ES2371360T3 - REDUCTION OF INTERFERENCES BETWEEN ADJUVANTS CONTAINING OIL AND ANTIGENS CONTAINING TENSIOACTIVE. - Google Patents

Abstract

A process for preparing an immunogenic composition, comprising the steps of combining (i) a viral surface antigen component that includes a surfactant and (ii) a fatty adjuvant component, to give a composition in which the weight ratio between the fatty adjuvant and the surfactant is less than 1000: 1.

Description

Reduction of interference between adjuvants containing oil and antigens containing surfactants

Technical field

The present invention is in the field of manufacturing adjuvant vaccines. Specifically, it refers to the use of fatty adjuvants during the manufacture of antigen-based vaccines containing surfactants.

Prior art

The use of adjuvants to enhance immune responses against vaccine antigens is well known (e.g., see references 1 and 2). For many years, the only adjuvants approved for human use have been aluminum salts III, however in many countries vaccines containing adjuvant MF59 and adjuvant RC-52 have been approved, including Italy (in the Chiron FLUAD product ™) and Argentina (in the product of Bern Biotech SUPERVAX ™) Other adjuvants in the last stages of experimentation for human use include mixtures of cholesterol and saponins, ISCOM, MPL ™, AS04, virosomes, SBA4, etc. MPL ™ or monophosphoryl lipid A 3-O-deacylated has been used with HBsAg, for example see WO 98/43670.

A common feature of some alternatives to aluminum salts is the presence of a fatty component. For example, adjuvant MF59 includes squalene (an oil) and MPL ™ contains a deacylated form of monophosphoryl lipid A having multiple fatty acid chains attached to a di-glucosamine structure.

The invention relates to the avoidance of interference that may occur between these fatty adjuvants and antigens that include a surfactant component.

SUMMARY OF THE INVENTION

The inventor has found that fatty adjuvants in vaccine compositions may be incompatible with antigens that include a surfactant component. However, in many situations it is still desirable to combine a fatty adjuvant and an antigen / surfactant mixture, and it is an object of the invention to provide ways of avoiding the difficulties that may arise in doing so. It is an additional object of providing methods for combining adjuvants and antigens, in which incompatibility is avoided.

To avoid these incompatibility problems when using antigens that are normally purified using surfactants, one approach would be to use a method to prepare an immunogenic composition, in which: (a) the composition comprises an antigen and a fatty adjuvant; and (b) the antigen is substantially purified in the absence of the surfactant. Purifying the antigen by an alternative route that avoids the use of surfactant would overcome any incompatibility with fatty adjuvants.

For clinical, historical or regulatory reasons it may not be possible to purify an antigen without the use of surfactants or to completely eliminate the surfactants from vaccines. In this situation, the invention avoids interference by reducing the proportion between the oil and the surfactant in the composition. A typical MF59 / HBsAg vaccine contains a huge excess of oil with respect to the surfactant, with an oil: surfactant ratio of more than 4000: 1 (by weight). The invention is directed to minimize the amount of fatty adjuvant in a composition and uses, at most, an excess of 1000 times by weight of oil. Therefore, the invention provides a process for preparing an immunogenic composition, comprising the steps of combining (i) a viral surface antigen component that includes a surfactant and (ii) a fatty adjuvant component, to give a composition in which The weight ratio between the fatty adjuvant and the surfactant is less than 1000: 1.

Fatty adjuvant

The invention uses fatty adjuvants, that is an adjuvant component that includes a fatty molecule. Typical fatty adjuvants comprise a metabolizable oil, a fatty acid and / or a molecule comprising a fatty acid moiety.

Two preferred fatty adjuvants are the adjuvants known as ’MF59’ and ’MPL’. ’MF59’ is a fatty adjuvant because it contains squalene, which is a metabolizable oil. ’MPL’ is a fatty adjuvant because it contains a disaccharide substituted with multiple fatty acid chains. As well as MF59, oil-in-water emulsion adjuvants can also be used.

Thus, preferred compositions of the invention include: (a) an oil-in-water emulsion, such as a submicron oil-in-water emulsion. comprising squalene and polyoxyethylene sorbitan monooelate (and, optionally, sorbitan trioelate); and / or (b) a 3-O-deacylated lipid monophosphoryl A. Polyoxyethylene sorbitan monooelate is also known as polysorbate 80.

In addition to ’MF59’ and ’MPL’, other fatty adjuvants that can be used with the invention include, among others: glucosamide phosphate derivatives; N-acyl-pseudopeptides; the soluble adjuvant derived from lipid A from Escherichia

55 E06795518 08-11-2011

coli known as "OM-174"; lipid-containing compounds bound to an acyclic structure containing phosphate; and analogs of acyclic synthetic lipid A.

When an oil-in-water emulsion adjuvant, such as MF59, is usually added to an equal volume of an aqueous antigenic composition, so that the emulsion is 50% by volume of the total composition. When a 3D-MPL adjuvant is used, a typical dose is between 25 μg / ml and 200 μg / ml, for example in the range of 50-150 μg / ml, 75-125 μg / ml, 90-110 μg / ml or about 100 μg / ml. It is usual to administer between 25-75 μg of 3D-MPL per dose, for example between 45-55 μg or approximately 50 μg of 3D-MPL per dose. An emulsion adjuvant such as MF59 will normally be provided in a container other than that of the antigen, for extemporaneous mixing at the time of use, or it may already be provided mixed with the antigen. Normally, a 3D-MPL adjuvant will already be mixed with the antigen before distribution to end users.

MF59

The adjuvant ’MF59’ is an oil-in-water emulsion formed by squalene, Tween 80 (polyoxyethylene sorbitan monooleate) and Span 85 (sorbitan trioleate). The emulsion is microfluidified to give an emulsion with a drop size in submicrons. The preparation of M59 was initially described in reference 3 and the product has been manufactured and marketed in Chiron Corpotation. Additional details can be found in Chapter 10 of ref. 2, Chapter 12 of ref. 1 and in references 4 to 6. The composition of MF59 by volume is 5% squalene, 0.5% polysorbate 80 and 0.5% Span 85. In terms of weight, these proportions become 4, 3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. The surfactant content of the same MF59 is not taken into account when calculating the oil: surfactant ratio, since the ratio is based on the content of antigen surfactant.

The M59 emulsion advantageously includes citrate ions, for example 10 mM sodium citrate buffer.

Other emulsions in oil in water

As an alternative to the MF59 adjuvant, other oil-in-water emulsions can be used. Normally, adjuvant emulsions include at least one oil and at least one surfactant, the oil (s) and the biodegradable (metabolizable) and biocompatible surfactant (s). Generally, the oil droplets in the emulsion have a diameter of less than 5 μm, usually with a submicron diameter. Small droplet sizes can be achieved with a microfluidizer to provide stable emulsions. Drops smaller than 220 nm are preferred, since they can be subjected to filter sterilization.

Emulsions can include oils such as those from an animal source (such as a fish) or a vegetable source, rather than mineral oils. Sources of vegetable oils include nuts, seeds and grains. Examples of walnut oils are peanut oil, soybean oil, coconut oil and olive oil, the most frequently available. Jojoba oil, obtained, for example, from jojoba seed can be used. Seed oils include safflower oil, cottonseed oil, sunflower oil and the like. In the grain group, corn oil is the most readily available, but oils from other cereal grains such as wheat, oats, rye, rice, millet, triticale and the like can also be used. Fatty acid esters of 6-10 carbon atoms of glycerol and 1,2-propanediol, although not produced naturally in seed oils, can be prepared by hydrolysis, separation and esterification of suitable materials from of the oils of nuts and seeds. Fats and oils of mammalian milk are metabolizable and therefore can be used in the practice of this invention. The processes of separation, purification, saponification and other means necessary to obtain pure oils from animal sources are well known in the art. Most fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils and whale oil, such as whale sperm, are examples of the various fish oils that can be used herein. A series of branched chain oils are biochemically synthesized in 5-carbon isoprene units and are usually referred to as terpenoids. Shark liver oil contains a branched unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane, which is particularly preferred herein. document. Squalane, the saturated analog of squalene, is also a preferred oil. Fish oils, including squalene and squalene, are readily available from commercial sources or can be obtained by methods known in the art. Other preferred oils are tocopherols (see below). Mixtures of oils can also be used.

Surfactants can be classified by their "EHL" (hydrophilic / lipophilic balance). Preferred surfactants of the invention have an EHL of at least 10, preferably at least 15 and more preferably at least 16. The invention can be used with surfactants that include, among others: polyoxyethylene sorbitan ester surfactants (usually referred to as the Tween), especially polysorbate 10 and polysorbate 80; copolymers of ethylene oxide (OE), propylene oxide (OP), and / or butylene oxide (OB), sold under the trade name of DOWFAX ™, such as OE / OP block copolymers, octoxinols, in which the number of repeating ethoxy (oxy-1,2-ethanediyl) groups may vary, of which octoxynol-9 (Triton X-100, or toctylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (usually known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Among the preferred surfactants to include in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. Can be used

5 mixtures of surfactants, for example mixtures of Tween 80 / Span 85 or mixtures of Tween 80 / Triton-X100.

Oil-in-water emulsion adjuvants useful with the invention include, among others:

• An emulsion of squalene, μ-tocopherol and Tween 80. The emulsion may include phosphate buffered saline. It can also include Span 85 (e.g., 1%) and / or lecithin. These emulsions can have 2 to 10% squalene, 2 to 10% tocopherol and 0.3 to 3% Tween 80, and the weight ratio of

10 squalene: tocopherol is preferably ≤ 1, since it provides a more stable emulsion. One of these emulsions can be prepared by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml of squalene) and, subsequently, microfluidifying the mixture. The resulting emulsion may have drops of oil in submicrons, for example with an average diameter of between 100 and 250 nm, preferably of

15 approximately 180 nnm.

•

An emulsion of squalene, α-tocopherol and a Triton detergent (eg, Triton X-100).

