AP771A

AP771A - Vaccines containing a saponin and a sterol.

Info

Publication number: AP771A
Application number: APAP/P/1997/001123A
Authority: AP
Inventors: Nathalie Marie-Josephe Claude Garcon-Johnson; Martin Friede
Original assignee: Smithkline Beecham Biologicals S A
Priority date: 1995-04-25
Filing date: 1996-04-01
Publication date: 1999-10-07
Also published as: DE69637254T2; ATE373487T1; BG101995A; CZ296216B6; HU227944B1; SI0822831T1; SA96170297B1; BG63491B1; DZ2026A1; NO974859D0; RO119068B1; KR100463372B1; EP0955059A3; CA2217178A1; CN1248737C; BR9608199B1; EP0955059B1; NO974859L; EP0822831B2; CN1289065C

Abstract

The invention relates to a vaccine composition comprising an antigen, an immunoiogically active saponin fraction and a sterol.

Description

VACCINES CONTAINING A SAPONIN AND A STEROL

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.

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 US patent 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.

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 β-sitosterol, sdgmasterol, 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

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 basemediated hydrolysis is enhanced in formulations containing cholesterol.

AP/P/ S 7 / 0 1 12 3

Preferred compositions of the invention are those forming a liposome structure. Compositions where the steroi/immunologically active saponin fraction forms an IS COM. structure also form an aspect of the invention.

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 gg to about 100 gg, preferably about 10 μ§ to about 50 μg per dose.

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. Tne liposomes may also contain a charged lipid which increases the stability of the lipsomeQS21 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%.

Preferably the compositions of the invention contain MPL (3-deacylated monophosphoryl 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.

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.

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.

AP.00771

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, anrigen or antigenic compositions derived from HIV-1, (such as gpl 20 or gpl 60), 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 orHSV2, cytomegalovirus (especially human) (such as gB or derivatives thereof), Varicella Zoster Virus (such as gpl, Π 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 US Patent 5,149,650 or chimeric polypeptides containing immunogenic fragments from HSRV proteins F and.

G, eg FG glycoprotein disclosed in US Patent 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 Bair 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.

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 F 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

AP/P/ 9 7/01 123 peptide which is cleaved to allow for the mature soluble 283 amino acid protein to be secreted from a host cell.

In another aspect of the invention, Hepatitis B surface antigen is a preferred vaccine antigen.

(

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-S l-preS2-S polypeptide described in EP 0 198 474 (Endocronics) or close analogues thereof such as those described in EP 0 304 578 (Me 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.

Normally the HBsAg will be in particle form. The particles may comprise for example S protein alone or may be composite panicles, 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.

The preparation of hepatitis B surface antigen S-protein is well documented- See for example, Harford et ai (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.

The formulations within the scope of the invention may also contain an anti-tumour antigen and be useful for imrnunotherapeutically treating cancers.

Vaccine preparation is generally described in New Trends and Developments in

Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A.

AP. Ο Ο 7 7 1

1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for' example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

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 meg of protein, preferably 2-100 meg, most preferably 4-40 meg. 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.

¹⁵

The formulations of the present invention maybe used for both prophylatic and therapeutic purposes.

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 ah 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.

The following examples and data illustrates the invention.

AP/P/ 9 7/01123

Examples

1.1 Method of preparation of liposomes:

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 pm filter. Extrusion or sonication could replace this step.

Typically the cholesterohphosphaddylcholine 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 ±e final liposomes contain MPL in .the membrane (referred to as MPL in).

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 500nm to 15 pm. The liposomes by themselves are stable over time and have no fusogenic capacity.

1.2 Formulation procedure:

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 lOOnm particles.

1.3 The lytic activity of QS21 is inhibited by liposomes containing cholesterol.

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.

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

AP. Ο Ο 7 7 1 liposomes do not contain cholesterol, inhibition of lysis requires a one thousand fold excess of phospholipid over QS21.

The same holds true using fluorescent liposomes to measure the lytic activity.

In the graph below, the lyric activity of 4 pg of QS21 treated with liposomes lacking cholesterol (1 mg eggyolk lecithin per ml) or containing cholesterol (1 mg lecithin, 500 pg cholesterol per ml) was measured by fluorescence.

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).

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. Polyoxyethanyl20 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.

AP/P/ 9 7/01 123

1.4 Increased stability of QS21 by liposomes containing cholesterol.

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 reversephase HPLC. For example, the graph below shows that at pH 9 and at a temperature of 37°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.

