U.S. patent application number 15/054801 was filed with the patent office on 2016-06-30 for quil a fraction with low toxicity and use thereof.
The applicant listed for this patent is NOVAVAX AB. Invention is credited to Jill Ekstrom, Kefei Hu, Karin Lovgren Bengtsson, Bror Morein, Katarina Ranlund.
Application Number | 20160184427 15/054801 |
Document ID | / |
Family ID | 27731119 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160184427 |
Kind Code |
A1 |
Morein; Bror ; et
al. |
June 30, 2016 |
QUIL A FRACTION WITH LOW TOXICITY AND USE THEREOF
Abstract
Fraction A of Quil A can be used together with at least one
other adjuvant for the preparation of an adjuvant composition,
where the included adjuvant components act synergistically to
enhance level of immune response and have synergistic
immunomodulating activity on the co-administered antigens or
immunogens. Other adjuvants can comprise saponins, naturally
occurring, synthetic or semisynthetic saponin molecules; e.g.
saponins and saponin fractions from Quil A, cell wall skeleton,
block polymers, TDM, lipopeptides, LPS and LPS-derivatives, Lipid A
from different bacterial species and derivatives thereof, e.g.,
monophosphoryl lipid A, CpG variants, CT and LT or fractions
thereof.
Inventors: |
Morein; Bror; (Uppsala,
SE) ; Lovgren Bengtsson; Karin; (Uppsala, SE)
; Ekstrom; Jill; (Uppsala, SE) ; Ranlund;
Katarina; (Uppsala, SE) ; Hu; Kefei; (Uppsala,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVAVAX AB |
Uppsala |
|
SE |
|
|
Family ID: |
27731119 |
Appl. No.: |
15/054801 |
Filed: |
February 26, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14714664 |
May 18, 2015 |
|
|
|
15054801 |
|
|
|
|
14445690 |
Jul 29, 2014 |
|
|
|
14714664 |
|
|
|
|
10562866 |
May 16, 2006 |
8821881 |
|
|
PCT/SE2004/001038 |
Jul 7, 2004 |
|
|
|
14445690 |
|
|
|
|
Current U.S.
Class: |
424/278.1 |
Current CPC
Class: |
A61K 2039/57 20130101;
A61K 39/39 20130101; A61K 2039/55577 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 7, 2003 |
SE |
0301998-1 |
Claims
1. Use of fraction A of Quil A together with at least one other
adjuvant for the preparation of an adjuvant composition with
synergistic effect including enhancement of immune responses and
immunomodulating activity.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
14/714,664, filed May 18, 2015, which is a continuation of
application Ser. No. 14/445,690, filed Jul. 29, 2014, now
abandoned, which is a continuation of application Ser. No.
10/562,866, filed May 16, 2006, now U.S. Pat. No. 8,821,881, which
is a national stage application of International Appl.
PCT/SE04/01038, filed Jul. 7, 2004 and which claims priority of
Swedish patent Appl. No. 0301998-1, filed Jul. 7, 2003, all of
which are hereby incorporated by reference.
FIELD OF INVENTION
[0002] The present invention relates to the use of fraction A of
Quil A together with at least one other adjuvant for the
preparation of an adjuvant composition with synergistic effects
including level of immune responses and immunomodulating
activity.
PRIOR ART
[0003] There is a great need for efficient adjuvant and vaccine
delivery systems both for man and animal to be used for immune
prophylactics or for immune therapy. For animal vaccines there are
a number of different adjuvants including iscom and iscom matrix
adjuvanted vaccines. However, only aluminium hydroxide and calcium
phosphate adjuvants are commercially available in human vaccines,
and an oil emulsion adjuvant (MF59) has recently been registered
for a human influenza vaccine. Thus, there is a lack of efficient
adjuvants, particularly for human vaccines. Adjuvants are not only
important for enhancing the level immune response but even more for
the quality or type of immune response, which has to match the type
of infection the vaccine is intended to protect against. With
regard to pathogens establishing themselves intracellularly like
viruses, but also some bacteria and parasites, a so-called Th1 type
of immune response is required for optimal immune protection, and
in many cases a Th1 type of response is a prerequisite for immune
protection. However, it is also now well established, that a pure
Th1 or Th2 type of response may cause side effects, since a balance
between the two types of the T helper cells are required for immune
regulation. I.e. the Th1 response regulate the Th2 response e.g. by
the production of IFN-.gamma.and the Th1 response is regulated by
the Th2 response e.g. by the production of the cytokine IL10. Thus,
the Th1-Th2 balance is essential to avoid side effects. To be able
to induce correct type of immune response for protection against
the various pathogens a number of adjuvants will be required. A Th1
response is reflected by the IgG2a antibody response, and therefore
used as a marker for Th1 t helper cell response. One important
aspect for adjuvants is the safety including the fact that the
immune response evoked shall have a quality to avoid side effects
when a subsequent infection occurs after the vaccination. Severe
side effects were the case with respiratory syncytial virus when an
aluminium hydroxide adjuvanted formalin inactivated respiratory
syncytial virus (RSV) vaccine was tried in children nearly 30 years
ago. The vaccinated children became sicker and there was a higher
death rate among them after natural infection with RSV than in
non-vaccinated children.
[0004] Acute toxicity or side effects have been major concerns for
both veterinary and particularly human use of quillaja saponins in
vaccine preparations. Theses goals were only partially met with
success, the purified fractions e.g., QA-21 (EP 0 362 279 B2) and
combinations of fractions A and C (WO 96/11711, lscotec-patent)
were indeed chemically defined compared to "Quillaja Saponaria
Molina" but they still caused some toxicity and side effects.
[0005] It has now turned out that fraction A of Quil A has a low
toxicity, and in low dose enhance and the level of immune responses
and the immunomodulatory capacity of other adjuvants in suboptimal
doses, which when used by themselves may be toxic or cause
side-effects in efficient doses. Thus, it facilitates the use of
other adjuvants which, when used by themselves, might be toxic in
doses they are efficient.
SUMMARY OF THE INVENTION
[0006] The present invention relates to the use of fraction A of
Quil A together with at least one other adjuvant for the
preparation of an adjuvant composition with synergistic effect to
enhance the level of immune responses and immunomodulating
activity. It especially concerns the use of fraction A of Quil A in
a composition comprising iscom particles wherein the different
fractions of Quil A are integrated into different iscom and iscom
matrix particles.
DESCRIPTION OF DRAWINGS
[0007] FIG. 1
[0008] High dose (50 .mu.g) of QHC in matrix is toxic, while a high
dose of QWT in 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 lgG2a subclass (B).
[0009] FIG. 2
[0010] Synergistic effects of QWT-matrix and QHC-matrix when
supplemented to OVA to enhance the antibody response in Balb/C mice
(see text). The dose of QWT-matrix and QHC-matrix ranged as follows
in group 1, no QWT or C; Gr. 2, 0.3 .mu.g QWT no C; Gr. 3, 0.3
.mu.g QWT +2 .mu.g C; Gr. 4, 10 .mu.g QWT no C; Gr. 5, 10 .mu.g QWT
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 OVA:
[0011] A Total IgG 3 weeks after the first immunisation.
