Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NOVEL ADJWANT COMPOSITIONS AND VACCINE FORMULATIONS
COMPRISING SAME
DESCRI~IION
Field Of The Invention
The present invention relates to adjuvant compositions and to vaccine
formulations comprising same, as well as to methods of making and using
such adjuvants and vaccines.
Description of the Related Art
The injection of an antigen into an animal has long been regarded as an
effective method of producing antisera (i.e., serum antibody) or increasing the
antisera levels in the animal either for the protection of the host animal (i.e.,
15 vaccination) or to produce antisera for isolation and use in other animals.
Vaccines occupy a unique place in health care because unlike most
therapies they are given to healthy people to prevent diseases. Because
vaccination use has been a primary factor in controlling many childhood
20 diseases, great effort is applied in expanding the use of vaccines. Vaccines are
being developed for many diseases including cholera, malaria, herpes, chicken
pox, and pneumonia.
It also has long been known that use of certain adjuvants can increase
25 the titer of antisera produced against a foreign antigen and provide prolonged
protection against the unwanted effect of the antigen itself or the pathogens
carrying such antigen. The adjuvant will influence the titer, duration, isotype,and avidity of the antibody as well as influence cell-mediated immunity.
Research and development efforts have focused on developing vaccine
30 adjuvants that enhance the body's immunological response to vaccines with
extended duration of effectiveness.
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Effective adjuvant formulations in the form of Freund's complete
adjuvant (FCA) or Freund's incomplete adjuvant (FIA) have been known
since the mid-1930's and have been used to improve the production of
5 antisera against heterologous antigens in laboratory animals.
The key characteristics of Freund's complete adjuvant and Freund's
incomplete adjuvant are the emulsification of the antigen in mineral oil to
ensure the formation of a slow-release depot of the antigen at the injection at
10 the injection site. Freund's complete adjuvant contains killed mycobacteria
and app~u~"Lly acts by ~Lef~r~lLtially inducing antibody against epitopes on
~lPn~hlred ~-oLeiL.s. The result is higher levels of antisera produced when
compared with antigen alone. However, Freund's complete and incomplete
adjuvant are known to produce signific ~nt toxic complications.
In ~ ition to causing chronic pain and suffering as an nn~l~sirable side
effect, FCA induces local granulomas and possibly m;llign~n~-ies. For these
reasons, FCA has never been approved for use in human or veterinary
vaccines in the United States.
A major goal in the area of vaccine development is the production of
vaccine formulations which include the efficacy and exclude the deleterious
side effects of adjuvants such as FCA. Attempts to reduce the toxicity while
retaining efficacy of adjuvants such as Freund's adjuvant have largely failed,
25 in part, from a lack of understanding of the specific biological mechanism(s) responsible for adjuvant efficacy.
Several commercial adjuvant products are available which, while safer
than Freund's adjuvant, have significantly lower effectiveness than Freund's
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adjuvant. For example, oil and water emulsions with the antigen adsorbed to
the oil phase having brief retention are commercially available. Also
commercially used is aluminum hydroxide where the antigen is adsorbed
directly on the aluminum hydroxide. In another commercial product for
5 experimental non-human use, available under the trademark Adjuvax, the
antigen is physically incorporated in a polysaccharide matrix of a glucan
polysaccharide .
The original adjuvants were substances of biological origin that
10 enhanced a specific antibody response. Since mycobacterium has been
effectively used as an adjuvant, attempts have been made to isolate those
biologically active components from mycobacterium cell walls that are
responsible for immunostimulation. The lipid fraction extracted from
mycobacteria contains trehalose dimycolate (TDM) as an active component
15 while the mycob~ct~rillm cell wall contains N-acetylmuramyl-L-~l~nine-D-
isoglutamine, also known as muramyl dipeptide (MDP), as an active
component. Lipopolysacrh~r~ s obtained from the cell wall of these gram-
negative bacteria exhibit immllnostimulating activity, but toxicity attributed to
its lipid A portion has precluded its use. Glucan, a ,~-1, 3-polyglucose from
20 Saccharomyces cerevisiae, a yeast, has been reported to induce antitumor
effects, improve resistance to microbial pathogens and stimulate antibody
response to a variety of antigens.
The toxicological issues associated with adjuvants of microbial origin
25 have resulted in research on nonmicrobial substances. These nonmicrobial
substances include deL~l~;e-lts, salts, sugars, polyribonucleotides, and naturalsubstances of m~mm~ n origin. Both nonionic and cationic d~L~lgellts have
achieved success as adjuvants, with more lipophilic deLelg~ts being more
effective. Saponins have amphipathic surface activity, so their mechanism for
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inrlll~ ing adjLIv~l-t activity may be .simil~r to that of deLer~,~l.ts. Saponins are
not used in human v~crines because of toxicological issues. Lymphokines and
monokines have a very short biological half-life, so pharmacokinetic concerns
preclude their use as adjuvants.