•

An emulsion of squalene, poloxamer 80 and poloxamer 401 ("Pluronic ™ L121"). The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful release vehicle for muramyl dipeptides and has been used with threonyl-MDP in the adjuvant " SAF-1 " [7] (0.05-1% Thr-MDP,

20 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). Also can be used are Thr-MDP, as in the adjuvant “AF " [8] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidification is preferred.

• An emulsion that has 0.5-50% of an oil, 0.1-10% of a phospholipid and 0.05-5% of a non-ionic surfactant. As described in reference 9, preferred phospholipid components are phosphatidylcholine,

Phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron drop sizes are advantageous.

• A submicron oil-in-water emulsion of a non-metabolizable oil (such as a light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a lipophilic saponin conjugate (such as GPI-0100, described in

Reference 19, produced by the addition of aliphatic amine to deacysaponin through the carboxyl group of glucuronic acid), dimethyliodooctadecylammonium bromide and / or N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanodiamine.

• An emulsion in which a saponin (eg, QuilA or QS21) and a sterol (eg, cholesterol) are associated in the form of helical micelles [11].

Preferably, the emulsions are mixed with antigen extemporaneously at the time of release. Therefore, the adjuvant and the antigen are normally stored separately in a packaged or distributed vaccine, prepared for formulation at the end at the time of use. In general, the antigen will be in aqueous form, so that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids to be mixed may vary (eg, between 5: 1 and 1: 5), but is usually about 1: 1.

When a composition includes a tocopherol, any of the tocopherols α, β, γ, δ, ε or ξtocopherols may be used, but α tocopherols are preferred. Tocopherol can take several forms, for example different salts and / or isomers. The salts include organic salts, such as succinate, acetate, nicotinate, etc. D-α-tocopherol and DL-α-tocopherol can also be used. A preferred α-tocopherol is DL-α-tocopherol and the preferred salt of this tocopherol is succinate.

45 3D-MPL

Monophosphoryl lipid A 3-O-deacylated (ED-MPL, also referred to as monophosphoryl lipid A 3-de-O-acylated or -3O-deacyl-4'-monophosphoryl lipid A) is a known adjuvant. The name indicates that position 3 of glucosamine at the reducing end of lipid monophosphoryl A is deacylated. ED-MPL has been prepared from a Salmonella minnesota liver-free mutant and is chemically similar to lipid A, but lacks a phosphoryl labile group.

50 acid and a base labile acyl group. Activates the monocyte / macrophage lineage cells and stimulates the release of several cytokines, including IL-1, IL-12, TNF-a and GM-CSF. The preparation of 3D-MPL was initially described in reference 12 and the product has been manufactured and marketed in Corixa Corporation under the trade name "MPL". Additional details can be found in references 13 to 16.

3D-MPL can take the form of a mixture of related molecules, which vary according to their acylation (e.g., 55 having 3, 4, 5 or 6 acyl chains, which can have different lengths). The two glucosamine monosaccharides (also known as 2-deoxy-2-amino-glucose) are N-acylated and their carbons in position 2 (i.e., in positions 2 and 2 ') and there is also O-acylation in the 3 'position. The group attached to carbon 2 has the formula -NH-CO-CH2-CR1R1. The group fixed to carbon 2 ’has the formula -NH-CO-CH2-CR2R2’. The group attached to carbon 3 ’has the formula -O-CO-CH2-CR3R3’. A representative structure is:

The groups R1, R2 and R3 are, each independently, - (CH2) n-CH3. The value of n is preferably between 8 and 16, more preferably between 9 and 12, and more preferably it is 10.

The R1 ’, R2’ and R3 ’groups can each be independently: (a) -H; (b) -OH; or (c) -O-CO-R4, in which R4 is -H or - (CH2) m-CH3, in which the value of m is preferably between 8 and 16, more preferably 10, 12 or 14 . In

10 position 2, m is preferably 14. In position 2 ', m is preferably 10. In position 3', m is preferably 12. Groups R1 ', R2' and R3 'are, therefore, preferably -O-acyl groups of dodecanoic acid, tetradecanoic acid or hexadecanoic acid.

When all R1 ’, R2’ and R3 ’are –H, the 3D-MPL has only 3 acyl chains (one in each of positions 2, 2’ and 3 ’). When only two of R1 ', R2' and R3 'are –H, the 3D-MPL can have 4 acyl chains. When only one 15 of R1 ', R2' and R3 'are –H, the 3D-MPL can have 5 acyl chains. When none of R1 ', R2' and R3 'is –H, the 3D-MPL can have 6 acyl chains. The 3D-MPL adjuvant used in accordance with the invention may be a mixture of these forms, with 3 to 6 acyl chains, but it is preferred that 3D-MPL with 6 acyl chains be included in the mixture and, in particular, to ensure that the hexaacyl chain form makes up at least 10% by weight of the total 3D-MPL, for example �20%, �30%, �40%, �50% or more. It has been discovered that 3DMPL with 6 acyl chains is

20 the most active adjuvant form.

Therefore, the most preferred form of 3D-MPL for inclusion in the compositions of the invention is: When 3D-MPL is used in the form of a mixture, references to amounts or concentrations of 3D-MPL in the compositions of the invention are refer to the species of 3D-MPL combined in the mixture.

5 Under aqueous conditions, 3D-MPL can form micellar aggregates or particles of different sizes, for example with a diameter <150 nm or> 500 nm. One or both can be used in the invention and the best particles can be selected by routine testing. Smaller particles (eg, small enough to give a clear aqueous suspension of 3D-MPL) are preferred for use according to the invention for their superior activity [17]. Preferred particles have an average diameter of less than 220 nm, more preferably

10 less than 200 nm or less than 150 nm or less than 120 nm, and may even have an average diameter of less than 100 nm. However, in most cases the average diameter will not be less than 50 nm. These particles are small enough to be suitable for sterilization by filtration. The particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals an average particle diameter. When a particle is said to have a diameter of x nm, they will generally have a particle distribution

15 around this average, but at least 50% in number (e.g., 60%, 70% 80%, 90%, or more) of the particles will have a diameter within the range of x ± 25%.

3D-MPL can be used alone as an adjuvant or in combination with one or more additional adjuvant compounds. For example, it is known to use ED-MPL in combination with QS21 saponin [18], with QS21 and an oil-in-water emulsion [19], with aluminum phosphate. [20], with aluminum hydroxide [21] or with saponins e

20 interleukin-12 [22].

50 E06795518 08-11-2011

A preferred adjuvant mixture, particularly for use with HBsAg, comprises a mixture of 3D-MPL and an aluminum salt, preferably aluminum phosphate. Advantageously, the 3D-MPL is adsorbed on the aluminum salt. Preferably, at least 50% (by weight) of 3D-MPL is adsorbed, for example, 60%, 70%, 80%,

90%, 95%, 98% or more.

Aluminum adjuvants currently in use are usually referred to as "aluminum hydroxide" adjuvants.

or "aluminum phosphate." However, these are consensus names, since none is a precise description of the actual chemical compound that is present (eg, see chapter 9 of reference 2). For the combination with a fatty adjuvant, such as 3D-MPL, the invention can use any of the "hydroxide" or "phosphate" salts that are generally used as adjuvants.

Adjuvants known as "aluminum hydroxide" are normally aluminum oxyhydroxide salts, which are usually at least partially crystalline. Aluminum oxyhydroxide, which can be represented by the formula AIO (OH), can be distinguished from other aluminum compounds, such as aluminum hydroxide Al (OH) 3, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070 cm-1 and one shoulder at 3090-3100cm-1 (chapter 9 of ref. 2).

Adjuvants known as "aluminum phosphate" are normally salts of aluminum hydroxyphosphates, which often also a small amount of sulfate. They can be obtained by precipitation and the reaction conditions and concentrations during precipitation influence the degree of hydroxyl phosphate substitution in the salt. In general, hydroxyphosphates have a molar ratio of PO4 / Al between 0.3 and 0.99. Hydroxyphosphates can be distinguished from AIPO4 by the presence of hydroxyl groups. For example, a band of the IR spectrum at 3164 cm-1 (eg, when sealed at 200 ° C) indicates the presence of structural hydroxyls (Chapter 9 of ref. 2).

The PO4 / Al3 + molar ratio of an aluminum phosphate adjuvant will, in general, be between 0.3 and 1.2, preferably between 0.8 and 1.2, and, more preferably, 0.95 ± 0.1. Aluminum phosphate will, in general, be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminum hydroxyphosphate with a PO4 / Al molar ratio between 0.84 and 0.92, including 0.6 mg of Al3 + / ml. Aluminum phosphate will, in general, be particulate. Typical particle diameters are in the range of 0.5-20 μm (eg, approximately 510 μm) after adsorption of any antigen.

The aluminum phosphate PZC is inversely related to the degree of hydroxyl phosphate substitution, and this degree of substitution may vary depending on the reaction conditions and the concentration of the reactants used for the preparation of the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate = PCZ more acid) or by adding a buffer such as a histidine buffer (makes the PZC more basic). The aluminum phosphates used in accordance with the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example about 5.7.

An aluminum phosphate solution used to prepare a composition of the invention may contain a buffer (eg, a phosphate or a histidine or a Tris buffer), but this is not always necessary. The aluminum phosphate solution is preferably sterile and non-pyrogenic. The aluminum phosphate solution may include free aqueous phosphate ions, for example present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM. The aluminum phosphate solution may also comprise sodium chloride. The concentration of sodium chloride is preferably in the range of 0.1 to 100 mg / ml (eg, 0.5-50 mg / ml, 1-20 mg / ml, 2-10 mg / ml) and, more preferably, it is about 3 ± 1 mg / ml. The presence of NaCl facilitates the correct measurement of the pH before the adsorption of antigens.

Preferably, the aluminum phosphate is used in the form of an aqueous solution to which 3D-MPL (and, optionally, an antigen) is added (NB: It is standard to refer to aqueous aluminum phosphate as a "solution", though, in a strict physicochemical view, the salt is insoluble and forms a suspension). It is preferred to dilute the aluminum phosphate to the required concentration and ensure a homogeneous solution before the addition of 3D-MPL and / or the antigen.