[0012] B IgG2a 2 weeks after the second immunisation.
[0013] C IgG1 2 weeks after the second immunisation.
[0014] FIG. 3
[0015] Toxicity of QWT and AC (i.e. 703) respiratory syncytial
virus (RSV) iscoms measured by survival rate in newborns (1 week
old) mice after one intraperitoneal injection with 1 .mu.g iscom
(protein). The protein/saponin ratio is 1/1.
[0016] FIG. 4
[0017] Antibody response of newborn (1 week old) and adult mice
after one intraperitoneal immunisation and a subsequent boost after
3 weeks with 1 .mu.g iscom (protein). The protein/saponin ratio is
1/1.
[0018] FIG. 5
[0019] Cytotoxic T cell (CTL) response after one intraperitoneal
immunisation with 1 .mu.g iscom (protein). The protein/saponin
ratio is 1/1. The spleen cells were collected 1 and 3 weeks after
the intraperitoneal immunisation.
[0020] FIG. 6
[0021] QWT matrix is less toxic on VERO cells (a monkey cell line)
than 703 matrix and C matrix after exposure for 72 hrs in culture
measured by growth rate proportional (%) to non-exposed cell
cultures. QWT matrix is well tolerated at all concentrations tested
i.e. up to 1300 .mu.g. No cell growth is recorded in cell cultures
exposed 800 .mu.g of 703 matrix or 45 .mu.g of QHC matrix.
[0022] A. Exposure of VERO cells to QWT matrix and 703 matrix as
indicated.
[0023] B. Exposure of VERO cells to QHC matrix as indicated.
[0024] FIG. 7
[0025] QWT matrix is less toxic on spleen cells obtained from mice
than C matrix after exposure for 72 hrs in culture measured by
growth rate measured by a colorimetric method as described in the
text. The growth rate is compared with spleen cells grown in medium
alone or together with mitogen Con A.
[0026] A. Exposure of spleen cells to QWT matrix in decreasing
doses from 10 to 1.25 .mu.g as indicated.
[0027] B. Exposure of spleen cells to QHC matrix in decreasing
doses from 10 to 1.25 .mu.g as indicated.
[0028] FIG. 8
[0029] This figure shows the preparation of fractions A, B and C by
HPLC;
[0030] FIG. 9
[0031] This figure shows synergistic effect of QWT-matrix and
QHC-matrix. Groups of 8 female Balb/c mice were immunised s.c. at
the base of the tail with 5 micrograms of ovalbumin (OVA) alone (Gr
1) or mixed with A-matrix (Gr 2) or C-matrix (Gr 3) respectively or
a mixture of A-matrix and C-matrix (Gr 4). The mice were immunised
at weeks 0 and 4, serum samples were taken at weeks 3 (prime) and 6
(booster). The sera were tested for antigen specific antibodies IgG
or subclasses (IgG 1 and IgG2a) in ELISA. The antibody response to
OVA (5 .mu.g) is strongly enhanced by a combination of QWT-Matrix
and QHC-Matrix compared to the use of either Matrix on its own.
Particularly the IgG2a response is enhanced. The enhancement (IgG
and IgG2a) is demonstrated three weeks after priming (A and B).
[0032] FIG. 10
[0033] With reference to FIG. 9, the enhancement of IgG also is
shown two weeks after booster.
[0034] FIG. 11
[0035] With reference to FIG. 9, the enhancement of IgG1 also is
shown two weeks after booster.
[0036] FIG. 12
[0037] With reference to FIG. 9, the enhancement of IgG2a also is
shown two weeks after booster.
[0038] FIG. 13
[0039] This figure shows antibody responses (total IgG and IgG2a)
to OVA after immunization with 5 mg of OVA alone (Gr 1) or mixed
with QWT (Gr2) or mixed with a combination of QWT and CT (Gr 3) or
QWT and MPL (Gr 4) respectively. The synergistic adjuvant effect of
QWT-Matrix given together with CT or MPL is demonstrated for a week
immunogen; OVA. Both the magnitude of the IgG response (A) and
particularly a specific enhancement (immunomodulation) of the IgG2a
subclass (B) should be noted.
[0040] FIG. 14
[0041] This figure shows antibody response (total IgG) to TT
(Tetanus Toxoid) after immunization with 2.5 Lf of TT alone or with
CT (1 or 0.2 .mu.g) or a combination of QWT and 0.2 .mu.g of CT
after first immunization.
[0042] FIG. 15
[0043] With reference to FIG. 14, antibody response (total IgG)
also is shown after booster.
[0044] FIG. 16
[0045] This figure shows antibody response (IgG2A) to TT (Tetanus
Toxoid) after immunization with 2.5 Lf of TT alone or with CT (1 or
0.2 .mu.g) or a combination of QWT and 0.2 .mu.g of CT after first
immunization.
[0046] FIG. 17
[0047] With reference to FIG. 16, antibody response (IgG2A) also is
shown after booster.
[0048] Antibody response to TT (Tetanus Toxoid) is enhanced and
modulated by addition of QWT-Matrix. The IgG response after
addition of QWT-Matrix to a low dose of CT (0.2 .mu.g) is in the
same range as that of 1 .mu.g of CT (FIG. 14 and FIG. 15). The
IgG2a response (FIG. 16 and FIG. 17) is however strongly enhanced,
indicating a synergistic modulatory effect of QWT-Matrix and
CT.
[0049] FIG. 18
[0050] This figure shows antibody response (total IgG) to TT
(Tetanus Toxoid) after immunization with 2.5 Lf of TT alone or with
MPL (50 or 10 .mu.g) or a combination of QWT and 10 .mu.g MPL after
first immunization.
[0051] FIG. 19
[0052] With reference to FIG. 18, antibody response (total IgG) to
TT (Tetanus Toxoid) also is shown after booster.
[0053] FIG. 20
[0054] This figure shows antibody response (IgG2a) to TT (Tetanus
Toxoid) after immunization with 2.5 Lf of TT alone or with MPL (50
or 10 .mu.g) or a combination of QWT and 10 .mu.g MPL after first
immunization.
[0055] FIG. 21
[0056] With reference to FIG. 20, antibody response (IgG2a) to TT
(Tetanus Toxoid) also is shown after booster.
[0057] The adjuvant effect of Monophosphoryl Lipid A (MPL),
measured as antibody response to Tetanus Toxoid (TT), is enhanced
and modulated by addition of QWT-Matrix. The IgG response after
addition of QWT-Matrix to a low dose of MPL (10 .mu.g) higher than
that of both 50 and 10 .mu.g of MPL (FIG. 18 and FIG. 19). The
IgG2a response (FIG. 20 and FIG. 21) is strongly enhanced,
indicating a synergistic modulatory effect of QWT-Matrix and
MPL.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The invention relates to the use of fraction A of Quil A
together with at least one other adjuvant for the preparation of an
adjuvant composition with synergistic effect to enhance the level
and quality immunomodulating activity. It especially relates to the
use of fraction A of Quil A together with one or more other
adjuvants where fraction A at a low and well tolerated dose
synergistically enhance the immuno enhancing effect of the co
administered adjuvant, which by its own is too toxic for
prophylactic or clinical use. I.e. a low well tolerated (otherwise
sub-optimal) dose of the co-administered adjuvant is rendered
efficient and feasible for use. Thus, the other adjuvants are
preferably those, which have a substantial toxicity and the dose of
which has to be lowered to be accepted for prophylactic and
clinical use, but also adjuvants which are weak and cannot by their
own enhance efficient levels of immune responses or exert efficient
qualitative immunomodulating capacity.
