CZ116799A3 - Adjuvant preparation and vaccine - Google Patents

Abstract

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

Description

Adjuvant and vaccine

Technical field

The present invention relates to novel vaccine compositions, methods for their preparation and their use in medicine. In particular, the present invention relates to vaccines comprising alum, an antigen, an immunologically active fraction from the bark of Quillaja saponaria molina such as QS21 and a sterol.

State of the art

Immunologically active saponin fractions having adjuvant activity obtained 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 tree Quillaja saponaria molina and a process for its production are described (as QA21) in U.S. Patent No. 5,057,540. Quillaja saponin has also been described as an adjuvant in Scott et al., Int. Archs. Allergy Appl. Immun. 1985, 77: 409. However, the use of QS21 as an adjuvant is associated with several disadvantages. For example, when QS21 is injected as a free molecule into mammals, necrosis, i.e., localized tissue death, has been observed at the injection site.

WO 96/33739 describes a vaccine comprising an antigen, an immunologically active fraction from the bark of Quillaja saponaria molina, such as QS21, and a sterol.

It has now been surprisingly found that the inclusion of alum in a vaccine containing MPL, QS21 and SUV increases both antibody and cellular responses and that vaccines containing MPL, QS21, SUV and alum are non-toxic with a good reactivity profile and that they have increased adjuvant activity. Furthermore, it appears that the combined adjuvant

• · · ·

they favor a TH1 response.

The essence of the invention

In a first aspect, the invention comprises a vaccine adjuvant comprising alum, an immunologically active saponin fraction and a sterol. By alum is meant aluminum hydroxide or aluminum phosphate.

Preferably, the adjuvant composition of the present invention comprises an immunologically active saponin fraction in a significantly purified form. Preferably, the composition of the present invention comprises QS21 in a significantly purified form, i.e., the QS21 is at least 90% pure, more preferably at least 95% pure, and most preferably at least 98% pure.

Other immunologically active saponin fractions useful in the compositions of the present invention are QA17/QS17. Compositions of the present invention containing QS21 and cholesterol show lower induced reactivity compared to compositions without cholesterol, while the adjuvant effect is maintained. In addition, it is known that QS21 is degraded under alkaline conditions when the pH is 7 or higher. Another advantage of the compositions of the present invention is that the stability of QS21 to alkaline hydrolysis is increased in compositions containing cholesterol.

Preferred sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. These sterols are well known in the art, for example cholesterol is described in the Merck Index, 11th edition, page 341, as a natural sterol from animal fat.

The most preferred sterol is cholesterol.

-

Preferred compositions of the present invention are compositions forming a liposomal structure, i.e. small unilamellar vesicles (SUVs). Compositions in which the sterol/immunologically active saponin fraction forms an ISCOM structure also form an aspect of the present invention.

The ratio of QS21:sterol will typically be 1:100 to 1:1 by weight. Preferably, an excess of sterol is used, and the ratio of QS21:sterol is at least 1:1 by weight:weight. In typical vaccines for administration to humans, QS21 and sterol will be in an amount of from about 1 µg to about 100 µg, preferably from 10 µg to about 50 µg per dose of QS21.

The liposomes preferably comprise a neutral lipid, for example phosphatidylcholine, which is preferably non-crystalline at room temperature, for example egg yolk phosphatidylcholine, dioleoylphosphatidylcholine or dilaurylphosphatidylcholine. The liposomes may also comprise 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 typically 1-20% w/w, preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), preferably 20-25%.

Particularly preferred according to the present invention are compositions containing MPL (3-deacylated mono-phosphoryl-lipid A, also known as 3D-MPL). 3D-MPL is known from GB 2220211 (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 described in International Patent Application 94/21292.

Suitable compositions according to the present invention are those in which liposomes are first prepared without MPL, and MPL is then added/preferably in the form of 100 nm particles. MPL is thus not contained in • · 4 4 · 4

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4 4 4 vesicle membrane (compositions known as MPL out). Compositions in which MPL is contained in the vesicle membrane (known as MPL in) also form an aspect of the present invention. The antigen may be contained in the vesicle membrane or may be contained outside the vesicle membrane. Preferably, soluble antigens are outside the membrane and hydrophobic or lipidated antigens are either in the membrane or outside the membrane.

