U.S. patent application number 16/823836 was filed with the patent office on 2020-07-09 for iscom preparation and use thereof.
The applicant listed for this patent is NOVAVAX AB. Invention is credited to Karin LOVGREN-BENGTSSON, Bror MOREIN.
Application Number | 20200215189 16/823836 |
Document ID | / |
Family ID | 20288449 |
Filed Date | 2020-07-09 |
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United States Patent
Application |
20200215189 |
Kind Code |
A1 |
MOREIN; Bror ; et
al. |
July 9, 2020 |
ISCOM PREPARATION AND USE THEREOF
Abstract
The invention relates to a composition comprising a mixture of
at least two iscom complexes each complex comprising essentially
one saponin fraction from Quillaja saponaria Molina. The complexes
may be iscom complexes or iscom matrix complexes. The invention
also pertains to the use of such a mixture for the preparation of
an immunomodulating pharmaceutical, and adjuvant, formulations for
immunization, e.g. for production of monoclonal antibodies, and a
vaccine. Kits of parts comprising at least two parts, wherein each
part comprises one iscom complex or one iscom matrix complex
according to the invention are also embraced.
Inventors: |
MOREIN; Bror; (Uppsala,
SE) ; LOVGREN-BENGTSSON; Karin; (Uppsala,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVAVAX AB |
Uppsala |
|
SE |
|
|
Family ID: |
20288449 |
Appl. No.: |
16/823836 |
Filed: |
March 19, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14587116 |
Dec 31, 2014 |
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16823836 |
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10520022 |
Jan 23, 2006 |
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PCT/SE03/01180 |
Jul 7, 2003 |
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14587116 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55577
20130101; A61P 37/04 20180101; A61P 31/22 20180101; A61K 39/39
20130101; A61P 31/12 20180101; A61P 31/14 20180101; A61P 31/20
20180101; A61P 31/16 20180101; A61P 43/00 20180101; A61P 31/04
20180101 |
International
Class: |
A61K 39/39 20060101
A61K039/39 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2002 |
SE |
02021103 |
Claims
1. A composition comprising a mixture of at least two iscom
complexes, chosen from iscom and iscom matrix complexes, each
complex comprising essentially one saponin fraction from Quillaja
saponaria Molina.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/587,116, filed Dec. 31, 2014, which is a
continuation application of U.S. application Ser. No. 10/520,022,
filed Jan. 23, 2006, which is a national stage application of
International Application PCT/SE03/01180, filed on Jul. 7, 2003 and
which claims priority of Swedish patent application No. 02021103,
filed Jul. 5, 2002, all of which are hereby incorporated by
reference.
FIELD OF INVENTION
[0002] This invention relates to a composition comprising a mixture
of at least two iscom complexes or iscom matrix complexes, each
complex comprising one saponin fraction from Quillaja saponaria
Molina, and the use thereof as immunomodulators or adjuvants in
formulations to be used for immunisations including vaccines.
Especially the invention relates to the use of purified,
semipurified or defined fractions of quillaja saponin in iscom and
iscom-matrix adjuvanted vaccines. The use of saponin preparations
according to this invention results in products with increased
tolerability and increased immunogenicity. The preparations may be
used in methods to tailor the immunogenicity with increased control
of inflammatory, hypersensitivity and allergic reactions.
PRIOR ART
[0003] The immune stimulatory properties of quillaja saponins have
been known for long (Ramon 1926) and quillaja saponins have been
used in free form, sometimes in combination with Al(OH).sub.3 in
commercial vaccines since 1950s (Dalsgaard 1978, Ma et al. 1994,
Espinet 1951). A substantially more efficient use of the quillaja
saponins compared to conventional free forms was described by
Morein et al., in 1984--the ISCOM technology (EP 0 109 942 B1, EP 0
242 380 B1 and EP 0 180 564 B1) and a few years later the
ISCOM-matrix technology (Lovgren and Morein 1988, EP 0 436 620
131). Using the iscom technology vaccine antigens are incorporated
into a 40 nm complex consisting of quillaja saponins, cholesterol
and phospholipid(s). The ISCOM-matrix technology employs the
quillaja saponin: cholesterol:phospholipid complex in mixture (not
associated) with the antigen(s). Both technologies decreases or
abolish the haemolytic activity of the quillaja saponins, a
property causing local side effects and adds to the overall
toxicity of quillaja saponin preparations (Bomfod et al 1992).
[0004] Quillaja saponin preparations are heterogeneous mixtures of
surface-active glycosides and serious problems in finding/defining
batches with predicted and consistent adjuvant activity led to the
isolation and characterisation of a "homogenous" fraction denoted
"Quillaja saponaria Molina" (Dalsgaard, 1974). This fraction was
later shown to contain a range of related structures that were
further purified into fractions/peaks by means of reversed phase
HPLC (Kensil 1988, 1991, Kersten 1990 EP 0 362 279 B2, EP 0 555 276
B1). The motivation for this purification was not only to produce
homogenous fractions of saponins that were readily characterised
and defined but also to define a less toxic product. Acute toxicity
or side effects have been major concerns for both veterinary and
particularly human use of quillaja saponins in vaccine
preparations. These goals were only partially met with success, the
purified fractions e.g., QA-21 (EP 0 362 279 132) and combinations
of fractions A and C (WO 96/11711, Iscotec-patent) were indeed
chemically defined compared to "Quillaja saponaria Molina" but they
still caused some toxicity and side effects. Despite the fact that
fraction A virtually lack toxicity, a mixture consisting of 70%
fraction A and 30% fraction C was not or only marginally less toxic
than 100% of fraction C of Quillaja saponaria Molina.