Incubation of 20 pg QS21 In pr*jenc· ot SUV containing cholesterol at pH ? for Ιό hra at arc

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°C to maintain adjuvant composition. The use of liposomes may overcome this requirement

1.5 Reactogenicity studies:

Mice injected in tibialis muscle with 5 pg QS21 (or digitonin) added to increasing quantities of liposomes (expressed in terms of pg cholesterol). Lytic activity is expressed as pg QS21 equivalent, which means that quantity of QS21 required to achieve the same hemolysis as the sample.

AP. Ο Ο 7 7 1

Redness, necrosis and toxicity in the muscle at the site of injection were scored visually after sacrificing the mice.

formulation	lytic activity pg	redness	. necrosis	toxicity
QS21 -PBS	5	-r-~-
QS21 +1 pg chol (SUV)	4		-i-
QS21 +5 pg chol (SUV)	0 .	-
QS21+25 pg chol (SUV	0		-	-r
SUV alone	0	-	-	-
digitonin	5	-	-
PBS	0	-	-	-

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.

1.6 Reactogenicity intra-muscularly in rabbits io

Values in U.I./L

Experiment	Formulation	DayO	hemolysis	Dayl	hemolysis	Day 3	hemolysis
Rabbit n°l Rabbit n°2 Rabbit n°3 Rabbit n°4 Rabbit n°5	QS21 50pg	1078 1116 660 592 3400		8650 4648 4819 5662 7528		1523 1435 684 684 1736
Mean SD	1369 1160		6261 1757		1212 495

AP/P/ 9 7/01123

SUBSTITUTE SHEET (RULE 26)

Experiment	Formulati on	DayO	hemolysis	Dayl	hemolysis	Day 3	hemolysis
Rabbit n°6 Rabbit n°7 Rabbit n°8 Rabbit n°9 Rabbit n°10	QS21 50pg Choi in SUV 50pg (i-.i)	596 540 611 521 1092		1670 602 1873 507 787		460 594 803 616 555
Mean SD	672 238		1088 636		606 125

Experiment	Formulati on	DayO	hemolysis	Dayl	hemolysis	Day3	hemolysis
Rabbit n° 11		332		344		387
Rabbit n°12		831		662		694
Rabbit n°13	QS21 50pg	464		356		519
Rabbit n°14	Choi in	528		720		614	e
	SUV 150pg
Rabbit n°15	(1:3)	1027		568		849
Mean		637		530		613
SD		285		173		175

Experiment	Formulation	DayO	hemolysis	Dayl	hemolysis	Day3	hemolysis
Rabbit n°16 Rabbit n°17 Rabbit n°l 8 Rabbit n°19 Rabbit n°20	QS21 50pg Choi in SUV 250pg (1:5)	540 498 442 822 3182	4-	769 404 717 801 2410		745 471 (4535) 925 960
Mean 1 SD	1097 1175		1020 793		775 224	(1527) (1692)

SUBSTiTUTE SHEET (RULE 26)

AP. ο Ο 7 7 1

Experiment	Formulation	DayO	hemolysis	Dayl	hemolysis	Day3	hemolysis
Rabbit n°21 Rabbit n°22 · Rabbit n°23 Rabbit n°24 Rabbit n°25	PBS	. 321 660 650 1395 429		290 535 603 (3545) 323		378 755 473 (5749) 263
Mean SD	691 419		438 155	(1059) (1396)	467 210	(1523) (2369)

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.

1.7 Binding of the liposome-QS21 complex to alum.

QS21 was incubated with neutral liposomes containing excess cholesterol as well as radioactive cholesterol and then incubated with alum (A1(OH)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.

This indicates that the QS21 has bound to the liposomes and permits the iiposomeQS21 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.

This indicates that compositions of the invention can be used in alum based vaccines.

1.8 Comparison of liposomal QS21/MPL and free QS21+MPL for antibody arid CMI induction

AP/P/ 9 7./ 0 1 1 2 3

SUV were prepared by extrusion (EYPC:chol:MPL 20:5:1).

SUBSTITUTE SHEET (RULE 26)

For MPL out, liposomes were prepared without MPL and MPL added as 100 nm. particles

QS21 was added prior to antigen. Chol:QS21 = 5:1 (w/w)

MLV were made by freeze-thawing SUV 3x prior to antigen addition.

To have theantigen entrapped, the antigen was added to SUV prior to freeze-thawing and QS21 added after freeze-thaw. Antigen encapsulation = 5% in, 95% out.

