking the lipid to a moiety of
appropriate net charge. For example, the neutral lipid cholesterol
may be linked to succinic acid (negative charge) to yield choles-
terol hemisuccinate (CHS) of negative charge. The tris(hydroxymeth-
yl)aminomethane form of CHS is designated CHStris and its application
to liposomes is more fully discussed in U.S. Patent No. 4,721,612.
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Summary of the Invention
This invention includes a composition comprising a liposome in
noncovalent association with an externally disposed antigen and in
one embodiment further comprising adjuvant or further comprising
aluminum hydroxide or Lipid A. Preferred antigens are
nonpartitioning. In some embodiments the antigen is hydrophilic or
lipophilic. Variously the affinity association is noncovalent
association and the like such as electrostatic, hydrophilic,
hydrogen bonding, or other bonding related to van der Waals forces
such as configurational stickiness. Liposomes of this invention may
be unilamellar or multilamellar.
In particular applications liposomes may comprise cholesterol
hemisuccinate, phosphatidylserine, phosphatidic acid, or
phosphatidylglycerol as well as aminodiglyceride, glyceridecholine,
stearylamine, trimethylstearylamine, dioctadecyl trimethylammonio
derivatives (e.g., 1,2 bis(oleoyloxy)-3-dioctadecyl trimethylammonio
propane -- "DOTAP") or any bilayer forming amphiphile having a
charged hydrophilic moiety.
Useful antigens include HIV or portion thereof with particular
reference to PBl. Antigen includes peptide, glycopeptide or
glycoprotein. In particular applications the antigen is influenza
or fragments thereof, herpes or fragments thereof, haemophilus B or
fragments thereof or malaria or fragments thereof. Antigens also
include isolated or bioengineered fragments of viruses, bacteria,
cancer cells, humoral cells and body fluid components.
In another embodiment the invention includes a method of producing a
vaccine composition comprising a liposome in affinity (noncovalent)
association with an externally disposed and preferably
nonpartitioning antigen. This method comprises contacting, in an
aqueous solution, an antigen and a liposome comprising said bilayer
forming material of reciprocal affinity to said antigen, such that
the antigen and the liposome form an affinity association.
Optionally this process may further include removing non-affinity
associated antigen.
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In the practice of this method the affinity between antigen and
liposome is electrostatic or hydrogen bonding or is configurational
stickiness. Additionally by this method the liposome is of net
negative charge and the antigen of net positive charge or the
liposome is of net positive charge and the antigen of net negative
charge. In the practice of this method in one embodiment the
liposome comprises CHS. In another embodiment the antigen comprises
PBl. In particular applications the liposomes are subjected to
shearing force.
This invention yet further includes a method of inducing an immune
response in an animal, including a human, comprising administering
to said animal a therapeutically effective amount of a composition
comprising a liposome in noncovalent affinity association with an
externally disposed preferably nonpartitioning antigen. The method
can further utilize adjuvant such as aluminum hydroxide or Lipid A.
Variously by this method antigen is hydrophilic or lipophilic.
Further by this method the affinity is electrostatic, hydrogen
bonding or configurational stickiness.
In the practice of this method of treatment in various
embodiments the liposome comprises cholesterol hemisuccinate,
phosphatidylserine, phosphatidic acid, or phosphatidylglycerol as
well as aminodiglyceride, glyceridecholine, stearylamine,
trimethylstearylamine, dioctadecyl trimethylammonio derivatives or
any bilayer forming amphiphile having a charged hydrophilic moiety.
Antigens can comprise HIV or portions thereof, particularly PBl.
Variously antigens are noted to be protein, peptide, glycopeptide,
or glycoprotein, polypeptide, or poly(amino acid) and will be
termed, collectively, "peptide". Particularly noted as antigens are
influenza or fragments thereof, herpes or fragments thereof,
haemophilus B or fragments thereof, or malaria or fragments
thereof, as well as isolated or bioengineered fragments of viruses,
bacteria, cancer cells, humoral cells and body fluid components.
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Detailed Description of the Invention
For clarity, in the discussion of this invention the following
5 definitions will be used:
"Adjuvant" shall mean a substance or material to potentiate an
immune response when used in conjunction with an antigen or
immunogen. Adjuvants are further used to elicit immune response
sooner, or a greater response, or with less antigen.
"Antigen" shall mean a substance or material that is recognized
specifically by an antibody and/or combines with an antibody.