[0059] The at least one other adjuvant may be chosen preferably
from saponins, naturally occurring, or derivatives thereof,
synthetic or semi synthetic saponin molecules derived from crude
saponin extract of Quillaja saponaria Molina; e.g. saponins and
saponin fractions from Quil A, cell wall skeleton, blockpolymers,
e.g. hydrophilic block copolymers, e.g. CRL-1005, TDM (Threhalose
di mucolate), lipopeptides, LPS and LPS-derivatives, Lipid A from
different bacterial species and derivatives thereof, e.g.,
monophosphoryl lipid A, muramyl di or tripeptide or derivatives
thereof, CpG variants, CpGODN variants, endogenous human animal
immunomodulators, e.g. GM-CSF, IL-2, adjuvant active bacterial
toxins, native or modified, e.g. cholera toxin CT, and its
subcomponents CTB and CTA1, thereto labile toxin (LT) of E. coli,
or Bordetella pertussis (BP) toxin and the filamentus
heamagglutenin of BP.
[0060] The saponin fractions from Quil A other than fraction A may
be the 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 0 362 279 B2, Q-VAC (Nor-Feed, AS Denmark),
Quillaja Saponaria Molina Spikoside (Isconova AB, Uppsala Science
Park, 75183 Uppsala, Sweden).
[0061] 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 0 362 279 B2, Especially QA-7, 17-18 and 21 may be used. They
are obtained as described in EP 0 362 279 B2, especially at page 6
and in Example 1 on page 8 and 9.
[0062] 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.
[0063] Preferably the at least other adjuvant is sub fragment C or
B from Quil A.
[0064] In one embodiment of the invention the adjuvant fraction A
of Quil A, in this text also referred to as QWT and the at least
one other adjuvant may be integrated into each one different iscom
particle or iscom matrix particles. They may also be integrated
into one and the same iscom particle or iscom matrix particles.
Thus the adjuvants may be integrated into each a different iscom
particle or different iscom matrix particles and then mixed in a
composition.
[0065] The iscom particle may be an iscom complex or an iscom
matrix complex made from any saponin. The adjuvant fraction A and
the other at least one adjuvant may also be coupled on to the
different or the same iscom particles or iscom matrix particles or
one or more of the adjuvants may be mixed with the iscom
particles.
[0066] In order to be integrated into iscom particles the adjuvants
need to have some hydrophobic molecule. Adjuvants that do not have
hydrophobic molecules may be coupled to such molecules. Hydrophobic
molecules and coupling methods are described in EP 180564.
Preferably the adjuvants are integrated into different iscom
particles.
[0067] In another embodiment of the invention the adjuvant fraction
A of Quil A is integrated into iscom particles, whereas the other
at least one adjuvant are not integrated into iscom particles and
are used in free form in the composition.
[0068] In another preferred embodiment of the invention the
adjuvant fractions of Quil A is integrated into iscom particles or
iscom matrix particles, whereas other adjuvants are not integrated
into iscom particles or iscom matrix particles and are used in free
form in the composition.
[0069] In another especially preferred embodiment the composition
comprises fraction A of Quit A integrated into iscom particles or
is corn matrix particles and at least one other adjuvant, which is
not integrated into iscom particles or iscom matrix particles.
[0070] In another preferred embodiment the at least other adjuvant
is MPL or cholera toxin CT. The MPL or cholera toxin may be
integrated into the same iscom particle or iscom matrix particle or
into each a different iscom particle or iscom matrix particle.
Preferably the MPL or cholera toxins are in free form.
[0071] In still another preferred embodiment the Quil A fraction A
is incorporated into an iscom particle or iscom matrix particle and
the at least one other adjuvant is incorporated into each a
different iscom particle or iscom matrix particle or the other at
least on other adjuvant is incorporated into the same iscom or
iscom matrix particle but different form the particle into which
the Quil A fraction A was incorporated or the other at least one
adjuvant is in free form.
[0072] The adjuvant fraction A and the other (co-administered) at
least one adjuvant may also be formulated in liposomes or with
oil-based adjuvant formulation or with a non-ionic block polymer or
presented in another particulate formulations such as PLG, starch,
Al(OH).sub.3 or in free form.
[0073] 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. These complexes 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.
[0074] 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.
[0075] In a preferred formulation iscoms and iscom matrix have been
formulated with fraction A and C of Quillaja in different iscom
particles, which cause minimal side effects (see the examples).
These iscoms have been compared with a formulation comprising 70%
of fraction A and 30% of fraction. C of Quil A called 703 and
produced according to WO 96/11711, which is in clinical trial in
man for a human influenza virus vaccine. According to WO 96/11711
the A and C fractions are integrated into the same particle. The
toxicity study was carried out in newborn mice, which arc much more
sensitive than adult mice. The study shows that the newborn mice
better tolerate the new iscoms produced from fraction A of Quil A
than the 703 formulation. Furthermore, the efficacy of the new
formulations according to the invention is tested with antigens
from a pathogen i.e. human respiratory syncytial virus hRSV and
with a weak antigen i.e. ovalbumin (OVA). A synergistic effect of
fraction A of Quil A in a matrix formulation named QWT is shown in
example 1. Also strong antigens like cholera toxin (CT) and tetanus
toxin can be modulated by the adjuvant formulations according to
the present invention by enhancing antibody increase, but above all
by potent immuno modulation as described in examples 8 and 9.
[0076] A composition according to the invention may comprise the
adjuvant fraction A from Quit A and the at least one other adjuvant
in any weight ratios. Preferably fraction A of Quil A is from
2-99.9 weight %, preferably 5-90 weight % and especially 50-90
weight% counted on the total amount of adjuvants. For e.g.
Al(OH).sub.3, oil adjuvants and block polymers the amount of
fraction A, of Quil A may be substantially lower.
[0077] One preferred iscom composition comprises 50-99.9% of
fragment A of Quil A and 0.1-50% of fragment C and/or fraction B
and/or other fractions or derivatives of Quit A (hereinafter non-A
Quil A fractions) counted on the total weight of fractions A and
non-A Quil A fractions. Especially the composition comprises
70-99.9% of fragment A of Quil A and 0.1-30% of non-A Quil A
fractions, preferably 75-99.9% of fragment A of Quil A and 0.1-25%
of non-A Quil A fractions and especially 80-99.9% of fragment A of
Quit A and 0.1-20% of non-A Quil A fractions counted on the total
weight of fraction A and non-A Quil A fractions. Most preferred
composition comprises 91-99.1% of fragment A of Quil A and 0.1-9%
of non-A Quil A fractions counted on the total weight of fractions
A and non-A Quil A fractions, especially 98.0 99.9% of fraction A
and 0.1-2.0% of non-A Quit A fractions counted on the total weight
of fractions A and non-A Quil A fractions.