-mice (balb/c for gD, B10BR for RTSs) were injected twice in the footpad.

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

ag = 10 pg RTSs	anti HBsAg Titres 15days post boost
formulation	IgGl	IgG2a	IgG2b
SUV/QS + MPL(out) + Ag	1175	10114	71753
MLV/QS + MPL(oui) + Ag	2247	11170	41755
MLV/QS/MPL(m) + As	969	7073	18827
MLV/QS/MPL<in)/Ag(m) + 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

ag = 20pg gD	anti- gD	CMI
formulation		IgG	IFN-y96 hr (pg/ml)	IL2 48hr 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/MPLfm)	+ Ag	676	373	15
MLV/QS/MPL(in)/Aa(in)	+ Ag	1064	715	15
QS + MPL	+ Ag	1177	764	15
	Ag	<100	567	44
SUV/QS + MPL(out)		<100	181	15
MLV/QS/MPLcin)		<100	814	105

AP. Ο Ο 7 Ί 1

The data shows that SUV/QS-MPLiout) induces high antibody titres at least as good 5 as QS21+MPL, as well as inducing IL2 a marker of cell mediated immunity, while quenching QS21 reactogenicity.

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:

Isotypes 7days post II

Formulation	antigen	IgG 7 post II (GMT)	IgG 14post II (GMT)	IgGl gg/ml	%	IgG2a gg/ml	%	IgG2b gg/ml		%
SUV/QS21 + MPL out	gD (5gg)	20290	16343	331	26	716	56		222	17
SUV/QS2i/MPLin	gD (5gg)	12566	10731	418	44	412	44		111	12
QS21+MPL	gD (5gg)	10504	10168	200	34	285	48		107	18
SUV/QS21 +MPL out	none	0	0	0	0	0	0		0	0
QS21	gD(5gg)	3468	4132	156	66	67	28		14	6
SUV/QS21	gD (5gg)	11253	11589	484	57	304	36		65	8

1.9 Comparison of gpl20 plus liposomal MPL/QS21 with free MPL/QS21

Liposomes = SUV containing MPL in the membrane

Chol:QS21 = 6:1 £ g U 0 / L 6 /d/dV

Response was tested two weeks after one immunisation

formulation	proliftn	IFN-g ng/ml	IL2 pg/ml	ILS pg/ml
SUV/MPL/QS21 + Ag	12606	16.6	59	476
MPL+QS21+Ag	16726	15.8	60	404

After second immunisation:

formulation	proliftn	IFN-g ng/ml	IL4 pg/ml	ILS pg/ml
SUV/MPL/QS21 + Ag	12606	135	0	250
MPL+QS21+Ag	16726	60	0	500

SUBSTITUTE SHEET (RULE 26)

The data shows that QS21 associated with cholesterol-containing liposomes and MPL induces Thl/ThO response equal to MPL+QS21.

At this ratio of cholesterol to QS21, QS21 is non-toxic in rabbits (by CPK).

In a second experiment balb/c mice were immunised intra-footpad with gpl20 in the presence of QS21 or QS21 + SUV containing cholesterol. The cytotoxic T-lymphocyte activity in spleen cells was measured.

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.

2. Vaccines

2.1 Formulation of HBsAg L*,S particles.

HBsAg L*,S particles may be formulated as follows:

10pg of HBsAg L*,S particles/dose are incubated lh. at room temperature under agitation. The volume is adjusted using water for injection and a PBS solution and completed to a final volume of 70μ1/ dose with an aqueous solution of QS21 (10μ g/dose). pH is kept at 7 ± 0.5.

Similar formulations may be prepared using 1 and 50pg of HBsAg L*,S and also using the HBsAg S antigen.

These formulations may be tested in the Woodchuck surrogate therapeutic modeL using

Woodchuck HB V antigens as a model.

AP.00771

Woodchuck model

DQ QS21 (i.e. QS21/cholesrerol 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 wi± 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.

2.2 Guinea Pig Model (HSV)

2.2.1 Prophylactic model

Groups of 12 female Hanley guinea pigs were either injected intramuscularly on day 0 and day 28 with the following formulations:

1st experiment:

gD 5 pg + QS21 50 pg + SUV containing 50 pg cholesterol gD 5 pg + QS21 100 pg + SUV containing 100 pg cholesterol gD 5 pg + QS21 50 pg + SUV containing 250 pg cholesterol gD 5 pg + QS21 50 pg 2nd experiment:

gD 5pg + MPL 12.5 pg + QS21 12.5pg + SUV containing 62.5 pg cholesterol, or left untreated.

