HK1119049A - Adjuvant composition comprising aluminium phosphate and 3d-mpl - Google Patents

Description

Adjuvant composition comprising aluminium phosphate and 3D-MPL

All documents cited herein are hereby incorporated by reference in their entireties.

Technical Field

The present invention relates to the field of vaccine adjuvants.

Background

Aluminum salts (often referred to as "alum") are classical vaccine adjuvants. Various other adjuvants have been described, and reference 1 and 2 are given for details. One of these adjuvants is 3' deacylated monophosphoryl lipid A (or "3D-MPL").

References 3-10 report the success of using an adjuvant system called "AS 04" in patients with non-responsive hepatitis, said adjuvant system to contain 3D-MPL and alum [11-14 ]. It is an object of the present invention to improve and enhance the adjuvant system.

Disclosure of Invention

The composition of the invention comprises an aluminium phosphate adjuvant and 3D-MPL adjuvant. This dual adjuvant combination has been described in the general terms of references 12-14, but the present invention discloses a number of improvements to this combination as follows:

(a) the osmolality of the composition is between 200-400 mOsm/kg.

(b) The pH of the composition should be between 5 and 7.5.

(c) The composition should contain a buffer.

(d) At least 50% of the 3D-MPL in the vaccine should be adsorbed to aluminium phosphate.

(e) The 3D-MPL in the vaccine should take the form of micellar structures with a diameter of less than 150 nm.

(f) The 3D-MPL in the vaccine should be a mixture of different acylated forms, preferably at least 10% in the 6-acyl-chain form.

(g) The composition may contain one or more of the following components: polyoxyethylene sorbitol monooleate; sorbitol; triethanolamine; triethylammonium ion (triethyllamonium ion); lactose; sucrose; trehalose; mannose.

These modifications may be used alone or in combination.

Accordingly, the present invention provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) 3-O-deacylated monophosphoryl lipid A adjuvant, characterized in that the osmolality of the composition is between 200 and 400 mOsm/kg.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) 3-O-deacylated monophosphoryl lipid A adjuvant, characterized in that the pH of the composition is between 5 and 7.5.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) 3-O-deacylated monophosphoryl lipid A adjuvant, characterised in that the composition contains a buffer, e.g. having a pH of between 5 and 7.5.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) 3-O-deacylated monophosphoryl lipid A adjuvant, characterized in that at least 50% of the 3-O-deacylated monophosphoryl lipid A is adsorbed to the aluminum phosphate.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) 3-O-deacylated monophosphoryl lipid A adjuvant, characterized in that unadsorbed 3-O-deacylated monophosphoryl lipid A in the composition is less than 50 μ g/ml.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) a 3-O-deacylated monophosphoryl lipid A adjuvant, characterized in that the 3-O-deacylated monophosphoryl lipid A is in the form of particles having a diameter of less than 150 nm.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) a 3-O-deacylated monophosphoryl lipid a adjuvant comprising a mixture of acylated disaccharides, wherein each disaccharide: (a) having two β -1', 6-linked 2-deoxy-2-glucosamine monosaccharide subunits; (b) phosphorylation at the 4' position; (c) unsubstituted in the 1, 3 and 6' positions; (d) acylation at the 3' position O; and (e) N-acylation at the 2 and 2' positions; wherein at least 10% by weight of the components of the acylated disaccharide mixture contain O-acyl substitutions on the aliphatic carbon atom of each acyl group itself in the 2, 2 'and 3' positions.

The present invention also provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) 3-O-deacylated monophosphoryl lipid A adjuvant; and at least one selected from the group consisting of sorbitol, triethanolamine ion, lactose, sucrose, trehalose, and mannose.

Various features of the above may be used in combination. Accordingly, the present invention provides an adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) a 3-O-deacylated monophosphoryl lipid a adjuvant, characterized in that the composition has one or more of the following properties:

(1) the osmolality is between 200 and 400 mOsm/kg;

(2) the pH is between 5 and 7.5.

(3) Contains a buffer solution;

(4) at least 50% of the 3-O-deacylated monophosphoryl lipid A is adsorbed to the aluminum phosphate;

(5) wherein unadsorbed 3-O-deacylated monophosphoryl lipid A is less than 50 μ g/ml;

(6) the 3-O-deacylated monophosphoryl lipid A adjuvant is in the form of particles having a diameter of less than 150 nm;

(7) the 3-O-deacylated monophosphoryl lipid a adjuvant comprises a mixture of acylated disaccharides, wherein each disaccharide: (a) having two β -1', 6-linked 2-deoxy-2-glucosamine monosaccharide subunits; (b) phosphorylation at the 4' position; (c) unsubstituted in the 1, 3 and 6' positions; (d) acylation at the 3' position O; and (e) N-acylation at the 2 and 2' positions; wherein the mixture of acylated disaccharides contains at least 10% by weight of the components in which the acyl groups in the 2, 2 'and 3' positions are themselves substituted with O-acyl groups on an aliphatic carbon atom; and/or

(8) Contains at least one substance selected from sorbitol, triethanolamine ion, lactose, sucrose, trehalose and mannose.

The invention also provides immunogenic compositions comprising an adjuvant composition of the invention, further comprising (iii) an antigen.

Aluminium phosphate adjuvants

The composition of the invention comprises an aluminium phosphate adjuvant and 3D-MPL adjuvant.