In a preferred aspect of the present invention, liposomes/SUVs are first added to QS21 and then this mixture is mixed with alum, resulting in a significant portion of QS21 being bound to the alum (by interaction with the liposomes). Such compositions, when injected, will result in a slower release of QS21 into the body, due to the effect of the alum, than if QS21 were free or not fixed to the liposomes. Compositions comprising MPL, QS21, SUVs and alum are particularly preferred because they are non-toxic and highly immunogenic.

Preferably, the vaccine compositions of the present invention will comprise an antigen or antigenic composition capable of eliciting an immune response against a human or animal pathogen. In a first aspect, therefore, the invention comprises a vaccine composition comprising alum, an antigen, an immunologically active saponin fraction and a sterol.

Antigens or antigenic compounds known in the art may be used in the compositions of the present invention, including polysaccharide antigens, antigens or antigenic compounds derived from HIV-1 (such as gp120 and gp160), any feline immunodeficiency virus, human or animal herpes viruses such as gD or derivatives thereof or from immediate early proteins such as ICP27 from HSV1 or HSV2, cytomegalovirus (particularly human) (such as gB or derivatives thereof), varicella zoster virus (such as gp1, II or III) or from hepatitis viruses such as hepatitis virus : ' r • · ·· • · · · · • · ·

B, for example, the surface antigen of hepatitis B virus or its derivatives, hepatitis A virus, hepatitis C virus or hepatitis E virus, or from other viral pathogens such as respiratory syncytial virus (for example, the F or G proteins of HSRV or immunogenic fragments thereof described in U.S. Patent 5,149,650 or chimeric polypeptides comprising immunogenic fragments from the F and G proteins of HSRV, for example, the FG glycoprotein described in U.S. Patent 5,194,595), antigens derived from meningitis strains such as meningitis A, B and C, Streptococcus pneumoniae, human papillomavirus, Haemophilus influenzae B (Hib), Epstein-Barr virus (EBV) or from bacterial pathogens such as Salmonella, Neisseria, Borrelia (for example, OspA or OspB or derivatives thereof), or Chalmydia or Bordetella, for example P.69, PT and FHA, or antigens derived from parasites such as Plasmodium or Toxoplasma.

The HSV glycoprotein D (gD) or a derivative thereof is a preferred vaccine antigen. It is located on the viral membrane and has also been demonstrated in the cytoplasm of infected cells (Eisenberg, R.J. et al., J. of Virol. 1980, 35: 428-435). It consists of 393 amino acids including the signal peptide and has a molecular weight of approximately 60 kDa. Of all the HSV envelope glycoproteins, it is probably the best characterized (Cohen et al., Virology 60: 157-166). It is known to play a central role in the binding of the virus to cell membranes in vivo. In addition, glycoprotein D has been shown to be able to induce the production of neutralizing antibodies in vivo and to protect animals from lethal infection. A truncated form of the gD molecule is truncated at the C-terminal anchoring region and can be produced by mammalian cells as a soluble protein that is secreted into the cell culture supernatant. Such soluble forms of gD are preferred. The production of truncated forms of gD is described in EP 0139417. Preferably, the gD is derived from HSV-2. One embodiment of the invention is a truncated HSV-2 glycoprotein D of 308 amino acids that comprises amino acids 1 to 306 of the native glycoprotein with the addition of asparagine and glutamine to the C-terminus of the truncated protein that has been stripped of the membrane anchoring region. This form of the protein contains a signal peptide that is cleaved to allow secretion of the mature soluble protein of 283 amino acids from the host cell.

In another aspect of the invention, a preferred antigen for the vaccine is the hepatitis B virus surface antigen.