[0078] The composition may further comprise a pharmaceutically
acceptable carrier, diluent, excipient or additive.
[0079] For the purposes of identification of Fractions A, B and C
referred to herein, reference may be made to the purification
procedure of Example 1. In general terms, in this procedure
Fractions A, B and C are prepared from the lipophilic fraction
obtained on chromatographic separation of the crude aqueous Quil A
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.
[0080] When prepared as described herein, Fractions A, B and C of
Quil A 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.
[0081] All publications mentioned herein are incorporated by
reference. By the expression "comprising" we understand including
but not limited to. The invention will now be described by the
following non-limiting examples. The scope of the invention is
rather what the skilled person would interpret from the disclosure
and found equivalent or a natural development thereof.
EXAMPLE 1
[0082] In this experiment it is emphasised that QWT in iscom and
matrix 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.
QWT is compared with QHC, since it is evaluated in human clinical
trials.
Materials and Methods
Formulation of QHC, QWT and 703-Matrix Iscoms
[0083] A mixture of phosphatidyl choline (PC) and cholesterol (C)
(15 mg/ml of each) is prepared in 20% MEGA-10 in water. The
preparation is heated to 60.degree. C. and treated with light
sonication until all lipid is solubilised.
[0084] Quillaja saponin is dissolved to 100 mg/ml in water. The 703
mixture contains 7 parts (by weight) of Fraction A and 3 parts of
Fraction C.
[0085] QWT saponin contains Fraction A alone.
[0086] 703-matrix. 5 ml of PBS is mixed with 10 mg of the PC/C
mixture (667 microliters), 35 mg 703 (350 microliters) is added,
the mixture is mixed and PBS is added to a final total volume of 10
ml. The mixture is extensively dialysed against PBS using a
Slide-A-Lyser (3-15 ml, Pierce) dialysis cassette.
[0087] QWT-matrix, and QHC-matrix 5 ml of PBS is mixed with 10 mg
of each PC and C (667 microliters), 40 mg QWT (400 microliters) and
30 mg of QHC (300 microliters) respectively is added, the mixture
is mixed and PBS is added to a final total volume of ml. The
mixture is extensively dialysed against PBS using a Slide-A-Lyser
(3-15 ml, Pierce) dialysis cassette.
Experimental Design
[0088] Female MNRI mice (18-20 g) were used in this Example. Group
1 consisted of 8 mice immunised twice 4 weeks apart subcutaneously
(s.c.) with 10 .mu.g OVA adjuvanted with 50 .mu.g QWT matrix. Group
2 had the same number of mice immunised by the same procedure but
the adjuvant was 50 .mu.g QHC matrix. Sera were collected before
first immunisation and 3 weeks after and 2 weeks after the
boost.
Antibody Determination
[0089] The specific OVA serum antibody responses were determined by
ELISA both for total IgG response (including all IgG subclasses)
and in the IgG2a subclasses as described before (Johansson, M and
Lovgren-Bengtsson (1999) Iscoms with different quillaja saponin
components differ in their immunomodulating activities. Vaccine 19,
2894-2900).
Results
[0090] All mice immunised with OVA adjuvanted with QWT matrix
survived and did not develop any sign of discomfort. Out of 8 mice
immunised with OVA adjuvanted with QHC matrix 4 mice (50%)
died.
[0091] There is no significant difference between the groups with
regard total antibody responses (FIG. 1(A)), but there is more
spread of the antibody titres between the animals in group 1, i.e.
mice immunised with OVA adjuvanted with QWT-matrix.
[0092] There was no difference in mean titres in the IgG2a subclass
between group 1 and 2 (FIG. 1(B)), but there is more spread of the
antibody titres between the animals in group 2, i.e. mice immunised
with OVA adjuvanted with QHC-matrix.
[0093] In the second experiment of this example it was explored
whether QWT matrix can benefit from the complementation of another
adjuvant, or it facilitate the use of a more toxic adjuvant. The
IgG2a response reflects that the Th2 type of lymphocytes are
involved. The dose of QWT-matrix and QHC-matrix ranged as follows;
in group 1, no QWT-matrix or QHC-matrix; Gr. 2, 0.3 .mu.g
QWT-matrix no QHC-matrix; Gr. 3, 0.3 .mu.g QWT-matrix+2 .mu.g QHC
matrix; Gr. 4, 10 .mu.g QWT-matrix no QHC; Gr. 5, 10 .mu.g
QWT-matrix+2 .mu.g QHC-matrix X. 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. (example 8 FIGS. 2(A), (B)
and (C)).
[0094] Sera were collected 3 weeks after the first immunisation and
2 weeks after the boost.
[0095] The OVA specific serum antibody responses were determined by
ELISA for total IgG response and in the IgG2a and IgG1 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
[0096] After the first immunisation no antibody response was
recorded in mice receiving non-adjuvanted OVA or OVA adjuvanted
with 0.3 .mu.g of QWT matrix with and without 2 .mu.g of QHC matrix
(FIG. 2(A)).
[0097] After the second immunisation a low response was detected in
3 out of 8 mice immunised with non-adjuvanted OVA in the IgG1
subclass (FIG. 2(B)), but no response was recorded in the IgG2a
subclass. Neither was antibody responses recorded in the IgG2a
subclass with the lowest adjuvant doses of QWT matrix i.e. 0.3
.mu.g with and without 2 .mu.g of QHC matrix (FIG. 2(B)). There was
a clear enhancement of the antibody response in the IgG2a subclass,
when the low dose of 2 .mu.g QHC matrix was added to the 10 .mu.g
of QWT matrix (FIG. 2(B)).
Conclusion
[0098] QWT has a low toxicity and still a strong modulatory effect
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 IgG1 subclass. It is also shown
that the QWT matrix synergies with a low dose of QHC matrix. This
fact is important, because QWT makes it possible to optimise the
adjuvant effect and minimise the side effects of other
adjuvants.
EXAMPLE 2
[0099] Respiratory syncytial virus (RSV) is a major pathogen for
young children (hRSV) but also for elderly. A closely related virus
(bRSV) is a pathogen for young calves causing severe disease and
high economical losses for calf breeders. The envelope proteins of
hRSV were selected as model antigens, because they represent
antigens from a pathogen for which a vaccine is lacking and for
which there is a great need. The newborn mouse represents a model
for the newborn, and a very sensitive animal, which requires a
vaccine formulation virtually free of side effects, and a model in
which important immunological reactions can be measured because of
available reagents techniques. An early vaccine against hRSV was
tested in children, but it did not protect against disease. On the
contrary it exacerbated disease when a subsequent natural infection
occurred. In this experiment we have selected 703 as a quillaja
component in the ISCOM to compare with the present invention,
because a 703 vaccine formulation is in human trials, thus a
candidate for human vaccines. In the present experiment the
toxicity of QWT iscoms and 703 iscoms is compared.