The term "aluminum phosphate" is conventional in the art, but the actual chemical compounds it represents are not described precisely [ see, e.g., chapter 9 of reference 2]. The present invention can be used with any "aluminum phosphate" adjuvant conventionally used as an adjuvant, typically aluminum hydroxyphosphate, which also often contains small amounts of sulfate (i.e., aluminum hydroxyphosphate sulfate). These adjuvants can be obtained by precipitation, the reaction conditions and concentration during precipitation affecting the extent to which phosphate takes up the hydroxyl groups of the salt. PO in hydroxy phosphate₄The molar ratio Al is generally between 0.3 and 1.2. The hydroxyphosphate differs from the strictly AlPO by the presence of the hydroxyl group₄. For example 3164cm^-1Indicating the presence of structural hydroxyl groups (e.g. when heated to 200 ℃) [ chapter 9 of reference 2]。

The aluminium salt may take any suitable physical form but is typically amorphous.

PO with aluminum phosphate adjuvant₄/Al³⁺The molar ratio is generally between 0.3 and 1.2, preferably between 0Between 8 and 1.2, more preferably 0.95. + -. 0.1. The aluminium phosphate is generally amorphous, in particular a hydroxy phosphate. A typical adjuvant is PO₄/Al³⁺The molar ratio is between 0.84 and 0.92, and 0.6mg of Al is contained³⁺Amorphous aluminum hydroxyphosphate per ml. The aluminum phosphate is typically particulate. Typical diameters of these particles after adsorption of any antigen and/or 3D-MPL are 0.5-20 μm (e.g., about 5-10 μm).

The PZC of aluminum phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, which varies depending on the reaction conditions and reactant concentrations used to precipitate the salt. PZC can also be varied by varying the concentration of free phosphate ions in the solution (more phosphate-more acidic PZC) or by adding buffers such as histidine buffer (making PZC more basic). The PZC of the aluminium phosphate used in the present invention is typically between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example about 5.7.

It is preferred to use aluminium phosphate in the form of an aqueous solution to which 3D-MPL (and optionally antigen) is added (note: although aqueous aluminium phosphate is normally referred to as a "solution", from a strict physicochemical point of view the salt is insoluble and forms a suspension). Preferably, the aluminium phosphate is diluted to the required concentration and the solution is made homogeneous before the addition of 3D-MPL and/or antigen.

3D-MPL and/or Al before antigen addition³⁺The concentration of (B) is generally between 0 and 10 mg/ml. The preferred concentration is between 0.5 and 3 mg/ml.

The aluminum phosphate solution used to prepare the compositions of the invention may contain a buffer (e.g., a phosphate or histidine or Tris buffer), but need not. The aluminum phosphate solution is preferably sterile, pyrogen-free. The aluminum phosphate solution can contain free aqueous phosphate ions, for example, at a concentration of between 1.0-20mM, preferably between 5-15mM, and more preferably about 10 mM. The aluminum phosphate solution may also contain sodium chloride. The concentration of sodium chloride is preferably 0.1-100mg/ml (e.g., 0.5-50mg/ml, 1-20mg/ml, 2-10mg/ml), more preferably about 3. + -.1 mg/ml. The presence of sodium chloride helps to correctly detect pH prior to adsorption of other components, and can also affect osmolality.

3D-MPL adjuvant

The composition of the invention comprises an aluminium phosphate adjuvant and 3D-MPL adjuvant.

3-O-deacylated monophosphoryl lipid A (3D-MPL) is also known as 3-de-O-acyl monophosphoryl lipid A or 3-O-deacyl-4' -monophosphoryl lipid A. This name indicates deacylation at the 3-position of the reducing terminal glucosamine in monophosphoryl lipid a. It was prepared from heptose-free (heposels) mutants of Salmonella minnesota (Salmonella minnesota), similar in chemical structure to lipid a but lacking an acid-labile phosphoryl group and a base-labile acyl group. It activates cells of the monocyte/macrophage lineage and stimulates the release of several cytokines, including IL-1, IL-2, TNF- α and GM-CSF. Preparation of 3D-MPL was initially described in reference 15, which is manufactured by Corixa Corporation and is sold under the trade name MPL^TMAnd (5) selling. See references 16-19 for additional details.

Typical compositions comprise 3D-MPL at a concentration of between 25 μ g/ml and 200 μ g/ml, such as 50-150 μ g/ml, 75-125 μ g/ml, 90-110 μ g/ml or about 100 μ g/ml, typically between 25 and 75 μ g of 3D-MPL is administered per dose, such as between 45 and 55 μ g, or about 50 μ g of 3D-MPL per dose.

Preferably, the 3D-MPL is adsorbed onto aluminium phosphate. Preferably at least 50% (by weight) of 3D-MPL is adsorbed, e.g.. gtoreq.60%,. gtoreq.70%,. gtoreq.80%,. gtoreq.90%,. gtoreq.95%,. gtoreq.98% or more. The percentage of adsorption can be determined in the same way as for the antigen (see below). In a composition having a total concentration of 3D-MPL of 100 μ g/ml, the concentration of unadsorbed 3D-MPL should be less than 50 μ g/ml, e.g.. ltoreq.40 μ g/ml,. ltoreq.35 μ g/ml,. ltoreq.30 μ g/ml,. ltoreq.25 μ g/ml,. ltoreq.20 μ g/ml,. ltoreq.15 μ g/ml,. ltoreq.10 μ g/ml,. ltoreq.5 μ g/ml,. ltoreq.2 μ g/ml,. ltoreq.1 μ g/ml, etc.

3D-MPL can take the form of a mixture of related molecules that are acylated to varying degrees (e.g., contain 3, 4, 5, or 6 acyl chains of varying lengths). The two glucosamine (also known as 2-deoxy-2-amino-glucose) monosaccharides have an N-acyl group at the 2-position (i.e., the 2 and 2 'positions) and an O-acyl group at the 3' position. The group attached to carbon 2 is of the formula-NH-CO-CH₂-CR¹R^1＇As shown. The group attached to carbon 2' is of the formula-NH-CO-CH₂-CR²R^2’As shown. The group 3' to the carbon is of the formula-O-CO-CH₂-CR³R^3'As shown. A representative structure is:

radical R¹、R²And R³Each independently is- (CH)₂)_n-CH₃. The value of n is preferably between 8 and 16, more preferably between 9 and 12, most preferably 10.

