U.S. patent application number 10/967395 was filed with the patent office on 2005-09-29 for vaccines containing a saponin and a sterol.
This patent application is currently assigned to SmithKline Beecham Biologicals s.a.. Invention is credited to Claude Garcon, Nathalie Marie-Josephe, Friede, Martin.
Application Number | 20050214322 10/967395 |
Document ID | / |
Family ID | 34068762 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050214322 |
Kind Code |
A1 |
Claude Garcon, Nathalie
Marie-Josephe ; et al. |
September 29, 2005 |
Vaccines containing a saponin and a sterol
Abstract
The invention relates to a vaccine composition comprising an
antigen, an immunologically active saponin fraction and a
sterol.
Inventors: |
Claude Garcon, Nathalie
Marie-Josephe; (Wavre, BE) ; Friede, Martin;
(Brussels, BE) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham Biologicals
s.a.
|
Family ID: |
34068762 |
Appl. No.: |
10/967395 |
Filed: |
October 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10967395 |
Oct 18, 2004 |
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09478705 |
Jan 6, 2000 |
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6846489 |
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09478705 |
Jan 6, 2000 |
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09102676 |
Jun 23, 1998 |
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Current U.S.
Class: |
424/202.1 ;
424/203.1 |
Current CPC
Class: |
A61K 2039/55577
20130101; A61K 39/245 20130101; A61K 39/39 20130101; C12N
2740/13034 20130101; A61K 39/292 20130101; A61K 2039/55505
20130101; A61K 39/12 20130101; A61K 2039/57 20130101; C12N
2710/16634 20130101; A61K 2039/55572 20130101; C12N 2730/10134
20130101; A61K 2039/55555 20130101; A61K 39/015 20130101 |
Class at
Publication: |
424/202.1 ;
424/203.1 |
International
Class: |
A61K 039/295; A61K
039/116 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 1995 |
GB |
GB 9508326.7 |
Jun 28, 1995 |
GB |
GB 9513107.4 |
Claims
1.-12. (canceled)
13. An adjuvant composition comprising a sterol and a substantially
pure preparation of QS21, characterized in that the adjuvant
composition is in the form of an ISCOM.
14. An adjuvant composition according to claim 1, wherein the QS21
is at least 90% pure.
15. An adjuvant composition according to claim 1, wherein the QS21
is at least 95% pure.
16. An adjuvant composition according to claim 1, wherein the QS21
is at least 98% pure.
Description
[0001] The present invention relates to novel vaccine formulations,
to methods of their production and to their use in medicine. In
particular, the present invention relates to vaccines containing an
antigen, an immunologically active fraction derived from the bark
of Quillaja Saponaria Molina such as QS21, and a sterol.
[0002] Immunologically active saponin fractions having adjuvant
activity derived from the bark of the South American tree Quillaja
Saponaria Molina are known in the art. For example QS21, also known
as QA21, an Hplc purified fraction from the Quillaja Saponaria
Molina tree and it's method of its production is disclosed (as
QA21) in U.S. Pat. No. 5,057,540. Quillaia saponin has also been
disclosed as an adjuvant by Scott et al, Int. Archs. Allergy Appl.
Immun., 1985, 77, 409. However, the use of QS21 as an adjuvant is
associated with certain disadvantages. For example when QS21 is
injected into a mammal as a free molecule it has been observed that
necrosis, that is to say, localised tissue death, occurs at the
injection site.
[0003] It has now surprisingly been found that necrosis at the
injection site can be avoided by use of formulations containing a
combination of QS21 and a sterol. Preferred sterols include
.beta.-sitosterol, stigmasterol, ergosterol, ergocalciferol and
cholesterol. These sterols are well known in the art, for example
cholesterol is disclosed in the Merck Index, 11th Edn., page 341,
as a naturally occuring sterol found in animal fat.
