Liposomal vaccine compositions comprising a polysaccharide antigen and a protein adjuvant

This application is a continuation claiming priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 12/088,672, filed Aug. 19, 2008, a U.S. national phase filing of PCT application PCT/EP2006/066935 having an international filing date of Sep. 29, 2006, which claims benefit of priority to European Application No. 05256160.2, filed Sep. 30, 2005. The contents of the above patent applications are incorporated by reference herein in their entirety.

The present invention relates to liposomal compositions, in particular compositions useful for eliciting an immune response against polysaccharide antigens, in particular derived from pathogenic microbes, such as pneumococcal and Hib polysaccharide antigens.

Bacterial infections caused by encapsulated bacteria are a major world health problem. The species Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis are difficult to vaccinate against due to the thymus independent nature of the major surface antigens, the capsular polysaccharides.

T-cell independent antigens present particular problems regarding the development of effective vaccines. Antibody production is low and is not normally boosted by re-immunisation. The antibody isotypes are restricted to IgM and other isotypes are generally of low affinity for a specific antigen.

A major problem lies in the response of young children to T-cell independent vaccines. These individuals are amongst the most vulnerable such bacterial infections. This age group responds most poorly to T-cell independent antigens.

Various methods have been attempted to adjuvantate polysaccharide antigens. For instance polysaccharides have been conjugated to carrier proteins such as tetanus toxoid, which results in some improvement in immunogenic effect. The polysaccharides have alternatively been formulated with liposomes, which also gives an enhanced immune response.

Burgeot, C. et al in Vaccine 2001, 19, 2092-2099, disclose immunopotentiation of polysaccharide vaccine from Staphylococcus aureus co-entrapped in liposomes with alpha toxin. The liposomes were formed of liposome forming compounds containing egg phosphatidylcholine, stearylamine and cholesterol in molar ratios of 7:2:1. The liposomes were formed using the dehydration rehydration method, with dehydration of a mixture of empty SUV and antigens suspended in a 10 mM Hepes (pH 7.4), 150 mM sodium chloride buffer. The ratio of polysaccharides to toxin protein ranged from 20 to 5. Alpha toxin is a highly toxic substance and is haemolytic. The authors reason that its activity in potentiating the antigenicity of the polysaccharide antigen is dependent upon the properties it exhibits as a toxin involving puncturing of the cell membrane. They show that the potentiation is not achieved with heat inactivated toxin derivative. Alpha toxin has a subunit molecular weight of about 33 kDa.

Pietrobon, P. J. F. et al in Immunomethods 4, 236-243 (1994) describe co-entrapment into liposomes of the T independent antigen LPS (which is not water-soluble) and a polypeptide with T-cell recognition sites, haemagglutinin (HA). The liposome sizes are not disclosed. The weight ratio of LPS to HA is in the range 2:1 to 1:10. The liposomes are made by forming a lipid film containing LPS and HA and hydrating this in aqueous suspending liquid containing n-octylglucopyranoside. HA is not inert, but binds to various cell surface receptors via sialic acid bearing glycons and stimulates an innate immune response. Compounds which stimulate an innate immune response may generate undesirable inflammation.

According to the invention there is provided a new liposomal composition comprising coentrapped within liposomes a polysaccharide antigen and a protein carrier; wherein the polysaccharide antigen is water-soluble and the protein carrier has at least one T-cell epitope, a molecular weight of at least 35 kDa, and is non-toxic; wherein the polysaccharide antigen and protein carrier are non-conjugated; and the liposomes have an average diameter in the range 50-700 nm and are formed from liposome forming compounds having, in combination, no overall ionic charge and comprising at least one phospholipid.

In the invention, the term “coentrapped” means that the two actives, namely the polysaccharide antigen and the protein carrier, must be associated with the same liposome. Although association may be solely with the outer surface of liposomes, it is preferred that the actives are at least in part entrapped in the intravesicular space of the liposomes. Both actives should be water-soluble and thus located in the aqueous phase of the liposomal formulations. Where the liposomes are multi-lamellar the actives may be between lamellae.

In the invention it is important that the polysaccharide antigen and the protein carrier are not covalently conjugated to one another. This has the great advantage of allowing coformulation of many ingredients especially different antigens to form a multivalent antigen composition and avoiding the chemical conjugation step that would be necessary for such conjugation.