Radical R^1'、R^2'And R^3'Each independently is: (a) -H; (b) -OH; or (c) -O-CO-R⁴Wherein R is⁴is-H or- (CH)₂)_m-CH₃Wherein m is preferably between 8 and 16, more preferably 10, 12 or 14. In the 2-position, m is preferably 14. In the 2' position, m is preferably 10. In the 3' position, m is preferably 12. Thus the group R^1'、R^2'And R^3'Preference is given to the-O-acyl radicals of dodecanoic acid, tetradecanoic acid and hexadecanoic acid.

When R is¹、R^2'And R^3’When all are-H, 3D-MPL has only 3 acyl chains (one at each of the 2, 2 'and 3' positions). When R is^1'、R^2'And R^3'When only two of them are-H, the 3D-MPL may have 4 acyl chains. When R is^1'、R^2'And R^3'When only one of them is-H, the 3D-MPL may have 5 acyl chains. When R is^1'、R^2'And R^3'When none of them is-H, the 3D-MPL may have 6 acyl chains. The 3D-MPL adjuvant used in the present invention may be a mixture of these forms having 3-6 acyl chains, but it is preferred that the mixture comprises 3D-MPL having 6 acyl chains, especially ensuring that the 6 acyl chain form is at least 10% by weight of the total 3D-MPL, e.g.. gtoreq.20%,. gtoreq.30%,. gtoreq.40%,. gtoreq.50% or more. 3D-MPL with 6 acyl chains was found to be the most active adjuvant form.

Thus, the most preferred form of 3D-MPL comprised by the composition of the invention is:

when 3D-MPL is used in the form of a mixture, reference to the amount or concentration of 3D-MPL in a composition of the invention refers to the amount or concentration of the various species of 3D-MPL mixed in the mixture.

Under aqueous conditions, 3D-MPL may form micellar aggregates or particles of varying sizes, e.g., <150nm or >500nm in diameter. Either or both may be used in the present invention and the preferred particles may be selected by routine experimentation. Smaller particles are preferred for use in the present invention because of their preferred activity (e.g., small enough to give a clear aqueous suspension of 3D-MPL) [20 ]. Preferably the particles have an average diameter of less than 150nm, more preferably less than 120nm, and may even have an average diameter of less than 100 nm. However, in most cases, the average diameter is not less than 50 nm.

Adsorption of 3D-MPL onto aluminium phosphate may not allow direct measurement of 3D-MPL particle size, but the particle size may be measured before adsorption.

Particle diameter can be assessed by conventional techniques of dynamic light scattering, which reveal the average particle diameter. When it is said that the diameter of the particles is xnm, it usually means that the particle distribution range is around this average value, but the diameter of particles at least 50% by number (e.g.. gtoreq.60%,. gtoreq.70%,. gtoreq.80%,. gtoreq.90% or more) is in the range of x.minus or plus 25%.

Optional antigens

The adjuvant system of the present invention is preferably used in combination with an antigen to enhance the immune response resulting from administration of the antigen.

Preferred antigens for use with the adjuvant system of the invention are viral antigens such as Hepatitis B Virus (HBV), Human Papilloma Virus (HPV) or Herpes Simplex Virus (HSV) antigens. The adjuvant system is also suitable for use in combination with a parasite antigen, such as Plasmodium falciparum.

The antigen concentration is typically between 5. mu.g/ml and 50. mu.g/ml, for example between 10 and 30. mu.g/ml, between 15 and 25. mu.g/ml or about 20. mu.g/ml. The amount of antigen per dose is also typically between 5. mu.g/dose and 50. mu.g/dose, for example between 10 and 30. mu.g/dose, between 15 and 25. mu.g/dose or about 20. mu.g/dose.

Preferably the antigen is adsorbed onto an aluminium phosphate adjuvant. The percentage of the specific antigen adsorbed in the composition is preferably at least 50% (by weight), such as 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, such as up to 100%. The adsorbed material can be separated from the unadsorbed material, for example by centrifugation, wherein the antigen adsorbed to the aluminium phosphate tends to form a precipitate, while the unadsorbed antigen remains in the supernatant, thereby allowing the percentage of adsorbed antigen in the composition to be conveniently detected. The amount of antigen in the supernatant is subtracted from the total amount of antigen in the composition (e.g., by ELISA assay) and the percentage of adsorbed antigen is calculated. Preferably the antigen is adsorbed completely, i.e. no antigen is detectable in the supernatant.

Hepatitis B Virus (HBV) is one of the known factors causing viral hepatitis. HBV viral particles consist of an inner core surrounded by an outer shell protein or capsid, which contains the viral DNA genome. The major component of the capsid is a protein called the HBV surface antigen or more commonly "HBsAg", which is a 226 amino acid peptide with a molecular weight of about 24 kDa. Hepatitis B viruses all contain HBsAg, which when administered to normal vaccinees stimulates the production of anti-HBsAg antibodies that protect against HBV infection.

Thus, the preferred HBV antigen is HBsAg. HBsAg can be adsorbed onto aluminium phosphate using the method described in reference 21. Adsorption onto aluminium phosphate is well known as ENGERIX-B^TMThe product (in which HBsAg is adsorbed to aluminium hydroxide) differs from HEPACCINE^TMAnd RECOMBIVAX^TMThe product is the same. As referred to in the textIn figure 22, for HBsAg, aluminium phosphate is an adjuvant over aluminium hydroxide.