The animals were bled at 14 and 28 days after the second immunisation, and the sera tested for their gD-specific ELISA antibody titres.

Animals were then challenged intravaginally with 105 pfu HSV-2 MS strain. They 30 were scored daily from day 4 to 12 for evaluation of primary herpetic lesions. Scoring was as follows:

Vaginal lesions:

- bleeding = 0.5

- redness for one or 2 days without bleeding = 0.5

- redness and bleeding for a day = 1

- redness without bleeding lasting at least 3 days = 1 External herpetic vesicles:

- < 4 small vesicles = 2

- >= 4 small vesicles or one big vesicle 4 >= 4 large lesions 8 fusing large lesions =

16

- fusing large lesions on all external genital area = 32.

AP/P/ 9 7/01123

The results are shown in the table below:

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AP/P/ 97 / 0 112 3

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.

2.2.2 Therapeutic Model

In the therapeutic model, female Hartley guinea pigs were first challenged with 10^ pfu HSV-2 MS strain. Animals with herpetic lesions were then randomly allotted to groups of 16.

On day 21 and day 42, they were either immunised with one of the following formulations:

- gD + MPL 50pg + QS21 50pg + SUV containing 250 pg cholesterol,

- gD + A1(OH)3 + MPL 50pg + QS21 50ug, + SUV containing 250 pg cholesterol or left untreated.

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:

(_Λ

MPL/ QS21/SUV wiih or without Alum had a marked effect on the median severity of recurrent disease. It also slightly reduced episode number and duration (see Table).

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Claims

Claims for National Phase

0 0 7 7 1 *

(.'m ine now jx'siiciij.iriv liescriHot! .nnt', . '.-·ο·.’ίΊ a ι lied iip./our Said i ;i v.;n. i· and in ' v. l·:;:·. . winner :lit* same is :n he pcii;n nie<.','

i. v.e dee lure that what I/\ve claim is —

1. A vaccine composition comprising an antigen, an immunologically active saponin fraction, and a sterol, characterised in that the ratio of saponin:sterol is from 1:1 to l:100(w/w).
2. A vaccine composition according to claim 1 wherein the ratio of saponimsterol is in the range from 1:1 to 1:5 (w/w).
3. A vaccine composition according to claim 1 wherein the ratio of saponimsterol is 1:2 (w/w).
4. A vaccine composition according to any of claims 1 to 3, wherein the immunologically active saponin fraction is QS21.
5., A vaccine composition according to any of claims 1 to 3, wherein the sterol is cholesterol.
6. A vaccine composition according to any of claims 1 to 5 which further contains 3D-MPL.

AP/P/ 9 7/01 123
7. A vaccine composition as claimed in any of claims 1 to 6 which further comprises a carrier.
8. A vaccine composition as claimed in claim 7, wherein the carrier is selected from the group comprising: oil in water emulsions, alum, microspheres and encapsulated antigen particles.
9. A vaccine composition as claimed in any of the claims 1 to 8, further comprising an antigen or antigenic composition derived from any of Human Immunodeficiency Virus, Feline Immunodeficiency Virus, Varicella Zoster virus, Herpes

AP .0 0 7 7 1

Simplex Virus type 1, Herpes Simplex virus type 2, Human cytomegalovirus, Hepatitis A, B, C or E, Respiratory Syncytial virus, human papilloma virus, Influenza virus, Hib, Meningitis virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium or Toxoplasma.'
10. A vaccine as claimed in any of the claims 1 to 8, further comprising an antigen derived from a tumour.
1 1. A composition as claimed in any of the preceding claims for use in medicine.
12. Use of composition as defined in any of the claims 1 to 9, for the manufacture of a vaccine for the prophylatic treatment of viral, bacterial or parasitic infections.
13. Use of composition as defined in any of the claims 1 to 10, for the manufacture of a /accine for the immunotherapeutic treatment of viral, bacterial, parasitic infections or cancer.
14. A method of treating a mammal suffering from or susceptible to a pathogenic infection comprising the administration of a safe and effective amount of a composition according to any of claims 1 to 9.
15. A method of treating a mammal suffering from or susceptible to cancer comprising the administration of a safe and effective amount of a composition as claimed in claim 10.
16. A process for making a vaccine composition as claimed in any of claims 1 to 10, comprising admixing an immunoiogically active saponin fraction and cholesterol with an antigen or antigenic composition.