N-acyl-pseudodipeptides

Preferably, an N-acyl-pseudodipeptide adjuvant includes a hydroxyl group that is esterified by a neutral or charged acid group. Acyl groups give pseudopeptides a fatty character. Preferably, the adjuvant has the formula (I)

in which:

-

R1 is an acyl group derived from a saturated or unsaturated straight or branched chain carboxylic acid having 2 to 24 carbon atoms, the carboxylic acid being unsubstituted or substituted with one or more substituents selected from the group consisting of the groups hydroxyl, alkyl, alkoxy, acyloxy, amino, acylamino, acylthio and (C1-24) alkyl thio;

5-R2 is an acyl group derived from a saturated or unsaturated straight or branched chain carboxylic acid having 2 to 24 carbon atoms, the carboxylic acid being unsubstituted or substituted with one or more substituents selected from the group consisting of the hydroxyl, alkyl, alkoxy, acyloxy, amino, acylamino, acylthio and (C1-24) alkyl thio groups;

-

X is a hydrogen atom or an acid group in neutral or charged form; 10 -Y is a hydrogen atom or an acid group in neutral or charged form;

-

A is an oxygen atom, a sulfur atom or an imino group -NH-;

-

B is an oxygen atom, a sulfur atom or an imino group -NH-;

-

in is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

-

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 15 -p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Y

-

is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

provided that at least one of the substituents X or Y designates an acidic group in neutral or ionic form.

The adjuvant can be used in acidic or salt form with an organic or mineral base. ’

Salt forming bases intended for therapeutic use mainly include alkali metal bases, such

20 as sodium, potassium or lithium hydroxide, ammonium salts; alkaline earth metal bases such as calcium or strontium hydroxide and magnesium salts; ferrous metal salts and the like; organic bases such as those derived from primary, secondary, tertiary amines, such as methylamine, diethylamine, monoethanolamine, diethanolamine, benzylamine, N-methylbenzylamine, veratrylamine, trimethoxybenzylamine, basic amino acids such as lysine and ornithine or aminoazúcares. Examples of bases not intended for therapeutic use are brucine,

Strychnine, agmatine, homarin, glucosamine, N-methylglucosamine or N-methylmorpholine.

Preferred substituents X and Y are selected from:

carboxy [alkyl (C1-5)]

-CH - [(CH2) rCOOH] [(CH2) sCOOH], in which: r is 0.1, 2, 3, 4 or 5; s is 0, 1, 2, 3, 4 or 5

-

 phosphono [C1-5 alkyl] 30-dihydroxyphosphoryloxy [C1-5 alkyl]

-

 dimethoxyphosphoryl

-

 phosphono

-

 hydroxysulfonyl

-

 hydroxysulfonyl [(C1-5) alkyl]

35-hydroxysulfonyloxy [(C1-5) alkyl]

When the x and / or Y substituents designate a neutral acid group, reference is made to the free carboxylic, sulfonic or phosphoric form. When the acid group is in a charged form, reference is made to the carboxylic, sulphonic or phosphoric salt form, namely, by the addition of an organic or mineral base, preferably one intended for therapeutic use. Similar considerations apply when X and / or Y designate a group

Carboxyalkyl, alkenyl biscarboxylic, hydroxysulfonyl, hydroxy sulfonylalkyl, hydroxysulfonyl oxyalkyl, phosphonoalkyl or a phosphoryloxyalkyl.

When m = 1 and n = 0, the adjuvant is derived from serine. When m = 2 and n = 0, the adjuvant is derived from homoserine. If m = 3 and n = 0, reference is made to a pentahomoserine compound. If m = 4 and n = 0, reference is made to a hexahomoserine compound.

When p = 3 and q = 1, the product of interest may be a compound of citrulline, ornithine or arginine. When p = 4 and q = 1, reference is made to a homoarginine or lysine compound.

R1 and R2 include saturated or unsaturated, branched or linear chain acyl derivatives, having a chain of varying size of a different or identical nature, which may have one or more substituents selected from the group consisting of alkyl, amino, acylamino groups, hydroxyl, alkoxy, acyloxy, acylthio and alkylthio.

Examples of such substituted acylated derivatives are ricinoleyl, 1,2-hydroxystearoyl, 2-hydroxy-3-methyl butyryl, 3-hydroxy-2-aminopentanoyl, palmitoyl, elaidyl radicals; eleostearoyl, araquidoyl, arachidonyl, gadoleyl, behenyl, erucilo, 8-methyldecanoyl, 9-methyldecanoyl, docosohexaenoyl or eicosapentaneoyl.

Preferred adjuvants have the formula (Ia), in which A and B are both oxygen atoms.

In formula (Ia), X and Y are preferably a hydrogen atom or a phosphono group.

Suitable adjuvants of formula (Ia) include:

-

3- (3-Dodecanoyloxytetradecanoylamino) 9- (3-hydroxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol 1 and / or 10 dihydrogen phosphate and its addition salts formed with an organic or mineral base,

-3- (3-Dodecanoyloxytetradecanoylamino) 9- (3-hydroxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol 1,1015 bis (dihydrogen phosphate) and its addition salts formed with an organic or mineral base,

-

3- (3-Hydroxytetradecanoylamino) 9- (3-dodecanoyloxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol 1,10-bis (dihydrogen phosphate) and its addition salts formed with an organic or mineral base,

-

3- (3-Dodecanoyloxytetradecanoylamino) 9- (3-hydroxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol 1 dihydrogen phosphate and its addition salts formed with an organic or mineral base,

20 - 3- (3-hydroxytetradecanoylamino) 9- (3-docecanoyloxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol 1 dihydrogen phosphate and its addition salts formed with an organic or mineral base,

-

3- (3-Hydroxytetradecanoylamino) 9- (3-docecanoyloxytetradecanoylamino) 4-oxo-5-azadecan-1,10-diol 10 dihydrogen phosphate and its addition salts formed with an organic or mineral base,

Procedures for synthesizing adjuvants of formula (I) are disclosed in detail in reference 23 and include

25 procedures comprising the steps of: Protecting the functional amino groups at positions (q + 1) and w of a diamino acid by blocking reagents that readily undergo acidolysis and hydrogenolysis, respectively; reacting the free carboxylic functional group with a reducing agent to give a corresponding alcohol; deprotect the functional amino group at position (q + 1) and then acylate by means of a functional carboxylic acid derivative of formula R2OH and subsequently release the functional amino group by hydrogenolysis, to

30 give a diamino alcohol of general formula II:

In which the amino alcohol is condensed in the presence of a peptide condensation agent in an inert solvent, together with a w-hydroxy, w-amino or w-thio amino acid compound of general formula III:

to provide a dipeptide compound of formula (IV)

Whose terminal free alcohol functional group may, if necessary, be protected (e.g., by an alkyl group

or acyl, or any other suitable protecting group) or otherwise substituted, if necessary, in the presence of a

coupling agent The protected alcohol can be subjected to catalytic hydrogenation or other deprotection treatment in order to obtain the derivative of general formula I.

Methods for synthesizing adjuvants of formula (II) are also disclosed in detail in reference 23 and include procedures comprising the steps of: protecting the functional amino groups at positions (q + 1) and w of a diamino acid of formula H2N (CH2 ) pCHNH2 (CH2) q + 1COOH protecting reagents that easily undergo acidolysis and hydrogenolysis, respectively; react the free carboxylic functional group with a

10 reducing agent to give a corresponding alcohol; deprotect the functional amino group in the position (q + 1) and then acylate by means of a carboxylic acid functional derivative of formula R2OH, then deprotect the functional amino group by hydrogenolysis to obtain an amino alcohol of general formula II, in wherein the amino alcohol is condensed in the presence of a peptide condensation agent in an inert solvent, together with a functional derivative of w-hydroxyamino acid of general formula IIIa:

wherein X is a dialkyloxy- or diaryloxy-phosphoryl radical of formula (RO) 2P-O, to give a peptide type compound of formula IVa:

wherein R is a radical that easily undergoes hydrogenolysis, whose other functional alcohol group can, if desired, be phosphorylated by a phosphorylation agent in the presence of a coupling agent, if necessary. The protected alcohol can be subjected to catalytic hydrogenation on the one hand in order to protect the functional alcohol group optionally present in the acyl group R2 and, on the other, release the functional phosphate group and,

Then, deprotect the second optionally present phosphate functional group by hydrogenolysis, in order to obtain the derivative of general formula V:

and optionally perform the additional salt formation stage by means of an organic or mineral base. The stereochemistry of chiral centers of acylamino groups is determined by amino acid configuration

30 used initially, while the stereochemistry of acylamino groups depends on the fatty acid configuration initially used. It can be started from a diamino acid that has an L or D configuration or a racemic nature. It can be started from a hydroxylated amino acid of L, D configuration or a racemic mixture. All these stereoisomers or diastereoisomers are included within the scope of the invention.

Particularly preferred adjuvants and remarkably mono and bis-phosphorylated compounds are those known as "OM-294-MP" (formula VI) and "OM-294-DP" (formula VII) [23]:

OM-174

OM-174 can be obtained by chemical synthesis and retains a triacyl motif of lipid A. It is described in more detail in references 24-26. Reference 26 provides the OM-174 formula as:

Therefore, OM-174 has a diglucosamine structure linked by (� (1-6) 1,4'-bisphosphorylated, as found in natural lipid A. OM-174 may be present as aggregates with a structure Hi micellar, that is

10 in which the lipid molecules are packaged with their structure on a cylindrical surface with the acyl chains directed inwards, the cylinders themselves being hexagonally packed. A cubic phase may be absent. An HII phase may also be absent.

Glucosaminide phosphate derivatives

Aminoalkylglucosaminide (AGP) derivatives (eg, RC-529 [27,28]) are fatty adjuvants for acyl chains. In general, these derivatives may have the following formula [29]:

in which:

-X represents an oxygen or sulfur atom in the axial or equatorial position; 5 -Y represents an oxygen atom or an NH group;

-

&quot; n &quot;, &quot; m &quot;, &quot; p &quot; and &quot; q &quot; they are the same or different and are whole numbers from 0 to 6;

-

R1, R2 and R3, which may be the same or different, are fatty acyl residues having 1 to 20 carbon atoms and in which R1, R2 or R3 is optionally hydrogen;

-

R4 and R5 are the same or different and are hydrogen or methyl;

R6 and R7 are the same or different and are hydrogen, hydroxy alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl or carboxy and esters and amides thereof;

-

R8 and R9 are the same or different and are phosphono or hydrogen, with, preferably, being at least one of R8 and R9 phosphono.