Particular note is made of both natural and bioengineered antigens
15 such as peptides, glycopeptides and glycoproteins. Specific
antigens include antivirals such as herpes, hepatitis, rabies,
parainfluenza, measles, mumps, respiratory syncytial virus;
antibacterials such as pneumonia, haemophilus B, staphylococcus,
meningococcus, Neisseria gonorrhea; and protozoa such as malaria or
20 fragments thereof.
"Epitope" shall mean the smallest part of an antigen
recognizable by the combining site of an immunoglobulin.
"Externally disposed" shall, in referring to antigen or
immunogen, mean, positioned by an "affinity association" so as to
bear an epitope external to the outermost lamella of an associated
liposome. Included are epitopes ionically associated with the
liposome, nonpartitioned into the outermost lamellae, and with
epitope exposed.
"Affinity association" shall mean noncovalent intermolecular
associations such as electrostatic association, hydrogen bonding,
and configurational stickiness.
"Configurational stickiness" shall be understood to mean
physical parameters that facilitate immobilization of antigen or
immunogen externally on a liposome such as van der Waals forces.
,,
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"Nonpartitioning", referring to antigen or immunogen, refers to
hydrophilic immunogens and those lipophilic immunogens the epitopes
of which are not substantially incorporated into the outermost
lamella of a liposome. Those lipophilic immunogens that can be
separated from liposomes by physical manipulation such as by
dialysis, charge manipulation or other eauilibrium based separations
are deemed to be not substantially incorporated into the lamellae
and nonpartitioning.
"Immune response" shall mean a specific response of the immune
system of an animal to antigen or immunogen. Immune response may
include the production of antibodies.
"Immunity" shall mean a state of resistance of a subject animal
to an infecting organism or substance. It will be understood that
infecting organism or substance is defined broadly and includes
parasites, toxic substances, cancers and cells as well as bacteria
and viruses. A Therapeutically Effective Immunization Course will
produce immune response to protect the organism against challenging
antigen.
"Immunization conditions" shall mean factors which affect an
immune response including amount and kind of antigen or adjuvant
delivered to a subject animal, method of delivery, number of
inoculations, interval of inoculation, the type of subject animal
and its presenting condition.
"Immunization dose" shall mean the amount of antigen or
immunogen needed to precipitate an immune response. This amount
will vary with the presence and effectiveness of various adjuvants.
This amount will vary with the animal and immunogen or antigen or
adjuvant but will generally be between about O.l ,ug/ml or less to
about 500 ,ug per inoculation. The immunization dose is easily
determined by methods well known to those skilled in the art, such
as by conducting statistically valid host animal immunization and
challenge studies. See, for example, Manual of Clinical ImmunoloaY,
1 334 1 65
10 --
H.R. Rose and H. Friedman, American Society for Microbiology,
Washington, D.C. (1980). In some instances several immunization
doses including booster doses will be administered to provide
immunity.
s
"Immunogen" shall mean a substance or material (including
antigens) that is able to induce an immune response alone or in
conjunction with an adjuvant. This will generally be a protein,
peptide, polysaccharide, nucleoprotein, lipoprotein, synthetic
polypeptide, or hapten linked to a protein, peptide, polysaccharide,
nucleoprotein, lipoprotein or synthetic polypeptide or other
bacterial, viral or protozoal fractions. It will be understood that
"immunogen" includes substances which do not generate an immune
response (or generate only a therapeutically ineffective immune
response) unless associated with an adjuvant (e.g., small peptides)
which will be referred to as "adjuvant-obligatory" immunogens.
"Infective agent" shall mean an disease causing agent including
fungi, bacteria, viruses and parasites.
"Mimetic", in relation to antigens, immunogens and epitopes,
shall refer to a moiety (either natural or synthetic) that
duplicates by structure, accessibility or reactivity the response of
B-cell immuno-receptors to the subject antigen in native state
configuration or epitope in native state configuration.
"Native state configuration" shall mean that organization of a
moiety as it is when present in situ in its usual condition, to be
distinguished from non-native state configuration (denatured)
wherein the moiety is altered as to immuno-reactivity from that of
the in situ organization.
"Vaccine" shall mean a pharmaceutical formulation which
induces immune response or immunity in a subject animal.