In the typical vaccine formulation comprising an antigen and a vehicle
(carrier) component, a clear distinction between the vehicle and the adjuvant
cannot always be made because many vehicles have adjuvant-like activity,
which may result from immunostimulation effects and/or slow release of
10 antigen. For example, aluminum salts are the most widely used vehicles in
v~rrinr~ licensed for human and veterinary use. The antigen is believed to
reside in the aluminum gel, releasing slowly over time to produce a continual
rh~llr-nge to the immune system. In addition to this clear vehicle effect,
~lllminum salts probably act as true adjuvants by virtue of their chemotactic
15 properties for various immunological cells. Other examples of vehicles with
adjuvant-like activities include water/oil emulsions, oil/water emulsions,
microencapsulation, and liposomes.
I;or more than 20 years, the ability of liposomes to stimulate antibody
20 response has been known, but issues in the development of a~Lo~liate
components for liposomes as r~ r~ for vaccines are still in debate. The
antigen can be encapsulated into the aqueous spaces of the liposome core or
attached to the external surface of the lipid bilayer. The adjuvant property of
liposomes can be further enhanced by the inclusion of certain
25 immunostimulants such as lipid A, lipopolysaccharide, or MDP. Liposomes
are believed to exhibit their adjuvant properties by being taken up
~leLei~lltially by macrophages, but liposomal delivery does not provide for a
sustained release of antigen.
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Nanoparticles, solid colloidal particles from 10 to 1000 nm of synthetic
polymers such as polymethylmethacrylate, are reported to be effective
adjuvants whether the antigen is encapsulated within the nanoparticle or
' adsorbed to the nanoparticle surface. The adjuvant effect of these vaccines
5 improves with increasing hydrophobicity and decreasing particle size. Because
the polymethylmethacrylate nanoparticles are slowly biodegradable, the
adjuvant effect may be caused by a continuing antigen challenge to the
immune ~y~le~
10A recent study evaluating ~ ellt adjuvants for their ability to induce
antibody in mice to HIV-2 split whole virus reported that
polymethylmethacrylate nanoparticles was the best overall adjuvant when
considering the immune response and observable toxic side-effects. ~owever,
the data also suggested that two or more different adjuvants may be necess~ry
15 to induce the required immune response against physically different antigens.An alternative explanation of the study data is that the immunological
response to each antigen is best augmented by a unique adjuvant. In either
case, the development of alternative adjuvants is critical to the successful
development of potent vaccine formulations.
The technical and patent literature describes various other attempts at
improved adjuvants.
U.S. Patent 5,273,965 describes compounds of the saponin family which
25 can be used to administer vaccines via nasal spray or eye drops.
Infection and Immunity, Sept. 1991, pp. 2978-2986, describes a poly(DL-
lactide-co-glycolide) microsphere useful as an adjuvant for Staph. enterotoxin
B toxoid.
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U.S. Patent 5, 057,503 to J.K. Czop, et al. discloses small molecular weight
biologically active oligosAc~h~ri~les which are interactive with ~-glucan
rece~Lois on m~mm~ n phageocytic cells. This unit ligand composition, a
5 heptaglucoside, is described as being derivatizable with 2-aminopyridine to
increase the capacity of the glucocide to stimtll~te ,13-glucan rece~Lo,a and
potentiate functions mediated by such re~lola. The heptaglucoside is
~l~cfrihed as useful for vaccine or other immunomodulating agent
preparations such as adjuvant therapy.
U.S. Patent 5, 189,028 to L.H. Nikl, et al. ~les~ es the stimulation of
immllnP :jy:,lellls of fish by administration of a ,13-1, 3-glucan, partit ~ rly a ,~
3-glucan having a ,6-1, 3-linked main chain with ,~-1, 6-linked single glucose
side rh~in~.
U.S. Patent 4,981,684 to N.M. MacKenzie, et al. disdoses the formulation
of adjuvant mAtri~es c~ ,ising a water-insoluble antigen which is
solubilized with a solubilizing agent, e.g., a deLer~3e,-t species, urea or
guanidine, then admixed with a glycoside, a sterol, and optionally, a
20 phospholipid, thereby forming an immuno-stimulating complex subst~nti~lly
without removal of the solubilizing agent.