For vaccine production, HBsAg can be prepared in two ways. The first method involves purification of the antigen from the plasma of chronic hepatitis B virus carriers in particulate form, since during HBV infection the liver synthesizes large amounts of HBsAg and releases it into the bloodstream. The second method involves expressing the protein by recombinant DNA methods. The HBsAg used in the present invention may be prepared by any method, but preferably by recombinant expression. Specifically, it is preferable to prepare HBsAg by expression of Saccharomyces cerevisiae (Saccharomyces cerevisiae). Unlike native HBsAg (i.e. plasma purified product), yeast-expressed HBsAg is generally non-glycosylated, which is the most preferred form of HBsAg for use in the present invention because of its high immunogenicity and because it is prepared without the risk of contamination of blood products. Yeast-expressed HBsAg is preferably in the form of substantially spherical particles (average diameter about 20nm) comprising a lipid matrix containing phospholipids.

After purification, the HBsAg may be dialyzed (e.g., with cysteine) to remove any preservatives containing mercury, such as thimerosal that may have been used during HBsAg preparation [23 ].

In addition to "S" sequences, the surface antigen may comprise all or part of a pre-S sequence, for example all or part of a pre-S1 and/or pre-S2 sequence.

A preferred HPV antigen for use in the present invention is the L1 capsid protein, which can be assembled to form a structure called a Virus Like Particle (VLP). VLPs can also be produced using yeast cells (e.g., Saccharomyces cerevisiae) or insect cells (e.g., moth (Spodoptera) cells, such as Spodoptera frugiperda (Sfrugiperda) or Drosophila (Drosophila) cells). For yeast cells, the plasmid vector may carry the L1 gene; for insect cells, the baculovirus vector may carry the L1 gene. More preferably the composition comprises L1 VLPs of HPV-16 and HPV-18 strains (produced). This bivalent combination has been shown to be highly effective [24 ]. In addition to HPV-16 and HPV-18 strains, L1 VLPs of HPV 6 and HPV-11 strains (produced) may also be included. Oncogenic HPV strains may also be utilized. The vaccine may comprise between 20-60 μ g/ml (e.g., about 40 μ g/ml) of L1 for each HPV strain.

A preferred HSV antigen for use in the present invention is the membrane glycoprotein gD. Preferably, gD ("gD 2" antigen) of the HSV-2 strain is used. The compositions may employ the gD form [25] deleted of the C-terminal membrane anchoring region, e.g., a truncated gD comprising amino acids 1-306 of the native protein and added asparagine and glutamine at the C-terminus. This form of the protein contains a signal peptide which is cleaved to yield a 283 amino acid mature protein. The absence of the anchor region allows the protein to be prepared in soluble form.

Preferred Plasmodium falciparum antigens for use in the present invention are based on the Circumsporozoite (CS) protein. This protein may take the form of a recombinant protein in which the CS protein portion is fused to hbsag, referred to as "RTS, S" or TRAP. A hybrid protein in which the C-terminal part of substantially all of CS contained in RTS is linked to HBsAg through the first four amino acids of S2 part of HBV surface antigen [26 ]. When expressed with yeast (in particular saccharomyces cerevisiae), the RTS produced is a lipoprotein particle (in particular comprising phospholipids) which, when co-expressed with the S antigen of HBV, produces a mixed particle known as RTS, S. The RTS to S ratio used was about 1 to 4. Reference 27 describes TRAP antigens.

Pharmaceutical composition

In addition to adjuvant and antigenic components, the compositions of the invention may also comprise other components. These components can be of various origins. For example, they may be one of the antigen or adjuvant components used during production, or may be added separately from the antigenic component.

Preferred compositions of the invention comprise one or more pharmaceutical carriers and/or excipients.

For controlling tonicity, it is preferred to include physiological salts, e.g., mineral salts, such as sodium salts. Preferably sodium chloride (NaCl) at a concentration of between 1 and 20 mg/ml. Salts may be present during mixing of the adjuvant and mixing of the antigen with the adjuvant.

The compositions typically have an osmolality of between 200mOsm/kg and 400mOsm/kg, preferably 240-360mOsm/kg, more preferably 290-300 mOsm/kg. Osmolality has previously been reported to have no effect on pain caused by vaccination [28], but it is still preferred to maintain osmolality within this range.

The compositions of the invention may comprise one or more buffers. Typical buffers include: a phosphate buffer; tris buffer solution; a boric acid buffer solution; a succinic acid buffer; histamine buffer or citric acid buffer. To avoid competition with the 3D-MPL phosphate group in the buffer, buffers other than phosphate buffer are preferred. The buffer content is usually 5-20 mM.

The pH of the compositions of the invention is generally between 5.0 and 7.5, more typically between 5.0 and 6.0, or between 6.0 and 7.0 for optimum stability.

Due to the adsorptive properties of the antigen, the final vaccine product may be a suspension with a cloudy appearance. This appearance means that microbial contamination is not readily observable, and therefore the vaccine preferably contains an antimicrobial agent. This is particularly important when the vaccine is packaged in a multi-dose container. Preferred antimicrobial agents are 2-phenoxyethanol and thimerosal. However, it is preferred that no mercury-containing preservatives (e.g., thimerosal) are used in the process of the present invention. However, if the antigen is treated with such a preservative prior to preparation of the composition of the invention, it is difficult to avoid the presence of trace amounts of the preservative. However, for safety reasons, it is preferred that the final composition contains less than about 25ng/ml mercury. Most preferably, the final composition is undetectable for thimerosal. This is generally achieved by removing the mercury-containing preservative from the antigen preparation prior to adding the antigen to the process of the invention or by avoiding the use of thimerosal during the preparation of the components used in the composition.

During preparation, the components are usually diluted with WFI (water for injection) to give the desired final concentration.