The configuration of the stereogenic centers in 3 ’to which normal fatty acyl residues are attached is

15 R or S, but preferably R. The stereochemistry of the carbon atoms to which R4 or R5 are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to enter into of the scope of the subject invention. Suitable adjuvants include the salts of these compounds.

A preferred AGP compound is ’RC-529’, which can be prepared as described in reference 30: Synthetic acyclic lipid A analogs

An analog of lipid A that can be used as an adjuvant in the compositions of the invention is ER-112022 [31], a phospholipid dimer connected by an acyclic structure:

Each monomer unit of ER-112022 contains 3 unique fatty groups that are indirectly bound to a phosphatodiester. Fatty groups include a 10-carbon ether chain, a 12-carbon unsaturated acyloxy chain attached to the ether chain and a 14-carbon b-oxo-amide chain closer linked to phosphodiester. Therefore, the compound has 3 unique characteristics compared to the natural lipid of E. coli: (i) ER-112022 lacks a cyclic carbohydrate structure; (ii) second, phosphates are phosphodiesters incorporated within the confines of the structural base, unlike phosphoesters in lipid A of E.coli; and (iii) the structure is symmetric (six fatty acids symmetrically organized in a structure that is not carbohydrate).

Similar useful compounds include those with the formula XIV, XV or XVI, or salts thereof:

as defined in reference 32, such as' ER 803058 ',' ER 803732 ',' ER 804053 ', ER 804058', 'ER 804059', 'ER 804442', 'ER 804680', 'ER 804764', ER 803022 or 'ER 804057', for example:

Lipid-containing compounds bound to an acyclic phosphate-containing structure can also be used as adjuvants, such as the TLR4 antagonist E5564 [33,34]:

40 E06795518 11-08-2011

The surfactant

The invention uses surfactant during purification of the antigen, but at controlled levels with respect to the fatty adjuvant.

Normally, the surfactant will be a non-ionic surfactant, particularly those already in the vaccine formulations. Organic surfactants are preferred. Typically, these are the product of the reaction of an alkylene oxide (eg, ethylene oxide) with a fatty alcohol, fatty acid, alkylphenol, alkylamino or other suitable compound having at least one active hydrogen atom. For most surfactants, the most common alcohols, amines and acids have a carbon chain length in the range of C8-C18. The most common alkylphenols are nonylphenol and octylphenol. Particularly preferred are surfactants containing poly (oxyethene) residues.

For example, the invention can be used with surfactants including, among others: polyoxyethylene sorbitan ester surfactants (usually referred to as the Tween), especially polysorbate 10 and polysorbate 80; copolymers of ethylene oxide (OE), propylene oxide (OP), and / or butylene oxide (OB), sold under the trade name of DOWFAX ™, such as OE / OP block copolymers, octoxinols, in which the number of repeating ethoxy (oxy-1,2-ethanediyl) groups may vary, of which octoxynol-9 (Triton X-100, or toctylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (usually known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.

Two specific surfactants of interest are Triton X-100 and polysorbate 20. Preferred embodiments of the invention use an antigenic component comprising one of these two non-ionic surfactants.

The invention is particularly suitable for use with polysorbate 20. This surfactant has an established safety profile for administration to humans, included within vaccines.

Surfactants can be classified by their "EHL" (hydrophilic / lipophilic balance). Preferred surfactants of the invention have an EHL of at least 10, preferably at least 15 and, more preferably at least 16.

In order to administer large doses of the surfactant to a patient it is preferred that the concentration of the surfactant in the composition does not exceed 50 μg / ml, for example � 40 μg / ml, � 35 μg / ml, � 30 μg / ml, � 25 μg / ml, � 20 μg / ml, � 15 μg / ml, � 10 μg / ml, � 5 μg / ml, etc. A concentration of � 20 μg / ml is preferred.

Antigen

The invention involves the use of a viral surface antigen. The invention is particularly suitable for use with antigens that are normally purified using surfactants. Usually, these antigens are lipophilic. They will normally include at least one transmembrane region, which functions in vivo to locate the antigen within a lipid bilayer, for example on the surface of a pathogen.

The antigen component includes a surfactant. Instead of being a simple mixture of antigen and surfactant, it is preferred that the antigen and the surfactant be in the form of a complex. Antigen / surfactant complexes include surfactant-stabilized liposomes containing antigen and niosomes, which are vesicles formed by synthetic non-ionic amphiphilic compounds. A preferred antigen / surfactant complex is a hepatitis B surface particulate antigen, purified in the presence of a surfactant. Reference 35 describes how recombinant HBsAg particles can retain the Tween 20 that was used during purification.

55 E06795518 08-11-2011

(up to 25 μg of Tween 20 per 100 μg of HBsAg).

The most preferred antigen for use with the invention is, therefore, the hepatitis B virus (HBV) surface antigen in the form of substantially spherical particles (mean diameter of approximately 20 nm), including a lipid matrix comprising phospholipids and a nonionic surfactant, such as polysorbate 20. The nonionic surfactant can be incorporated into the particle during purification.

Hepatitis B virus (HBV) is one of the known agents that causes viral hepatitis. The HBV virion consists of an internal nucleus surrounded by an external protein or capsid, and the viral nucleus contains the DNA genome of the virus. The main component of the capsid is a protein known as HBV surface antigen or, more commonly, ’HBsAg’, which is normally a 226 amino acid polypeptide with a molecular weight of -24 kDa. All existing hepatitis B vaccines contain HBsAg and, when this antigen is administered to a normal vaccine, it stimulates the production of anti-HBsAg antibodies, which protect against HBV infection.

For the manufacture of vaccines, HBsAg can be formed in two ways: The first procedure involves purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers, since large amounts of HBsAg are synthesized in the liver and released in the bloodstream during an HBV infection. The second mode involves expressing the protein by recombinant DNA procedures. HBsAg for use with the process of the invention is expressed recombinantly in yeast cells. Suitable yeasts include Saccharomyces hosts (such as S. cerevisiae), Pichia (such as P. pastoris) or Hanensula (such as H. polymorpha). Alternatively, the antigen can be expressed in recombinant mammals (e.g., in Chinese hamster ovary (CHO) cells, COS cells, Bu3 cells etc.), from insect (e.g., using baculovirus vectors) or vegetables cells. However, yeast expression is generally used.

The HBsAg expressed in yeast is preferably not glycosylated. Unlike native HBsAg (i.e., as in the purified plasma product), HBsAg that is expressed in yeast is, in general, non-glycosylated and this is the most preferred form of HBsAg for use with the invention, because it is highly immunogenic and can be prepared without the risk of contamination of the blood product. The HBsAg particles expressed in yeasts may include phosphatidinylinositol, which is not found in natural HBV virions. The particles may also include a non-toxic amount of LPS in order to stimulate the immune system [36].

The main procedures for purifying HBsAg are known in the art (e.g., see references 37-62) .: Of these various methods, the invention uses a non-ionic surfactant during purification, so that the surfactant is incorporated in the final particulate HBsAg product. Using polysorbate 20 during the alteration of recombinant yeast cells at the beginning of purification is a preferred way of introducing the surfactant into HBsAg particles.

A preferred method for the purification of HBsAg involves, after cell rupture: ultrafiltration, size exclusion chromatography; anion exchange chromatography; ultracentrifugation; desalination and sterilization by filtration. Lysates can be precipitated after cell breakage (eg, using a polyethylene glycol), leaving the HBsAg in solution, prepared for ultrafiltration. Before or after sterilization by filtration, it is possible to stabilize the HBsAg particles by treatment with formaldehyde. Then, excess formaldehyde can be removed by ultrafiltration or by chromatography. Additional filtration can be sterilized.

Preferably, the HBsAg comes from HBV, subtype adw2.

In addition to the "S" sequence, a surface antigen may include all or part of a pre-S sequence, such as all or part of a pre-S1 and / or pre-S2 sequence.

Other viral surface antigens that can be used in the invention will be, in general, coat glycoproteins from the coated viruses. Viral surface antigens for use with the invention may include, among others:

-

A retroviridae protein. Retroviridae includes lentivirus and foamvirus. Viruses of interest include HTLV-I, HTLVII, feline immunodeficiency virus (IVF), human immunodeficiency virus (HIV, including HIV-1 and HIV-2), simian immunodeficiency virus (SIV), foamy virus of chimpanzee and human foamvirus.

-

A paramyxoviridae protein, such as the F. Paramixoviridae protein includes (a) Paramyxovirinae, which includes Paramyxovirus, Rubulavirus and Morbillivirus and (b) Pneumovirinae, which includes Pneumovirus. Viruses of interest include parainfluenza virus (PIV), human paramyxovirus, Rinderpest virus, small ruminant pest virus, measles virus, mumps virus, respiratory syncytial virus (RSV), Nipah virus, Hendra virus, equine morbilivirus (EMV), Lyssavirus and Menangle virus.

-

A filoviridae protein. Filoviridae include the Marburg and Ebola viruses.

-

A portion of glycoproteins from a coronaviridae Coronaviridae include coronavirus and torovirus. Viruses of interest include human coronaviruses (including SDRA coronavirus), the infectious bronchitis virus

avian, feline infectious peritonitis virus, murine hepatitis virus, swine epidemic diarrhea virus, swine hemagglutinant encephalomyelitis virus, swine transmissible gastroenteritis virus and Berne virus.

-

A rhabdoviridae protein, such as the G. Rhabdoviridae protein include Rhabdovirus, Vesiculovirus,

5 Lyssavirus, Ephemerovirus, Cytorhabdovirus and Nucleorhabdovirus. Viruses of interest include vesicular stomatitis virus, rabies virus, mokola virus, bovine ephemeral fever virus.