Materials and Methods
Formulation of 703 and QWT RSV-ISCOMs
[0100] RSV iscoms with different Quillaja saponin compositions (A,
C and AC i.e., ISCOPREPTM703) were prepared from sucrose gradient
purified HRSV, essentially using the method described previously
[17,18]. Briefly, 2 ml (1.6 mg/ml) purified RSV was solubilized
with OG (1-O-n-Octyl-.beta.-D-glueopyranosid, C14H28O6, Boehringer,
Mannheim, GmbH, FRG) at a final concentration of 2% (w/v) for 1 h
at 37.degree. C. under constant agitation. The solubilized virus
was applied onto a discontinuous sucrose gradient of 2 ml 20%
sucrose layer containing 0.5% OG, over a cushion of 50% sucrose.
After centrifugation at 210,000 g at 4.degree. C. in a Kontron
TST-41 rotor for 1 h, the sample volume together with the 20%
sucrose layer containing viral proteins were collected, and extra
lipids I.e. cholesterol and phosphatidylcholine, and Quillaja
saponin, i.e. QH-A or QH-C or ISCOPREPTM703 was added in
proportions of protein: cholesterol: phosphatidylcholine: Quillaja
saponin=1:1:1:5 calculated by weight. After extensive dialysis
against 0.15 M ammonium acetate at 4.degree. C. for 72 h, the
ISCOMs were purified by centrifugation through 10% sucrose at
210,000 g in Kontron TST-41 rotor at 10.degree. C. for 18 h. The
pellet containing the iscoms was re-suspended in 200 .mu.l PBS.
Protein concentration was determined by amino acid analysis
(Aminosyraanalyslaboratoriet, Uppsala, Sweden). Samples were
submitted for negative staining electron microscopy. No
morphological differences were observed among the three iscoms. All
showed typical iscom structures, i.e. cage-like spherical particles
with a diameter of around 40 nm. The RSV antigens and iscom
structures were found in the same fraction of a sucrose gradient
after centrifugation.
Experimental Design
[0101] One litter of at least 7 newborn (one week old) mice per
group were injected intraperitoneally (i.p.) once with either a
formulation 703 iscoms or QWT iscoms The dose groups of each
quillaja component ranged between 0.11 .mu.g and 1 .mu.g measured
as protein content (FIG. 3). The weight ratio QWT or 703 (quillaja
saponin) to protein is 1/1. The pups were observed for 15 days
after i.p. injection. It should be noted, that the i.p. injection
is a rough mode of administration and mice are much more sensitive
for i.p. injection than for intramuscular and subcutaneous modes of
administrations.
Results
[0102] Doses of 0.66 and 1 .mu.g killed 65 resp. 50% oldie mice
injected with the 703 iscoms, while all the mice injected with the
QWT iscoms survived including those receiving 1 .mu.g of QWT
iscoms.
Conclusion
[0103] The QWT iscom is well tolerated even by a harsh route as the
i.p. route in a very sensitive animal model. It is better tolerated
than a formulation being in human trials.
EXAMPLE 3
[0104] In this example the serum antibody response was tested with
the envelope proteins G and F of hRSV as a model for vaccine
antigen. The hRSV antigens were selected because hRSV represents
antigens from a pathogen for which a vaccine is lacking and for
which there is a great need. The newborn mouse represents a model
for the newborns, which are immunologically immature requiring an
adjuvant system with potent immune modulatory capacity
(WO97/30727). Furthermore, a newborn mouse represents an animal
system, which is very sensitive and requires a vaccine formulation
virtually free of side effects. Similar vaccine formulations were
tested as described in example 2, i.e. the QWT AND 703 iscoms.
Materials and Methods
Formulation of QWT and 703 RSV-Iscoms
[0105] See example 2
Experimental Design
[0106] One-week-old mice and adult mice (BALB/C) were distributed
into 2 groups of newborns and 2 groups of adults. One litter of
newborns with minimum of 7 animals per group and 8 adults were
immunised i.p. with 1 .mu.g of hRSV in the QWT iscoms or in the 703
iscom formulation. One group of newborns and 1 group of adult mice
were immunised once, while 1 group of newborns and 1 group of adult
mice were boosted 3 weeks after the first immunisation with the
same formulations by the same mode. All experiments were repeated
once.
[0107] Sera were collected before boost and week 7 of life i.e. 3
weeks after boost. Because of the small size of the newborns the
sera were pooled from one group.
Antibody Determination
[0108] The specific RSV serum antibody responses were determined by
ELISA in both IgG1 and IgG2a subclasses as described using 0.1
.mu.l of formalin killed RSV virus as coating antigen (Johansson. M
and Lovgren-Bengtsson (1999) Iscoms with different quillaja saponin
components differ in their immunomodulating activities. Vaccine 19,
2894-2900).
Results
[0109] The results are illustrated in FIG. 4. After one
immunisation both adults and newborns responded with RSV specific
IgG1 antibodies measured by ELISA. After one immunisation the QWT
iscoms induced higher RSV specific IgG1 antibody response in the
newborn than the 703 iscom. Otherwise, there were no clear
differences between the two iscom formulations as regards to their
capacity to induce IgG1 and IgG2a RSV specific antibody responses
in adults or in newborns. The antibody titres in general were
10-fold higher in the adults than in newborns. The IgG2a response
to RSV was insignificant after one immunisation in newborns
regardless they were immunised with QWT or 703 iscoms. RSV specific
IgG2a were clearly detected after one immunisation in adults.
Conclusion
[0110] The serum antibody responses were at least as high after 1
as well as after 2 immunisations of newborns or adults with the QWT
iscom formulation as after the same immunisation schedules with the
703 iscom. In view of the results of example 2, showing that the
QWT iscom has a considerably lower toxicity than the 703 iscom, the
QWT iscom is preferred for vaccine formulation.
EXAMPLE 4
[0111] Cytotoxic T lymphocytes (CTL) are essential for the immune
defence against intracellular pathogens. Above all virus-infected
cells are targets for CTL by killing the infected cells.
Consequently, CTL is an important arm of the immune defence against
viral infections. This example shows that QWT iscoms containing
hRSV envelope antigens specifically induce and efficiently prime
for memory CTL both in newborn and adult mice. It is surprising,
that the QWT iscoms induced CTL memory as efficiently in the
newborns as in adults in view of their immature immune system.
Materials and Methods
Formulation of QWT and 703 RSV Iscoms
[0112] The QWT and 703 ISCOMs were prepared as described in example
2.
Animals and Experimental Design
[0113] One litter of newborns with at least 7 animals were used for
each experiment (8 adult BALB/C(H-2Kd) mice). Each experiment was
carried out twice. One-week-old mice or adult mice were injected
i.p. with 1 .mu.g of QWT iscoms. One week resp. 3 weeks after
immunisation spleen cells (effector cells) were cultured
(restimulated) for 6 days in vitro with HRSV infected
(BCH4)-fibroblast (target cells). The ratio of effector/target
(E/T) ranged from 2 to 100 (FIG. 5). The target cell lysis was
measured by Cr51 release and expressed as % specific lysis (% SL)
according to standard procedure. 100% lysis was measured as Cr51
release from detergent treated cells. The background was the lysis
caused by uninfected fibroblasts (BC) (see FIG. 5).