According to Al³⁺It is stated that the concentration of aluminum phosphate in the compositions of the present invention is preferably less than 5mg/ml, e.g., 4mg/ml or less, 3mg/ml or less, 2mg/ml or less, 1mg/ml or less, and the like.

The concentration of 3D-MPL in the compositions of the invention is preferably less than 200. mu.g/ml, e.g.. ltoreq.150. mu.g/ml,. ltoreq.125. mu.g/ml,. ltoreq.110. mu.g/ml,. ltoreq.100. mu.g/ml etc.

The concentration of each antigen in the compositions of the invention is preferably less than 60. mu.g/ml, e.g.. ltoreq.55. mu.g/ml,. ltoreq.50. mu.g/ml,. ltoreq.45. mu.g/ml,. ltoreq.40. mu.g/ml etc.

Preferably, the patient is administered a 0.5ml dose of the composition of the invention. References to a dose of 0.5ml are to be understood as including normal differences, for example 0.5ml ± 0.1ml, 0.5ml ± 0.05ml, etc.

Preferred compositions contain about 50. mu.g of 3D-MPL and about 0.5mg of aluminum adjuvant per dose.

The present invention can provide bulk material suitable for packaging into unit doses for subsequent distribution to a patient. The above mentioned concentrations are typically those in the final packaged dose, and thus the bulk vaccine concentration may be higher (e.g. by dilution to reduce the final concentration).

The compositions of the invention are typically in aqueous form.

Other components present in the compositions of the present invention may include: polyoxyethylene sorbitol monooleate ("tween 80") [20] which has been used to prevent the agglomeration of 3D-MPL; sorbitol, which has been used to prevent 3D-MPL agglomeration; triethanolamine which has been used to dissolve 3D-MPL; triethylammonium ion for dissolving 3D-MPL; lactose; sucrose; trehalose and/or mannose.

The method of the invention

The present invention provides a method of producing an adjuvant composition of the invention comprising mixing (i) an aluminium phosphate adjuvant; and (ii) a 3-O-deacylated monophosphoryl lipid A adjuvant.

The invention also provides a method of producing a composition of the invention comprising admixing (i) an antigen; (ii) an aluminum phosphate adjuvant; and (iii) a 3-O-deacylated monophosphoryl lipid A adjuvant. The components (i), (ii) and iii) may be mixed in any order, but preferably the antigen and aluminium phosphate are mixed first, and then 3D-MPL is added to the antigen/aluminium phosphate mixture. Alternatively, 3D-MPL and aluminium phosphate may be mixed prior to addition of antigen to the adjuvant mixture.

The present invention provides a process for producing the composition of the present invention comprising the steps of: (a) expressing the antigen in a recombinant host; (b) purifying the antigen; and (c) mixing the purified antigen with (i) an aluminum phosphate adjuvant and (ii) a 3-O-deacylated monophosphoryl lipid A adjuvant. As mentioned above, the three components mixed in step (c) may be mixed in any order. Preferred recombinant hosts are the yeast and insect cells described above.

The present invention provides a process for producing the composition of the present invention comprising the steps of: (a) mixing an antigen, an aluminum phosphate adjuvant and a 3-O-deacylated monophosphoryl lipid A adjuvant; (b) detecting the osmolality of the composition; and, if the osmolality is outside the range of 200-400mOsm/kg, (c) adjusting the osmolality to within the range of 200-400 mOsm/kg. Conditioning may include the addition of physiological salts, for example sodium salts, such as sodium chloride.

The present invention provides a process for producing the composition of the present invention comprising the steps of: (a) mixing an antigen, an aluminum phosphate adjuvant and a 3-O-deacylated monophosphoryl lipid A adjuvant; (b) detecting the pH of the composition; and, if the pH is outside the range of 5.0-7.5, (c) adjusting the pH to be in the range of 5.0-7.5. The adjustment may include the addition of an acid or base.

The present invention provides a process for producing a composition of the present invention comprising mixing: (i) an antigen, (ii) an aluminum phosphate adjuvant and (iii) a 3-O-deacylated monophosphoryl lipid a adjuvant, wherein the 3-O-deacylated monophosphoryl lipid a adjuvant of component (iii) is in the form of particles having a diameter of less than 150 nm. Components (i), (ii) and (iii) may be mixed in any order. Component (iii) may also comprise polyoxyethylene sorbitol monooleate and/or sorbitol.

After mixing the antigen and the adjuvant, the method of the present invention may comprise the steps of extracting and packaging a 0.5ml sample of the mixture into a container. For multiple dose cases, multiple doses of the dose may be extracted and packaged together in a single container.

The method of the invention may include the further step of packaging the vaccine into a container for use. Suitable containers include vials and disposable syringes (preferably sterile).

Packaging the compositions of the invention

To package the compositions of the present invention in vials, the vials are preferably made of glass or plastic material. Preferably, the composition is added after the vials are sterilized. To avoid the problem of allergy to latex for the patient, the vial is preferably sealed with a latex-free stopper. The vials may contain a single dose of vaccine, or may contain multiple doses ("multidose" vials), e.g., 10 doses. When using multi-dose vials, the individual doses should be withdrawn under strictly sterile conditions using sterile needles and syringes, taking care not to contaminate the vial contents. The vials are preferably made of colorless glass.

In the case of packaging the composition in a syringe, the syringe will typically not have a needle attached to it, although the syringe and needle may be provided separately and assembled at the time of use. Preferably a safety needle (safety needle). Typical needles are 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge. The syringe may be labelled with a peelable label which is printed with the batch number and expiry date of the contents to assist in keeping the record (record keeping). The plunger of the syringe is preferably fitted with a stopper to prevent accidental dislodgement of the plunger during aspiration. The syringe may be fitted with a latex cap and/or a plunger. The disposable syringe may contain a single dose of vaccine. The syringe is typically fitted with a cap (tip cap) preferably made of butyl rubber to seal the tip prior to needle attachment. If the syringe and needle are packaged separately, the needle is preferably fitted with a butyl rubber boot. Grey butyl rubber is preferred. A preferred syringe is under the trade name Tip-Lok^TMThose syringes that are put on the market.