-

A protein of togaviridae. Togaviridae includes alphavirus and rubivirus. Viruses of interest include Sindbis virus, Eastern and Western encephalitis virus, Semliki forest virus, Rubella virus, Aura virus, Babanki virus, Barmah avis-A forest virus, Bebaru, the Buggy Creek virus, the virus

10 chikungunya, the Everglades virus, the Fort Morgan virus, the getah virus, the J Highlands virus, the Kyzylagach virus, the Mayaro virus, the Middelburg virus, the Mucambo virus, the Ndumu virus, the Ockelbo virus. o’nyong-nyong virus, Pixuna virus, Ross River virus, Sagiyama virus, A virus, Venezuelan equine encephalitis virus and Whataroa virus.

-

A cover glycoprotein ('E') of a flaviviridae. Flaviviridae include Flavivirus, Pestivirus and Hepacivirus.

15 Viruses of interest include dengue virus, hepatitis C virus, yellow fever virus, Japanese encephalitis virus. West Nile virus, St. Louis encephalitis virus, bovine diarrhea virus and tick-borne encephalitis virus (TBE).

-

A bunyaviridae protein Bunyaviridae include Bunyavirus, Nairovirus, Phlebovirus, Hantavirus and Tospovirus. Viruses of interest include bunyavirus, Bunyamwera virus, California encephalitis virus,

20 La Cross virus, Hantaan virus, Sin Nombre virus, Crimea-Congo hemorrhagic fever virus, Sandfly Sicilian fever virus and Rift Valley fever virus.

-

A protein of arenaviridae. Arenaviridae include lymphocytic choriomeningitis virus, ippy virus and lassa virus.

-

A protein of hepadnaviridae (including HBsAg). Hepadnaviridae include orthohepadnavirus and

25 avihepadnavirus. In addition to the human hepatitis B virus, this family of viruses includes the ground squirrel hepatitis B virus, the groundhog hepatitis B virus, the wooly monkey hepatitis B virus, the hepatitis virus of the Arctic squirrel, the hepatitis B duck virus, the heron hepatitis B virus and the Ross B goose hepatitis B virus.

-

A herpesviridae protein. Herpesviridae include Simplexvirus, Varicellovirus, Roseolovirus,

30 Cytomegalovirus, Muromegalovirus, Linfocriptovirus and Rhadinovirus. Viruses of interest include human herpes viruses, including herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human Herpesvirus 6 (HHV6), human herpesvirus 7 (HBV7) and human Herpesvirus 8 (HHV8), etc. Suitable antigens can be selected from gB, gC, gD and gH glycoproteins (eg, in HSV). Particularly preferred is HSV gD2.

35 - A protein of Ortomixoviridae. Ortomixoviridae include influenza and thogoto viruses. Antigens of influenza viruses (influenza A, B or C viruses), including surface antigens hemagglutinin (HA) and / or neuraminidase (NA) are preferred.

These viral surface antigens are normally purified using surfactants and, therefore, the antigen component may include a surfactant, particularly if it is present in particulate form that includes lipids.

The invention is also useful with particulate antigens based on hybrid or fusion proteins comprising a viral surface antigen and a heterologous antigen. For example, it is known to condense the HBsAg sequence with heterologous antigens to exploit the ability of HBsAg to assemble into particles.

For example, reference 63 reports fusions of HIV-1 gp120 with HBsAg to give a protein that spontaneously assembles into particles that resemble the particles of native HBsAg in size and

45 density, consistent with a lipid composition of approximately 25% and a gp120 content of approximately 100 per particle. The gp120 could be folded into its native conformation in the fusion and still retain its biological activity. Similarly, HIV gp41 epitopes have been improved by preparing internal fusions with HBsAg and the fusion protein was self-assembled in yeasts in 22 nm lipoprotein particles [64].

50 This approach has also been used for malaria vaccines. Reference 65 reports that epitopes of up to 61 aa of the malaria gp190 antigen were inserted into the HBsAg sequence and that the expressed hybrid particles could elicit an anti-gp190 immune response in animals. Reference 66 indicates a protein that has 16 repeats of a 4-mer sequence of the circumsporozoite protein expressed in the form of a fusion protein with HBsAg. Reference 67 indicates the yeast production of virus-like particles

55 composed of PfsI6 condensed with HBsAg. Reference 68 discloses a hybrid antigen in which the circumsporozoite protein is condensed with HBsAg. Reference 69 reveals a merger of the region in the C

50 E06795518 08-11-2011

terminal of P. 1ivax merozoite surface protein 1, which formed immunogenic particles 20-45 nm in size. Therefore, the use of HBsAg to present malaria antigens in particulate form of self-assembly is well known in the art.

Therefore, the invention can be used with hybrid antigens comprising a viral surface antigen and a heterologous antigen. The heterologous antigen can be inserted into the viral surface antigen sequence or can be condensed with the N-terminus or the C-terminus of the viral surface antigen sequence. If the native viral surface antigen can be assembled into particles (eg, HBsAg), insertion or fusion will not impede this assembly.

In these hybrid proteins, the heterologous antigen can come from a bacterium, a fungus, a parasite, a virus (but, by definition, the heterologous antigen is not HBsAg), etc. It is possible to include a complete heterologous antigen in the hybrid protein, but it is more common to include an antigen fragment of the antigen.

Particularly, when the viral surface antigen is HBsAg, the heterologous antigen can be derived from HIV, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae or Plasmodium ovale. Suitable HIV antigens for preparing hybrids with HBsAg include the gp120 envelope glycoprotein or antigen fragments thereof [63]. Suitable P.falciparum antigens for preparing HBsAg hybrids may be based on a subunit of the circumsporozoite surface antigen ("CSP"), for example, may include between 3 and 20 repetitions of their NANP motif (SEQ ID NO: 2) and / or may include the C-terminal region of the CSP (but usually does not include the final 12 amino acids of the C-terminal). For example, the invention may use the antigen known as "RTS", which contains a large portion of the C-terminal of the CSP of NF54 or 7G8 isolation of P. falciparum (amino acids 210 to 398, which includes 19 NANP repeats and the epitope region of T cells in amino acids 367 to 390), condensed with the N-terminus of HBsAg into four amino acids of the preS2 portion of HBsAg. When expressed in yeasts, RTS forms particles that include lipids (mainly phospholipids) in addition to the protein. Therefore, the RTS sequence may contain: (i) a methionine residue at the N-terminus;

(ii) Met-Ala-Pro; (iii) 189 amino acids corresponding either to amino acids 210-398 of the P. falciparum 7G8 CS protein or to amino acids 207-395 of the P. falciparum NF54 CS protein; (iv) Arg or Gly; (v) Pro-Val-Thr-Asn of the Pre-S2 hepatitis B protein; and (vi) HBsAg. From isolate 7G8, the full-length RTS has the sequence provided as SEQ ID No. 1 in reference 70 (see also Figure 5 of reference 71):

The hybrid protein can be expressed in yeasts using a coding sequence SEQ ID NO. 1. It is preferred to co-express the hybrid protein in yeasts with normal HBsAg. This approach has previously been used with RTS and the co-expression product is called "RTS, S". A ratio of RTS: S of approximately 1: 4 is useful. Expression in S. cerevisiae yeast is preferred, using a plasmid having a sequence encoding the hybrid protein, and includes: (1) an earlier sequence of a glyceraldehyde-3-phosphate dehydrogenase gene to control the expression of the coding sequence; and (2) an ARG3 transcription terminator after the coding sequence. Also, plasmids will normally include: (3) a LEU2 selection marker; (4) a 2P plasmid sequence; and (5) an origin of functional replication in Escherichia coli.

RTS, S can be combined with malaria antigen ears, such as anonymous thrombospondin-related protein (TRAP). A preferred fatty adjuvant for use with RTS, S includes an oil-in-water emulsion, 3D-MPL and a QS-21 saponin.

Proportion between fatty adjuvant and surfactant

The weight ratio in the composition of the fatty adjuvant and the antigen surfactant is less than 1000: 1.

For an MF59 adjuvant, the weight ratio is based on the amount of squalene. For a composition containing 0.1 μg / ml of surfactant in an antigenic component, the concentration of squalene will be less than 100 μg / ml. For a composition containing 1 μg / ml of surfactant in an antigenic component, the concentration of squalene will be less than 1 μg / ml. For a composition containing 10 μg / ml of surfactant in an antigenic component, the concentration of squalene will be less than 10 μg / ml, while the concentration of squalene in a typical composition adjuvant with MF59 would be 43 mg / ml (i.e. 4300: 1).

For a 3D-MPL adjuvant, the weight ratio is based on the total amount of 3D-MPL, that is, including all the different acyl forms that may be present. For a composition containing 0.1 μg / ml of surfactant in an antigen component, the concentration of 3D-MPL will be less than 100 μg / ml. For one

50 E06795518 08-11-2011

Composition containing 1 μg / ml of surfactant in an antigenic component, the concentration of 3D-MPL will be less than 1 μg / ml.

The 1000: 1 ratio is the maximum. To further reduce the potential for interference between the oil in an adjuvant and the surfactant in an antigen, the proportion can be reduced. Therefore, the ratio can be 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, or even 25: 1. At a ratio of 100: 1, a composition containing 10 μg / ml of surfactant in an antigenic component would have an oil content (eg, squalene or 3D-PML) or 1 mg / ml. At a ratio of 25: 1, a composition containing 10 μg / ml of surfactant in an antigenic component would have an oil content (e.g., squalene or 3D-PML content) of 250 μg / ml, on the contrary , a composition comprising 100 μg / ml of 3D-MPL would have no less than 4 μg / ml of surfactant as part of the antigen.

The ratio is preferably 1.5: 1 for example 2: 1, 2.5: 1, 3: 1, 4: 1, 5: 1 or more.

For 3D-MPL, a ratio between 2.5: 1 and 25: 1 is preferred, more preferably between 2.5: 1 and 10: 1, and even more preferably between 2.5: 1 and 5: 1. Therefore, a composition containing 10 μg / ml of surfactant in an antigenic component would have a 3D-MPL content of between 25 μg / ml and 250 μg / ml, preferably between 25 μg / ml and 100 μg / ml, and even more preferably between 25 μg / ml and 50 μg / ml, on the contrary, a composition comprising 100 μg / ml of 3D-MPL would include an antigen having a surfactant content of between 4 μg / ml and 40 μg / ml, and even more preferably between 20 μg / ml and 40 μg / ml.