[0004] In a first aspect the present invention therefore provides a
vaccine composition comprising an antigen, an immunologically
active saponin fraction and a sterol. Preferably the compositions
of the invention contain the immunologically active saponin
fraction in substantially pure form. Preferably the compositions of
the invention contain QS21 in substantially pure form, that is to
say, the QS21 is at least 90% pure, preferably at least 95% pure
and most preferably at least 98% pure. Other immunologically active
saponin fractions useful in compositions of the invention include
QA17/QS17. Compositions of the invention comprising QS21 and
cholesterol show decreased reactogenicity when compared to
compositions in which the cholesterol is absent, while the adjuvant
effect is maintained. In addition it is known that QS21 degrades
under basic conditions where the pH is about 7 or greater. A
further advantage of the present compositions is that the stability
of QS21 to base-mediated hydrolysis is enhanced in formulations
containing cholesterol.
[0005] Preferred compositions of the invention are those forming a
liposome structure. Compositions where the sterol/immunologically
active saponin fraction forms an ISCOM structure also form an
aspect of the invention.
[0006] The ratio of QS21:sterol will typically be in the order of
1:100 to 1:1 weight to weight. Preferably excess sterol is present,
the ratio of QS21:sterol being at least 1:2 w/w. Typically for
human administration QS21 and sterol will be present in a vaccine
in the range of about 1 .mu.g to about 100 .mu.g, preferably about
10 .mu.g to about 50 .mu.g per dose.
[0007] The liposomes preferably contain a neutral lipid, for
example phosphatidylcholine, which is preferably non-crystalline at
room temperature, for example eggyolk phosphatidylcholine, dioleoyl
phosphatidylcholine or dilauryl phosphatidylcholine. The liposomes
may also contain a charged lipid which increases the stability of
the liposome-QS21 structure for liposomes composed of saturated
lipids. In these cases the amount of charged lipid is preferably
1-20% w/w, most preferably 5-10%. The ratio of sterol to
phospholipid is 1-50% (mol/mol), most preferably 20-25%.
[0008] Preferably the compositions of the invention contain MPL
(3-deacylated mono-phosphoryl lipid A, also known as 3D-MPL).
3D-MPL is known from GB 2 220 211 (Ribi) as a mixture of 3 types of
De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains
and is manufactured by Ribi Immunochem, Montana. A preferred form
is disclosed in International Patent Application 92/116556.
[0009] Suitable compositions of the invention are those wherein
liposomes are initially prepared without MPL, and MPL is then
added, preferably as 100 nm particles. The MPL is therefore not
contained within the vesicle membrane (known as MPL out).
Compositions where the MPL is contained within the vesicle membrane
(known as MPL in) also form an aspect of the invention. The antigen
can be contained within the vesicle membrane or contained outside
the vesicle membrane. Preferably soluble antigens are outside and
hydrophobic or lipidated antigens are either contained inside or
outside the membrane.
[0010] Often the vaccines of the invention will not require any
specific carrier and be formulated in an aqueous or other
pharmaceutically acceptable buffer. In some cases it may be
advantageous that the vaccines of the present invention will
further contain alum or be presented in an oil in water emulsion,
or other suitable vehicle, such as for example, liposomes,
microspheres or encapsulated antigen particles.
[0011] Preferably the vaccine formulations will contain an antigen
or antigenic composition capable of eliciting an immune response
against a human or animal pathogen. Antigen or antigenic
compositions known in the art can be used in the compositions of
the invention, including polysaccharide antigens, antigen or
antigenic compositions derived from HIV-1, (such as gp 120 or gp
160), any of Feline Immunodeficiency virus, human or animal herpes
viruses, such as gD or derivatives thereof or Immediate Early
protein such as ICP27 from HSV1 or HSV2, cytomegalovirus
(especially human) (such as gB or derivatives thereof), Varicella
Zoster Virus (such as gpI, II or III), or from a hepatitis virus
such as hepatitis B virus for example Hepatitis B Surface antigen
or a derivative thereof, hepatitis A virus, hepatitis C virus and
hepatitis E virus, or from other viral pathogens, such as
Respiratory Syncytial virus (for example HSRV F and G proteins or
immunogenic fragments thereof disclosed in U.S. Pat. No. 5,149,650
or chimeric polypeptides containing immunogenic fragments from HSRV
proteins F and G, eg FG glycoprotein disclosed in U.S. Pat. No.
5,194,595), antigens derived from meningitis strains such as
meningitis A, B and C, Streptoccoccus Pneumonia, human papilloma
virus, Influenza virus, Haemophilus Influenza B (Hib), Epstein Barr
Virus (EBV), or derived from bacterial pathogens such as
Salmonella, Neisseria, Borrelia (for example OspA or OspB or
derivatives thereof), or Chlamydia, or Bordetella for example P.69,
PT and FHA, or derived from parasites such as plasmodium or
toxoplasma.