As used herein, the term hepatitis B virus surface antigen or HBsAg includes any HBsAg antigen or fragment thereof exhibiting the antigenic properties of the HBs surface antigen. It should be understood that in addition to the 226 amino acid sequence of HBsAg (see Tiollais et al., Nature, 317: 489 (1985) and references therein), HBsAg as described herein may comprise all or part of the pre-S sequence as described in the above references and in EP-A-0278940. In particular, 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 HBsAg L-protein according to the open reading frame of hepatitis B virus serotype ad (this polypeptide is designated L*; see EP 0414374). The HBsAg of the present invention may also include the pre-S1-preS2-S polypeptide described in EP 0198474 (Endotronics) or close analogs thereof, for example as described in EP 0304578 (McCormic and Jones). The HBsAg as used herein may also include mutants, for example the escape mutant described in WO 91/14703 or European Patent Application No. 0511855A1, in particular HBsAg having an amino acid substitution at position 145 from glycine to arginine.

HBsAg is usually in the form of particles. The particles may contain, for example, the S protein alone, or they may be composite particles, for example (L*, S), where L* is defined above and S denotes

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HBsAg S-protein. Said particle is preferably in the form in which it is expressed in yeast.

The preparation of the S-protein of the hepatitis B virus surface antigen is well known in the literature. See, for example, Harford et al., (1983) in

Develop. Biol. Standard 54, p. 125, Gregg et al., (1987), v

Biotechnology 5, p. 479, EP 0226846, EP 0299108 and references cited therein.

In another embodiment, the antigen for the vaccine is an RSV antigen, namely the F/G antigen. U.S. Patent 5,194,595 (Upjohn) describes chimeric glycoproteins comprising immunogenic segments of the F and G glycoproteins of RSV and suggests that such proteins can be expressed in a variety of systems including bacterial, yeast, mammalian (e.g., CHO cells), and insect cells (e.g., using baculovirus).

Wathen et al., (J. Gen. Virol. 1989, 70: 2625-2635) describe a certain RSV FG chimeric glycoprotein expressed by a baculovirus vector consisting of amino acids 1-489 of the F protein linked to amino acids 97-279 of the G protein.

The compositions of the present invention may also contain an anti-tumor antigen and may be used for immunotherapy of tumors.

The preparation of vaccines is generally described in New Trends and Developments in Vaccines, edited by Voliér et al., University Park Press, Baltimore, Maryland, USA, 1978. Liposome packaging is described, for example, in Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is described, for example, in Likhite, U.S. Patent 4,372,945 and Armor et al., U.S. Patent 4,474,757.

· • •00

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The protein content in each dose of vaccine is selected as the amount of protein that induces an immunoprotective response without significant adverse side effects in conventional vaccines. Such content will vary depending on the particular immunogen used and how it is presented. In general, it is expected that each dose will contain 1-1000 ug of protein, preferably 2-100 ug, most preferably 4-40 ug. The optimal amount for a particular vaccine can be determined by standard studies involving observation of the immune response of subjects. After the initial vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

The compositions of the present invention can be used for both prophylactic and therapeutic purposes.

In another aspect, the invention therefore includes the use of a vaccine composition according to the present invention for the treatment of human patients. The invention includes a method of treatment comprising administering an effective amount of a vaccine according to the present invention to a patient. In particular, the invention includes a method for the treatment of viral, bacterial and parasitic infections or tumors comprising administering an effective amount of a vaccine according to the present invention to a patient.

The following examples and data illustrate the present invention.

Examples of embodiments of the invention

Example 1: Preparation of a vaccine containing alum, SUV .MPL and QS21

1.1. SUV preparation method

A mixture of lipids (such as phosphatidylcholine from either egg yolk, • ·

• · · · ··· • · · · * • ·

:phosphatidylcholine 1:4 (w/w) and the aqueous solution is added in such an amount as to obtain a final cholesterol concentration of 5 to 50 mg/ml.

1.2. Antigen (1-500 µg, preferably 10-100 µg) is added to alum (e.g. aluminium hydroxide or aluminium phosphate) (100-500 µg) in water. The volume of water is chosen such that the volume of the final preparation is 500 µl. After incubation for 15-30 minutes, 50 µg of MPL in the form of small particle size MPL (WO 94/21292) is added. The MPL is allowed to adsorb onto the alum for 15-30 minutes at room temperature. 10-fold concentrated phosphate buffered saline (1.5 M sodium chloride, 0.5 M sodium phosphate, pH 7.5) is added in such a volume that the final preparation is isotonic. This preparation is incubated at room temperature for 15-30 minutes.