Preferably, the syringe is packaged (with the vaccine) such that the pre-filled syringe is received by the physician or patient.

When glass containers (e.g., syringes or vials) are used, it is preferred to use borosilicate glass rather than soda lime glass.

After the composition is packaged in a container, the container can be packaged in a box for dispensing, e.g., a cardboard box, which can be labeled with vaccine details, e.g., its trade name, a list of antigens in the vaccine (e.g., "recombinant hepatitis B virus," etc.), a packaging container (e.g., "single-use prefilled Tip-Lok syringe" or "10 x 0.5ml monodose vial"), its dosages (e.g., "each containing a 0.5ml dose"), precautions (e.g., "for adults only"), expiration dates, etc. Each cassette may contain more than one packaged vaccine, for example 5-10 packaged vaccines (especially for vials). If the vaccine is packaged in a syringe, the packaging (material) may display the design of the syringe.

The vaccine can be packaged together (e.g., in the same box) with an insert containing the vaccine details (e.g., instructions for administration, details of the antigen in the vaccine, etc.). Instructions for use may also include precautions such as preparing the epinephrine solution to prevent allergic reactions after vaccination, and the like.

The packaged vaccine material is preferably sterile.

The packaged vaccine material is preferably pyrogen-free, e.g. containing <1 EU (endotoxin unit, standard measure) per dose, preferably <0.1 EU.

The packaged vaccine material is preferably gluten-free.

The pH of any packaged aqueous vaccine material is preferably between 5 and 8, for example between 5.5 and 6.5. Thus, the method of the invention may comprise the steps of adjusting the pH of the bulk vaccine prior to packaging.

The packaged vaccine is preferably stored at a temperature between 2 ℃ and 8 ℃. Should not be frozen.

Methods of treatment and administration of vaccines

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

The present invention also provides a composition of the invention for use in medicine.

The invention also provides (i) an antigen; (ii) an aluminum phosphate adjuvant; and (iii 3-O-deacylated monophosphoryl lipid A adjuvant for use in the manufacture of a medicament for administration to a patient.

The methods and uses of the invention are particularly suited for eliciting an immune response to protect against and/or treat the following disorders after administration to a patient: HBV infection; HSV infection; genital herpes caused by HSV; HPV infection; genital warts caused by HPV; HPV-induced cervical cancer and/or malaria.

The immunogenic compositions of the invention are preferably vaccines for the prevention and/or treatment of infection.

To achieve full efficacy, a typical immunization regimen may include administration of multiple doses. For example, administration can be at months 0 and 6 (0 being the time of first administration); dosing at 0, 1, 2 and 6 months; on days 0, 21, followed by a third dose between months 6-12; or at 0, 1, 2, 6 and 12 months.

The compositions of the invention may be administered by intramuscular injection, for example, into the arm or leg.

Since the composition of the present invention contains an aluminum adjuvant, precipitation of components may occur during storage. Thus, the composition should be shaken before administration to the patient. The shaken composition will be a turbid white suspension.

Other antigenic Components

In addition to comprising HBsAg, HPVL1, HSVgD and/or malaria antigens, the compositions of the invention may contain one or more further antigens. For example, one or more of the following antigens may be present: hepatitis a virus antigen; diphtheria toxoid; tetanus toxoid; an inactivated poliovirus antigen; cellular pertussis antigens; cellular pertussis antigens including detoxified pertussis toxin, filamentous hemagglutinin, and optionally a 69kDa antigen; conjugated haemophilus influenzae b capsular saccharides, typically comprising tetanus toxoid as a carrier protein; a conjugated serogroup a neisseria meningitidis capsular saccharide; a conjugated serogroup C neisseria meningitidis capsular saccharide; a conjugated serogroup Y neisseria meningitidis capsular saccharide; conjugated serogroup W135 neisseria meningitidis; conjugated streptococcus pneumoniae (s. pneumoconiae) capsular saccharide.

Alternatives to aluminum phosphate

For some applications, the aluminum phosphate adjuvant may be replaced with an aluminum hydroxide adjuvant, or a combination of aluminum hydroxide and aluminum phosphate adjuvants may be used. For example, aluminum hydroxide is preferred over aluminum phosphate in HPV and HSV vaccines. Accordingly, the above definition of the invention may be amended accordingly.

The term "aluminum hydroxide" is conventional in the art, but the actual chemical compound that it represents is not precisely described [ see, e.g., chapter 9 of reference 2]. The present invention may be used with any "aluminium hydroxide" adjuvant conventionally used as an adjuvant, typically aluminium oxyhydroxide, which is usually at least partially crystalline. Aluminum oxyhydroxides of the formula AlO (OH) and other aluminum compounds, e.g. aluminum hydroxides Al (OH)₃In the Infrared (IR) spectrum, in particular at 1070cm^-1The absorption band and 3090-^-1Strong acromion [ chapter 9 of reference 2]]. The degree of crystallization of the aluminum hydroxide adjuvant is reflected in the width of the half-height diffraction band (WHH), and poorly crystallized particles show greater line broadening (linerodading) due to smaller crystals. The surface area increases with increasing WHH, and adjuvants with higher WHH values have been found to have greater antigen adsorption capacity. The typical morphology of aluminum hydroxide adjuvants is fibrous (e.g., as observed by transmission electron micrographs). The pI of aluminum hydroxide is typically about 11, i.e., the adjuvant itself is positively charged at physiological pH. It is reported that the adsorption capacity of the aluminum hydroxide adjuvant per mgAl at pH7.4⁺⁺⁺Between 1.8 and 2.6mg protein.