[0012] HSV Glycoprotein D (gD) or derivatives thereof is a
preferred vaccine antigen. It is located on the viral membrane, and
is also found in the cytoplasm of infected cells (Eisenberg R. J.
et al; J of Virol 1980 35 428-435). It comprises 393 amino acids
including a signal peptide and has a molecular weight of
approximately 60 kD. Of all the HSV envelope glycoproteins this is
probably the best characterised (Cohen et al J. Virology 60
157-166). In vivo it is known to play a central role in viral
attachment to cell membranes. Moreover, glycoprotein D has been
shown to be able to elicit neutralising antibodies in vivo and
protect animals from lethal challenge. A truncated form of the gD
molecule is devoid of the C terminal anchor region and can be
produced in mammalian cells as a soluble protein which is exported
into the cell culture supernatant. Such soluble forms of gD are
preferred. The production of truncated forms of gD is described in
EP 0 139 417. Preferably the gD is derived from HSV-2. An
embodiment of the invention is a truncated HSV-2 glycoprotein D of
308 amino acids which comprises amino acids 1 through 306 naturally
occuring glycoprotein with the addition Asparagine and Glutamine at
the C terminal end of the truncated protein devoid of its membrane
anchor region. This form of the protein includes the signal peptide
which is cleaved to allow for the mature soluble 283 amino acid
protein to be secreted from a host cell.
[0013] In another aspect of the invention, Hepatitis B surface
antigen is a preferred vaccine antigen.
[0014] As used herein the expression `Hepatitis B surface antigen`
or `HBsAg` includes any HBsAg antigen or fragment thereof
displaying the antigenicity of HBV surface antigen. It will be
understood that in addition to the 226 amino acid sequence of the
HBsAg antigen (see Tiollais et al, Nature, 317, 489 (1985) and
references therein) HBsAg as herein described may, if desired,
contain all or part of a pre-S sequence as described in the above
references and in EP-A-0 278 940. In particular the HBsAg may
comprise a polypeptide comprising an amino acid sequence comprising
residues 12-52 followed by residues 133-145 followed by residues
175-400 of the L-protein of HBsAg relative to the open reading
frame on a Hepatitis B virus of ad serotype (this polypeptide is
referred to as L*; see EP 0 414 374). HBsAg within the scope of the
invention may also include the pre-S1-preS2-S polypeptide described
in EP 0 198 474 (Endotronics) or close analogues thereof such as
those described in EP 0 304 578 (Mc Cormick and Jones). HBsAg as
herein described can also refer to mutants, for example the `escape
mutant` described in WO 91/14703 or European Patent Application
Number 0 511 855A1, especially HBsAg wherein the amino acid
substitution at position 145 is to arginine from glycine.
[0015] Normally the HBsAg will be in particle form. The particles
may comprise for example S protein alone or may be composite
particles, for example (L*,S) where L* is as defined above and S
denotes the S-protein of HBsAg. The said particle is advantageously
in the form in which it is expressed in yeast.
[0016] The preparation of hepatitis B surface antigen S-protein is
well documented. See for example, Harford et al (1983) in Develop.
Biol. Standard 54, page 125, Gregg et al (1987) in Biotechnology,
5, page 479, EP 0 226 846, EP 0 299 108 and references therein.
[0017] The formulations within the scope of the invention may also
contain an anti-tumour antigen and be useful for
immunotherapeutically treating cancers.
[0018] Vaccine preparation is generally described in New Trends and
Developments in Vaccines, edited by Voller et al., University Park
Press, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes
is described, for example, by Fullerton, U.S. Pat. No. 4,235,877.
Conjugation of proteins to macromolecules is disclosed, for
example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al.,
U.S. Pat. No. 4,474,757.
[0019] The amount of protein in each vaccine dose is selected as an
amount which induces an immunoprotective response without
significant, adverse side effects in typical vaccinees. Such amount
will vary depending upon which specific immunogen is employed and
how it is presented. Generally, it is expected that each dose will
comprise 1-1000 mcg of protein, preferably 2-100 mcg, most
preferably 4-40 mcg. An optimal amount for a particular vaccine can
be ascertained by standard studies involving observation of
appropriate immune responses in subjects. Following an initial
vaccination, subjects may receive one or several booster
immunisation adequately spaced.