The protein carrier is non-toxic to a mammalian body and should not stimulate an innate immune response when administered thereto. Thus, the carrier is effectively ‘inert’ in the mammalian body. This is in contrast to protein adjuvants which are not biologically inert but engage (either directly themselves, or via components that they release from killed host cells) host receptors to elicit cellular responses from the cells of the immune system in the form of an innate immune response.

The carrier protein has a molecular weight high enough for it to act as a T-cell dependent antigen. The molecular weight is at least 35 kDa, for instance up to 1000 kDa, eg in the range 75-400 kDa. The present invention can use proteins which have been used as carriers for T-cell independent antigens in the prior art, such as ovalbumin, tetanus toxoid and diphtheria toxoid. The protein should generally be non-haemolytic, and non-toxic. In terms of non-toxicity, for instance, the protein should have an LD50 in excess of 4 mg/kg (in mice, iv or sc). Preferably the protein should be level 6 on the Hodge & Sterner scale, ie be Relatively Harmless.

Suitable proteins include ovalbumin, tetanus toxoid, diphtheria toxoid and diphtheria CRM197 (a genetic mutant of diphtheria toxin). Tetanus toxoid has a molecular weight of about 100 kDa. Tetanus toxoid and diphtheria CRM197 (a genetic mutant of the toxin) fail to elicit the significant immune response elicited by the corresponding toxins. This is thought to be due to attenuation by formaldehyde or genetic mutation. These carriers do, however, elicit an adaptive immune response by activation and the subsequent proliferation of T-helper cells that are needed for antibody responses.

Preferably, the liposomal composition is in unit dosage form and comprises 1-30 μg of the protein carrier, more preferably 10-30 μg. Such dosages are believed to be suitable for administration to a subject in need of immunisation, such as a human.

In the invention, the polysaccharide antigen is preferably derived from an infectious agent, preferably a pathogenic bacterium, for instance selected from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, E. coli , or group B Streptococcus . Most preferably the antigen is derived from Haemophilus influenzae or Streptococcus pneumoniae . The polysaccharide antigen is preferably a T-cell independent antigen. In the composition of the invention, the weight ratio of polysaccharide antigen to protein carrier is preferably in the range 6:1 to 1:6, more preferably 4:1 to 1:3. Preferably, there is an excess of polysaccharide to protein in the composition.

In the invention, there may be single polysaccharide antigen but preferably the composition is multivalent, that is it comprises several polysaccharide antigens in admixture. The composition may comprise two or more, three or more, five or more, seven or more, for instance up to 40, preferably in the range 7 to 23 polysaccharide antigens. Preferably, the antigens are derived from the same bacterial species. The antigens in such a multivalent composition may be co-encapsulated in liposomes, or there may be several populations, the liposomes of which each contain a separate antigen or antigen mixture distinct from the other populations. Preferably the antigens are separately entrapped in liposomes, that there is a district popoulation for each polysaccharide antigen. This allows optimum flexibility in terms of generating a variety of different thereof vaccines from common starting-materials.

The liposomes comprise compounds having no overall ionic charge. The compounds are preferably neutral, including zwitterionic compounds with one anionic and one cationic charge, but may contain small quantities of anionic or cationic compounds provided these are charge-balanced with oppositely charged compounds. Preferably the compounds comprise phosphatidylcholine compounds and/or phosphatidylethanolamine compounds. The liposome-forming compounds are usually amphiphilic, i.e. consist of a hydrophobic component and a hydrophilic component. The hydrophobic components are generally provided by acyl chains but the liposome-forming compounds may alternatively be glycerol-ether based lipid compounds. Non lipidic compounds, that is not based on glycerol compounds, may be included if desired, for instance non-ionic surfactant type materials. Preferably the compositions comprise cholesterol, for instance in a molar amount of at least 10%, preferably at least 25%, based on total liposome forming components.

The composition of the invention may be in the form of an aqueous suspension, that is in which the liposomes are suspended in a continuous aqueous medium. Alternatively the composition may be a precursor of such an aqueous composition, which may be diluted with water or an aqueous liquid to form the aqueous suspension. Such precursors may be in the form of dried materials, especially in powder form, for instance provided by spray drying or freeze drying (lyophilisation).