[0005] In work leading to the present invention it was also shown
that the different quillaja saponin fractions had not only
different toxicity but also different immuno-modulating properties
(Johansson et al., EP 0 362 279 B2). By combining these fractions
different immunomodulating capacities were obtained e.g. a Th1
driving or a Th2 driving capacity. It is however desirable to
reduce the side effects that limits the amount of each fraction to
be used in a tolerable formulation.
SUMMARY OF THE INVENTION
[0006] The present invention relates to the use of at least two
purified peaks or defined fractions of quillaja saponin in iscom
and iscom-matrix as separate entities (particles). I.e. these
fractions are not combined in the very same iscom or iscom matrix
particles, and the particles with different loads are mixed
together to constitute a formulation for immunisation. It has
surprisingly turned out that a mixture of iscom or iscom matrixes
each comprising a different fraction of Quillaja saponaria Molina
has lower toxicity than when these Quillaja saponaria Molina
fractions are integrated into the same iscom or iscom matrix
particle. For example the mixture of fraction A-matrix and fraction
C-matrix, or the use of fraction A-matrix or fraction C-matrix
alone were considerably less toxic in mice than when the same
fractions were integrated in the same iscom matrix (Example 4,
table 1). Further, the immunogenicity or immune modulating
properties are easier to tailor, and the possibilities are
considerably enhanced to make improved vaccine formulations
optimised both for the target species and the needs/requirements of
the vaccine antigens.
[0007] Mice are particularly sensitive to quillaja saponins and
overdosing leads to death within 4 days, often within 24 hours.
Therefor mice were used to monitor the effects of toxicity and
immunogenicity of the formulations prepared according to this
invention. The interspecies variation in sensitivity to quillaja
saponin is huge and reflects the needs for species optimisation to
obtain tolerable formulations, but also for steering to obtain
optimal immunogenicity of vaccine formulations. E.g. equines do not
die from large doses of quillaja saponin, but they are prone to
develop fever and local side effects after injection with free
Quillaja saponaria Molina, iscoms and iscom-matrix produced from
Quillaja saponaria Molina or mixed fractions of Quillaja saponaria
Molina.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The invention is illustrated by the following figures of
which:
[0009] FIG. 1 shows the preparation of fractions A, B and C by
HPLC;
[0010] FIG. 2 shows antigen specific antibody responses against
influenza virus micelles as described in the text were tested in
ELISA (log Titre) in the IgG1 (A) and IgG2a (B) subclasses, Mice
(female NMRI) were immunised weeks 0 and 4 with the vaccine
formulations described in Table 2 i.e. groups I through 8. Mice
were bled weeks 3 and 6. The antibody responses were tested from
bleeding collected at week 6.
[0011] FIG. 3 shows the cell mediated immune response measured as
the production of the cytokines IL-5 (A) and IFN-.gamma. (B) by
spleen cells collected week 6 after immunisation as described in
FIG. 2 after stimulation in vitro with influenza virus micelles as
described in the text.
[0012] FIG. 4 shows high dose (50 .mu.g) of QHC in matrix is toxic,
while a high dose of QHA in ISCOM-MATRIX is non-toxic, when
supplemented to OVA to enhance the antibody response in Balb/C mice
(see text). Both formulations enhance similar specific antibody
responses against OVA as measured 3 weeks after the second
immunisation by ELISA for the total IgG response (A) and in the
IgG2a subclass (B).
[0013] FIG. 5 shows synergistic effects of QHA and QHC matrices
when supplemented to OVA to enhance the antibody response in Balb/C
mice (see text). The dose of QHA and C matrices ranged as follows
in group 1, no A or C; Gr. 2, 0.3 .mu.g A no C; Gr. 3, 0.3 .mu.g
A+2 .mu.g C; Gr. 4, 10 .mu.g A no C; Gr. 5, 10 .mu.g A 2 .mu.g C.
The dose of OVA was 10 .mu.g. There were 8 mice per group, which
were immunised twice 4 weeks apart s.c. with respective
formulation. The antibody titres were measured by ELISA against:
[0014] A Total IgG 3 weeks after the first immunisation; [0015] B
IgG2a 2 weeks after the second immunisation; and [0016] C IgG1 2
weeks after the second immunisation.
[0017] There is a highly significant difference between groups 4
and 5 (p<0.0001).
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to a composition comprising a
mixture of at least two iscom complexes each complex comprising
essentially one saponin fraction from Quillaja saponaria Molina.
The iscom complex may be an iscom matrix complex or an iscom
complex.
[0019] Iscom contains at least one glycoside, at least one lipid
and at least one type of antigen substance. The lipid is at least a
sterol such as cholesterol and optionally also phosphatidyl
choline. This complex may also contain one or more other
immunomodulatory (adjuvant-active) substances, and may be produced
as described in EP 0 109 942 B1, EP 0 242 380 B1 and EP 0 180 564
B1.