U.S. Patent 5,032,401 to S. Jamas describes a pharmaceutical composition
comprising whole glucan particles and a p~rm~cologically active substance
25 such as a drug or antigen contained within, uniformly dispersed with, or
ch~mif~lly linked to the whole glucan particles.
U.S. Patents 5,091,187 and 5,091,188 to D.H. Haynes disclose
phospholipid-coated microcrystal or microparticle compositions providing an
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injectible delivery form for administration of water-insoluble drugs to a
m~mm~ n host for sustained release. The pharm~ce~ lly effective agent
is produced in solid form coated with a membrane-forming lipid which
stabilizes the active ingredient material by hydrophobic and hydrophilic
5 interactions. The active solid ingredient-containing particles are formed in
small finely divided form, by sonication or other process inducing high shear.
U.S. Patent 5,246,707 discloses the sustained release delivery of water-soluble
biomolecules and drugs using phospholipid-coated microcrystals,
microdroplets and high-concentration liposomes. The phospholipid-coated
10 microcrystal and the phospholipid-coated microdroplet are described as
useable as vaccine adjuvants.
It therefol~ is an object of the present invention to provide an
improved adjuvant having an immunostimulating character, but without
15 toxic side effects.
It is another object of the present invention to provide a vaccine
composition comprising such adjuvant which is safe and effective in use.
Other objects and advantages of the invention will be more fully
apparent from the ensuing disclosure and appended rl~irns
SUMMARY OF THE INVENTION
The present invention relates to an adjuvant composition, which may
be usefully employed with an antigen or an antigen-based vaccine, to enhance
immunostimulative response.
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In a broad composition aspect, the invention relates to an adjuvant
comprising a polys~crhari~le-phospholipid conjugate.
Parti~lllarly ~lef~:lled polysaccharides of the adjuvant of the invention
5 include ~-glucan, chitosan, gal~ctomanans, and alginates.
In another aspect, the present invention relates to a method of
synthesizing an adjuvant from a polysaccharide.
The adjuvant may be synthesized from a polysaccharide and
phospholipid, using any suitable reagents, including bifunctional or other
polyfunctional reagents.
The adjuvant may for example be synthesized by the steps of:
rPa~-ting the poly~ar~haricl~ with an oxidizing agent to form
aldehyde functionality on the polysaccharide;
reacting the aldehyde-functionalized polysaccharide with an
a~lo~liate bifunctional reagent, to yield a polysaccharide function-
alized with a linking functionality which is reactive with a
phospholipid to further yield a polysaccharide-phospholipid
conjugate; and
2~ reacting the functionalized poly~c~hari~7e with a phospholipid
to yield the polysaccharide-phospholipid conjugate.
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The invention in a further aspect comprises a vaccine composition
including the adjuvant of the invention and an antigen for producing
antibodies in an animal.
,~
Further, the invention relates to inducing an immunological response
in an animal comprising administering the vaccine including the adjuvant in
an amount sufficient to produce an antibody response in such animal.
Other aspects and features of the invention will be more fully apparent
from the ensuing disclosure and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a graph of the antibody titer in mice, for vaccination with
bovine serum albumin (BSA), BSA in microdroplet form (BSA-MD), BSA in
microdroplet form with a ,13-glucan conjugate adjuvant according to one
embodiment of the present invention (BSA-GMD), and BSA with Freund's
Complete Adjuvant (FCA).
FIGURE 2 is a graph of Ig~ titer in mice plasma at 1:4096 dilution, at
one, two and three months after injection, for vaccination with bovine serum
albumin (BSA), BSA in microdroplet form with a ,13-glucan conjugate
adjuvant according to one embodiment of the present invention (BSA-MD),
and BSA with Freund's Complete Adjuvant (BSA-Freund's).
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED MODES
OF CARRYING OUT SAME
The present invention is based on the surprising and unexpected
discovery that polysaccharides when conjugated with phospholipids form
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adjuvants which (i) increase the titer, duration, isotype and avidity of the
antibody produced in a host animal, and (ii) have low toxicity and good
effectiveness and safety characteristics in the host animal when compared to
Freund's adjuvant.
The polysaccharides which are used to form the adjuvants of the
invention may comprise any suitable polysaccharides, e.g., a polysacchaAde
having an immunostimulative activity.