The immunogenic composition

In addition to containing antigen (and a fatty adjuvant (and a low level of surfactant), the compositions of the invention may comprise vehicles, adjuvants, excipients, buffers etc., as described in greater detail below. Non-antigenic components may proceed from various sources, for example, they may be present in one of the antigen or adjuvant materials used during manufacturing or may be used separately from these components.

Preferred compositions of the invention include one or more pharmaceutical vehicles and / or excipients.

To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg / ml.

Generally, the compositions will have an osmolality of between 200 mOsm / kg and 400 mOsm / kg, preferably between 240-360 mOsm / kg and, more preferably, will fall within the range of 280-320 mOsm / kg. It has been previously reported that osmolality does not have an impact on pain caused by vaccination [72], but nevertheless, it is preferred to keep osmolality within this range.

The compositions of the invention may include one or more buffers. Typical buffers include: A phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Normally, the buffers will be included in the range of 5-50 mM and, preferably, in the range of 5-20 mM.

Generally, the pH of a composition of the invention will be between 5.0 and 7.5, and more typically between 5.0 and 6.0, for optimum stability or between 6.0 and 7.0. Therefore, the process of the invention may include a step of adjusting the pH of the crude vaccine before packaging in the dosage containers. A vaccine containing diphtheria and tetanus toxoids preferably has a pH 6, to avoid the risk of reversal of the toxicity of toxoids (particularly in diphtheria toxoid) and, therefore, a vaccine containing toxoids. and HBsAg antigens preferably have a pH between 6.0 and 7.0. For other vaccines, including monovalent HBsAg vaccines, a pH <6 may be acceptable.

Preferably, the compositions of the invention are sterile.

Preferably, the compositions of the invention are non-pyrogenic, for example containing <1 EU (endotoxin unit, a standard measure) per dose and, preferably, <0.1 EU per dose.

Preferably, the compositions of the invention have no glute.

If adsorbed HBsAg is used, the final vaccine product may be a suspension with a cloudy appearance. This aspect means that microbial contamination cannot be easily seen and, therefore, the vaccine preferably contains an antimicrobial agent. This is particularly important when the vaccine is packaged in multi-dose containers. Preferred preservatives to include are 2-phenoxyethanol and / or thimerosal. However, it is recommended not to use mercury preservatives during the process of the invention, although thimerosal has been found to be in many existing HBV vaccines. However, for safety, it is preferred that the final composition contains less than about 25 ng / ml of mercury. More preferably, the fine vaccine product does not contain detectable thimerosal. In general, this will be achieved by removing the mercury preservative from an antigen preparation prior to its addition in the process of the invention or avoiding the use of

50 E06795518 08-11-2011

Thimerosal during the preparation of the components used to make the composition. HBsAg can be dialyzed (eg, with cysteine) to remove any mercury preservative, such as thimerosal, that may have been used during the preparation of HBsAg [73,74].

During manufacturing, dilution of the components to give desired final concentrations will usually be done with API (water for injections)

The concentration of any aluminum phosphate in a composition of the invention, expressed in terms of Al3 + is preferably less than 5 mg / ml, for example 4 mg / ml, 3 mg / ml, mg / ml, 1 mg / ml , etc.

The concentration of HBsAg in a composition of the invention is preferably less than 100 μg / ml, for example 90 μg / ml, 80 μg / ml, 70 μg / ml, 65 μg / ml, 60 μg / ml, 55 μg / ml, 50 μg / ml, 45 μg / ml, 40 μg / ml, etc. A concentration of approximately 40 μg / ml or approximately 20 μg / ml is typical.

Preferably, the compositions of the invention are administered to patients in doses of 0.5 ml. It will be understood that references to 0.5 ml doses include normal variations, for example 0.5 ml ± 0.05 ml.

The invention can provide bulk material, which is suitable for packaging in individual doses, which can then be distributed for administration to patients. The concentrations mentioned above are normally concentrations in the final packaged dose, and therefore, the concentrations of the crude vaccine may be higher (eg, to reduce to the final concentrations by dilution).

Generally, the compositions of the invention will be in aqueous form.

Packaging of the compositions of the invention

After combining the antigen and the adjuvant, the methods of the invention may comprise a step of extracting and packaging a 0.5 ml sample of the mixture in a container. For multidose situations, quantities for multiple doses will be extracted and packaged together in a single container. As mentioned above, in an alternative arrangement the antigen and the adjuvant are packaged separately, for extemporaneous mixing at the time of use.

The methods of the invention may comprise the additional step of packaging the vaccine in containers for use. Suitable containers include vials and disposable syringes (preferably sterile).

When a composition of the invention is packaged in a vial, the vial will preferably be made of glass or plastic material. Preferably, the vial is sterilized before the composition is added. To avoid problems with latex-sensitive patients, the vials are preferably sealed with a latex-free cap. The vial may include a single dose of vaccine or may include more than one dose (a "multidose" vial), for example 10 doses. When a multidose vial is used, each dose will be extracted with a sterile needle and syringe under strict aseptic conditions, being careful to avoid contamination of the vial contents. Preferred vials are made of colorless glass.

A vial may have a cap (e.g., a Luer-type closure) adapted so that a pre-filled syringe can be inserted into the cap, expel the contents of the syringe into the vial and so that the vial contents can be reintroduced into the syringe. After the syringe is removed from the vial, a needle can be inserted and the composition can be administered to a patient. Preferably, the cover is inside a seal or cover, so that the seal or cover has to be removed before the cover can be accessed.

When the composition is packaged inside a syringe, preferably, normally, the syringe will not carry the needle in place, although a needle can be supplied separately with the syringe for placement and use. Safety needles are preferred. Typical 2.54 cm and 0.61 mm, 2.54 cm and 0.51 mm and 1.6 cm and 0.51 mm needles are typical. The syringes can be provided with labels that can be detached in which the batch number and expiration date of the contents will be printed, in order to facilitate the maintenance of a record. Preferably, the syringe plunger has a cap to prevent the plunger from being accidentally removed during aspiration. A butyl rubber plunger is preferred. The syringes may have a cap and / or a rubber latex plunger. Disposable syringes contain a single dose of vaccine. Generally, the syringe has a cap on the tip to seal the tip before inserting a needle and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately, the needle will preferably be equipped with a butyl rubber cover. Gray butyl rubber is preferred. Preferred syringes are those marketed under the brand name "Tip-Lok".

When a glass container (e.g., a syringe or a vial) is used, it is preferred to use a container made of borosilicate glass instead of a glass of soda lime.

Once a composition is packaged in a container, the container can be placed inside a box for distribution, for example inside a cardboard box, and the box will be labeled with the details of the vaccine, for example its commercial name, a list of the antigens in the vaccine (eg, "recombinant hepatitis B" etc.), the

55 E06795518 08-11-2011

presentation container (eg, “Disposable pre-filled syringes of the Tip-Lok type or single-dose vials of 10 x 0.5 ml), their dose (eg, each with a dose of 0.5 ml), warnings (eg, "For adult use only" or "For pediatric use only)", an expiration date, an indication (e.g., "active immunization against hepatitis B virus (HBV) caused by all known subtypes for patients with renal insufficiency (including pre-hemodialysis and hemodialysis patients) from 15 years of age &quot; etc.), a patent number etc. Each box could contain more than one packaged vaccine, for example five or ten packaged vaccines (particularly for vials). If the vaccine is inside a syringe, the box may show an image of the syringe.

The vaccine can be distributed together with (e.g., in the same box) a booklet that includes the details of the vaccine, for example administration instructions, details of the antigens that the vaccine carries etc. Also, the instructions may contain warnings for, for example, having an adrenaline solution readily available in the event of a severe anaphylactic reaction following vaccination, etc.

The packaged vaccine is stored (preferably) at a temperature between 2 ° C and 8 ° C. It should not freeze.

Vaccine treatment and administration procedures

The compositions of the invention are suitable for administration to human patients and the invention provides a method for raising an immune response in a patient, comprising the step of administering such a composition of the invention to the patient.

The invention also provides the use of (i) a viral surface antigen component that includes a surfactant and (ii) a fatty adjuvant in the manufacture of a medicament for administration to a patient, in which the weight ratio between the adjuvant fatty in (ii) and the surfactant in (i) is less than 1000: 1.

The immunogenic compositions of the invention are preferably vaccines for, for example, use in the prevention and treatment of hepatitis B virus infection. Patients who have received compositions of the invention preferably have an anti-titer title. -HBsAg GM serum 500 mUI / ml, measured 6 weeks after the first immunization. More preferably, the titer is 500 mIU / ml, measured after 12 months.

The compositions are particularly useful for protecting against hepatitis B virus infections and / or treating them in patients for whom existing adjuvant vaccines (such as ENGERIX B ™ product) are ineffective. Subgroups of patients for whom the compositions are particularly suitable include: Immunocompromised patients, hemodialysis patients, patients on pre-hemodialysis, patients with renal insufficiency, patients with early renal insufficiency before requiring hemodialysis, patients awaiting a transplant of liver, for example on waiting lists, patients with end-stage renal failure. Patients who have received an organ transplant (particularly a liver transplant), for example in the period of 6 months prior to a first dose of the vaccine of the invention, receiving patients (or who have received in, for example, the period 6 months prior to the first dose of the vaccine of the invention, treatment with hepatitis B immunoglobulin (HBIg), patients with an HLA DQ2 haplotype [75]; patients with an HLA DR3 haplotype [75]; patients with a haplotype HLA DR7 [75]; patients with the HLA DQB 1 * 0202 allele [76], HIV-infected patients, chronic carriers of HBV, patients who have recently received a blood transfusion, patients receiving immunosuppressive drugs, patients suffering AIDS, patients with ascites, patients with cirrhosis, patients with encephalopathy, patients who are receiving interferon therapy, and, in particular ifn-a, cigarette smoking patients, cigar smoking patients, pac Patients with a body mass index of 30 kg / m2 and patients who have received an HBsAg vaccine but have not become seroconverted (e.g. eg, they have a serum anti-HBsAg titer <10 mUI / ml after a conventional primary dosing schedule, such as 3 doses of ENGERIX B ™).