Antigens or immunogens may have a net positive or negative charge,
or be neutral. The net charges are easily determined by a number of
techniques well known in the art. For peptides the net charge may
be
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assessed through determination of the isoelectric point. Peptides
of isoelectric points of about 5 or less have net negative charge
and the peptides are soluble in basic solution. Isoelectric points
of about 8 or more indicate a net positive charge and the peptides
are soluble in acid solution. Isoelectric points from 5 to 8
indicate generally neutral peptides (from slightly acidic to
slightly basic). The determination of isoelectric points is well
known in the art and is discussed in "Relationship Between in vivo
Degradative Rates and Isoelectric Points of Proteins", Dice, et al.,
Proc. Natl. Acad. Sci. USA, 72:3895-97 (1975).
Net charges on peptides can be manipulated by a number of methods
well known in the art including adding charge bearing amino acids or
amino acid segments. By way of example ly~ine and arginine add
positive charge while aspartic and glutamic acid add negative
charge. It is important to note in any manipulation of antigens or
immunogens including addition of amino acids or exposure to acidic
or basic solutions or temperature variation or light that conditions
must not be such as to compromise immunogenicity or native state
configuration.
A particularly useful immunogen is the PBl fraction of the HIV 1
virus (Repligen Corporation, Cambridge, MA). This fraction is
characterized in "HTLV-III/LAV-Neutralizing Antibodies of an E.
coli-Produced Fragment of the Virus Envelope", Putney, et al.,
Science, 234:1392-5 (1986). PBl peptide is soluble in aqueous
solution containing solubilizing substances such as urea and
detergents and was supplied in such solution. Such solubilization
substances were incompatible with pharmaceutical preparations and a
dialysis process was utilized to remove them.
Antigens or immunogens which partition into the liposome lamellae
such as melanoma antigen in CHS liposomes may not yield a sufficient
immunogenic response without repeated inoculation and additional
adjuvant. Without being bound by any particular theory it is
L
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believed that this partitioning results in the limitation of
exposure of epitopes externally to the adjuvant liposomes.
Modification (e.g., conjugation with a nonpartitioning moiety) of
such an antigen or immunogen while preserving native state
configuration acts so as to prevent partitioning. In addition it
can be advantageous to utilize both affinity associated antigen and
entrapped antigen in a particular composition, vaccine or dosage
form.
A preferred vaccine of this invention is a liposome organized so as
to have an immunogen in electrostatic association with the exterior
of said liposome. The preferred liposome may be multilamellar
(e.g., multilamellar vesicles or stable plurilamellar vesicles). In
a particular embodiment the vaccine contains externally disposed
antigen or immunogen in such electrostatic association.
In some embodiments the most successful immunogens will be those
purified from (or duplicative or mimetic of) the infective agent
while maintaining immunogen native state configuration. It is
understood that some vaccines are prepared without resort to living
infective organisms due to the potential for contamination of the
final vaccine. It is thus useful to produce immunogens
synthetically (including bioengineering) such as through techniques
of recombinant DNA technology, recombinant RNA technology or other
synthesis in host cells, but it is preferable that even
synthetically produced immunogens be of native state configuration.
Selection of lipid and antigen of reciprocal affinity is simply
accomplished by those skilled in the art. Depending upon the type
of noncovalent affinity association applicable various association
techniques are used. For hydrogen bonding the elevation of
temperature of the aqueous solution suspending antigen and liposomes
above the transition temperature of the affinity bonded entities is
effective. For van der Waals, or electrostatic or configurational
stickiness admixing of the antigen and liposomes in aqueous solution
is sufficient, but as to electrostatic affinity the liposome and
antigen are of opposite charge.
~.
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Degree of affinity association of nonpartitioning antigen or
immunogen is simply determined by methods well known in the art. By
one method the relative attachment and nonattachment is determined
by centrifuging the liposomes and antigen or immunogen admixture and
then determining the amount of antigen or immunogen in the
supernatant. The amount not in the supernatant being a measure of
the amount affinity attached.
Also useful is priming in generation of the immune response. A
vaccine may generate a weak immune response that is not greatly
potentiated on secondary challenge. However, after a first
administration of the adjuvant-associated immunogen, a substantial
immune response is generated upon application of a second
inoculation of the adjuvant free immunogen in solution.
The efficacy and utility of this invention is disclosed in Tables 1
and 2.
Table 1 presents the data of an experiment in which CH~riS
formulation was compared with other liposome formulations.
Liposomes made of CHStriS (Table 1, group F) were prepared by
multilamellar vesicles (MLV) procedure. This formulation involves
the coating of the surface of negatively charged liposomes with the
positively charged antigen.
Liposomes made of dimyristoylphosphatidylcholine/cholesterol
(DMPC/CHOL) were prepared using either stable plurilamellar vesicle
(SPLV) procedure (D-W, D) or monophasic vesicle (MPV) procedure (D-
E, D-T).