10Polysaccharides which may be used in adjuvant compositions in the
broad practice of the present invention include species described in
"Carbohydrate Chemistry," ed. byJohn F. Kennedy, Clarendon Press, Oxford,
1988; "The Carbohydrates, Chemistry and Biochemistry," ed. by W. Pigman
and D. Horton, ArA~l~mi~ Press, Inc., 1970; and "Chitin, Chitosan, and R~l~te~l
15Enzymes," ed. by John P. Zikakis, A~-A~7~m;C Press, Inc., 1984. Partic~-lArly
er~ d polysAc~hArici~ species include ,~-gl~ n~, chitosan, galactomanans,
and AlginAtP~, with ,~-glucans being cul-el-Lly most ~ler~lled.
AS fii~ sed hereinabove, the adjuvants of the present invention may
20 be synth~ci7e~1 from a polysaccharide and phospholipid, via any suitable
synthetic method, and using any suitable reagents, including bifunctional or
other polyfunctional reagents.
Most generally, the polys~c~h~rici~ is complexed by conjugation with a
25 phospholipid by reaction, with may comprise oxidation of the polysa~rh~ric~e,or other functionalizing reaction, to produce a functionalized poly~c~hAride
which is of a form that is conjugatable with a phospholipid.
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The adjuvant may for example be synthesized by reacting the
polysaccharide with an oxidizing agent to form aldehyde functionality on the
polysaccharide, following which the aldehyde-functionalized polysaccharide is
reacted with an a~Lo~liate bifunctional reagent, to yield a polysaccharide
5 functionalized with a linking functionality. The linking functionality is
reactive with a selected phospholipid to further yield a polysaccharide-
phospholipid conjugate.
In one specific synthesis method within the scope of the broad
10 invention, the bifunctional reagent in the above-described synthesis method
comprises a thiol hydrazide compound, which is employed in the synthesis
procedure to yield a thiol-functionalized poly~c~h~ricle. The thiol-
functionalized polysaccharide subsequently is reacted with a phospholipid, to
yield a polysAt-~ h~ le-phospholipid conjugate as the aforementioned
i 5 adjuvant.
As a further specific example of the synthesis of an adjuvant i n
accordance with the present invention, the starting polysaccharide is reacted
with an oxidizing reagent such as a periodate compound, to convert oxidizable
20 functional groups of the polysaccharide to corresponding aldehyde
functionality (-CHO pendant groups). The resulting aldehyde-functionalized
polysaccharide then is reacted with a mercaptohydrazide compound, such as
for example 2-acetamido-4-mercapto-butyric acid hydrazide (AMBH), or other
suitable bifunctional reagent, to provide a suitable reactive moiety (end group)25 on the functionalized polysaccharide for linking of a phospholipid conjugate
thereto.
In the synthesis of the adjuvant conjugates of the present invention,
reagents other than the bifunctional reagents described in the preceding
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paragraph may be advantageously employed, including for example reagents
which may not require initial oxidation of the polysaccharide. A phospholipid
may be conjllg~te~i to an existing functional group on the polysAc( h~ri~le, such
as an amino or a hydroxyl function, by methods known in the art of synthetic
5 chemistry.
The phospholipid conjugate which is used to form the polysaccharide-
phospholipid adjuvant of the present invention may comprise any suitable
phospholipid, which is coordinatable, e.g., by covalent, ionic, hydrogen,
10 associative, or other conjugative bonding, to form a ph~rm~cologically stable complex with the polysaccharide which renders the polys~c~ h;~ricl~
bioavailable in the host system to produce the desired immunostimulative
response.
Within the broad practice of the present invention, the phospholipid
component of the polys~c~rh~ride-phospholipid coniugate may be rendered
into conjugatable form by reaction with suitable reagent(s), e.g., an a~lo~liatebifunctional reagent. In some instances of the practice of the present
invention, it may be advantageous to functionalize a phospholipid so that it is
20 directly conjugatable with the polysaccharide, and so that no intermediate
reaction(s) involving the polysaccharide are nec-o~s~ry prior to conjugating thepolysaccharide with a phospholipid. In other instances, only the
polysaccharide may be modified to render it in conjugatable form, and in still
other instances, both the polysaccharide and the phospholipid starting
25 materials are modified to render them conjugatable, viz-a-vis one another.
As one specific example of rendering a phospholipid component in
suitable form for conjugation with a polysaccharide, by reacting a bifunctional
reagent with a phospholipid, the bifunctional reagent is N-succinimidyl-3-(2-
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pyridyldithio)-propionate, sometimes hereinafter referred to as SPDP, and the
phospholipid is dipalmatoylphosphatidyl-ethanolamine, sometimes
hereinafter referred to DPPE. SPDP and DPPE may be reacted with one another
in a suitable solvent medium, e.g., chloroform. The chloroform in the
5 reaction volume advantageously is replaced, via evaporation of the
chloroform under nitrogen atmosphere, with a suitable water-miscible
solvent such as acetonitrile, to yield the DPPE-SPDP conjugate as the
phospholipid component for subsequent reaction with the modified
polys~rrh~ e. The modified polysaccharide, having for example a thiol
10 functionality (as a result of reaction with a mercaptohydrazide compound)
then is reacted with the SPDP-derivatized phospholipid to form a
polysaccharide-phospholipid conjugate as the adjuvant product.