QS21 (50 μg) is then added to SUV (containing between 50 and 250 μg of cholesterol). The mixture is added to the alum/antigen/MPL/buffer mixture above. If desired, a bacteriostatic agent such as thiomersal (50 μg) is added.

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Example 2. ;

Table 1 shows the binding of QS21 to alum in ^the presence and absence of liposomes containing 25% (wt/wt) of QS21 ^.

ΦΦ φ „ _ φφφ ΦΦ· φ « φ φ φ φ φ φ φ φ ··· · · · · φ ······· · Φ···ΦΦ·· φφφ φφφ Φ·

ΦΦ φ · · φ ·· ·· · · dioleoylphosphatidylcholine and using a 5-fold excess of cholesterol relative to QS21.

Means SUV binding QS21 (Mg) 500 pg alum + 50 pg QS21 0 < 10 500 pg alum + 50 pg QS21 250 pg chol + 1 mg DOPC > 10

To increase the binding of QS21 to alum, the amount of liposomes can be reduced. This reduces the cholesterol:QS21 ratio, however, QS21 has been found to be non-toxic at cholesterol:QS21 ratios of 1:1 and higher. Table 2 shows that if the amount of alum is reduced (from 500 pg to 100 pg), the amount of QS21 bound is significantly reduced and the amount of MPL bound is also reduced. By adding less liposomes, while maintaining a cholesterol:QS21 ratio of 1:1 or higher, higher amounts of QS21 and MPL can be bound to alum.

Agent Chol/QS21 binding QS21 binding MPL (Mg) (Mg)

500 pg alum + + 50 pg MPL 50 Mg QS21 + 5/1 42 >48 100 pg alum + 50 Mg QS21 + 5/1 17 >40 + 50 pg MPL 100 pg alum + 50 Mg QS2i + 2/1 30 >45 + 50 pg MPL 100 pg alum + 50 Mg QS21 + 1/1 40 >45

+ 50 pg MPL

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Example 3

The adjuvant effect of the combination of antigen (gD2t from Herpes simplex virus-2 - expressed in CHO cells and containing 283 amino acids of the mature N-terminus of the mature glycoprotein) with MPL and QS21 in combination with liposomes is tested with and without alum. The preparations are tested in vervet monkeys.

Green monkeys are immunized twice (day 0, 28) with 20 pg gD2t plus 50 μg QS21 with or without liposomes (250 pg cholesterol plus 1 mg DOPC) and with or without 500 pg alum. The immune response is analyzed on day 42.

The results are shown in Fig. 1 to 4.

The antibody response is measured as IgG against the gD protein. Figure 1 shows that the combination of MPL + QS21 + SUV + alum induces higher antibody titers than the combination without alum. Figure 2 shows that the composition of the present invention induces the highest antigen-specific proliferation.

The data show that the inclusion of aluminum hydroxide in vaccines containing MPL and QS21 and SUV increases both antibody and cellular responses. This is an unexpected finding, as it is generally accepted that aluminum as an adjuvant favors a Th2 type of response, whereas the results presented here show that the response contains a significant Th1 component that is not suppressed by the addition of alum.

The composition containing MPL and QS21 and SUV and alum is non-toxic and highly immunogenic.

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Example 4; Production of RSV FG proteins in CHO cells

Plasmid pEE14-FG containing a chimeric construct containing a fusion between the amino acid sequences F (1 - 525) and G (69 - 298) was obtained in collaboration with A. Bollen (ULB/CRI, Belgium). This FG fusion protein contains a total of 755 amino acids. It starts at the N-terminal signal sequence of F and lacks the C-terminal transmembrane domain (525 - 574) - the anchoring domain - of the F glycoprotein. Furthermore, it contains, after the extracellular region of the G glycoprotein, without the amino-terminal region, the signal/anchoring domain of the G protein, which is a typical class II glycoprotein.