[0020] An iscom matrix comprises at least one glycoside and at
least one lipid. The lipid is at least a sterol such as cholesterol
and optionally also phosphatidyl choline. The iscom complexes may
also contain one or more other immunomodulatory (adjuvant-active)
substances, not necessarily a saponin, and may be produced as
described in EP 0 436 620 B1.
[0021] The composition according to the invention may comprise
iscom or iscom matrix complexes only or mixtures of iscom complex
and iscom matrix complex. Different iscom and/or iscom matrix may
be mixed wherein different saponin fractions from Quillaja
saponaria Molina are used.
[0022] The invention also covers the use of a mixture of at least
two iscom or iscom matrix complexes each comprising one saponin
fraction from Quillaja saponaria Molina for the preparation of an
immunomodulating pharmaceutical.
[0023] Another aspect of the invention is the use of a mixture of
at least two iscom or iscom matrix complexes according to claim 1
each comprising one saponin fraction from Quillaja saponaria Molina
and at least one antigen for the preparation of a vaccine.
[0024] A further aspect of the invention is the use of a mixture of
at least two iscom matrix complexes according to claim 1 each
comprising one saponin fraction from Quillaja saponaria Molina for
the preparation of an adjuvant.
[0025] The immunogen which is incorporated into or associated with
the iscom matrix in accordance with this invention may be any
chemical entity which can induce an immune response in an
individual such as (but not limited to) a human or other animal,
including but not limited to a humoral and/or cell-mediated immune
response to bacteria, viruses, mycoplasma or other micro-organisms.
The specific immunogen can be a protein or peptide, a carbohydrate,
polysaccharide, a lipopolysaccharide or a lipopeptide; or it can be
a combination of any of these.
[0026] Particularly, the specific immunogen can include a native
protein or protein fragment, or a synthetic protein or protein
fragment or peptide; it can include glycoprotein, glycopeptide,
lipoprotein, lipopeptide, nucleoprotein, nucleopeptide; it can
include a peptide-peptide conjugate; it can include a recombinant
nucleic acid expression product.
[0027] Examples of such immunogens are cited in EP 0 109 942 B1 and
include, but are not limited to, those that are capable of
eliciting an immune response against viral or bacterial hepatitis,
influenza, diphtheria, tetanus, pertussis, measles, mumps, rubella,
polio, pneumococcus, herpes, respiratory syncytial virus,
haemophilias influenza, chlamydia, varicella-zoster virus, rabies
or human immunodeficiency virus.
[0028] The antigens may be incorporated into iscom or coupled on to
iscom or iscom matrix or mixed with iscom and/or iscom matrix. Any
mixtures of such iscom or iscom matrix may be used. One or more
antigens may be used and a transport and passenger antigen may be
used as described in EP 9600647-3 (PCT/SE97/00289).
[0029] The lipids used are particularly those described in the
applicant's patent EP 0 109 942 B1 in particular on p. 3 and in
patent EP 0 436 620 B1 on p. 7 lines 7-24. Especially sterols such
as cholesterol and phospholipids such as phosphatidylethanolamine
and phosphatidylcholine are used. Lipid-containing receptors that
bind to the cell-binding components, such as glycolipids including
the cholera toxin's receptor, which is the ganglioside GM1, and
fucosed blood group antigen may be used. The cell-binding
components can then function as mucus targeting molecule and be
bound to the lipid-containing substances through simply mixing them
with complexes that contain them. Iscom complexes comprising such
receptors and receptors are described in WO 97/30728.
[0030] The term "one saponin fraction from Quillaja saponaria
Molina" is used throughout this specification and in the claims as
a generic description of a semi-purified or defined saponin
fraction of Quillaja saponaria or a substantially pure fraction. It
is important that the fraction does not contain as much of any
other fraction to negatively affect the good results that are
obtained when the mixtures of iscom or iscom matrix comprising
essentially one fraction is used. The saponin preparation may, if
desired, include minor amounts for example up to 40% by weight,
such as up to 30% by weight, up to 25% by weight, up to 20% by
weight, up to 15% by weight, up to 10% by weight, up to 7% by
weight, up to 5% by weight, up to 2% by weight, up to 1% by weight,
up to 0.5% by weight up to 0.1% by weight of other compounds such
as other saponins or other adjuvant materials.
[0031] The saponin fractions according to the invention may be the
A, B and C fractions described in WO 96/11711, the B3, B4 and B4b
fractions described in EP 0 436 620, the fractions QA1-22 described
in EP 0 3632 279 132 Q-VAC (Nor-Feed, AS Denmark), Quillaja
saponaria Molina Spikoside (Isconova AB, Ultunaallen 2B, 756 51
Uppsala, Sweden).
[0032] The fractions
QA-1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20-21 and 22 of
EP 0 3632 279 B2, especially QA-7, 17-18 and 21 may be used. They
are obtained as described in EP 0 3632 279 B2, especially at page 6
and in Example 1 on page 8 and 9.
[0033] Fractions A, B and C described in WO 96/11711 are prepared
from the lipophilic fraction obtained on chromatographic separation
of the crude aqueous Quillaja saponaria Molina extract and elution
with 70% acetonitrile in water to recover the lipophilic fraction.