[0020] The formulations of the present invention maybe used for
both prophylatic and therapeutic purposes.
[0021] Accordingly in a further aspect, the invention therefore
provides use of a vaccine of the invention for the treatment of
human patients. The invention provides a method of treatment
comprising administering an effective amount of a vaccine of the
present invention to a patient. In particular, the invention
provides a method of treating viral, bacterial, parasitic
infections or cancer which comprises administering an effective
amount of a vaccine of the present invention to a patient.
[0022] The following examples and data illustrates the
invention.
EXAMPLES
[0023] 1.1 Method of Preparation of Liposomes:
[0024] A mixture of lipid (such as phosphatidylcholine either from
egg-yolk or synthetic) and cholesterol in organic solvent, is dried
down under vacuum (or alternatively under a stream of inert gas).
An aqueous solution (such as phosphate buffered saline) is then
added, and the vessel agitated until all the lipid is in
suspension. This suspension is then microfluidised until the
liposome size is reduced to 100 nm, and then sterile filtered
through a 0.2 .mu.m filter. Extrusion or sonication could replace
this step.
[0025] Typically the cholesterol:phosphatidylcholine ratio is 1:4
(w/w), and the aqueous solution is added to give a final
cholesterol concentration of 5 to 50 mg/ml. If MPL in organic
solution is added to the lipid in organic solution the final
liposomes contain MPL in the membrane (referred to as MPL in).
[0026] The liposomes have a defined size of 100 nm and are referred
to as SUV (for small unilamelar vesicles). If this solution is
repeatedly frozen and thawed the vesicles fuse to form large
multilamellar structures (MLV) of size ranging from 500 nm to 15
.mu.m The liposomes by themselves are stable over time and have no
fusogenic capacity.
[0027] 1.2 Formulation Procedure:
[0028] QS21 in aqueous solution is added to the liposomes. This
mixture is then added to the antigen solution which may if desired
contain MPL in the form of 100 nm particles.
[0029] 1.3 The Lytic Activity of QS21 is Inhibited by Liposomes
Containing Cholesterol.
[0030] When QS21 is added to erythrocytes, they lyse them releasing
hemoglobin. This lytic activity can also be measured using
liposomes which contain cholesterol in their membrane and an
entrapped fluorescent dye, carboxyfluorescein--as the liposomes are
lysed the dye is released which can be monitored by fluorescence
spectroscopy. If the fluorescent liposomes do not contain
cholesterol in their membrane no lysis of the liposomes is
observed.
[0031] If the QS21 is incubated with liposomes containing
cholesterol prior to adding it to erythrocytes, the lysis of the
erythrocytes is diminished depending on the ratio of cholesterol to
QS21. If a 1:1 ratio is used no lytic activity is detected. If the
liposomes do not contain cholesterol, inhibition of lysis requires
a one thousand fold excess of phospholipid over QS21.
[0032] The same holds true using fluorescent liposomes to measure
the lytic activity. In the graph below, the lytic activity of 4
.mu.g of QS21 treated with liposomes lacking cholesterol (1 mg
eggyolk lecithin per ml) or containing cholesterol (1 mg lecithin,
500 .mu.g cholesterol per ml) was measured by fluorescence.
[0033] The data shows that QS21 associates in a specific manner
with cholesterol in a membrane, thus causing lysis (of cells or
fluorescent liposomes). If the QS21 first associates with
cholesterol in liposomes it is no longer lytic towards cells or
other liposomes. This requires a minimum ratio of 0.5:1
chol:QS21(w/w).
[0034] Cholesterol is insoluble in aqueous solutions and does not
form a stable suspension. In the presence of phospholipids the
cholesterol resides within the phospholipid bilayer which can form
a stable suspension of vesicles called liposomes. To avoid the
requirement to add phospholipids a soluble derivative was tried.
Polyoxyethanyl-cholesterol sebacate is soluble in water at 60 mg/ml
however even at a 2000 fold excess (w/w) over QS21 no reduction in
the lytic activity of QS21 was detected.