These patients may have a creatinine clearance rate of less than 30 ml / min (the normal healthy range is 100-140 ml / min in men and 90-130 ml / min in women). Preferably, patients will be at least 15 years old, for example between 15-40 years of age, between 15-60 years of age, between 40-60 years of age or even more than 60 years of age. Patients older than 55 years can be treated in a useful way regardless of any underlying disease.

The compositions of the invention can be administered by intramuscular injection in, for example, the deltoid.

When the compositions of the invention include an aluminum-based adjuvant, sedimentation of the components may occur during storage. Therefore, the composition should be agitated before administration to a patient. The stirred composition will be a cloudy white suspension.

Preferred methods and vaccines of the invention

A preferred method for preparing an immunogenic composition comprises the steps of combining (i) an HBsAg component that includes polysorbate 20 and (ii) an adjuvant component comprising 3D-MPL

45 E06795518 08-11-2011

adsorbed on an aluminum phosphate, to give a composition in which the weight ratio between 3D-MPL and polysorbate 20 is less than 1000: 1.

A preferred immunogenic composition is one in which: (a) the composition comprises HBsAg, polysorbate 20, 3D-MPL and an aluminum phosphate adjuvant, in which polysorbate 20 is incorporated into HBsAg particles during purification; and (b) the weight ratio between 3D-MPL and polysorbate 20 is less than 1000: 1. Preferably, 3D-MPL and HBsAg are adsorbed in aluminum phosphate. The concentration of HBsAg is approximately 40 μg / ml. The concentration of 3D-MPL is approximately 40 μg / ml. The concentration of Al3 + is approximately 1mg / ml.

Another preferred composition comprises (i) HBsAg, purified from S. cerevisiae and (ii) an adjuvant comprising a mixture of aluminum phosphate and 3D-MPL. The concentration of HBsAg is approximately 40 μg / ml. The concentration of 3D-MPL is approximately 100 μg / ml. The concentration of Al3 + is approximately 1mg / ml. HBsAg includes polysorbate 20 and the weight ratio of oil: surfactant (i.e., the weight ratio of 3D-MPL: polysorbate 20) is between 2.5: 1 and 100: 1 ie polysorbate 20 is present at a level between 1 μg / ml and 40 μg / ml (that is, between 2.5 μg and 100 μg of polysorbate 20 per 100 μg of HBsAg). Preferably, the ratio is between 2.5: 1 and 25: 1, that is, polysorbate 20 is present at a level between 4 μg / ml and 40 μg / ml. Both 3D-MPL and HBsAg are adsorbed in aluminum phosphate.

general

The term "comprising &quot; it encompasses (which includes "in addition to" constituted by ", for example a composition" comprising "X may be constituted only by X or may include something additional, for example X + Y.

The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. When necessary, the word "substantially" may be omitted from the definition of the invention.

The term "approximately" in relation to a numerical value x means, for example, x ± 10%.

Unless specifically indicated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Therefore, the components can be mixed in any order. When there are three components, two components can be combined with each other and then the combination can be combined with the third component, etc. As described above, the components can be mixed during manufacturing or extemporaneously at the time of use.

When an antigen is described as "adsorbed" in an adjuvant, it is preferred that at least 50% (by weight) of said antigen is adsorbed, for example 50%, 60%, 70%, 80%, 90%, 95 %, 98% or more. It is preferred that HBsAg is fully adsorbed, that is, nothing detectable in the supernatant.

When animal (and particularly bovine) materials are used in cell culture, they should be obtained from sources that are free of transmissible spongiform encephalopathies (EST) and, in particular, free of bovine spongiform encephalopathy (BSE).

Butyl rubbers include chlorobutyl and bromobutyl rubbers.

It will be appreciated that ionizable groups may exist in the neutral form shown in the formulas herein or they may exist in a charged form, for example depending on the Ph. Therefore, a phosphate group may be shown as -PO- (OH) 2, this The formula is simply representative of the neutral phosphate group and other charged forms fall within the invention. Similarly, sugar rings may exist in an open and closed form and that, while the closed forms are shown in the structural formulas of the present specification, the open forms are also encompassed by the invention.

MODES FOR CARRYING OUT THE INVENTION

Three different HBsAg preparations were studied:

•

HBsAg expressed in purified S. cerevisiae cells using non-ionic surfactant.

•

HBsAg expressed in purified Hanensula yeast cells using non-ionic surfactant.

•

HBsAg expressed in CHO cells, with the preS2 sequence, purified without using surfactants.

Two different adjuvants were studied:

•

Aluminum hydroxide (1 mg / ml)

•

MF59 in citrate buffer (13 mM)

E06795518 08-11-2011

Six formulations were prepared, each with 20 μg / ml HBsAg, 0.15M NaCl and 0.01% methiolate:

TO

B C D AND F

HBsAg

CHO  CHO Hanensula Hanensula S. cerevisiae S. cerevisiae

Adjuvant

Al hydroxide MF59 Al hydroxide MF59 Al hydroxide MF59

NaCl

0.15M  0.15M 0.15M  0.15M  0.15M  0.15M

Mertiolate

0.01%  0.01% 0.01%  0.01%  0.01% 0.01%

Surfactant

- - + + + +

It has been reported that MF59 enhances the antibody response in primates to recombinant HBsAg [77]. Formulations A to D were used to immunize African green monkeys. Groups of 6 monkeys were immunized intramuscularly on day 0 and day 28. Blood was drawn at time 0 and then every 2 weeks until week 8, and anti-HBsAg titres (GMT) were measured by ELISA. The titles were normalized to 1.0, which was the value of day 14 in each group, and the normalized results were as follows:

TO

B C D

Guest

CHO  CHO Yeast Yeast

Adjuvant

Al hydroxide MF59 Al hydroxide MF59

Surfactant

- - + +

Day 14

1.0  1.0 1.0 1.0

Day 28

0.9  0.8 1.3 0.4

Day 42

19.2  159.8 108.8 85.2

Day 56

10.8  32.6 61.3 24.5

As for the titles of day 42 and day 56, these data show that adjuvant MF59 increased the titles

10 much more markedly than the aluminum adjuvant for the antigen expressed in CHO (160-fold increase versus 20-fold increase on day 42), but the situation was reversed for the antigen expressed in Hanensula (85-fold increase versus increase in 110 times on day 42).

Similar experiments were performed on baboons using formulations B, E and F. The GMT values were measured 14 days after the first injection and with respect to the value of group F were the following:

TO

B AND F

Surfactant

- - + +

Adjuvant

Al hydroxide MF59 Al hydroxide MF59

Day 14

- 13.5 4,5 1.0

Here, the use of MF59 instead of the aluminum adjuvant leads to a lower GMT response (compare groups E and F), while an increase could be expected (group B and reference 77).

Anti-HBsAg responses 28 days after the first injection using formulations E and F were also compared in rhesus monkeys, African green monkeys and C3H mice (note: mice received 60% of the

20 doses of HBsAg). In addition to measuring GMT values, the number of responders was also measured, ie animals that show a titer of> 10 ml U / ml. The normalized results for group F were the following:

40 E06795518 11-08-2011

GMT

Responders

AND

F  AND F

Adjuvant

Al hydroxide MF59 Al hydroxide MF59

Rhesus

4.2 1.0 60% twenty %

AGM

110.0 1.0 100% fifty %

Mice

0.1 1.0 94% 100%

In contrast to mice, the use of MF59 as an adjuvant in primates resulted in a considerable decrease in GMT values and respondent levels for HBsAg expressed in S. cerevisiae.

Finally, the increase in GMT resulting from booster vaccination with formulations E and F was measured in seropositive African green monkeys. Titles were measured before the booster dose and, again 28 days later, and the titre increase was as follows:

Group

Adjuvant Increment (x)

AND

Al hydroxide 91.5 X

F

MF59  19.1 X

In addition to providing a smaller increase than the aluminum hydroxide adjuvant, MF59 gave a lower GMT value 28 days after the booster dose.

Overall, MF59 appears to be an adjuvant worse than aluminum hydroxide for HBsAg expressed in yeasts in primates, but a better adjuvant for HBsAg expressed in CHO. These results can be explained by an interaction between (a) residual surfactant in purified yeast HBsAg and (b) excess squalene oil in adjuvant MF59. As an oil, squalene is subject to the action of surfactants under aqueous conditions, and conversely, and it has been proposed that the surfactant in the antigen is incompatible with the oil in the adjuvant. This incompatibility can be treated with HBsAg purification without using detergents (e.g., as in the material expressed in CHO) or by ensuring that the oil is not present in an excess such that it interferes with the surfactant in the antigen.

In an HBsAg composition adjuvant with MF59, a volume of MF59 is combined with a volume of antigenic solution. Therefore, in a volume of 100 ml, there will be a solution of 50 ml of MF59 and 50 ml of HBsAg. These 100 ml contain 2.15 g of squalene from MF59 and, at a concentration of HBsAg of 40 μg / ml, contain 2 mg of HBsAg. At a concentration of polysorbate 20 of 25 μg per 100 μg of HBsAg [35], the 100 ml contains 0.5 mg of polysorbate 20. The oil-surfactant ratio in a typical vaccine adjuvant with MF59 is therefore 4300: 1 . By reducing the excess oil to less than 1000: 1, the interaction between the oil in the adjuvant and the surfactant in the antigen can be substantially reduced, so that interference is reduced.

It should be understood that the invention has been described by way of example only and that modifications can be made while remaining within the scope of the invention.

REFERENCES

[1] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.

[2] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).

[3] Document WO90 / 14837.

[4] Podda &amp; Del Giudice (2003) Expert Rev Vaccines 2: 197-203.

[5] US Pat. 6,299,884.

[6] US Pat. 6,451,325.