This lipophilic fraction is then separated by semipreparative HPLC
with elution using a gradient of from 25% to 60% acetonitrile in
acidic water. The fraction referred to herein as "Fraction A" or
"QH-A" is, or corresponds to, the fraction, which is eluted at
approximately 39% acetonitrile. The fraction referred to herein as
"Fraction B" or "QH-B" is, or corresponds to, the fraction, which
is eluted at approximately 47% acetonitrile. The fraction referred
to herein as "Fraction C" or "QH-C" is, or corresponds to, the
fraction, which is eluted at approximately 49% acetonitrile.
[0034] By combining iscom or iscom-matrix complexes comprising
different fractions of Quillaja saponaria Molina it is possible to
produce preparations that are less toxic. It has also turned out
that the effect of the compositions seems to be receptor mediated
i.e. to receptors on the antigen presenting cells (APC) recognising
the complexes. Thus, when two different fractions of Quillaja
saponaria Molina are integrated in the same iscom complex this
complex may bind to receptors with affinity to fraction 1 plus
receptors with affinity to fraction 2, i.e. two sets of receptors.
Whereas when the fractions are in separate iscom particles or iscom
matrix particles each particle will bind to the corresponding
receptor(s) and limited to the receptors for which it has affinity.
When two sets of receptors on the APC are triggered by the same
particle that may cause strong effects leading to side effects.
Moreover, the way that the complexes effect their action via
receptors may be different in different species. Therefore, any
combination of weight % of iscom complexes based on their content
of different fractions of Quillaja saponaria Molina may be
used.
[0035] The use of saponin preparations according to this invention
results in products with increased tolerability, increased
immunogenicity. The preparations may be used in methods to tailor
the immunogenicity including increased control of inflammatory,
hypersensitivity and allergic reactions. This tailor making may be
species dependent and may affect toxicity, tolerability and
immunogenicity.
[0036] Any ratio of subfragments of Quillaja saponaria Molina
saponins may be used. Also, any combination of subfragments of
Quillaja saponaria Molina may be used. Thus, two or more sub
fragments may each be integrated into iscom or iscom matrix complex
and used in the mixture according to the invention.
[0037] Preferably mixtures of iscom and/or matrix are used in which
the fraction Quillaja saponaria Molina and fraction Quil C are
separately incorporated into different iscom complexes or matrix.
As mentioned above any combinations of weight % of the different
iscom complexes based on their content of fraction A and C of
Quillaja saponaria Molina respectively may be used. The mixtures
may comprise from, 0.1 to 99.9 by weight, 5 to 95% by weight, 10 to
90% by weight 15 to 85% by weight, 20 to 80% by weight, 25 to 75%
by weight, 30 to 70% by weight, 35 to 65% by weight, 40 to 60% by
weight, 45 to 55% by weight, 40 to 60%, by weight, 50 to 50% by
weight, 55 to 45% by weight, 60 to 40% by weight, 65 to 35% by
weight, 70 to 30% by weight, 75 to 25% by weight, 80 to 20% by
weight, 85 to 15% by weight, 90 to 10% by weight, 95 to 05% by
weight, of iscom complexes comprising fraction A of Quillaja
saponaria Molina (as herein defined) and the rest up to 100% in
each case of interval of iscom complexes comprising fraction C of
Quillaja saponaria Molina (as herein defined), counted on the
content of the sum fractions A and C of Quillaja saponaria Molina
in the iscom complexes.
[0038] The mixture may comprise from 75% to 99.5% by weight of
fraction A and 0.5% to 25% by weight of fraction C. Preferably, the
mixture comprises from 90% to 99% by weight of fraction A and 1% to
10% by weight of fraction C. A particularly preferred preparation
comprises about 91% to 98% by weight of fraction A and about 2% to
9% by weight of fraction C, especially about 92% to 96% by weight
of fraction A and about 4% to 8% by weight of complexes of fraction
C counted on the content of the sum fractions A and C of Quillaja
saponaria Molina in the iscom complexes.
[0039] All intervals mentioned above may be used for any
combination of any fraction of Quillaja saponaria Molina in
formulations for administration to any type of human or animal
species. Examples of animal species to which the formulations
according to the invention may be administrated are companion
animals such as cats, dogs, horses, birds such as parrots,
economical important species such as cattle, e.g. bovine species,
swines, sheep, goats. Preferably more than 50% by weight of
fraction C is used in combination with any of the other fractions
and especially in combination with fraction A. Thus, from
50.5-99.5% by weight of C and 0.5-49.5% by weight of A may be
used.
[0040] When prepared as described herein, Fractions A, B and C of
Quillaja saponaria Molina each represent groups or families of
chemically closely related molecules with definable properties. The
chromatographic conditions under which they are obtained are such
that the batch-to-batch reproducibility in terms of elution profile
and biological activity is highly consistent.
[0041] The present invention also extends to a vaccine composition
comprising as the active component thereof either (i) an
immunogenic iscom as broadly described above or (ii) an iscom
matrix as broadly described above and at least one immunogen,
together with one or more pharmaceutically acceptable carrier
and/or diluents.