[0035] 1.4 Increased Stability of QS21 by Liposomes Containing
Cholesterol.
[0036] QS21 is very susceptible to hydrolysis at a pH above 7. This
hydrolysis can be monitored by measuring the decrease in the peak
corresponding to QS21 on reverse-phase HPLC. For example, the graph
below shows that at pH 9 and at a temperature of 37.degree. C., 90%
of QS21 is hydrolysed within 16 hours. If liposomes containing
cholesterol are added to the QS21 at a ratio of 2:1 (chol:QS21 w/w)
no hydrolysis of the QS21 is detected under the same conditions. If
the ratio is 1:1 10% of the QS21 is degraded.
[0037] It is concluded that when QS21 associates with liposomes
containing cholesterol it becomes much less susceptible to
base-mediated hydrolysis. The hydrolysis product is described as
having no adjuvant activity when given parenterally, hence vaccines
containing QS21 have to be formulated at acidic pH and kept at
4.degree. C. to maintain adjuvant composition. The use of liposomes
may overcome this requirement.
[0038] 1.5 Reactogenicity Studies:
[0039] Mice injected in tibialis muscle with 5 .mu.g QS21 (or
digitonin) added to increasing quantities of liposomes (expressed
in terms of .mu.g cholesterol). Lytic activity is expressed as
.mu.g QS21 equivalent, which means that quantity of QS21 required
to achieve the same hemolysis as the sample.
[0040] Redness, necrosis and toxicity in the muscle at the site of
injection were scored visually after sacrificing the mice.
1 lytic formulation activity .mu.g redness necrosis toxicity QS21 +
PBS 5 +++ .+-. +++ QS21 + 1 .mu.g chol (SUV) 4 +++ + ++++ QS21 + 5
.mu.g chol (SUV) 0 - - .+-. QS21 + 25 .mu.g chol (SUV 0 .+-. - +
SUV alone 0 - - - digitonin 5 - - .+-. PBS 0 - - -
[0041] The data shows that when the lytic activity is abolished by
the addition of liposomes containing cholesterol the toxicity due
to the QS21 is also abolished.
[0042] 1.6 Reactogenicity Intramuscularly in Rabbits
2 Values in U.I./L Experiment Formulation Day 0 hemolysis Day 1
hemolysis Day 3 hemolysis Rabbit n.degree. 1 QS21 50 .mu.g 1078
.+-. 8650 1523 Rabbit n.degree. 2 1116 4648 1435 Rabbit n.degree. 3
660 4819 684 Rabbit n.degree. 4 592 5662 684 Rabbit n.degree. 5
3400 7528 1736 Mean 1369 6261 1212 SD 1160 1757 495 Rabbit
n.degree. 6 QS21 50 .mu.g 596 1670 460 Rabbit n.degree. 7 Chol in
540 602 594 Rabbit n.degree. 8 SUV 50 .mu.g 611 1873 803 Rabbit
n.degree. 9 (1:1) 521 507 616 Rabbit n.degree. 10 1092 .+-. 787 555
Mean 672 1088 606 SD 238 636 125 Rabbit n.degree. 11 QS21 50 .mu.g
332 344 387 Rabbit n.degree. 12 Chol in 831 662 694 Rabbit
n.degree. 13 SUV 150 .mu.g 464 356 519 Rabbit n.degree. 14 (1:3)
528 720 614 Rabbit n.degree. 15 1027 568 849 Mean 637 530 613 SD
285 173 175 Rabbit n.degree. 16 QS21 50 .mu.g 540 769 745 Rabbit
n.degree. 17 Chol in 498 404 471 Rabbit n.degree. 18 SUV 250 .mu.g
442 717 (4535) Rabbit n.degree. 19 (1:5) 822 801 925 Rabbit
n.degree. 20 3182 .+-. 2410 960 Mean 1097 1020 775 (1527) SD 1175
793 224 (1692) Rabbit n.degree. 21 PBS 321 290 378 Rabbit n.degree.
22 660 535 755 Rabbit n.degree. 23 650 603 473 Rabbit n.degree. 24
1395 (3545) (5749) Rabbit n.degree. 25 429 .+-. 323 263 Mean 691
438 (1059) 467 (1523) SD 419 155 (1396) 210 (2369)
[0043] The data shows that the addition of cholesterol-containing
liposomes to the formulation significantly reduces the elevation in
CPK (creatine phospho kinase) caused by the QS21. Since the CPK
increase is a measure of muscle damage this indicates decreased
muscle damage and is confirmed by the histopathology.