The pEE14-FG expression plasmid was generated by inserting the FG coding sequence from pNIV2857 (A. Bollen, ULB/CRI, Belgium) as an Asp7181 (blunt end) 5' - HindIII (blunt end) 3' restriction fragment (2188 bp) into the Smal site of pEE14 (Celltech). The Kozak sequence from the FG start ATG codon was generated into the pNIV 2857 construct as follows:

pEE14-----ccc gtacc ATG GAG-----x----CAG TAG aagct ggg---pEE14 (Smál) Metl

Asp7181 (Klenow)

Gln(298)Stop

HindllII (Klenow)

5'F(1-525)

G(69-298)3'

The F sequence in pEE14-FG is from the SS2 RSV strain and was kindly provided by Dr. Pringle as a cDNA construct in a Vaccinia vector (Baybutt and Pringle, J. Gen. Virol. 1987, 2789-2796). The G sequence is from the A2 RSV strain and was produced from recombinant vaccinia G virus, which was obtained from Dr. G. Wertz (Alabama, USA).

Transfection of CHO K1 and stable expression of FG protein

CHO K1 cells derived from MCB 024M (Celltech) are transfected

²⁰ μ9 pEE14-FG plasmid DNA with double CsCl, which was purified by the Ca-phosphate-glycerol transfection procedure. Cell clones were selected according to the GS (glutamine synthetase) expression system procedure (Crocett et al., BioTechn., 1990, vol. 8, 662) and amplified in the presence of 25 μmol methionine sulfoximine (MSX) in G MEM medium lacking glutamine and supplemented with 10% dialyzed FBS (fetal bovine serum). After transfection, 39 MSX resistant clones were screened in 24-well plates and their supernatants were assayed for secretion of the FG fusion protein. All transfectants were found to be positive for expression of F antigens by a specific sandwich ELISA (i.e. rabbit polyclonal anti-FG serum/antigen/mAB19). The monoclonal antibody recognizes the conformational F1 epitope and is neutralizing.

The three best FG-producing clones (no. 7, 13 and 37) are subcloned one cell each in a limiting dilution assay using 0.07 cells per well in a 96-well plate. A total of 59 positive subclones are obtained and the 16 best FG-producing clones are further characterized by Western blot and ELISA. Again, the 8 best FG-producing clones are further amplified and their FG expression is evaluated in the presence and absence of sodium butyrate (2 mM) or DMSO (1 or 2%). Six subclones are amplified, placed in tubes and stored at -80 °C in liquid N ₂ . Finally, the three best FG-subclones are selected. These subclones are CHOK1 FG no. 7.18, 13.1 and 37.2.

Western blot analysis (under non-reducing conditions) with monoclonal mAb19 showed a major FG band of approximately 135 kDa. Purified FG protein from recombinant baculovirus-FG infected cells (Upjohn) appeared as a major band at +/- 100 kDa and an additional band at +/- 70 kDa under similar assay conditions.

• · ··· ·

Addition of sodium butyrate to the culture medium of CHO-FG cells increased the level of FG expression 3- to 12-fold, depending on the subclone and the culture conditions used. Namely, the CHO-FG subclone 13.1 expressed 8- to 10-fold more FG protein in the presence of butyrate (WB/ELISA).

The expression level was determined by ELISA (mAB19 or MoAb AK13) using purified FG baculo protein as a standard, as well as by Western blot analysis using serial dilutions.

According to ELISA test and culture conditions, CHO-FG 13.1 expression is 5 - 12 ug FG/ml after butyrate treatment. Under accumulation conditions and medium exchange (3 to 5 days) 16 - 28 ug secreted FG protein/ml is obtained.

The CHOK1 FG 13.1 cell line was adapted to growth in suspension and serum-free (S/SF) conditions using a special culture medium. The CHO FG 13.1 S/SF cell line cultured in butyrate-free medium expressed similar amounts of protein as the original adherent cell line cultured in butyrate-free medium. Addition of butyrate to the CHO FG 13.1 S/SF medium had little effect on FG production (1.5-2-fold increase).