The compositions of the invention may further comprise (a) diluent(s) and/or excipient(s). Since the compositions are intended to be used directly or after intermediate formulation steps, e.g. diluation, to be administered to mammalian subjects, the diluents or the excipients are preferably pharmaceutically acceptable. Suitable excipients are known.

It is particularly preferred that the composition comprises sugars. Sugars may assist stabilisation of the liposomes during formation of the liposomes and/or in storage. Preferably the liposomes are formed by the dehydration-rehydration method, empty small unilamellar vesicles (SUV's) (e.g. made by hydration of dry lipid film to form multi-lamellar vesicles (MLV) and sonication to SUV) and active (polysaccharide antigen and protein carrier) are suspended in an aqueous liquid prior to drying preferably by lyophilisation. The dried product is then rehydrated and optionally subjected to steps to remove non-entrapped material, over-sized liposomes or to reduce the average liposome size, by methods known to the person skilled in the art.

For improved control of rehydrated vesicle size, the method used includes sugar in the suspending liquid of the dehydration step, as discussed in WO99/65465. The sugar may be selected from monosaccharides such as glucose and fructose, disaccharides such as lactose and sucrose as well as polysaccharides. A particularly preferred sugar is a di-saccharide such as trehalose, sucrose or lactose or a mono-saccharide such as glucose. In particular the preferred sugar is sucrose.

In such methods, the amount of sugar is such that mass ratio of sugar to liposome forming compound is in the range of from 1:1 to 6:1 w/w, most preferably from 1:1 to 5:1. With higher levels of sugar, the encapsulation efficiency of the method is reduced. With lower amounts of sugar, however, the control over the size of the final liposomes is lost.

The invention further provides a process for process for producing the new composition in which the polysaccharide antigen and protein carrier are co-encapsulated in liposomes.

Preferably the methods described above are used for the encapsulation. Sugar need not be used but preferably is used in these dehydration-rehydration methods. The empty liposomes are preferably small or medium sized and may be multilamellar or unilamellar.

The product liposomes have average diameter in the range 50 to 700 nm, preferably in the range 80 to 500 nm, more preferably in the range 80 to 300 nm. Preferably the composition contains very low levels of liposomes with diameters greater than 1500 nm, even more preferably very low levels of liposomes with diameters greater than 1000 nm. The level of very small liposomes, e.g. less than 20 nm should also be kept as low as possible. The sizes are measured by photon correlation spectroscopy.

The invention also provides the use of the liposomes and compositions in the manufacture of a medicament for administration to a mammal to elicit an immune response to the polysaccharide antigen.

Preferably the immune response involves production of IgG at least, to the polysaccharide antigen, and additionally preferably IgM and IgA, preferably so that a protective effect to a challenge from the infectious micro-organism is achieved in the recipient.

Preferably the composition is administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intranasally, by inhalation, intravaginally, buccally or orally. Most preferably the composition is administered subcutaneously.

Generally the administration is for protective purposes to provide a response against infection by an infectious microbe, especially an infectious bacterium, of the type mentioned above. The subject to which the composition is administered may be a mammal of any age, in need of having protective immunity. The invention is of most value for treating humans. For instance administration may be to provide resistance to seasonal outbreaks, specific outbreaks of the infections, or may be part of health programmes especially for infants. The invention is of particular value for the treatment of human infants having reduced immune responses to T-cell independent antigens, for instance polysaccharide antigens, for instance being less than two years of age.

The invention further provides methods of administering the compositions.

Further preferred embodiments of the invention are mentioned in the claims.

The following examples illustrates the invention and refer to the following figures, in which

FIG. 1 shows the Anti-PS (PRP) antibody response of mice when administered formulations ID1, ID2 & 3 and ACT-HIB® without cholesterol (Example 1);

FIG. 2 shows the Anti-PS (PRP) antibody response of mice when administered formulations ID4, ID1, and ACT-HIB® with cholesterol (Example 1); and

FIG. 3 shows the IgG antibody response pre and post immunisation to seven serotypes tested (Example 2).