While any suitable phospholipid constituent may be employed in the
5 broad prac~ce of the invention, one particular class o~ phosphoiipid
compounds which may be advantageously employed includes fatty acid
phosphatidylethanolamine compounds, whose fatty acid component includes
two fatty acid moieties each of which is independently s~lecte-l from the group
consisting of lauroyl, palmatoyl, myristyl, oleyl, and stearyl, which in the
20 subsequent discussion are designated by the letters L, P, M, O, and S,
respectively, and in which the phosphatidylethanolamine moiety is
designated PE. Thus, illustrative phospholipid species based on the above-
mentioned fatty acid functional groups, which may be potentially usefully
employed in the practice of the present invention include those identified in
25 Table I below.
13
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Table I
Compound Df~ n~tion
dipalmitoylphosphatidylethanolamine DPPE
dilauroylphosphatidylethanolamine DLPE
dimyristoylphosphatidylethanol ~ mi n~ DMPE
dioleyl~t~sphatidylethanolamine DOPE
distearylphosphatidylethanolamine DSPE
lauroylpalmitoylphosphatidylethanolamine LPPE
lauroylmyristoylphosphatidylethanolamine LMPE
lauroyloleylphosphatidylethanolamine LOPE
lauroylstearylphosphatidylethanolamine LSPE
palmitoylmyristoylphosphatidylethanolamine PMPE
palmitoyloleylphosphatidylethanolamine POPE
palmitoylstearylphosphatidylethanolamine PSPE
oleylstearylphosphatidylethanolamine OSPE
oleylmyristoylphosphatidylethanolamine OMPE
myristoylstearylphosphatidylethanolamine MSPE
By conjugation of the phospholipid to the polysaccharide, there is
formed an adjuvant which is a-lministerable to a host animal by any of a
variety of administration routes to provide a slow and controlled
enhancement of immunological response.
The resulting adjuvant may be then be compounded for formulation
purposes with any suitable antigens, r~rri~rS, excipients, stabilizers, additives,
etc. and the formulation may be processed as nec~s~ry for end use or
a-lministration purposes. For example, the adjuvant formulation may be
lyophilized to form a powder formulation which is amenable to
administration by nebulization to a pulmonary locus of a host animal.
Alternatively, the fomulation may be subjected to sonication or other shear
treatment, to yield a microparticle composition for convenient
adrninistration.
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As a further variation of the compositions of the present invention,
suitable antigen or antigens may be coordinately linked to the phospholipid
and/or the polysaccharide moieties of the adjuvant, to provide an integrated
5 vaccine formulation for effecting enhanced immunostimulative response
from the host animal.
The host animals to which the adjuvant and adjuvant-containing
vaccine formulations of the present invention are usefully administered
10 include human as well as non-human m~mm~l~, fish, reptiles, etc.
In formulations of the adjuvant of the present invention, it may be
useful in some applications to employ an antigen covalently linked to a
phospholipid and/or polys~erh~ 1e moiety of the poIys~rrh~r1fle-
15 phospholipid conjugate. Alternatively, an antigen may be employed inmixture with the adjuvant of the invention. The specific formulation of
therapeutically effective compositions of the present invention may thus be
r~rne~l out in any suitable manner which will render the adjuvant
bioavailable, safe and effective in the subject to whom the formulation is
20 administered.
The invention broadly contemplates therapeutic adjuvant
formulations, which may for example comprise (i) at least one therapeutically
effective antigen or vaccine; and (ii) at least one polysaccharide-phospholipid
25 conjugate according to the invention.
Such therapeutic composition may for example comprise at least one
antigenic agent selected from the group consisting of:
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(A) viruses, b~ctPri~, mycoplasmas, fungi, and protozoa;
(B) fragments, extracts, subunits, metabolites and recombinant
constructs of (A);
(C) fr~gmPnts, subunits, metabolites and recombinant constructs
of m~mm~ n proteins and glycoproteins; and
(D) tumor-specific antigens.