Long-term expression evaluation and preliminary genetic characterization showed that the CHO FG 13.1 and S/SF adapted 13.1 cell lines are stable, containing intact FG expression cassettes that give rise to a single mRNA band of approximately 3000 nucleotides in length (Southern and Northern analyses). CHO FG 13.1 S/SF was further used for production of FG antigen.

Use of alum/MPL/QS21/SUV to enhance immune responses in vervet monkeys against the FG protein from RSV (respiratory syncytial virus)

FG protein (fusion protein containing F- and G-proteins from RSV) was expressed in CHO cells and purified. 20 µg of the purified protein was adsorbed onto alum (500 µg) to which monophosphoryl lipid A (MPL: 50 µg) was added. After incubation for 30 minutes at room temperature, phosphate buffered saline was added. Then, either SUV alone or a mixture of SUV and QS21 (50 µg QS21, SUV containing 250 µg cholesterol and 1 mg DOPC) was added. Green monkeys were given three injections of this preparation or FG on alum alone or FG mixed with MPL, SUV and QS21 in the absence of alum.

Fig. 5 further shows the RSV neutralizing titers and FG-ELISA titers obtained for each formulation. It is clear that the alum/MPL/QS 21/SUV group induces the highest titers.

Example 5: Comparison of Hepatitis B Vaccine Formulations Containing SL* Antigen with QS21/SUV with Formulations Containing Alum

Introduction

SL* is produced according to the process of European Patent Application No. 414374.

An immunogenicity study is performed in Balb/c mice to compare the antibody responses induced by compositions containing QS21-SUV in the presence or absence of Al(OH) ₃ . The dose of MPL is 5 µg, QS21 5 µg, SUV contains 25 µg cholesterol and 100 µg DOPC.

The experimental protocol is described in the materials and methods section. Briefly, mice are immunized intramuscularly in the paw area twice at an interval of 4 weeks with SL* vaccine containing either vehicle, • · ·

• · · · · 9>

• · immunostimulant or a combination thereof. The anti-HBs antibody response (IgG and isotypes) is analyzed.

The following groups will be used in the experiment:

1. SL* (2 MG) 2 . SL* (2 3 . SL* (2 M9) 4 . SL* (2 Mg)

Al(OH) _a (50 µg)

A1(OH) ₃ (50 μg)/MPL/QS21 A1(OH) ₃ (50 Mg)/QS21-SUV MPL/QS21-SUV

Results

Antibody responses are measured by ELISA as described in the Materials and Methods section. Two time points are analyzed: 28 days after the first injection (28 post I) and 14 days after the booster injection (14 post II).

Post I and post II anti-HBs responses detected from the collected serum are shown in Fig. 6.

These data show that comparable titers are induced in the primary response by all QS21-SUV containing compositions, while weaker responses are observed when Al(OH) ₃ is used alone.

In the secondary response, the weakest antibody response was also induced by the vaccine containing A1(OH) ₃ . However, not all formulations containing QS21-SUV elicited the same response.

The two formulations containing Al(OH) ₃ QS21-SUV (+/-) MPL induced the strongest antibody response (2x higher than MPL/QS21-SUV).

Although statistical analysis was not performed, the results obtained from individual sera confirm this observation.

·· ♦ • ·

The combination of Al(OH) ₃ and QS21-SUV (+/- MPL) also quantitatively affected the immune response, as shown by the isotype profile of the antibody response (Fig. 7).

Al(OH) induces a distinctly TH2 type immune response (only 3% IgG2a), while the composition containing Al(OH) ₃ /QS21-SUV (+/- MPL) induces more than 46% IgG2a.

Conclusion

The combination of alum with QS21-SUV (+/- MPL) induces a higher antibody titer than a formulation containing vehicle or immunostimulant alone.

Material and methods

Immunization of groups of 5 female Balb/c mice (6-8 weeks) were immunized intramuscularly in the paw area (gastrocnemicus muscle) twice at an interval of 4 weeks with 50 g of vaccine containing 2 g of SL* prepared in Al(OH) ₃ (50 g of Al ³ * equivalent), Al(OH) ₃ /QS21-SUV,

Al(OH) ₃ /MPL/QS21-SUV, MPL/QS21-SUV. A dose of 5 µg of immunostimulant was used.

