Title:  Vaccine adjuvants

United States Patent:  6,328,965

Assignee:  American Cyanamid Company (Madison, NJ)

Filed:  September 15, 1997

PCT NO:  PCT/BE95/00119

102(e) Date:  September 15, 1997

PCT PUB. Date:  July 4, 1996

Foreign Application Priority Data:  Dec 27, 1994[BE] (9401174)

Vaccine adjuvants comprising a sulpholipid polysaccharide in combination with an interface-forming constituent. The invention also provides a method for preparing a vaccine by emulsifying an aqueous solution of an antigen and a sulpholipid polysaccharide. The adjuvants are stable at high temperatures, and are at least as effective as conventional adjuvants. Their local toxicity, i.e. their reactogenicity, is generally lower than that of conventional adjuvants.

Description of the Invention

The present invention relates to novel vaccine adjuvants.

An antigen is defined as a foreign substance for a given organism, which when administered, for example, parenterally, induces an immune response, for example, the formation of antibodies.

Antibodies are substances contained in the blood and other fluids of the body, as well as in the tissues, and which bind to the antigen to make it innocuous. Antibodies constitute one of the natural defense mechanisms of the body. They are highly specific and they can kill, bind or make innocuous the antigen which has induced their formation.

The antigen, in contact with the immune system, thus activates a complex series of cellular interactions to eliminate the antigen and/or to reestablish the preceding equilibrium.

Two of the characteristic features of antigens are their immunogenicity, that is their capacity to induce an immune response in vivo (including the formation of specific antibodies), and their antigenicity, that is their capacity to be selectively recognized by the antibodies whose origins are the antigens.

It is known how to stimulate the immune response deliberately by administrating a specific antigen by means of a vaccine. The procedure allows the obtention of a state of immune response in the organism which allows a more rapid and more effective response of the organism during subsequent contact with the antigen.

However, some antigens have only a weak immunogenicity and they induce an insufficient immune response to procure an effective protection for the organism.

The immunogenicity of an antigen can be increased by administering it in a mixture with substances, called adjuvants, which increase the response against the antigen either by directly acting on the immunological system or by modifying the pharmacokinetic characteristics of the antigen and by thus increasing the interaction time between the latter with the immune system.

At this time, a great number of veterinary vaccines use adjuvants still comprising the standard emulsions of mineral oil, such as the adjuvant of the water in mineral oil type (W/O) or of the mineral oil in water type (O/W). For several years, research studies have been carried out to find alternatives having a similar efficacy at a reduced toxicity. The injections of these standard adjuvants based on mineral oil are often accompanied by local reactions whose severity depends to a large extent on the type of the emulsion and the nature of oil used. The use of adjuvants based on mineral oil is consequently limited to domestic animals (pigs, hens, ruminants, etc.) and laboratory animals.

Earlier, it has been shown that a synthetic copolymer of polysucrose and epichloridrin [sic; epichlorohydrin]--Ficoll--bearing sulfate and lipid groups (SL-Ficoll), incorporated in an emulsion of squalane (S) in water (S/W), had high adjuvant effect on different animal species--including pigs--against different types of antigen, including a few important viral antigens (Vaccine, Vol. 12, pp. 653-660 (1994) and Vaccine, Vol. 12, pp. 661-665 (1994), EPO 0,549,074). These formulations of Ficoll-based adjuvants are sufficiently effective to replace the standard formulations of mineral oil in water used in different porcine vaccines.

However, the local toxicity, that is the reactogenicity of these Ficoll-based formulations in pigs and mice did not turn out to be weaker than that of the standard formulations of mineral oil of the O/W type.

In addition, for the Ficoll-based formulations, the temperature has a pronounced effect on the stability of the emulsions. Some of these emulsions were stable for years at 4^oC., but the aqueous phase and the oil phase separate within a few days at 37^oC., and within approximately 10 min at 60^oC.

The purpose of the present invention is to propose an effective adjuvant for vaccines having an increased stability at high temperature and presenting a lower local toxicity.

This purpose is achieved by an adjuvant for vaccines comprising a sulfolipid polysaccharide combined with an interface-forming constituent (for example, an emulsion of the oil/water type (O/W)).

One of the advantages of the adjuvant according to the present invention is that it is more stable at high temperature than the Ficoll-based adjuvants.