The therapeutic composition may therefore utilize any su*able antigen
or vaccine component in combination with the polyc~rrh~ricle-phospholipid
conjugate of the invention, e.g., an antigenic agent sPlerte~ from the group
consisting of antigens from pathogenic and non-pathogenic org~ni~m~,
15 viruses, and fungi, in combination with a polys~crh~ricip-phospholipid
conjugate.
As a futher example, such therapeutic composition may suitably
comprise ~n~Lei,ls, peptides, antigens and vaccines which are
20 ph~rm~cologically active for disease states and conditions such as smallpox,
yellow fever, distemper, cholera, fowl pox, scarlet fever, diphtheria, tetanus,
whooping cough, influenza, rabies, mumps, measles, foot and mouth disease,
and poliomyelitis. In the resulting vaccine formulation, comprising (i) an
antigen, and (ii) the polysaccharide-phospholipid conjugate, the antigen and
25 adjuvant are each present in an amount effective to elicit an immune
response when the formulation is administered to a host Anim~l, embryo, or
ovum v~crin~ted therewith.
16
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The resulting vaccine formulations, including (i) an antigen, and (ii)
the polysaccharide-phospholipid conjugate, are usefully employed to induce
an immunological response in an animal, by administering to such animal
the vaccine formulation, in an amount sl1ffi~i~nt to produce an antibody
5 response in such animal.
The modes of administration may comprise the use of any suitable
means and/or methods for delivering the adjuvant or adjuvant-containing
vaccine to a corporeal locus of the host ~nimal where the adjuvant and
10 associated antigens are immumostimn1Atively effective. Delivery modes may
include, without limitation, parenteral administration methods, such as
subcutaneous (SC) injection, intravenous (IV) injection, nasal, ophthalmic,
trans~l~rmal, intramuscular (IM), intradermal (ID), intraperitoneal (IP),
intravaginal, pulmonary, and rectal administration, as well as non-parenteral,
15 e.g., oral, administration.
The dose rate and suitable dosage forms for the adjuvant and vaccine
compositions of the present invention may be readily riet~rmin~d by those of
ordinary skill in the art without undue experimentation, by use of
20 conventional antibody titer determination techniques and conventional
bioefficacy/ biocompatibility protocols, and depending on the particular
antigen or therapeutic agent employed with the adjuvant, the desired
therapeutic effect, and the desired time span of bioactivity.
The adjuvant of the present invention may be usefully administered to
the host animal with any other suitable pharmacologically or physiologically
active agents, e.g., antigenic and/or other biologically active substances.
17
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The features and advantages of the invention will be more fully
illlustrated by the following non-limiting examples, wherein all parts and
percentages are by weight, unless otherwise expressly stated.
Example I
A ,13-glucan-pho5pholipid conjugate in accordance with the present
invention was formulated by the synthesis procedure described below.
10 Modification of ~-Glucan
An was conjugated to AMBH as follows.
,~-Glucan was treated with sodium periodate to induce aldehyde
form~*~ n in the polys~rh~n~l~ 50 ~ll (5~1mole) of 0.I M sodium periodate
15 was added to a suspension of ,~-glucan (20 mg) in lml of water. The reaction
took place over 15 hours at room temperature. To the resulting suspension
was added 50 ~l (5 ~lmole) of 0.I M 2-Aret~mi~ -4-melcd~Lobutyric acid
hydrazide dissolved in acetonitrile (AMBH, Molecular Probes, Inc.). After
stirring at room temperature for 24 hours, the AMBH conjugated to ,~-glucan
20 suspension was used as such for conjugation with SPDP derivatized
phospholipid.
Svnthesis of SPDP Conjugated Dipalmitovlphosphatidvlethanolamine
Dipalmitoylphosphatidylethanolamine (173 mg; 250 ~umoles) was
25 dissolved in chloroform. Triethylamine (20 ~ll) and N-succinimidyl-3-(2-
pyridylthio)-propionate (78 mg; 250 ~lmoles) (Pierce Chemical Company
(Rockford, IL)), denoted hereinafter as SlJL~P, were added in order. The
mixture was gently stirred for 24 hours at room temperature. The chloroform
was evaporated using nitrogen. The residue was dissolved in 4 ml of
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acetonitrile to provide a solution containing 62 ,umoles of Sl~DP conjugated
dipalmitoylphosphatidylethanolamine per ml of solution.
Preparation of ,13-Glucan-Phospholipid Conjugate
SPDP conjugated dipalmitoylphosphatidylethanolamine (100 ~ll
containing 6.2 ~lmoles) was added to 1 ml of the suspension of AMBH-
conjugated ~I~-glucan. The mixture was stirred gently for 24 hours at room
temperature to yield the conjugate product.