Results
[0114] Already 1 week after priming of newborn and adult mice with
QWT iscoms their splenocytes generated to restimulation in vitro
with hRSV infected fibroblasts (BCH4) strong cytotoxic T cell
response (FIG. 5). No lysis was observed against uninfected target
cells (BC in FIG. 5).
Conclusion
[0115] RSV-QWT iscoms induce strong cytotoxic T cell responses in
1-week-old mice and in adult mice. Strong specific cytotoxicity is
observed already 1 week after one immunisation. In view of the
strong adjuvant effect of QWT iscoms and its low toxicity, this
vaccine delivery and adjuvant system is very likely to be valuable
for both human and animal vaccines.
EXAMPLE 5
[0116] Quillaja saponins have been shown to have strong adjuvant
effects, but they have caused side effects by their lytic
properties, which can be measured by lysis of red blood cells.
Toxic effects of any kind prevent the cell growth or proliferation
of living cells. It is well established that QHC and less purified
quillaja saponins like Quil A lyses red blood cells (Ronnberg B,
Fekadu M and Morein B, Adjuvant activity of non-toxic Quillaja
saponaria Molina components for use in iscom matrix, Vaccine, 1995
13, (14): 1375-82.). It is also clear that lytic effect of quillaja
saponins causes local reactions when injected. One way to avoid
lytic effects of saponins is to include them into ISCOM matrix.
Furthermore, the side effects can be reduced by selection of
quillaja saponin, which causes comparatively low side effect. In
this example the effect of QWT matrix is tested on VERO cells,
which is a primate cell line, and it is compared with QHC and 703
matrix formulations. In a second experiment spleen cells from mice
were exposed to QWT and QHC matrices. The spleen cells are
representative for the lymphatic system essential for the induction
of immune responses. The AlamarBlue Assay is used, which measures
quantitatively the proliferation of the cells based on detection of
metabolic activity.
Material and Methods
[0117] Cells and cell growth. Vero cells were cultured in RPMI 1640
medium (National Veterinary Institute Uppsala Sweden) supplemented
with 7% fetal calf serum (obtained as above). After outgrowth on 75
cm.sup.2 flasks (Corning-Costar, Acton Mass., USA) the cells are
detached from the plastic surface and diluted to 25 to 30 000 per
ml, and distributed in 100 .mu.l portions per well in 96 well cell
culture plates (Nunc A/S, Roskilde, Denmark). The cultures are
incubated in CO.sub.2 atmosphere for 24, 48 and 72 hours. Matrix
prepared with QWT, or 703 or QHC were diluted in medium from 0 to
1300 .mu.g per ml. The cell cultures were emptied from medium and
the matrix dilutions were added to the wells. As control only
medium was used. The test was carried out with the formulations to
be tested for incubation periods of 24, 48 and 72 hours. Most
suitable time period was 72 hours, which is presented here. The
controls are considered as 100% growth.
[0118] Recording of cell growth. The AlamarBlue assay (Serotec Ltd,
Oxford UK), which measures quantitatively the proliferation of the
cells based o detection of metabolic activity was used according to
the description of the manufacturer.
Results
[0119] After 72 hours incubation of the cell cultures with QWT
matrix at a concentration 1300 .mu.g per ml a cell growth of 80%
was recorded compared to the control cultures, while the cell
growth had declined to 0% when exposed to 703 matrix at a
concentration of 800 .mu.g per ml. The cell growth had declined to
0% when exposed to QHC matrix at concentration of 40 .mu.g per ml.
FIG. 6 illustrates one experiment out of 3 with similar
results.
Conclusion
[0120] QWT matrix is well tolerated by the cells and has very low
cell toxic effect.
[0121] In a second experiment spleen cells were exposed to QWT and
QHC matrices.
Material and Methods
[0122] Cells and cell growth. Spleen cells from Balb/C mice were
cultured in RPMI 1640 medium (National Veterinary Institute,
Uppsala, Sweden) supplemented with 7% fetal calf serum in 96-well
cell culture plates (Nunc, Roskilde Denmark). The test was carried
out on the spleen cells with the formulations QWT-iscom and
QHC-iscom for incubation periods of 24, 48 and 72 hours. Most
suitable period was 72 hours, which is presented here. The controls
are considered as 100% growth.
[0123] Recording of cell growth. The AlamarBlue Assay is used,
which measures quantitatively the proliferation of the cells based
on detection of metabolic activity was used according to the
description of the manufacture.
Results
[0124] After 72 hours exposure of the spleen cell cultures to QWT
matrix at a concentration 10 .mu.g per ml a cell growth of 80% was
recorded compared to the non-exposed spleen cell (control)
cultures, while the cell growth had declined close to 0% when
exposed to QVC matrix at a concentration of 2 .mu.g per ml (FIGS.
7(A) and (B)). FIG. 7 illustrates one experiment out of 3 with
similar results.
EXAMPLE 5
Preparation of Quillaja Saponaria Molina Subfragment Saponins
[0125] 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, Mass.).
[0126] 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.
[0127] 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).
[0128] 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.
[0129] 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. 6.
EXAMPLE 6
[0130] OVA is a weak antigen requiring adjuvant for induction of
potent immune response. Prospective adjuvants are, therefore, often
tested together with OVA to show the immune enhancement
quantitatively by measuring level of antibody or qualitatively by
measuring the immune modulatory effect. The modulatory effect is
e.g. recorded by the capacity to drive antigen specific IgG
subclass responses. A response dominated by IgG1 antibody is
significant for Th2 while IgG2a is significant for Th1 type of
response. A response in both IgG1 and IgG2a implicates the balance
of the immune modulation between Th1 and Th2. This example it is
carried out to demonstrate that QWT-Matrix acts synergistic with
the more toxic QHC-Matrix to allow the use of a comparatively low
and well tolerated dose of QHC-Matrix with optimized effect.
Materials and Methods
QWT and QHC-Matrix
[0131] These Quillaja saponin components (see Example 5) were
obtained and formulated into ISCOM-Matrix as described in Example
1. Ovalbumin (OVA) was obtained from Sigma (St Louis, USA).
Experimental Design
[0132] All mice were immunised s.c. at the base of the tail with a
total volume of 100 .mu.l. Group 1 consisted of 8 Balb/c mice
immunised twice 4 weeks apart, with 5 .mu.g OVA without addition of
adjuvant. Group 2 consisted of 8 mice immunised twice 4 weeks
apart, with 5 .mu.g OVA adjuvanted with 6 .mu.g QWT-Matrix. Group 3
consisted of 8 mice immunised twice 4 weeks apart with 5 .mu.g OVA
adjuvanted with 6 .mu.g QHC-Matrix. Group 4 consisted of 8 mice
immunised twice 4 weeks apart with 5 .mu.g OVA adjuvanted with low
dose of QHC-Matrix (2 .mu.g) supplemented and supplemented with 6
.mu.g QWT-Matrix.
[0133] Sera were collected before first immunisation and 3 weeks
after priming and 2 weeks after the boost.
Antibody Determination
[0134] Serum antibody determination, including total IgG, and
subclasses IgG1 and IgG2a, was carried in out in ELISA as described
in example 1.