[0044] 1.7 Binding of the Liposome-QS21 Complex to Alum.
[0045] QS21 was incubated with neutral liposomes containing excess
cholesterol as well as radioactive cholesterol and then incubated
with alum (Al(OH).sub.3) in PBS. Alone, neither neutral liposomes
nor QS21 bind to alum in PBS, yet negatively charged liposomes do.
When together however, QS21 and neutral liposomes bind to alum. The
supernatant contained neither QS21 (assayed by orcinol test) nor
radioactive cholesterol.
[0046] This indicates that the QS21 has bound to the liposomes and
permits the liposome-QS21 combination to bind to the alum. This may
arise from a negative charge being imposed on the liposomes by the
QS21, or to an exposure of hydrophobic regions on the liposomes.
The results also imply that QS21 does not extract cholesterol from
the membrane.
[0047] This indicates that compositions of the invention can be
used in alum based vaccines.
[0048] 1.8 Comparison of Liposomal QS21/MPL and Free QS21+MPL for
Antibody and CMI Induction
[0049] SUV were prepared by extrusion (EYPC:chol:MPL 20:5:1).
[0050] For MPL out, liposomes were prepared without MPL and MPL
added as 100 nm. particles
[0051] QS21 was added prior to antigen. Chol:QS21=5:1 (w/w)
[0052] MLV were made by freeze-thawing SUV 3.times. prior to
antigen addition.
[0053] To have the antigen entrapped, the antigen was added to SUV
prior to freeze-thawing and QS21 added after freeze-thaw. Antigen
encapsulation=5% in, 95% out.
[0054] mice (balb/c for gD, B10BR for RTSs) were injected twice in
the footpad.
[0055] gD is the glycoprotein D from Herpes Simplex virus. RTSs is
the Hepatitis B surface antigen (HBsAg) genetically modified to
contain an epitope from the Plasmodiium falciparum sporozoit.
3 anti HBsAg Titres ag = 10 .mu.g RTSs 15 days post boost
formulation IgG1 IgG2a IgG2b SUV/QS + MPL(out) + Ag 1175 10114
71753 MLV/QS + MPL(out) + Ag 2247 11170 41755 MLV/QS/MPL(in) + Ag
969 7073 18827 MLV/QS/MPL(in)/Ag(in) + Ag 1812 2853 9393 QS + MPL +
Ag 372 9294 44457 Ag <100 <100 <100 SUV/QS + MPL(out)
<100 <100 <100 MLV/QS/MPL(in) <100 <100 <100
[0056]
4 anti- CMI ag = 20 .mu.g gD gD IFN-.gamma.96 hr IL2 48 hr
formulation IgG (pg/ml) pg/ml SUV/QS + MPL(out) + Ag 2347 1572 960
SUV/QS/MPL(in) + Ag 2036 1113 15 MLV/QS + MPL(out) + Ag 1578 863 15
MLV/QS/MPL(in) + Ag 676 373 15 MLV/QS/MPL(in)/Ag(in) + Ag 1064 715
15 QS + MPL + Ag 1177 764 15 Ag <100 567 44 SUV/QS + MPL(out)
<100 181 15 MLV/QS/MPL(in) <100 814 105
[0057] The data shows that SUV/QS+MPL.sub.(out) induces high
antibody titres at least as good as QS21+MPL, as well as inducing
IL2 a marker of cell mediated immunity, while quenching QS21
reactogenicity.