Example II
,~-Glucan-Phospholipid Conjugate BSA Formulation (BSA-GLl
A mixture of 0.5 ml of the ,~-glucan-phospholipid conjugate product
suspension of Example I and 0.2 ml of aqueous BSA (1 mg/ml), 140 mg of egg
15 phosphatidylcholine, 70 mg of vitamin E, 70 mg of Squalene, and 0.75 ml of
phosphate bLIL~l~ed saline, was sonicated with a probe sonicator for 15 minutes
at 4 degrees Centrigrade, to form the ,~-glucan-phospholipid conjugate-BSA
vaccine emulsion formulation.
Example III
Microdroplet Adjuvallt Form~ tion of BSA (BSA-MD)
A microdroplet emulsion formulation of the BSA was performed in
accordance with the teachings of the aforementioned Haynes U.S. Patent
25 5,246,707. This vaccine formulation was used for comparison purposes.
A mixture of 0.2 ml of aqueous BSA (lmg/ ml), 140 mg of egg
phosphatidylcholine, 70 mg of vitamin E, 70 mg of s~ualene, and 1.25 ml of
phosphate buffered saline, was sonit ~te~l with a probe sonicator for 15 minutes
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WO 97/01330 PCT/US96/11051
at 4 degrees Centrigrade, to form the BSA-MD microdroplet vaccine
formulation.
Example IV
Adjuvant Studies in Mice
The adjuvant properties of an adjuvant composition of the present
invention were evaluated in vaccine formulations containing bovine serum
albumin (BSA) antigen. The comparative studies were r~ l out in mice,
10 and the results included a de~e"nillation of the antibody titer produced by
vaccination with the respective vaccine formulations.
The tests included vaccination of respective test ~nim~l~ with the
following vaccine formlllAti~ ns: (i) bovine serum albumin (BSA) in saline, (ii)15 BSA in microdroplet Pmlll~ic n form (BSA-MD), formulated in accordance
with Example III above, (iii) BSA with a ~-glucan-phospholipid conjugate
adjuvant accol.ling to one embo~iment of the present invention, prepared ~y
the procedure of Example II (BSA-GL), and (iv) BSA with Freund's Complete
Adjuvant (FCA). The results, discussed hereinafter in greater detail, are
20 shown in the graph of Figure 1.
Experimental Design
CF-1 mice (Charles River) approximately 25 grams in weight were used.
Mice (n=5 per group) were injected i.p. with 50 ~l of each formulation
25 containing the same amount of antigen on day 0 and given a booster injection
of the same quantity as the original injection on day 14, and serum samples
from each mouse were analyzed on day 28.
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WO 97/01330 PCT/US96/11051
Scre~nin~ the Sera Samples
The wells of 96-well microtiter plates were coated with a 0.5 mg/ml
solution of BSA used as antigen (50 ~ll aliquots added to the wells and allowed
to dry in the freezer). The wells were washed 3 times with wash buffer (Tris 20
mM, NaCl 0.8 M, 0.055~O Tween-20, pH 7.4) followed by filling each well with
an aqueous solution containing 1 mg/ml gelatin for 30 minutes followed by
three washes with the wash buffer. Serial dilutions of the sera (100 ~ll) were
added to the wells and kept overnight at 4~C. The wells were washed 3 times
with wash buffer and 100 ~ll of a 1:10,000 dilution of goat anti-mouse IgG-
10 alkaline phosphatase conjugate (Organon Teknika Corporation, Charlotte,
NC) was added. After 1 hour at room temperature, the wells were washed
three times with wash buffer. Freshly prepared solution (200 ~ll) of the
substrate (p-nitrophenyl phosphate disodium) 1 mg/ml in diethanolamine
buffer pH 9.8 (97 ml diethanolamine, 0.2 g NaN3, 100 mg MgCl2 ~6H2O in 1 liter
15 water) was added and kept at room temperature in the absence of light for 2
hours. The color development was quantitated at 405 nanometers in a
mi~l~,LiL~:, plate reader.
Results
The results are given in Figure 1. The non-formulated antigen,
designated as BSA (as expected) had the minimum antibody titer while the
microdroplet-formulated antigen (BSA-MD) provides better response than the
non-formulated antigen at various dilutions of the anti-sera. The immune
response of the BSA-glucan formulation (BSA-GL), comprising a ~-glucan-
25 phospholipid conjugate according to the present invention, was found to be
even better than the BSA-microdroplet formulation (BSA-MD) and as good as
that of the antigen formulated in Freund's Complete Adjuvant at all dilutions
of the anti-sera tested.