[0042] The formulation of such vaccine compositions is well known
to persons skilled in this field. Suitable pharmaceutically
acceptable carriers and/or diluents include any and all
conventional solvents, dispersion media, fillers, solid carriers,
aqueous solutions, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like. The use of
such media and agents for pharmaceutically active substances is
well known in the art, and it is described, by way of example, in
Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing
Company, Pennsylvania, USA. Except insofar as any conventional
media or agent is incompatible with the active ingredient, use
thereof in the pharmaceutical compositions of the present invention
is contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0043] The iscom or iscom matrix complex according to the invention
comprising each essentially one fraction of Quillaja saponaria
Molina may be administrated as a mixture or separately at the same
administration site or at different administration sites at the
same or at different times. Different fractions of Quillaja
saponaria Molina may be used in the different iscom complexes and
matrix complexes and in the different compositions.
[0044] The invention therefore also relates to a kit of parts
comprising at least two parts, wherein each part comprises one
iscom complex or one iscom matrix complex each complex comprising
one saponin fraction from Quillaja saponaria Molina. Different
fractions of Quillaja saponaria Molina may be used in the different
iscom complexes and matrix complexes in the different compositions
in the different parts.
[0045] The compositions and kit of parts according to the invention
may also comprise at least one other adjuvant than fractions from
Quillaja saponaria Molina. These adjuvants may be mixed with the
iscom and/or iscom matrix complexes or be integrated into the
complexes.
[0046] Examples of other adjuvants that can be incorporated in the
iscom and iscom matrix are any adjuvant, natural or synthetic, with
desired immunomodulatory effect, e.g. muramyl dipeptide
(MDP)-derivatives, such as fatty acid, substituted MDP, threonyl
analogues of MDP; DDA, poly anions such as Dextran sulphate,
lipopolysaccharides such as saponins (other than Quil A). ("Future
prospects for vaccine adjuvants", Warren, H. S. (1988) CRC Crit.
Rev. Immunol. 8:2, 83-101; "Characterisation of a non-toxic
monophosphoryl lipid A", (1987) Johnson, A. G. et al, Rev. Infect.
Dis. 9:5, 5512-5516; "Developmental status of synthetic
immunomodulators", Berendt, M. J. et al (1985), Year Immunol.
193-201; "Immunopotentiating conjugates", Stewart-Tull, D. E.,
Vaccine, 85, 3:1, 40-44).
[0047] It is especially advantageous to formulate compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the human subjects to
be treated; each unit containing a predetermined quantity of active
ingredient calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carder and/or
diluent.
[0048] In yet another aspect, the present invention extends to a
method of eliciting or inducing an immune response in an
individual, which comprises administering to the individual an
immunologically effective amount of a vaccine composition as
broadly described above.
[0049] As previously mentioned, the individual may be a human or
other animal, including a livestock animal (e.g. sheep, cow or
horse), laboratory test animal (e.g. mouse, rat, rabbit or guinea
pig), companion animal (e.g. dog or cat) or wild animal.
[0050] An immunologically effective amount means that amount
necessary at least partly to attain the desired immune response, or
to delay the onset of, inhibit the progression of, or halt
altogether, the onset or progression of the particular condition
being treated. This amount varies depending upon the health and
physical condition of the individual to be treated, the taxonomic
group of individual to be treated, the capacity of the individual's
immune system to synthesise antibodies, the degree of protection
desired the formulation of the vaccine, the assessment of the
medical situation, and other relevant factors. It is expected that
the amount will fall in a relatively broad range that can be
determined through routine trials.
[0051] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated component or group of components
but not the exclusion of any other components or group of
components.
[0052] All publication mentioned herein are hereby incorporated as
reference. The invention will now be described by the following
non-limiting examples.
EXAMPLE 1
Preparation of Quillaja saponaria Molina Subfragment Saponins
[0053] Purification of crude Quillaja saponaria Molina extract to
fractions A, B and C. A solution (0.5 ml) of crude Quillaja bark
extract in water (0.5 g/ml) is pre-treated on a sep-pak column
(Waters Associates, MA).
[0054] The pre-treatment involves washing of the loaded sep-pak
column with 10% acetonitrile in acidic water in order to remove
hydrophilic substances. Lipophilic substances including QH-A, QH-B
and QH-C are then eluted by 70% acetonitrile in water.
[0055] The lipophilic fraction from the sep-pak column is then
separated by a semipreparative HPLC column (CT-sil, C8,
10.times.250 mm, ChromTech, Sweden). The sample is eluted through
the column by a gradient from 25% to 60% acetonitrile in acidic
water. Three fractions are collected from the :HPLC column during
the separation. The residues after evaporation of these three
fractions constitute QH-A, QH-B and QH-C.
[0056] The fractions designated QH-A, QH-B and QH-C were eluted at
approximately 39, 47 and 49% acetonitrile respectively. The exact
elution profile and conditions are shown in FIG. 1.