This purpose is achieved by an adjuvant for vaccines comprising a sulfolipid polysaccharide combined with an interface-forming constituent.

The expression "polysaccharide" denotes a compound having at least three repeating sugar units connected covalently to each other.

The expression "sulfolipid polysaccharide" denotes a compound having at least three repeating sugar units connected covalently to each other, at least one sulfate group and at least one lipid group.

Preferably, the sulfolipid polysaccharide is a hydrophobic polysaccharide.

The expression "hydrophobic polysaccharide" denotes a polysaccharide which is less soluble in an aqueous phase than an a polar organic phase.

According to a first advantageous embodiment, the sulfolipid polysaccharide is selected from the group consisting of cyclodextrin, maltodextrin, insulin, Ficoll and Pullulan.

Preferably, the sulfolipid polysaccharide is selected from the group consisting of cyclodextrin, maltodextrin and insulin.

The preferred sulfolipid polysaccharide is cyclodextrin.

The sulfolipid polysaccharide contains, on average, at least 0.01 sulfate group per monosaccharide, while maintaining its hydrophobic character. Preferably, the sulfolipid polysaccharide contains, on average, at least 0.12 sulfate group per monosaccharide, while maintaining its hydrophobic character.

The sulfolipid polysaccharide on average contains not more than 1.0 sulfate group per monosaccharide, while maintaining its hydrophobic character. Preferably, the sulfolipid polysaccharide contains, on average, not more than 0.23 sulfate group per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is maltodextrin, it contains, on average, approximately 0.23 sulfate group per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is cyclodextrin, it contains, on average, approximately 0.20 sulfate group per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is insulin, it contains, on average, approximately 0.19 sulfate group per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is pullulan, it contains, on average, approximately 0.16 sulfate group per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is Ficoll, it contains, on average, approximately 0.12 sulfate group per monosaccharide, while maintaining its hydrophobic character.

On average, the sulfolipid polysaccharide contains at least 0.01 lipid group per monosaccharide, while maintaining its hydrophobic character. Preferably, the sulfolipid polysaccharide contains, on average, at least 1.05 lipid group per monosaccharide, while maintaining its hydrophobic character.

On average, the sulfolipid polysaccharide contains not more than 2.0 lipid groups per monosaccharide, while maintaining its hydrophobic character. Preferably, the sulfolipid polysaccharide contains, on average, not more than 1.29 lipid groups per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is maltodextrin, it contains, on average, approximately 1.29 lipid groups per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is cyclodextrin, it contains, on average, approximately 1.05 lipid groups per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is insulin, it contains, on average, approximately 1.24 lipid groups per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is pullulan, it contains, on average, approximately 1.24 lipid groups per monosaccharide, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is Ficoll, it contains, on average, approximately 1.22 lipid groups per monosaccharide, while maintaining its hydrophobic character.

The lipid groups contain, preferably, 4-22 carbon atoms.

The ratio of the sulfate groups to the lipid groups is advantageously 0.01-2 sulfate groups per lipid groups. Preferably, the ratio of the sulfate groups to the lipid groups is 0.10-0.19 sulfate group per lipid group, while maintaining the hydrophobic character of the compound.

Preferably, when the sulfolipid polysaccharide is maltodextrin, the ratio of the sulfate groups to the lipid groups is approximately 0.18 sulfate group per lipid group, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is cyclodextrin, the ratio of the sulfate groups to the lipid groups is approximately 0.19 sulfate group per lipid group, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is insulin, the ratio of the sulfate groups to the lipid groups is approximately 0.15 sulfate group per lipid group, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is pullulan, the ratio of the sulfate groups to the lipid groups is approximately 0.13 sulfate group per lipid group, while maintaining its hydrophobic character.

Preferably, when the sulfolipid polysaccharide is Ficoll, the ratio of the sulfate groups to the lipid groups is approximately 0.10 sulfate group per lipid group, while maintaining its hydrophobic character.

The expression "interface-forming constituent" (or "IFC") denotes a substance which forms, in an aqueous medium, a physical interface between the substance and the aqueous phase.

The interface-forming constituent is selected from the group consisting of a water-immiscible liquid (for example: squalane, soybean oil, mineral oil, hexadecane) or a solid which is insoluble in the aqueous phase.