Results
[0135] After priming (FIG. 9), there was an IgG response over 1:100
in 1 out of 8 mice immunised with OVA+QWT-Matrix (group 2), in 2
out of 8 mice in the group immunised with OVA+QHC-matrix (Group 3).
In contrast all 8 mice in the group immunised with low dose of
QHC-matrix complemented with QWT-Matrix (Group 4) responded with an
IgG response. None of the mice immunised with OVA alone responded
with a titre >1:100 after the primary immunisation.
[0136] After priming 2 out of 8 mice in the group immunised with
the combination of QWT-Matrix Matrix and a low dose of QHC-matrix
(Group 4) responded with an antigen specific IgG2a response
>1:100. No IgG2a response was recorded after priming in the
other groups.
[0137] After booster (FIG. 10, FIG. 11, and FIG. 12), all mice in
groups 2, 3 and 4 responded with IgG tires >1:100. However, the
titres in Group 2 and Group 3 varied over 2 logs (3 700-295 000 and
3 100-400 000 respectively), while the titres in group 4 varied
within 1/10 of a log (260 000-350 000).
[0138] The IgG1 results after priming were mirrored by that of the
IgG (total) response. Thus, these results arc not depicted by a
figure.
[0139] The antigen specific IgG2a response after booster was
negligible in Group 2 given OVA+WAIT-Matrix. Groups 1 and 3 showed
variable responses of the IgG2a subclass while in Group 4, given
OVA+QWT-Matrix and 2 .mu.g of QHC-matrix all responded with high
IgG2a titres, all within one log.
Conclusion
[0140] QWT-Matrix in a low dose is well tolerated and without
measurable side effects in the dose used in this example, but it is
also tolerated in a many-fold higher dose as shown in example 1. In
this example QHC-Matrix was used in a low and well tolerated but
sub-optimal dose for adjuvant use by its own. It is clearly
documented in this experiment, that QWT-Matrix and QHC-Matrix acts
synergistically in a well tolerated adjuvant formulation. It should
be emphasised that both the QWT-Matrix and QHC-Matrix doses, as
used in the combined formulation in this example, are too low to be
effective by their own, implicating synergism.
EXAMPLE 7
[0141] In this experiment the synergistic effect of QWT-Matrix is
tested on potent adjuvant active bacterial derived compounds; mono
phosphoryl lipid A (MPL) and cholera toxin (CT). It is evaluated
with regard to enhancement of the immunogenicity of the week
antigen, OVA. The NMRI out-bred mice were used, which in contrast
Balb/C mice readily respond with TH1 as well as TH2 type of
immunity reflected by the IgG2a (Th1) and IgG1 (Th2) antibody
levels.
Materials and Methods
OVA QWT-Iscoms
[0142] These ISCOMs were prepared essentially as described for
QWT-Matrix in example 1, with the exception that palmitified OVA
(pOVA) was added to the preparation at a concentration of 1 mg per
mg cholesterol. The preparation of pOVA-iscoms have been described
by Johansson and Lovgren-Bengtsson in Vaccine 17 (1999), p 2894. CT
was commercially obtained from KeLab, Gothenburg, Sweden. MPL
(L6895) and OVA were from Sigma (St. Louis, USA).
Experimental Design
[0143] All mice were immunised s.c. at the base of the tail with a
total volume of 100 Group 1 consisted of 8 NMRI mice immunised
twice, 4 weeks apart with 5 .mu.g OVA without addition of adjuvant.
Group 2 consisted of 8 mice immunised twice 4 weeks apart with 5
.mu.g OVA incorporated into QWT-iscoms containing bug QWT and no
additional adjuvant. Group 3 consisted of 8 mice immunised twice 4
weeks apart with 5 .mu.g OVA (as in group 1) adjuvanted with high
dose CT (1 .mu.g). Group 4 consisted of 8 mice immunised twice 4
weeks apart with 5 .mu.g OVA (as in group 1) adjuvanted with high
dose of MPL (50 .mu.g). Group 5 consisted of 8 mice immunised with
5 .mu.g OVA in QWT-ISCOMs (as in Group 2) supplemented with low
dose (0.2 .mu.g) CT. Group 6 consisted of 8 mice immunised twice 4
weeks apart with 5 .mu.g OVA in QWT-iscoms (as in Group 2)
adjuvanted with low dose (10 .mu.g) MPL.
[0144] Sera were collected 3 weeks after priming and 2 weeks after
the booster injection.
Antibody Determination
[0145] Serum antibody determination, including total IgG, and
subclasses IgG1 and IgG2a, was carried in out in ELISA as described
in example 1.
Results
[0146] The results are depicted in FIG. 13.
[0147] After the first immunisation the total IgG levels were
comparable for groups 2 to 6 and IgG2a antibody response to OVA was
low for mice in all groups (not shown).
[0148] After booster (FIG. 13(A)) the mice immunised with OVA alone
reacted with low serum levels of IgG. The OVA in QWT-ISCOM
supplemented with low dose of CT (0.2 .mu.g) induced 7-fold higher
IgG levels than the OVA adjuvanted with high dose (1 .mu.g) of CT.
The OVA in QWT-ISCOM supplemented with low dose of MPL (10 .mu.g)
induced 2 fold higher IgG levels than the OVA adjuvanted with high
dose (50 .mu.g) of MPL.
[0149] The IgG1 were essentially reflected by the total IgG
antibody responses, and are not shown.
[0150] Greater differences were recorded when measuring the
antibody responses within the IgG2a subclass (FIG. 13(B)). The MPL
low dose was enhanced about 10-fold in IgG2a serum antibody levels
with OVA in QWT-ISCOM formulation compared to OVA adjuvanted with
the high dose of MPL. Even more striking is the 100-fold
enhancement of the IgG2a response by the OVA-QWT-ISCOM with low
dose CT compared to OVA formulated with a high dose CT.
Conclusion
[0151] OVA in QWT-ISCOM formulated with low dose of CT or MPL were
considerably more immunogenic than the corresponding MPL or CT high
doses formulations excluding QWT. The QWT-ISCOM enhancement of the
immunogenicity of OVA was most striking in the IgG2a subclass
showing strong immune modulatory effects of the QWT component in
the respective formulations. Although the Th1 modulation was more
striking than that of Th2, the modulation geared by QWT-ISCOM was
balanced. The Th1 driving effect was more prominent over CT
explained by the fact that CT is more Th2 driving than MPL.
EXAMPLE 8
[0152] Vaccines are often composed of antigens in particulate forms
as is often the case with vaccines against bacteria or viruses.
Toxins on the other hand are soluble antigens detoxified by
conversion to toxoids e.g. by treatment with formalin. In example 1
(FIG. 2) and in examples 6 and 7 it is shown that the
immunogenicity of a weak soluble antigen OVA is strongly enhanced
by the synergistic effect of QWT-Matrix, when the QWT-Matrix is
used to complement a low and well tolerated dose of QHC-Matrix, CT
or MPL.
[0153] In this example a commercial soluble but immunogenic vaccine
antigen, Tetanus Toxoid (TT) is supplemented with Cholera Toxin
(CT) being a strong adjuvant driving a Th2 type of response, but
also toxic in comparatively low doses. Included in the example is
also a group of mice immunised with TT and adjuvanted with CT
complemented with QWT-Matrix. QWT-Matrix is added to show that
modulation of the CT response can be achieved with a low dose of CT
and that a well tolerated CT/QWT formulation can be obtained, which
is well tolerated due to a synergistic effect.