[0058] Additional results from a second experiment comparing QS21
and QS21 in the presence of cholesterol (SUV) in balb/c mice with
HSV gD as antigen are shown below:
5 IgG 7 post IgG 14 post Isotypes 7 days post II II II IgG1 IgG2a
IgG2b Formulation antigen (GMT) (GMT) .mu.g/ml % .mu.g/ml %
.mu.g/ml % SUV/QS21 + MPL out gD (5 .mu.g) 20290 16343 331 26 716
56 222 17 SUV/QS21/MPLin gD (5 .mu.g) 12566 10731 418 44 412 44 111
12 QS21 + MPL gD (5 .mu.g) 10504 10168 200 34 285 48 107 18
SUV/QS21 + MPL out none 0 0 0 0 0 0 0 0 QS21 gD (5 .mu.g) 3468 4132
156 66 67 28 14 6 SUV/QS21 gD (5 .mu.g) 11253 11589 484 57 304 36
65 8
[0059] 1.9 Comparison of gp120 Plus Liposomal MPL/QS21 With Free
MPL/QS21
[0060] Liposomes=SUV containing MPL in the membrane
Chol:QS21=6:1
[0061] Response was tested two weeks after one immunisation
6 IFN-g IL2 IL5 formulation proliftn ng/ml pg/ml pg/ml SUV/MPL/QS21
+ Ag 12606 16.6 59 476 MPL + QS21 + Ag 16726 15.8 60 404
[0062] After second immunisation:
7 IFN-g IL4 IL5 formulation proliftn ng/ml pg/ml pg/ml SUV/MPL/QS21
+ Ag 12606 135 0 250 MPL + QS21 + Ag 16726 60 0 500
[0063] The data shows that QS21 associated with
cholesterol-containing liposomes and MPL induces Th1/Th0 response
equal to MPL+QS21.
[0064] At this ratio of cholesterol to QS21, QS21 is non-toxic in
rabbits (by CPK).
[0065] In a second experiment balb/c mice were immunised
intra-footpad with gp120 in the presence of QS21 or QS21+SUV
containing cholesterol. The cytotoxic T-lymphocyte activity in
spleen cells was measured.
[0066] This demonstrates that QS21 alone induces CTL activity, and
that QS21 in the presence of liposomes containing cholesterol
induces CTL activity at least as good as, or better than, QS21
alone.
[0067] 2. Vaccines
[0068] 2.1 Formulation of HBsAg L*,S Particles.
[0069] HBsAg L*,S particles may be formulated as follows:
[0070] 10 .mu.g of HBsAg L*,S particles/dose are incubated 1 h. at
room temperature under agitation. The volume is adjusted using
water for injection and a PBS solution and completed to a final
volume of 70 .mu.l/dose with an aqueous solution of QS21 (10
.mu.g/dose). pH is kept at 7.+-.0.5.
[0071] Similar formulations may be prepared using 1 and 50 .mu.g of
HBsAg L*,S and also using the HBsAg S antigen.
[0072] These formulations may be tested in the Woodchuck surrogate
therapeutic model using Woodchuck HBV antigens as a model.
[0073] Woodchuck Model
[0074] DQ QS21 (i.e. QS21/cholesterol or quenched QS21) may be
tested in the woodchuck therapeutic model where animals are
chronically infected with the virus. Specific woodchuck hepatitis
virus vaccine may be add mixed with QS21 as such or DQ and with or
without MPL and administered to the animals every months for 6
months. Effectiveness of the vaccine may be assess through viral
DNA clearance.
[0075] 2.2 Guinea Pig Model (HSV)
[0076] 2.2.1 Prophylactic Model
[0077] Groups of 12 female Hartley guinea pigs were either injected
intramuscularly on day 0 and day 28 with the following
formulations:
[0078] 1st experiment:
gD 5 .mu.g+QS21 50 .mu.g+SUV containing 50 .mu.g cholesterol
gD 5 .mu.g+QS21 100 .mu.g+SUV containing 100 .mu.g cholesterol
gD 5 .mu.g+QS21 50 .mu.g+SUV containing 250 .mu.g cholesterol
gD 5 .mu.g+QS21 50 .mu.g
[0079] 2nd experiment:
gD 5 .mu.g+MPL 12.5 .mu.g+QS21 12.5 .mu.g+SUV containing 62.5 .mu.g
cholesterol, or left untreated.
[0080] The animals were bled at 14 and 28 days after the second
immunisation, and the sera tested for their gD-specific ELISA
antibody titres.