A. SPLV Liposome D-W
DMPC (80 mg) and cholesterol (20 mg) in 1 ml chloroform were
rotoevaporated under vacuum to a dry film and further
solubilized in 5 ml ether + 0.5 ml ethanol at 40C for a few
seconds until the solution was clear. The PBl antigen (100 ,ug)
in 0.5 ml 1% acetic acid was added to this solution and the
14 l 3 3 4 t 65
resulting mixture was sonicated under a stream of N2 at 40C
for about 1 minute until dryness under N2 stream (2-3
minutes). The resulting dry material was resuspended in 10 ml
phosphate buffered saline minus Ca++ and Mg++ (KCl 2g/L,
KH2PO4 2 g/L, NaCl 80 g/L, NaPO4'7H2O 21.6 g/L) ("PBS
minus") to a milky suspension which was then centrifuged at
10,000 rpm for 10 minutes at 4C. The pellet of liposomes was
resuspended in 2 ml PBS minus to a final concentration of 50 mg
lipid/ml.
B. SPLV, Liposome D
This liposome was prepared as described above for the D-W
liposome except that the centrifugation step was omitted.
Subsequently the material dried by N2 stream was resuspended
in 2 ml PBS minus to a final concentration of 50 mg lipid and
50 ,ug antigen/ml.
C. MPV, Liposome D-E
Cholesterol (20 mg) was dissolved in 1 ml ethanol containing
80 mg solubilized DMPC by brief heating to 40C (10-20 sec.) and
stirring. The solution of antigen (1 ml) was prepared
separately by mixing 100 ,ug PBl in 0.13 ml 1% acetic acid and
0.87 ml ethanol and immediately mixed with the lipid solution.
The mixture was rotoevaporated under vacuum at 40C and the
resulting material was resuspended in 2 ml PBS minus by
sonication for 10 seconds at 40C to a final suspension of 50 mg
lipid and 50 ,ug antigen/ml.
D. MPV, Liposome D-T
This liposome was prepared as described above for the D-E
liposome except that the ethanol was replaced by Tert-butyl-
alcohol (TBA).
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E. Liposome, H-D mixture
This preparation was done by mixing 0.5 ml of SPLV, liposome D
made of DMPC/CHOL and 0.5 ml of empty MLV liposome H made of
CHStris as described below. The mixture contained 75 mg lipids
(50 mg CHStris, 25 mg DMPC/CHOL) and 25 ,ug PBl antigen/ml.
F. MLV, Liposome H
Empty CHStris liposomes were prepared by hydration of 100 mg
CHStris with 1 ml of PBS minus for 2 hr. at room temperature and
intermittent vigorous vortex-mixing. Separately, a fresh
dialysate of PBl antigen in 1~ acetic acid was diluted in PBS
minus at 100 ,ug antigen/ml and 0.5 ml of this solution was
immediately mixed with 0.5 ml of empty CHStris liposomes to
allow affinity association between vesicles and antigen. The
mixture contained 50 mg lipid and 50 ~g antigen/ml.
G. Control of Complete Freund Adiuvant (CFA) and dialyzed (d)
antiaen
Equal 1 ml volumes of complete Freund's adjuvant (CFA) oil and
PBl dialyzed against 1% acetic acid and further diluted in 1
acetic acid to 100 ,ug antigen/ml, were mixed thoroughly and
emulsified by repeated passage through a 16G needle connecting
two 5 ml syringes. This emulsion contained 50 ,ug antigen/ml.
H. Control of CFA and non-dialyzed antiaen
This emulsion was prepared as described above except that PBl
antigen was not dialyzed.
I. Control of non-dialyzed antiaen in solution
PBl at a concentration of 0.77 mg/ml in a 50 mM phosphate
buffer, pH 6.8, containing 2 mM EDTA, 10 mM DTT and 8 M urea was
diluted in PBS minus to 50 ~g/ml.
1 3341 65
- 16 -
J. Control of dialyzed antiaen in solution
Stock PBl solution (0.77 mg/ml in a 50 ~_ phosphate buffer, pH
6.8, containing 2 m_ EDTA, 10 m_ dithiothreitol and 8 _ urea)
was dialyzed against 1~ acetic acid (pH 2.5) and the dialyzed
antigen diluted before use in PBS minus at 50 ,ug/ml.