Materials and Methods
QWT-Matrix
[0154] QWT-Matrix was formulated as described in Example 1.
TT and CT
[0155] TT was commercially obtained from The State SERUM Institute,
Copenhagen, Denmark.
[0156] CT was commercially obtained from KeLab, Gothenburg,
Sweden.
Experimental Design
[0157] All mice were immunised s.c. at the base of the tail with
100 .mu.l of vaccine. Group 1 consisted of 6 outbred NMRI mice
immunised twice 4 weeks apart with 2.5 Lf TT without addition of
adjuvant. Group 2 consisted of 8 mice immunised twice 4 weeks apart
with 2.5 Lf TT adjuvanted with high dose of CT (1 .mu.g). Group 3
consisted of 8 mice immunised twice 4 weeks apart with 2.5 Lf TT
adjuvanted with low dose of CT (0.2 .mu.g). Group 4 consisted of 8
mice immunised twice 4 weeks apart with 2.5 Lf TT adjuvanted with
low dose of CT (0.2 .mu.g) supplemented with 10 .mu.g of
QWT-Matrix.
[0158] Sera were collected 3 weeks after the priming and 2 weeks
after the booster.
Antibody Determination
[0159] This was carried in out in ELISA as described in example 1
except that the antigen was TT coated to ELISA plates (Nunc) at a
concentration of 1 .mu.g/ml.
Results
[0160] A clearcut primary antibody response, measured as
antigen-specific IgG, was recorded in all four groups showing that
TT is a comparatively strong immunogen. TT adjuvanted with low dose
of CT (0.2 .mu.g) supplemented with QWT-Matrix induced a 3-fold
higher primary IgG response compared to the other formulations
(FIG. 14).
[0161] After booster, the total IgG response increased in all
groups where the TT was supplemented with adjuvant (FIG. 15), while
the second immunisation did not significantly increase the antibody
level in mice immunised with TT alone.
[0162] After one immunisation the IgG2a response, indicating a Th1
type of immune response, was only induced in mice (group 4)
immunised with TT adjuvanted with low dose of CT (0.2 .mu.g)
supplemented with QWT-Matrix (FIG. 16).
[0163] After booster, the QWT-Matrix group of mice responded with
the highest IgG2a titres (FIG. 17). Mice in group 3 immunised with
TT adjuvanted with the low dose of CT (0.2 .mu.g) dose responded
with negligible or very low titres of TT-specific IgG2
antibody.
Conclusion
[0164] TT is a comparatively strong soluble immunogen promoting a
Th2 type of response. CT is a strong toxin with strong adjuvant
effect also promoting a Th2 type of response. In this experiment it
is shown that QWT-Matrix strongly promotes (modulates) the host to
respond also with antigen-specific IgG2a antibody when added to the
TT antigen supplemented with low dose of CT. It is interesting to
note the strong Th2 driving adjuvant effect of CT is modulated by
QWT-Matrix towards Th1. Thus, the QWT-Matrix has a strong immune
modulatory effect combined with CT as adjuvant.
EXAMPLE 9
[0165] In this example a commercial soluble vaccine antigen Tetanus
Toxoid (TT) is supplemented with monophosphoryl lipid A (MPL) being
a strong adjuvant driving a Th1 type of response. A low dose of MPL
was complemented with QWT-Matrix to demonstrate the modulatory and
synergistic effect of QWT-Matrix on the TT antigen in the presence
of MPL.
Materials and Methods
QWT-Matrix
[0166] QWT-Matrix was formulated as described in Example 1.
TT and MPL
[0167] TT was commercially obtained from The State SERUM Institute,
Copenhagen, Denmark.
[0168] MPL (L6895) was from Sigma (St. Louis, USA)
Tetanus Toxoid (TT)
[0169] TT was commercially obtained from The State SERUM Institute,
Copenhagen, Denmark.
Experimental Design
[0170] All mice were immunised s.c. at the base of the tail with
100 .mu.l of vaccine. Group 1 consisted of 6 outbred NMRI mice
immunised twice 4 weeks apart with 2.5 Lf TT without addition of
adjuvant. Group 2 consisted of 8 mice immunised twice 4 weeks apart
with 2.5 Lf TT adjuvanted with high dose of MPL (50 .mu.g). Group 3
consisted of 8 mice immunised twice 4 weeks apart with 2.5 Lf TT
adjuvanted with low dose of MPL (10 .mu.g). Group 4 consisted of 8
mice immunised twice 4 weeks apart with 2.5 Lf IT adjuvanted with
low dose of MPL (10 .mu.g) supplemented with 10 .mu.g of
QWT-Matrix.
[0171] Sera were collected 3 weeks after the priming and 2 weeks
after the booster.
Antibody Determination
[0172] This was carried out as described in example 8.
Results
[0173] A clearcut primary antibody response, measured as
antigen-specific IgG, was recorded in all four groups showing that
TT is a comparatively strong immunogen (FIG. 18). TT adjuvanted
with low dose of MPL (10 .mu.g) supplemented with QWT-Matrix
induced about 2-fold higher primary IgG response than the
formulation TT adjuvanted with 10 .mu.g MPL.
[0174] After booster the total IgG antibody response was
substantially increased in all groups where the TT was supplemented
with adjuvant (FIG. 21), while the second immunisation did not
significantly increase the antibody level in mice immunised with TT
alone. The mice immunised with TT adjuvanted with MPL (10 .mu.g)
supplemented with QWT-Matrix responded with more than a 100 fold
specific IgG response (FIG. 19), which was about 8-fold higher than
the response induced by TT supplemented low dose of MPL (10 .mu.g),
but no QWT-Matrix.
[0175] The IgG1 response showed the same profile as the total IgG
response both after the primary and second immunisation.
[0176] Mice immunised TT adjuvanted low dose of MPL (10 .mu.g)
supplemented with QWT-Matrix responded with 10 fold higher IgG2a
titres than mice immunised with TT supplemented with low dose of
MPL (FIG. 20). Mice in other groups did not develop significant
primary IgG2a response.
[0177] After booster the mice immunised with TT adjuvanted with low
dose of MPL (10 .mu.g) supplemented with QWT-Matrix responded with
the highest IgG2a titres being more than 100-fold higher than mice
in other groups (FIG. 21).
Conclusion
[0178] QWT-Matrix with antigen and/or MPL potently enhanced IgG2a
antibody response, but also IgG1 indicating a strong balanced
immune modulatory effect on the TT antigen in the presence of MPL.
The strong immunogenicity of TT is emphasised by the fact that MPL
by its own did not or only marginally enhance the total IgG or
IgG2a or IgG1 responses to the TT antigen. This indicates that a
strong adjuvant, like MPL, might have a limited immune modulatory
effect in the presence of a strong immunogen like TT. In contrast,
QWT is symbiotic in the effect with MPL demonstrated by the fact
that this combination has a strong immunomodulatory effect.
* * * * *