General terms

The term "comprising" includes "and" consists of … …, "e.g., a composition" comprising "X may consist of X alone or may comprise other materials, e.g., X + Y.

The word "substantially" does not exclude "completely", e.g., a composition that is "substantially free" of Y may be completely free of Y. "substantially" may be deleted from the definition of the invention as desired.

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

Unless specifically stated, a method comprising the step of combining two or more components does not require any particular order of mixing. Thus, the components may be mixed in any order. When there are three components, the two components can be mixed with each other first, and the mixture can be mixed with the third component, and so on.

It will be appreciated that the ionizable group may exist in a neutral form, as shown by the formulae herein, or in a charged form, depending, for example, on pH. Thus, the phosphate group may be as-P-O- (OH)₂As shown, this formula represents only a neutral phosphate group, and other charged forms are encompassed by the present invention. Similarly, sugar rings may exist in both open and closed forms, although the formulae herein show closed forms, open forms are also within the invention.

Modes for carrying out the invention

The method for purifying the recombinant saccharomyces cerevisiae expression HbsAg comprises: recovery of cells, precipitation, ultrafiltration, gel filtration, ion exchange, ultracentrifugation and desalting. The purified antigen was non-glycosylated and was observed to be in the form of substantially spherical particles (average diameter about 20 nm).

The antigen was maintained in phosphate buffer solution and allowed to adsorb onto amorphous aluminum phosphate adjuvant (concentration 3-6mg/ml Al) by stirring at room temperature for 1 hour⁺⁺⁺In between). The mixture was stored at room temperature for two weeks and then stored in a refrigerator. Corixa's 3D-MPL adjuvant was then added and allowed to adsorb onto the aluminium phosphate adjuvant, with water for injection and sterile saline making any dilution necessary to obtain the desired final antigen concentration. The bulk vaccine is then packaged in individual doses in disposable syringes.

First in healthy adolescents and adultsThe vaccines produced in this way were tested. The vaccine elicits a stronger immune response in all age groups than ENGERIX B^TMThe product (seroprotection rate) is up to 100%, and the GMT value is higher).

When the vaccine was administered in a two dose regimen (0 and 6 months), a 98.6% seroprotection rate was observed with the aluminum phosphate/3 dMPL adjuvant mixture, superior to the ENGERIX B administered at months 0, 1 and 6^TM96.8% of the total. GMT about 7800 (using ENGERIX B)^TMIs 3700). After the preliminary examination, a pre-hemodialysis patient aged 15 years or more (mean age 58) and a patient who had undergone hemodialysis were examined. These patients were naive to HBV. Compare a single dose of the vaccine (20. mu.g HBsAg) with a double dose of ENGERIX B^TMAdministered at months 0, 1, 2 and 6. The seroprotection (%) and anti-HBsAg GMT (mIU/ml) are as follows:

	adjuvants in vaccines	Time after first administration (month)
								2	6	7	12	24	30
		SP(％)	AP+3DMPL	49	82	91	86							86	70
AH	22							66	84	77	77	53

GMT(mIU/ml)	AP+3DMPL	80	250	3560	910	350	180
								AH	60	90	930	320	210	100

Thus, these vaccines consistently produce an immune response in hemodialysis adults superior to the main commercial ENGERIX B^TMA vaccine. In addition, protection began faster (e.g., 75% of patients were seroprotected with ENGERIX B at month 3)^TMIs 52%, p<0.005) for a longer period of time.

Another trial in naive patients with HBV awaiting liver transplantation showed similar results. Vaccine administration on days 0 and 21 (plus administration of ENGERIX B on day 7)^TM) And then the last dose was given between months 6 and 12.

	Adjuvant	Post-dose detection
				Day 28	Final dosage
		SP(％)	AP+3DMPL			32	60
AH	21			32
			GMT(mIU/ml)		AP+3 DMPL	20	480
AH	40	280

The serum protection was higher with the aluminium phosphate/3 dMPL mixture (60% vs 32%, p < 0.035).

Satisfactory safety and reactogenicity was observed in all patients. Transient local discomfort with the vaccine of the invention is greater, but subsides rapidly, with acceptable side effects compared to therapeutic benefit.

It is to be understood that the present invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention.

References (the contents of which are incorporated herein by reference)

[1] Vaccine additives, Preparation Methods and Research Protocols (Vaccine Adjuvants: cardboard Methods and Research Protocols), (Methods in Molecular Medicine series (Vol. 42), ISBN: 1-59259-083-7, ed. O' Hagan.

[2] Vaccine Design: the Subunit and Adjuvant Approach (vaccine design: Subunit and Adjuvant Approach), (compiled by Powell and Newman), Plenum Press 1995(ISBN 0-306-44867-X).

[3] Desombere et al, (2002) Vaccine 20: 2597-.

[4] Levie et al, (2002) Scand J Infect Dis 34: 610-4.

[5] Kong et al, (2003) Abstract from 11th International Symposium on viral hepatitis and Liver Disease (Abstract of the 11th International Symposium of viral hepatitis and Liver Disease), 6-10 th month in 2003, Sydney, Australia.

[6] Boland et al, (2003) Abstract from 11th International Symposium on viral hepatitis and Liver Disease (Abstract of the 11th International Symposium of viral hepatitis and Liver Disease), 6-10 th month of 2003, Sydney, Australia.

[7] Starkel et al, (2003) Abstract 61 from 55th Annual Meeting of the American Association for the Study of Liver Diseases ("Abstract 61 of the American society for Liver Diseases research"), 10 months 29 to 11 months 2, Boston, Mass.