Immunization: Balb/C (Jackson), 6-8 weeks old female mice were
inoculated intramuscularly ("IM") at day 0, 15, 35 and blood was
taken at day 0, 14, 28, 49, 63 and 79. The dose of antigen was
5 ,ug/0.1 ml/inoculum except group E which received 2.5 ,ug.
Determination of humoral immune response: PBl specific antibody
(IgG and IgM) was determined by a standard ELISA procedure.
Data presented in Table 1 indicated that the adjuvant effect
provided by CHS liposomes with affinity associated immunogen (group
F) was similar with that provided by complete Freund's adjuvant
"CFA" (groups G and H), a potent but highly toxic adjuvant.
Out of six liposomal formulations, five induced a positive response
in all mice tested 49 days or more after primary inoculation,
indicating an enhancement by liposomes of the secondary IgG memory
response and involvement of helper T-cells. The magnitude of the
response, however, was highly dependent on the nature of liposome
formulation and varied from low (groups A, B, C), medium (group E)
to high (group F).
Since the dose of antigen in group E was 2.5 ,ug/0.1 ml/inoculum
(rather than 5 ,ug) the adjuvant effect provided by this formulation
was probably underestimated. Both the original and acid dialyzed
PBl in PBS minus were weak immunogens (group I and J). In contrast,
the CHS liposome affinity associated preparation (group F) induced a
high antibody response and in addition no or minimal reaction was
observed at the site of inoculation.
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TABLE 2
In order to compare the effect of CHS affinity associated liposomes
with that of Alum, a standard adjuvant used in vaccines for humans,
groups of five mice were immunized with 20, 5, 1.25 and 0.31 ,ug PBl
in either Alum or CHS formulation.
The amount of Alum was kept constant at 0.4 mg/ml of physiological
saline (USP). The CHS liposome-PBl formulations were prepared as
described previously (group F, table 3). A formulation containing
50 ,ug antigen/ml was further diluted in a suspension of empty CHS
liposomes in PBS minus such that the amount of lipids was kept
constant at 50 mg/ml and the antigen decreased to 2.5, 1.25 and
0.31 ,ug/ml. Another CHS formulation at 50 mg lipid and 20 ~g
antigen was subjected to shearing force via vortexing and sonicating
at low energy intermittently for 2 hours at room temperature in
order to achieve a suitable suspension.
The results (Table 2) indicated that the CHS liposome with affinity
associated immunogen was a much stronger adjuvant than Alum. The
difference in immune titers were in general higher than one order of
magnitude with the exception of lowest dose (0.31 ,ug PBl) in which
the response remained low.
In addition, the comparison between CHS formulation and CFA
formulation both at 1.25 ,ug antigen reinforced the conclusion of the
previous experiment (Table 1) indicating a higher antibody response
by CHS affinity associated liposomes than by CFA.
Vaccines are conveniently administered in a dosage form. A "dosage
form" will be understood to mean any pharmaceutically form of
adminlstering a vacclne including subcutaneous, oral, intramuscular,
and ocular administration and utilizing vaccines in live, attenuated
or synthetic or bioengineered or partial forms along with adjuvants
and optionally immunomodulators such as cytokines. The combinations
of the foregoing elements are prepared so that the dosage form is
adapted to produce a therapeutically effective immune response in
the subject animal including a human as easily and effectively as
possible.
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The dosage forms including liposomal dosage forms resulting from the
method of the present invention can be used therapeutically in
mammals, including a human, in the treatment of infections or
conditions which require the delivery of immunogen in its bioactive
form. Such conditions include but are not limited to disease states
such as those that can be treated or prevented with vaccines.
Dosage forms also include use of adjuvant as well as vaccine
incorporated into gel such as aluminum gels, lipids such as Lipid A,
liquid crystals, powders, precipitates and solutions. In particular
embodiments the dosage form can be a unit dosage form configured and
adapted to a single administration.
The mode of administration of the dosage form may determine the
sites and cells in the organism to which the dosage form will be
delivered. The dosage forms including liposomal dosage forms of the
present invention can be administered alone but will generally be
administered in admixture with a pharmaceutical carrier selected
with regard to the intended route of administration and standard
pharmaceutical practice. The dosage forms may be injected
intramuscularly, subcutaneously or intradermally. The dosage forms
may also be administered via oral routes. For parenteral
administration, they can be used, for example, in the form of a
sterile aqueous solution which may contain other solutes, for
example, enough salts or glucose to make the solution isotonic.
Other uses, depending upon the particular properties of the
preparation, may be envisioned by those skilled in the art.