CA 02219904 1997-10-30 . - ,-
Example V
In separate tests comparing adjuvant properties of (1) unmodified ~-glucan
(not conjugated with phospholipid) with t2) a modified ,~-glucan-
phospholipid conjugate according to the present invention, the adjuvant-
containing unmodified ~-glucan did not provide a similar enhancement of
antibody titer achieved by the modified ,~-glucan-phospholipid conjugate.
Example VI
The adjuvant properties of an adjuvant composition of the present
invention were evaluated in vaccine formulations containing bovine serum
albumin (BSA) antigen. The comparative studies were carried out in mice,
and the results included a determination of the antibody titer produced by
vaccination with the respective vaccine formulations.
The tests included vaccination of respective test animals with the
following vaccine formulations: (i) BSA (u) a BSA-containing adjuvant
formulation representative of the present invention, prepared by the
procedure of Example II (BSA-GL), and (iv) BSA with Freund's Complete
Adjuvant (BSA-Freund's). The results, discussed below in ~,realel detail, are
shown in the graph of Figure 2.
Adjuvant Studies in Mice
Six week old BALB/c (Charles River) mice were injected s.c. with 200~11
of the adjuvant preparations. Each of the administered adjuvants
preparations contained 200 llg/ml of BSA. In addition, a normal saline
solution of BSA was prepared at 200 ~lg/ml. On day 14, each animal was
~A~ED SH~Fr
CA 02219904 1997-10-30
~ J;
' 2 4 ~A~
boosted with freshly prepared adjuvant preparations, made with the same
concentrations as the initial preparations. As in normal practice for boosting,
FIA was used instead of FCA. At 1 month, 2 months or 3 months post-initial
treatment, the mice were bled.
s
Screening the Sera Samples
The wells of 96-well microtiter plates were coated with a 1 mg/mL
solution of BSA (50,uL aliquots added to the wells and overnight at 4~C). The
wells were washed three times with PBS/1% Tween 20. 100~L of PBS
containing 10% goat serum and 1% Tween 20 were then incubated in the wells
for 1 hour at 37~C; plates were then washed 3 times with PBS/1% Tween 20.
Test sera were serially diluted (1:32 to 1:4096) in 100,uL of PBS/ 10% goat
serum/1% Tween 20 and added to each well and incubated overnight at 4~ C or
for 2 hours at 37~ C; the plates were then washed 6 times with PBS/1% Tween
20. Goat anti-mouse IgG or IgM coupled HRP antibody were diluted 1:1000 in
PBS/1% Tween 20 and incubated in the plates for 2 hours at 37~C; plates were
then washed 6 times with PBS/1% Tween 20. ABTS Peroxidase Substrate and
Peroxidase Solution were used for development of the peroxidase reaction.
ELISA readings were performed with a Fisher Biotech, Microkinetics BT 2000
20 plates reader (405 nm wavelength).
Results
The results are given in Figure 2, which is a graph of IgG titer in mice
plasma at 1:4096 dilution, at one, two and three months after injection, for
25 vaccination with the respective BSA, BSA-GL, and BSA-Freund's
compositions. As shown, the non-formulated antigen (BSA) had the
minimum titer, while the microdroplet-formulated antigen (BSA-GL)
representative of the present invention provided better response than
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WO 97101330 PCT/US96/11051
Freund's Adjuvant with BSA (BSA-Freund's) through 3 months post-initial
treatment.
5 INDUSTRIAL APPLICABILITY
The adjuvant compositions of the present invention are usefully
employed with an antigen or an antigen-based vaccine, for administration to a
host ~nim~l, embryo or ovum, to enhance immunostimulative response of
10 the recipient host.
Such therapeutic compositions may for example comprise one or more
antigenic agents such as (A) viruses, b~ t~ri~, mycopl~m~s, fungi, and
~roLo,oa; (B) fragments, extracts, subunits, metabolites and recombinant
15 constructs of (A); (C) fragments, subunits, metabolites and recombinant
constructs of m~mmAli~n proteins and glyco~roLeills; and (D) tumor-specific
antigens, and such therapeutic compositions may be pharmacologically active
for disease states and conditions such as smallpox, yellow fever, distemper,
cholera, fowl pox, scarlet fever, diphtheria, tetanus, whooping cough,
20 influenza, rabies, mumps, measles, foot and mouth disease, and poliomyelitis,wherein the antigen and adjuvant are each present in an amount effective to
elicit an immunological response when the formulation is administered to
the host animal, embryo, or ovum vaccinated therewith.
24