EXAMPLE 2
Preparation of Iscom Matrix
[0057] Materials:
[0058] Cholesterol, e.g., Sigma C 8503
[0059] Phosphatidyl choline (egg derived) e.g., Sigma P 3556
[0060] MEGA-10 (Bachem AG, Switzerland)
[0061] Quillaja saponin fractions A and C (patent WO9611711)
[0062] 0.22 .mu.m Sterile filters (Acrodisc)
[0063] PBS (10 mM phosphate buffered 150 mM saline, pH 6.8-7.4)
[0064] Slide-A-Lyzer cassettes MW cut off 12-14.000 (Pierce)
[0065] MEGA-10 (Stock Solution):
[0066] Make a 20% (w/w) stock solution by adding 8 ml distilled
water to 2.0 g of dry solid MEGA-10. Dissolve by gentle heating
(30-50.degree. C.) Filter through a 0.22 nm sterile filter, aliquot
and store at 20.degree. C.
[0067] Lipid Mixture (15 mg/ml):
[0068] Dissolve 100 mg of each cholesterol and phosphatidyl choline
in 10 ml 20% MEGA-10. The lipids dissolve slowly at 30-60.degree.
C. with slow stirring. Filter through a 0.22 nm sterile filter,
aliquot and store at -20.degree. C. After freezing, the lipid
mixtures need to be heated up to 40.degree. C. until clear.
Temperate all solutions to 24.+-.1.degree. C.
[0069] Saponin Stock Solutions (100 mg/ml):
[0070] 1.0 gram of Quillaja saponaria Molina fractions (A or C is
dissolved in sterile distilled water. Keep aliquots frozen at
-20.degree. C.). Filter through a 0.22 run sterile filter, aliquot
and store at -20.degree. C.
[0071] The different iscom-matrix is preparations are produced as
outlined in Table 1.
[0072] Prepare the mixtures as follows.
[0073] Add 2 ml PBS to a 50 ml falcon tube
[0074] 1 Add the lipid mixture and mix thoroughly
[0075] 2 Add saponin and mix thoroughly
[0076] 3 Add PBS to a final volume of 12.0 ml, mix thoroughly
[0077] 4 Incubate for 30 minutes
[0078] 5 Fill into Slide-A-Lyzer
[0079] 6 Dialyse against 4 changes of 2 liters of PBS
(24.+-.1.degree. C.) (for 48-60 hours)
[0080] 7 Aspirate from Slide-A-Lyzer and filter through 0.22 nm
sterile filter.
[0081] The formation of iscom-matrix was verified by negative
staining electron microscopy and the resulting concentrations of
quillaja saponin was determined by HPLC.
TABLE-US-00001 TABLE 1 Lipid-mixture Quillaja saponin PBS Amount
Volume Amount Volume Volume Preparation (mg) (.mu.l) (mg) (.mu.l)
(ml) A-matrix 12 800 48 480 2.0 + 8.72 C-matrix 12 800 30 300 2.0 +
8.90 703-matrix 12 800 42 420 2.0 + 8.78
EXAMPLE 3
Preparation of PR-8 Protein Micelles
[0082] 1 Dilute 12 mg of PR-8 monomers (1.5 mg/ml) with PBS to a
final protein concentration of 1.0 mg/ml.
[0083] 2 Filter through 0.22 nm sterile filter
[0084] 3 Fill into Slide-A-lyzer
[0085] 4 Dialyse against 4 changes of 2 liters of PBS
(24.+-.1.degree. C.) (for 48-60 hours)
[0086] 5 Aspirate from Slide-A-Lyzer
EXAMPLE 4
Immunisation Study
[0087] This example was carried out to show in a comparative study
that iscom-matrix composed of a mixture matrix particles provokes
minimal degree of side effects. One set of particles contain QHA as
the only saponin and the other set of particles contain QHC as the
only saponin and were prepared according to Example 2. This
formulation is named matrix with a "mixture of particles". The
comparison is done with an iscom matrix as described in Paten WO
96/11711 i.e. each particle contain both QHA and QHC in e.g. a
ratio of 70% QHA and 30% of QHC. This is a matrix with all in one
particle.
[0088] Balb/C mice were immunised days 0 and 42 with 1 .mu.g of PR8
micelles (prepared as described in example 3) mixed with the iscom
matrix formulation matrix with a mixture of particles and compared
with the iscom matrix with all in one particle, or with iscom
matrix containing 100% QHA or 100% QHC as described in Table 2.
Groups in which more than 50% of the mice died or suffered from
unacceptable side effects by the treatment were culled and excluded
from further investigations.
[0089] Serum samples were taken from all mice in groups 1-7 day 56,
two weeks after the booster administration. The sera were screened
for antigen specific antibodies of IgG 1 (A) and IgG2a (B)
subclasses. Group 8 in the figure represents unvaccinated mice. The
results are shown in FIG. 2.
[0090] After the second bleeding spleens were taken from two mice
per group (groups 2, 4, 5, 6 and 7). The spleen cells were
stimulated with PR8 micelles in vitro and the antigen specific
induction of IL-5 (A) and IFN-.gamma. (B) was measured. Group 8 in
the figure represents unvaccinated mice. The results are shown in
FIG. 3.