For administration to humans in the preventive or curative treatment
of disease states responding to vaccine based therapy, the
prescribing physician will ultimately determine the appropriate
therapeutically effective dosage for a given human subject, and this
can be expected to vary according to the age, weight, and response
of the individual as well as the nature and severity of the
patient's disease.
- - 19 1 3341 65
EXAMPLE 1
Affinity Attachment of Antiaen to Liposome
S PBl is a positively charged peptide and thus dialysis of PBl from
stock solution was performed against a 1% acetic acid at a pH of
2.5. Upon dialysis the PBl remained soluble for several hours in 1%
acetic acid. It was noted that, were the pH to approach neutral pH,
the PBl antigen would rapidly precipitate. Affinity attachment of
the PBl to liposomes was accomplished by utilizing a liposome of
reciprocal (here negative) charge. CHStriS liposomes having a
negative charge were used, suspended in neutral (pH 7.2) buffer and
then mixed with the PBl in 1% acetic acid solution. Care was taken
to avoid CHStriS precipitation as CHStriS liposome suspension is
lS rapidly precipitated by solutions below about pH 5. Therefore in
order to maintain the integrity of the liposome suspension, upon
addition of the PBl in acetic acid solution the pH of the mixture
was maintained above 5. Affinity association occurred upon mixing
of liposomes and antigen.
- -20_ 1 3 3 4 1 6 5
TA8LE 1
SERUM ANTIBODY TITERS AFTER IMMU~IZATIO~
OF BALB/C MICE WIT~ LIPOSOMAL PBl FORMULATIO~S
Group and Geometrlc Mean Titer (EIA Unit~/ml~
For~ulation Dav 14 D~y 28DaY 49 DaY 63 ~aY 79
A. Liposome D-~ - a~ - 66.08 140.85 74.00
(0/5) (0/5)(5~5) (5/5) (4/5)
B. Liposome D - - 144.47 95.68 70.77
(0/5) (0/5)(5/5) (5/5) (4/5)
C. Liposome D-E - 64.62113.00 154.21 180.90
(O/5) (4/5)(5/5) (4/4) (4/4)
D. Llposome D-T - - - 35.00
(0/5) (0/5)(0/5) (1/5) (0/5)
E. Liposome H-D - 20.49663.15 640.55 618.41
(0/5) (4/5)(5/5) (4/4) (4/4)
F. Liposome H - 1,135.786,593.49~11,465.316,104.65
(0~5) (5/5)(4/4) (4/4) (3/3)
G. CFA-PBl - 1,138.S8>11,329.05>11,465.3114,610.05
(0/5) (5/5)(5/5) (4/4) (4/4)
H. CFA-PBl - 321.032,253.232,875.41 3,134.13
(0/5) (5/5)(5/5) (5/5) (5/5)
I. PBI _ _ 300.00 43.47
(0/5) (0/5)(l/5) (2/5) (0/4)
J. PB1 - - 54.77 94.87 60
(0/5) (0/5)(2/5) (2/5) (1/4)
a/ Number of responting mice per group. Mean titer vas calculated from
the individual titer~ of the respontin~ mice only.
-21- 1 3341 65
TABLE 2
SERUM ANTIBODY TITERS AFTER IMMUNIZATION OF BALB/C MICE WITH
VARIOUS DOSES OF PBI IN LIPOSOMAL AND ALUM FORMULATIONS
.
Dose Geometric Mean Titer (EIA Units/ml~
Formulation (UQ) Dav 14 Dav 28 Dav 49
Alum 20.0 - a/ 161.341,212.31
(0/4) (4/4) (4/4)
Liposome 20.0 21.79 2,411.184,355.87
(2/4) (4/4) (4/4)
Alum 5.0 - 77.31 349.27
(0/4) (5/5) (5/5)
Liposome 5.0 38.73 931.977,949.76
(2/5) (5/5) (5/5)
Alum 1.25 - - 79.06
(0/5) (0/5) (2/5)
Liposome 1.25 - 152.02 917.78
(0/5) (4/5) (4/5)
Control (PBl 1.25
(0/5) (0/5) (0/5)
Control (CFA) 1.25 - 38.91 365.73
(0/5) (4/5) (5/5)
Alum 0.31 - 27.00 30.00
(0/5) (1/5) (1/5)
Liposome 0.31 - - 42.17
(0/5) (0/5) (3/5)
a/ Number of respondlng mice per group. Mean titer was calculated from
the individual titers of the responding mice only.