Animals were bled on day 28 (28 post I) and day 42 (14 post II) for ELISA antibody titer determination.

Resources

Components for individual batches will be used.

Preparation of the remedy

SL* (2 µg) is adsorbed or not for 15 minutes on 50 µg of Al(OH) diluted with water.

If necessary, 5 µg of MPL as a suspension of 100 nm particles (MPL out) is added to the formulation for 15 minutes. If necessary, a ten-fold concentrated buffer is added before adding 5 µg of QS21 mixed with liposomes at a weight ratio of QS21:cholesterol of 1:5.

Thiomersal is added to the formulations 15 minutes after the addition of QS21/SUV.

The formulations containing QS21-SUV are buffered with PBS pH 7.4 and the others are prepared in PBS pH 6.8.

Serology

Quantitative evaluation of anti-HBs antibodies is performed by ELISA using HBs (Hep286) as the coating antigen. Antigen and antibody solutions are used in a volume of 50 µl per well. The antigen is diluted to a final concentration of 1 µg/ml in PBS and adsorbed overnight at 4°C onto the wells of a 96-well microtiter plate (Maxisorb Immuno-plate, Nunc, Denmark). The plates are then incubated for 1 hour at 37°C with PBS containing 1% bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-fold dilutions of serum (with an initial dilution of 1/100) in saturation buffer are added to the HBs-coated plates and incubated for 1 hour 30 minutes at 37°C. The plate is washed four times with PBS 0.1% Tween 20 and biotin-conjugated anti-mouse IgG1, IgG2a,

IgG2b or a mixture of three antibodies (Amersham, UK) diluted 1/1000 in saturation buffer and incubated for 1.5 hours at 37 °C. After washing, streptavidin-biotinylated _·· ···· • · · · · · · · · · · · · · · · · · » • · ····· _· The reaction is terminated with 2N HSO and the

4 at 492/620 nm. ELISA titers are calculated with SoftmaxPro (using a four-parameter equation) and expressed in EU/ml.

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Claims (14)

An adjuvant composition comprising alum, QS21 and a sterol, wherein the alum is bound to QS21. An adjuvant composition according to claim 1, wherein the saponin is associated with liposomes or an Iscom containing a phospholipid and a sterol. An adjuvant composition according to any one of claims 1 or 2, wherein the sterol is cholesterol. Adjuvant composition according to any one of claims 1 to 3, characterized in that the ratio QS21: sterol is from 1: 100 to 1: 1. The adjuvant composition of any one of claims 1 to 5, further comprising 3-de-O-acylated monophosphoryl lipid A. A vaccine comprising the adjuvant of claims 1 to 6 and an antigen. The vaccine of claim 6 comprising an antigen or antigenic compound derived from any of human immunodeficiency virus, feline immunodeficiency virus, varicella zoster virus, herpes simplex type 1 virus, herpes simplex type 2 virus, 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, containing the adjuvant of claims 1 to 6. ·· · · · · · 99 ··· · ·· The vaccine of claim 6, wherein the antigen is a tumor antigen. The vaccine of claim 6, wherein the antigen is selected from the group consisting of SL * derived from hepatitis B virus, HSV gD ^ t or FG chimeric RSV protein. Use of a composition according to any one of claims 1 to 5 for the manufacture of a vaccine for the prophylactic treatment of viral, bacterial or parasitic infections. Use of a composition according to any one of claims 1 to 5 for the manufacture of a vaccine for the immunotherapeutic treatment of viral, bacterial or parasitic infections or tumors. A method for treating a mammal infected or susceptible to a pathogen infection comprising administering a safe and effective amount of a composition according to any one of claims 1 to 4. A method for treating a mammal with cancer comprising administering a safe and effective amount of a composition according to any one of claims 1 to 4 14. A method for producing an adjuvant composition according to claim 1, comprising mixing the QS21 and cholesterol fractions and binding the QS21 to the alum.