[7] Allison &amp; Byars (1992) Res Immunol 143: 519-25.

[8] Hariharan et al., (1995) Cancer Res 55: 3486-9.

[9] WO95 / 11700.

[10] US Pat. 6,080,725.

[11] WO2005 / 097181.

[12] UK patent application GB-A-2220211.

[13] Myers et al. (1990) pages 145-156 of Cellular and molecular aspects of endotoxin reaction.

[14] Ulrich (2000) Chapter 16 (pages 273-282) of reference 1.

[15] Johnson et al. (1999) J Med Chem 42: 4640-9. 5 [16] Baldrick et al., (2002) Regulatory Toxicol Pharmacol 35: 398-413.

[17] WO 94/21292.

[18] WO94 / 00153.

[19] WO97 / 01640 /

[20] WO96 / 26741.

[21] WO93 / 19780.

[22] US Pat. 6,375,945.

[23] WO00 / 00462.

[24] Meraldi et al., (2003) Vaccine 21: 2485-2491.

[25] Pajak et al., (2003) Vaccine 21: 836-842. 15 [26] Brandenburg et al., (2000) Eur J Biochem 267: 3370-7.

[27] Johnson et al., (1999) Bioorg Med Chem Lett 9: 2273-2278.

[28] Evans et al., (2003) Expert Rev Vaccines 2: 219-229.

[29] US Pat. 6,764,840.

[30] US Pat. application 2005/0107600.

[31] Lien et al., (2001) J. Biol. Chem 276: 1873-80.

[32] WO03 / 011223.

[33] Wong et al., (2003) J Clin Pharmacol 43 (7): 735-42.

[34] US2005 / 0215517.

[35] Costa et al., (1997) Rev. Inst. Med. Trop. S. Paulo v.39 no.1 São Paulo Jan. 25 [36] Vanlandschoot et al. (2005) J Gen Virol 86: 323-31.

[37] Hilleman (1993) pages 17-40 of Hepatitis B vaccines in clinical practice ISBN 0-8247-8780-3.

[38] Gerin et al., (1969) J Virol 4: 763.

[39] Gerin et al., (1969) J Virol 7: 569.

[40] Siebke et al., (1972) Acta Pathol Microbiol Scan sect B 80: 935.

[41] Pillot et al., (1976) J Clin Microbiol 4: 205.

[42] Duimel &amp; Krijnen (1972) Vox Sang 23: 249.

[43] Neurath et al., (1974) PNAS USA 71: 2663.

[44] Charm &amp; Wong (1975) Biotechnol Bioeng 16: 593.

[45] US Pat. 4,857,317. 35 [46] US Pat. 4,683,294.

[47] Houwen et al., (1975) J Immunol Methods 8: 185.

[48] Sitrin et al. (1993) pages 83-101 of Hepatitis B vaccines in clinical practice ISBN 0-8247-8780-3.

[49] Wampler et al., (1985) PNAS USA 82: 6830-4.

[50] Chi et al., (1994) Ann N and Acad Sci 721: 365-73.

[51] Agraz et al., (1994) J Chromatogr A 672: 25-33.

[52] Mason et al., (1992) PNAS USA 89: 11745-9.

[53] Deml et al., (1999) J Virol Methods 79: 205-17.

[54] Ibarra et al., (1999) J Chromatogr B Biomed Sci Appl 735: 271-7.

[55] WO90 / 10058. 45 [56] Japanese patent application JP63239234.

[57] US Pat. 4,694,074.

[58] EP-0341733.

[59] US Pat. 5,242,812.

[60] Russian patent application RU-2128707.

[61] Hardy et al., (2000) J Biotechnol 77: 157-67.

[62] Dogan et al., (2000) Biotechnol Prog 16: 435-41.

[63] Berkower et al., (2004) Virology 321 (1): 75-86.

[64] Eckhart et al., (1996) J Gen Virol 77: 2001-8.

[65] von Brunn et al., (1991) Vaccine 9 (7): 477-84. 55 [66] Vreden et al. (1991) Am J Trop Med Hyg 45 (5): 533-8.

[67] Moelans et al., (1995) Mol Biochem Parasitol 72 (1-2): 179-92.

[68] Stoute et al., (1997) N Engl J Med 336 (2): 86-91.

[69] Wunderlich &amp; del Portillo (2000) Mol Med 6 (3): 238-45.

[70] US Pat. 5,928,902

[71] WO93 / 10152.

[72] Nony et al., (2001) Vaccine 27: 3645-51.

[73] WO02 / 12287

[74] WO03 / 066094.

[75] Desombere et al., (1998) Tissue Antigens 51: 593-604. 65 [76] McDermott et al., (1997) Tissue Antigens. 50: 8-14.

[77] Traquina et al., (1996) J Infect Dis 174: 1168-75.

SEQUENCE LIST

<110> NOVARTIS VACCINES AND DIAGNOSTICS SRL CONTORNI Mario

<120> REDUCTION OF INTERFERENCES BETWEEN ADJUVANTS THAT ANTIGENS THAT CONTAIN TENSIOACTIVE

CONTAIN OIL Y

5

<130> PD41598W0

<140> PCT / IB2006 / YYYYYY

<141> 2006-08-02

<150> GB-0515906.6

<151> 2005-08-02

10

<150> GB-0522599.0

<151> 2005-11-04

<150> GB-0523923.1

<151> 2005-11-24

<160> 2

fifteen

<170> SeqWin99, version 1.02

<210> 1

<211> 4

<212> PRT

<213> Plasmodium falciparum

twenty

<400> 1

<210> 2

<211> 423

<212> PRT 25 <213> Plasmodium falciparum

<400> 2

Claims (31)

1. A process for preparing an immunogenic composition, comprising the steps of combining (i) a viral surface antigen component that includes a surfactant and (ii) a fatty adjuvant component, to give a composition in which the weight ratio between the fatty adjuvant and the surfactant is less than 1000: 1.

The method of claim 1, wherein the fatty adjuvant comprises a metabolizable oil.

3.

 The process of claim 2, wherein the fatty adjuvant comprises a submicron emulsion of oil in squalene water and polysorbate 80.

Four.

 The process of claim 1, wherein the fatty adjuvant comprises a molecule comprising a fatty acid moiety.

The method of claim 4, wherein the fatty adjuvant is a monophosphoryl lipid A 3-deoxycylated (‘3D-MPL’).

6.

 The method of claim 5, wherein the 3D-MPL is a mixture of molecules that vary according to their acylation.

7.

 The process of claim 5 or claim 6, wherein the fatty adjuvant includes:

8.

 The method of any one of claims 5 to 7, wherein the ED-MPL is in the form of particles.

9.

 The method of claim 8, wherein the particles can be sterilized by filtration.

10. The method of any one of claims 5 to 9, wherein the 3D-MPL is in a combination 20 with an aluminum salt.

eleven.

 The process of claim 10, wherein the aluminum salt is an aluminum phosphate.

12.

 The method of claim 11, wherein the 3D-MPL is adsorbed onto the aluminum salt.

40 E06795518 11-08-2011

13.

 The process of any of the preceding claims, wherein the surfactant is a polyoxyethylene sorbitan ester.

14.

 The method of claim 13, wherein the ester is polysorbate 20.

fifteen.

 The process of claim 13 or claim 14, wherein the concentration of surfactant is not more than 50 µg / ml.

16.

 The method of any of the preceding claims, wherein the viral surface antigen is a hepatitis B surface particulate antigen, purified in the presence of a surfactant.

17.

 The method of claim 16, wherein the HBsAg is expressed in a yeast cell.

18.

 The method of claim 17, wherein the yeast is Saccharomyces cerevisiae.

19.

 The process of any one of claims 16 to 18, wherein the HBsAg is not glycosylated and / or includes phosphatidylinositol.

twenty.

 The method of any one of claims 16 to 19, wherein the HBsAg is derived from the adw1 subtype of the hepatitis B virus.

twenty-one.

 The method of any of the preceding claims, wherein the antigen is a hybrid protein comprising a viral surface antigen and a heterologous antigen.

22

 The method of claim 21, wherein the viral surface antigen is HBsAg and the heterologous antigen is a malaria antigen.

2. 3.

 The method of claim 22, wherein the hybrid protein comprises HBsAg and a fragment of the plasmodium falciparum circumsporozoite protein.

24.

 The method of claim 23, wherein the hybrid protein comprises a C-terminal portion of a Cirmodium falciparum circumsporozoite protein, four or more tandem repeats of the immunodominant region of the circumsporozoite protein and HBsAg.

25.

 The process of any preceding claim, wherein the weight ratio of the fatty adjuvant and the surfactant is less than 500: 1.

26.

 The process of any preceding claim, wherein the weight ratio of the fatty adjuvant and the surfactant is less than 50: 1.

27.

 The method of any preceding claim, wherein the fatty adjuvant is a 3D-MPL and the weight ratio of the fatty adjuvant and the surfactant is between 2.5: 1 and 25: 1.

28.

 The method of any preceding claim, wherein the composition has an osmolality of 200 mOsm / kg to 400 mOsm / kg.

29.

 The process of any preceding claim, wherein the composition includes a phosphate buffer.

30

 The process of any preceding claim, wherein the composition has a pH between 6.5 and 7.0.

31.

 The use of (i) a viral surface antigen component that includes a surfactant and (ii) a fatty adjuvant in the manufacture of a medicament for administration to a patient, in which the weight ratio between the fatty adjuvant in (ii ) and the surfactant in (i) is less than 1000: 1.

32

 A process for preparing an immunogenic composition, comprising the steps of combining (i) an HBsAg component that includes polysorbate 20 and (ii) an adjuvant component comprising 3D-MPL adsorbed on an aluminum phosphate, to give a composition in the that the weight ratio between 3D-MPL and polysorbate 20 is less than 1000: 1.

33.

 An immunogenic composition, in which: (a) the composition comprises HBsAg, polysorbate 20, 3D-MPL and an aluminum phosphate adjuvant, in which polysorbate 20 is incorporated into HBsAg particles during purification; and (b) the weight ratio between 3D-MPL and polysorbate 20 is less than 1000: 1.