TABLE-US-00002 TABLE 2 Balb/C mice were immunised days 0 and 42
with the iscom matrix formulation matrix with a mixture particles
(MIX groups 1, 2, and 3) and compared with, the iscom matrix with
all in one particle (CONV groups 7, 8, 9, 10 and 11), or with iscom
matrix containing 100% QHA (groups 4 and 5) or 100% QHC (group 6
and 12). Group Amount A:C MIX/ No of Dead#/ no (.mu.g) (ratio) CONV
mice total 1 50 80:20 MIX 8 2/8 2 50 92:8 MIX 8 0/8 3 50 96:4 MIX 8
0/8 4 50 100% A 8 0/8 5 10 100% A 8 0/8 6 10 100% C 8 0/8 7 10
70:30 CONV 8 0/8 8 50 80:20 CONV 8 8/8 9 50 92:8 CONV 8 6/8 10 50
96:4 CONV 8 5/8 11 50 70:30 CONV 8 8/8 12 50 100% C 8 8/8 #Mice
that were euthanized or died within 24 h after administration. A:C
ratio (weight) of quillaja saponin fraction A and C
Results
[0091] Mice immunised PR8 micelles adjuvanted with high dose (50
.mu.g) of iscom matrix containing 80% QHA and 20% of QHC i.e. 10
.mu.g died within 1 or 2 days. Likewise did mice immunised with 50
.mu.g of the formulation 100% QHC died within 2 days.
[0092] In contrast mice immunised with 50 .mu.g of the formulation
100% QHA survive without any noticed adverse reactions. I.e. the
lower dose of QHC was sufficient to kill mice when incorporated in
the same matrix particle as QHA (CONV group 8 in Table 2). Even
when a low amount as 4 .mu.g (8(%) of QHC in combination with 46
.mu.g (92%) of QHA in a CONV matrix killed 6 out of 8 mice (group 9
Table 2). Also 2 .mu.g (4%) QHC killed mice when combined with QHA
in CONV matrix (group 10 Table 2). Mice in group 6 (Table 2)
received 10 .mu.g QHC being 100% (i.e. no QHA) in the matrix and
all mice survived.
[0093] Thus, the mice were more sensitive to QHC when combined with
QHA in the same CONV matrix particle (groups 8, 9, and 10).
[0094] Mice receiving low dose matrix i.e. 10 .mu.g of total
saponin divided on 70% of QHA and 30% of QHC survived all. In this
case the mice received 3 .mu.g QHC.
[0095] Mice immunised with PR8 micelles adjuvanted with a
formulation containing different matrix particles i.e. a mixture of
particles (MIX) including one set of QHA and one set of QHC
survived much higher doses of this matrix formulation than the CONV
formulation. Mice injected with the formulation with 92% QHA (46
.mu.g) and 8% QHC (4 .mu.g) group 2 in Table 2 (92:8) or with the
formulation 96:4 (Group 3 in Table 2) containing 2 .mu.g of QHC
survived all. This outcome shall be compared with that of
corresponding amounts of QHA and QHC in CONV matrix (groups 9 and
10 Table 2) which caused high mortality.
[0096] Thus, mortality-toxicity can be avoided by physical
separation of QHC from QHA and distribute them into different
matrix particles.
Enhancement of Antibody Response
[0097] The results are shown in FIG. 2. Antigen specific response
divided into IgG subclasses. Mice (female NMRI) were immunised
weeks 0 and 4 with the vaccine formulations described in Table 2.
Mice were bled weeks 3 and 6. The IgG1 (.sup.10log Elisa titres)
response at week 6 is shown in A and the corresponding IgG2
response in B.
[0098] An important finding in this experiment is that the immune
enhancing capacity is retained or enhanced as measured by antibody
responses when QHA and QHC are separated into different sets of
particles, as demonstrated in FIG. 2.
[0099] In FIG. 2 it is shown that a mixture of particles (MIX)
enhance the same level of IgG1 antibody (FIG. 2A) to PR8 micelles
as the same dose of QHA and QHC in the same proportions when
incorporated in the same particle i.e. CONV particles. However,
higher levels of IgG2a antibody antibodies were enhanced by the MIX
formulation (FIG. 2B). The groups 2 and 3 (MIX) shall be compared
with the groups 9 and 10 (CONV) QHA-QHC matrix and with 100% of low
dose QHC matrix (group 6) and with 100% of QHA high dose matrix
(group 4 Table 2). Mice in group 7 injected with a low dose of 10
.mu.g (CONV 70:30), i.e. the dose the mice can accept, responded
with a potent IgG1 (FIG. 2A) response, but a low IgG2a response
(FIG. 2B).
[0100] Thus, the invention with a matrix formulation with a mixture
of matrix particles can be given in high doses evading side
effects, enhance the antibody response to higher levels than those
with than the CONV matrix. Particularly the IgG2a response is
enhanced. The IgG2a response is e.g. particularly important for
defence against intracellular parasites e.g. viruses.
Enhancement of Cell Mediated Immune Response
[0101] The CONV matrix formulations have inferior capacity to
enhance cell mediated immunity in the doses tolerated than the MIX
formulations (FIGS. 3A and B). The MIX formulation (92:8, group 2)
enhance considerably higher IL-5 levels than the CONV (70:30 group
7), QHA-QHC formulation, or the 100% QHC matrix. Formulation (group
6). The mix (92:8, group 9) formulation also enhances the
IFN-.gamma. considerably better than the QHC 100% matrix (group 6)
or the CONV (70:30, group 7) formulation.