The insoluble solids in the aqueous phase of the present invention comprise insoluble salts (for example, Al(OH)₃, AlPO₄, alum, calcium oxalate), microparticles, nanoparticles, microspheres and nanospheres of one or more polymers, copolymers (for example, polyacrylate, poly(methyl methacrylate), polycyanoacrylate, polylactide, polyglycolide), or lipid bi-layers or lipophilic agents (for example, phospholipids) or micelles of surfactants.

Preferably, the "IFC" component is a water-immiscible liquid.

Advantageously, the interface-forming constituent is selected from the group consisting of soybean oil, squalane and hexadecane.

The stable adjuvants for vaccines are those which comprise a (hydrophobic) derivative of a sulfolipid polysaccharide, an interface-forming constituent and an emulsifier.

In addition, the efficacy of the adjuvants according to the present invention is comparable or even greater than that of the standard adjuvants. The local toxicity, that is the reactogenicity of the adjuvants according to the present invention is, in general, less than that of the standard adjuvants and less than that of the adjuvants based on Ficoll.

It would seem that the sulfolipid polysaccharides with a relatively lower molecular weight have a reduced local toxicity.

According to another aspect of the present invention, it is proposed to use sulfolipid polysaccharide as an adjuvant in vaccines.

According to yet another aspect of the present invention, a method is proposed to prepare a vaccine in an emulsion, which is characterized in that an aqueous solution of an antigen is emulsified in the presence of a sulfolipid polysaccharide, an emulsifier and an interface-forming constituent.

According to another aspect of the present invention, a vaccine is proposed which comprises an immunogenic quantity of an antigen (immunogen) and an adjuvant according to the present invention.

Preferably, the concentration of adjuvants is 0.1-100 mg/mL, preferably 2-20 mg/mL.

The vaccine comprises, besides the adjuvant, antigens, for example, of inactivated virus, live virus, bacteria, a subunit, protein, peptide and inactivated influenza virus of strain MRC-11, ovalbumin (OVA), inactivated influenza virus of strain A/Swine and/or inactivated pseudorabies virus (iPRV).

The measured antibody titers were higher than those obtained by the emulsions of mineral oil in water used in commercial products. It has been shown that there is a strong synergistic activity between the SL-Ficoll and the emulsions, which is more pronounced in pigs than in mice (Hilgers et al., Vaccine, Vol. 12, pp. 661-664, 1994). The antibody response in mice was increased significantly (Hilgers et al., Vaccine, Vol. 12, pp. 653-660, 1994).

The conclusion is drawn that the reactogenicity depends on the type of polysaccharide and oil included in the formulations, and that the molecular weight of the polysaccharide is one of the important factors.

The cyclodextrins and their derivatives are well known for their capacity to form inclusion complexes that enclose other substances, such as, for example, substances of pharmaceutical interest, in the hollow formed by their cyclic structure.

The advantages offered by these inclusion complexes include improvement of the solubility, of the bioavailability and/or the chemical stability, increase in length of the half-life, decrease of the secondary effects as well as certain advantages during production, such as the easier obtention of a dry powder to be used as a starting point for liquid preparations.

The sulfolipid polysaccharides (SLP) based on cyclodextrin can thus have interesting applications in addition to their use as adjuvant, which is described above.

Indeed, the SL-cyclodextrins are surfactants because of the simultaneous presence in these molecules of anionic groups (sulfates) and hydrophobic groups (aliphatic chains). This property is at the origin of the formation of micelles in an aqueous phase or mixed micelles in the presence of other surfactants, as well as the formation of emulsions of water-immiscible liquids, suspensions in an aqueous phase of insoluble particles or the formation of interfaces between an aqueous phase and a liquid or solid insoluble substance.

Thus, the SL-cyclodextrins present the double advantage of being able to form inclusion complexes and of being surfactants.

They can thus be considered to form a family of products presenting novel properties which lend themselves to original applications in the pharmaceutical field.

Numerous different SL-cyclodextrins have been obtained; they differ in their physicochemical properties resulting from the type of cyclodextrin (for example, .alpha., .beta., .gamma.), the content of sulfate groups as well as the content and the nature of the lipid groups.

Claim 1 of 33 Claims

What is claimed is:

1. Adjuvant for vaccines comprising a sulfolipid polysaccharide combined with an interface-forming constituent characterized in that the sulfolipid polysaccharide is a hydrophobic polysaccharide which is selected from the group consisting of cyclodextrin, maltodextrin, insulin, and pullulan.

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