[0081] Animals were then challenged intravaginally with 10.sup.5
pfu HSV-2 MS strain. They were scored daily from day 4 to 12 for
evaluation of primary herpetic lesions. Scoring was as follows:
[0082] Vaginal lesions:
[0083] bleeding=0.5
[0084] redness for one or 2 days without bleeding=0.5
[0085] redness and bleeding for a day=1
[0086] redness without bleeding lasting at least 3 days=1
[0087] External herpetic vesicles:
[0088] <4 small vesicles=2
[0089] >=4 small vesicles or one big vesicle 4>=4 large
lesions 8 fusing large lesions=16
[0090] fusing large lesions on all external genital area=32.
[0091] The results are shown in the table below:
8 Prophylactic Model Experiment 1 (chol refers to SUV containing
cholesterol) PRIMARY DISEASE Animal Vaginal External Lesion without
lesions lesions PI reduction severity * n FORMULATION lesion %
incidence % incidence % Index ** vs Control Median n 12 gD/QS21 50
.mu.g 50 33 17 73 93% 1.50 6 11 gD/QS21 50 .mu.g-chol 1/5 64 18 18
67 93% 2.50 4 12 gD/QS21 50 .mu.g-chol 1/1 100 0 0 0 100% -- -- 12
gD/QS21 50 .mu.g-chol 1/1 50 33 17 54 95% 0.75 6 12 UNTREATED 25 0
75 996 -- 55.00 9 Experiment 2 PRIMARY DISEASE Ab titres (GMT)
Animal Vaginal External ELISA NEUTRA without lesions lesions Lesion
day 14 day 28 day 28 lesion incidence incidence PI reduction
severity * n FORMULATION post II post II post II % % % Index ** vs
Control Median n 12 gD/QS21/SUV/MPL 47006 31574 449 58.33 33.33
8.33 37.50 94% 1.00 5 12 UNTREATED <400 <400 <50 16.67
8.33 75.00 587.50 -- 11.50 10 * Sum of the lesion scores for the
days 4 to 12 post-infection (animals without lesion are not
considered). Lesion scores: no lesion (0), vaginal lesions (0.5 or
1), external skin vesicles (2, 4, 8 or 16) ** Primary infection
index = SUM (Max. score i) .times. (Incidence %); with i = 0, 0.5,
1, 2, 4, 8 or 16
[0092] The table and graph show that in the prophylactic model, a
very high level of protection against primary disease was induced
upon immunisation with gD/MPL/QS21/SUV. Both the incidence of
external lesions and the lesion severity appeared highly reduced in
the group of animals immunised with gD/MPL/QS21/SUV.
[0093] 2.2.2 Therapeutic Model
[0094] In the therapeutic model, female Hartley guinea pigs were
first challenged with 10.sup.5 pfu HSV-2 MS strain. Animals with
herpetic lesions were then randomly allotted to groups of 16.
[0095] On day 21 and day 42, they were either immunised with one of
the following formulations:
gD+MPL 50 .mu.g+QS21 50 .mu.g+SUV containing 250 .mu.g
cholesterol,
gD+Al(OH).sub.3+MPL 50 .mu.g+QS21 50 .mu.g,+SUV containing 250
.mu.g cholesterol or left untreated.
[0096] They were monitored daily from day 22 to 75 for evaluation
of recurrent disease. Scoring was as described for the prophylactic
model. The results are shown in the table and graph below:
9 Therapeutic Model SEVERITY * DURATION ** EPISODE NBER *** %
reduction % reduction % reduction n FORMULATIONS Median vs Control
Median vs Control Median vs Control 16 gD + MPL + QS21 + SUV 9.00
43% 7.00 18% 3.00 14% 15 gD + Al(OH)3 + MPL + QS21 + SUV 8.50 46%
7.00 18% 3.00 14% 16 Untreated 15.75 -- 8.50 -- 3.50 -- * Sum of
the lesion scores for the days 22 to 75 post-infection. ** Total
days animals experienced recurrent lesions for the days 22 to 75
post infection *** Recurrence episode number for the days 22 to 75
post infection. One episode is preceded and followed by a day
without lesion and characterized by at least two days with erythema
(score = 0.5) or one day with external vesicle (score >= 2)
Immunotherapeutical treatment performed on days 21 and 42.
[0097] The results show that good levels of protection were also
induced in the therapeutic model of HSV-2 infection. Immunization
with gD/MPL/QS21/SUV with or without Alum had a marked effect on
the median severity of recurrent disease. It also slightly reduced
episode number and duration (see Table).
* * * * *