[0102] To note is that QHA has a strong capacity to enhance cell
mediated immune responses measured by IL-5 and IFN-.gamma.
production, but a low capacity to enhance antibody response.
[0103] In conclusion the invention defines a concept for iscom and
iscom matrix formulations that considerably reduce toxicity and
side effects allowing potent doses of the adjuvant active molecules
without loosing capacity to enhance immune response.
[0104] Moreover, while a low but acceptable dose of a QHC matrix
formulation has good capacity to enhance and IgG1 response it is
lower with regard to the important IgG2a response. The capacity of
QHC matrix to induce cell mediated immunity is also comparatively
low to that of the invention.
[0105] The QHA matrix potently enhances cell mediated immunity, but
is inferior to the invention to enhance antibody mediated
immunity.
[0106] The invention with mixed matrix particles is superior to
matrix formulations containing QHA and QHC in the same particle
(CONV) measured by IgG2a antibody response and measured by cell
mediated responses.
[0107] The new invention enhances a complete immune response and is
therefore superior to the earlier described matrix formulations,
which this example 4 shows.
EXAMPLE 5
[0108] In this experiment it is emphasised that that QHA is well
tolerated and has a strong immune enhancing and immune modulatory
capacity. Ovalbumin (OVA) is used because it is a weak antigen and
as such it does not induce a Th1 type of response. QHA is compared
with QHC, since the latter is evaluated in human clinical
trial.
Materials and Methods
[0109] Preparation of Quillaja saponaria Molina sub fragments is
described in EXAMPLE 1.
[0110] Preparation of Iscom matrix is described in EXAMPLE 2.
Experimental Design
[0111] Group 1 consisted of 8 mice immunised twice 4 weeks apart
subcutaneously (s.c.) with 10 .mu.g OVA adjuvanted with 50 .mu.g
QHA. Group 2 had the same number of mice immunised by the same
procedure but the adjuvant was 50 .mu.g QHC.
[0112] The shown antibody responses are from sera collected 2 weeks
after the boost.
Antibody Determination
[0113] The specific OVA serum antibody responses were determined by
ELISA both for total IgG response and in the IgG2a subclasses as
described in EXAMPLE 4 using a standard procedure with 10 .mu.g of
OVA per ml for coating the ELISA plates as test antigen.
Results
[0114] All mice immunised with OVA adjuvanted with QHA matrix
survived and did not develop any sign of discomfort. Out of 8 mice
immunised with OVA and juvanted with QHC matrix 4 mice died i.e.
50%.
[0115] There is no significant difference between the groups with
regard total antibody responses (FIG. 4A), but the ELISA titres
varied more between the animals in group 1, i.e. mice immunised
with QHA.
[0116] There was no difference in mean titres in the IgG2a subclass
between group 1 and 2 except that the ELISA titres varied more
between individual animals in group 2 i.e. mice immunised with QHC
(FIG. 4B).
[0117] In the second experiment of this example it was explored
whether QHA matrix can benefit from the complementation with
another adjuvant. The dose of QHA matrix and C matrix ranged as
follows in group 1, no A or C; Gr. 2, 0.3 .mu.g A no C; Gr. 3, 0.3
.mu.g A+2 .mu.g C; Gr. 4, 10 .mu.g A no C; Gr. 5, 10 .mu.g A 2
.mu.g C. The dose of OVA was 10 .mu.g. There were 8 mice per group,
which were immunised twice 4 weeks apart s.c. with respective
formulation. (FIG. 5 A, B and C).
[0118] Sera were collected 3 weeks after the first immunisation and
2 weeks after the boost.
[0119] The specific OVA serum antibody responses were determined by
ELISA for total IgG response and in the IgG2a and IgG 1 subclasses
as described (Johansson, M and Lovgren-Bengtsson (1999) Iscoms with
different quillaja saponin components differ in their
immunomodulating activities. Vaccine 19, 2894-2900).
Results
[0120] After the first immunisation no antibody response was
recorded in mice receiving non-adjuvanted OVA or OVA adjuvanted
with 0.3 .mu.g of QHA matrix with and without 2 .mu.g of QHC matrix
(FIG. 5A).
[0121] After the second immunisation a low response was detected in
3 out of 8 mice immunised with non-adjuvanted OVA in the IgG 1
subclass but no response was recorded in the IgG2a subclass.
Neither was antibody responses recorded with the lowest adjuvant
doses of QHA matrix i.e. 0.3 .mu.g with and without 2 .mu.g of QHC
matrix. There was a clear enhancement of the antibody response in
the IgG2a subclass when the low dose of 2 .mu.g QHC was added to
the 10 .mu.g of QHA (FIG. 5B).
Conclusion
[0122] QHA MATRIX has a very low toxicity and still a strong
modulatory effect, when included in ISCOMATRIX as shown by
promoting a strong TH1 type of response, in contrast to the
non-adjuvanted or the very low adjuvanted OVA, which only elicited
antibody response in the IgG 1 subclass. It is also shown that the
QHA matrix synergies with a low dose of QHC matrix. These results
are important, because it makes it possible to optimise the
adjuvant effect and minimise the side effects in a simple manner.
As shown for a weak antigen as OVA requiring potent adjuvant.
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