[8] Jacques et al, (2002) Vaccine 20: 3644-9.

[9] Tong et al, (2005) Kidney International 68: 2298-.

[10] Boland et al, (2004) Vaccine 23: 316-20.

[11]WO93/19780.

[12]WO96/26741.

[13] Us patent 5,972,346.

[14] Us patent 6,488,934.

[15] British patent application GB-A-2220211.

[16] Myers et al, (1990), Cellular and molecular assays of endotoxin reactions (Cellular and molecular aspects of endotoxin reactions), pp.145-156.

[17] Ulrich, (2000), chapter 16 of reference 1 (pages 273 and 282).

[18] Johnson et al, (1999) J Med Chem 42: 4640-9.

[19] Baldrick et al, (2002) Regulation Toxicol Pharmacol 35: 398-.

[20]WO 94/21292.

[21] Us patent 6013264.

[22] Us patent 4,624,918.

[23]WO03/066094.

[24] Harper et al (2004) Lancet 364(9447) 1757-65.

[25]EP-A-0139417.

[26] Us patent 5928902.

[27]WO 90/01496.

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

Claims (26)

1. An adjuvant composition comprising: (i) an aluminum phosphate adjuvant; and (ii) a 3-O-deacylated monophosphoryl lipid A adjuvant, wherein at least 50% of the 3-O-deacylated monophosphoryl lipid A is adsorbed onto the aluminum phosphate adjuvant.

2. The adjuvant composition of claim 1, wherein the composition has less than 5 μ g/ml of unadsorbed 3-O-deacylated monophosphoryl lipid a.

3. The adjuvant composition of any preceding claim, wherein at least 95% of 3-O-deacylated monophosphoryl lipid a is adsorbed onto the aluminium phosphate adjuvant.

4. The adjuvant composition of any preceding claim, wherein the 3-O-deacylated monophosphoryl lipid a adjuvant comprises a mixture of acylated disaccharides, wherein each disaccharide: (a) having two β -1', 6-linked 2-deoxy-2-glucosamine monosaccharide subunits; (b) phosphorylation at the 4' position; (c) unsubstituted in the 1, 3 and 6' positions; (d) o-acylation at the 3' position; and (e) N-acylation at the 2 and 2' positions; wherein at least 10% by weight of the components of said acylated disaccharide mixture contain an O-acyl substitution of the aliphatic carbon atom of each acyl group itself in the 2, 2 'and 3' positions.

5. An adjuvant composition according to any preceding claim, further comprising triethylammonium ions.

6. The adjuvant composition of any preceding claim, wherein the osmolality of the composition is between 200 and 400 mOsm/kg.

7. The adjuvant composition of any preceding claim, wherein the pH of the composition is between 5 and 7.5.

8. An immunogenic composition comprising: (i) an aluminum phosphate adjuvant; (ii) 3-O-deacylated monophosphoryl lipid a adjuvant; and (iii) an antigen, wherein at least 50% of said 3-O-deacylated monophosphoryl lipid A is adsorbed to said aluminum phosphate adjuvant.

9. The adjuvant composition of any preceding claim, wherein the aluminium phosphate adjuvant is amorphous.

10. The composition of claim 9, wherein the antigen is hepatitis b virus surface antigen (HBsAg).

11. The composition of claim 10, wherein at least 50%, preferably at least 90% of the HBsAg is adsorbed to the aluminium phosphate adjuvant.

12. The composition of claim 10 or 11, wherein the antigen is yeast-expressed HBsAg in the form of substantially spherical particles comprising a lipid matrix comprising phospholipids.

13. The composition of claim 12, wherein said yeast is saccharomyces cerevisiae.

14. The composition of any one of claims 10 to 13, wherein a 0.5ml dose of the composition comprises about 50 μ g of 3-O-deacylated monophosphoryl lipid a; about 0.5mg aluminum phosphate (as Al)³⁺Represents); and about 20. mu.g/ml HBsAg.

15. The composition of claim 8 or 9, wherein the antigen is a yeast-expressed mixed particle (RTS, S) comprising: (a) RTS, a hybrid protein containing the C-terminal portion of substantially all of the plasmodium falciparum CS protein linked to HBsAg by the first four amino acids of the S2 portion of the HBV surface antigen; and (b) S as a surface antigen of hepatitis B virus.

16. The composition of any one of claims 8 to 15, packaged into a syringe.

17. The composition of claim 16, wherein the syringe is made of borosilicate glass and is provided with a cap made of butyl rubber.

18. A process for preparing a composition according to any one of claims 8 to 15, comprising the steps of: (a) mixing the antigen with the aluminum phosphate adjuvant; and then (b) mixing the 3-O deacylated monophosphoryl lipid A adjuvant with the antigen/aluminum phosphate mixture.

19. The method of claim 18, wherein the antigen is adsorbed onto the aluminum phosphate adjuvant in step (a).

20. The method of claim 18 or 19, further comprising the step of withdrawing a 0.5ml sample of the mixture and injecting into the container after step (b).

21. The method of claim 20, wherein the container is a glass syringe.

(i) an antigen; (ii) an aluminum phosphate adjuvant; and (iii) the use of a 3-O-deacylated monophosphoryl lipid a adjuvant in the preparation of a vaccine for administration to a patient, wherein at least 50% of the 3-O-deacylated monophosphoryl lipid a is adsorbed onto the aluminum phosphate adjuvant.

23. The use of claim 22, wherein the vaccine is for intramuscular injection.

24. The use of claim 22 or 23, wherein the antigen is HBsAg.

25. The use of claim 24, wherein the composition is administered using an immunization regimen with administration at months 0, 1, 2, and 6, wherein 0 is the time of first administration.

26. The use of claim 24 or 25, wherein the patient is a hemodialysis adult.