WO2005102394A1 - Uric acid as adjuvant - Google Patents

Abstract

The present invention provides an adjuvant for polynucleotide vaccines, and in particular the present invention provides polynucleotide vaccines that comprise, or are administered in association with, a composition that is a breakdown product of a purine, which in particular, may be uric acid. The polynucleotide vaccines of the present invention are vaccines that encode an antigen against which it is desired to generate an immune response, and in particular the polynucleotide vaccine may be a DNA vaccine. Also provided by the present invention is the use of uric acid in the manufacture of a polynucleotide vaccine composition for the purpose of enhancing the immune response against the specific antigen that is encoded by the polynucleotide vaccine. Vaccine compositions, kits comprising separate polynucleotide composition and adjuvant compositions for separate administration, methods of manufacture of the vaccines and kits, and methods of treatment of individuals with the vaccine compositions of the present invention, are provided.

Description

URIC ACID AS ADJUVANT

The present invention provides a novel adjuvant for polynucleotide vaccines, and in particular the present invention provides polynucleotide vaccines that comprise, or are administered in association with, a composition that is a breakdown product of a purine, which in particular, may be uric acid. The polynucleotide vaccines of the present invention are vaccines that encode an antigen against which it is desired to generate an immune response, and in particular the polynucleotide vaccine may be a DNA vaccine. Also provided by the present invention is the use of uric acid in the manufacture of a polynucleotide vaccine composition for the purpose of enhancing the immune response against the specific antigen that is encoded by the polynucleotide vaccine. Vaccine compositions, kits comprising separate polynucleotide composition and adjuvant compositions for separate administration, methods of manufacture of the vaccines and kits, and methods of treatment of individuals with the vaccine compositions of the present invention, are provided.

Background of the Invention Uric acid, or 2,6,8-trihydroxypurine, in its purified form exists as a microcrystalline powder, is colourless, odourless, tasteless, almost insoluble in water, and decomposes above 250 °C (Sharp, D.W.A. (ed.) (2003) The Penguin Dictionary of Chemistry, 3rd edition. Penguin Books Ltd, London). Uric acid has the following molecular structure:

In humans, uric acid is formed as the end product of purine catabolism (Hitchings, G.H. (1978) Uric acid: Chemistry and synthesis. In: Uric Acid (Kelley,

W.N. & Weiner, I.M. (eds.)) Springer-Nerlag Berlin Heidelberg, New York. pp. 1-20), and commonly forms urate salts in the blood. In humans and primates it is present in small quantities in the urine; in most other animals and fish it is oxidised and catabolised further to yield urea or ammonia, which are excreted. Humans and primates accumulate approximately 10-fold higher levels of uric acid than most other mammals due to urate re-absorption in the kidneys and an evolutionary loss of urate oxidase (which metabolises uric acid to allantoin; Mousavizadeh, K. et al. (2003) Trends in Pharmacological Sciences 24: 563-4; Jeha, S. (2001) Seminars in Hematology 38(suppl. 10): 4-8). In contrast, in uricotelic animals (including birds, insects, lizards, snakes and some desert mammals) it is the major nitrogenous excretion product and occurs in the faeces in the form of a urate salt. The insolubility of uric acid in water allows metabolic nitrogen to be excreted in this solid form while water is conserved (Hitchings, G.H. (1978) Uric acid: Chemistry and synthesis. In: Uric Acid (Kelley, W.N. & Weiner, I.M. (eds.)) Springer-Nerlag Berlin Heidelberg, New York. pp. 1-20). Uric acid is the end product of the catabolism of adenosine and guanosine in humans. Uric acid is produced from xanthine (by the enzyme xanthine oxidase), which is produced from either guanine (the first breakdown product of guanosine) or hypoxanthine (which is the breakdown product of inosine, the first breakdown product of adenosine (Mathews, C.K., van Holde, K.E., & Ahern, K.G. (2000). Biochemistry, 3rd edition. Benjamin/Cummings, San Francisco, CA. p. 803). Uric acid has been shown to be a strong scavenger of carbon-centred and peroxyl radicals (Muraoka, S. & Miura, T. (2003) Pharmacology & Toxicology 93: 284- 9), as well as reactive nitrogen oxide species such as nitric oxide (NO) and peroxynitrite (ONOO^") (Ghafourifar, P. et al. (1999) Journal of Biological Chemistry 274: 31185-8). Along with vitamin C (ascorbic acid) and vitamin E (α-tocopherol), it is regarded as an important antioxidant in the blood; with the observation that serum uric acid concentration in humans is approximately six times that of vitamin C, it could also be said to be the most prominent (Hediger, M.A. (2002) Nature 417: 393-395). The ability of uric acid to scavenge free radicals suggests that it may help to protect against oxidative damage. Several studies have illustrated the ability of uric acid to protect protein, DNA and lipids from oxidative attack (Anderson, R.F. & Harris, T.A. (2003) Free Radical Research 37: 1131-6; Muraoka, S. & Miura, T. (2003) Pharmacology & Toxicology 93: 284-9). Administration of uric acid is also thought to prevent the formation of tolerance to nitroglycerin (glyceryl trinitrite), which is used in the therapy of cardiovascular diseases such as angina, congestive heart failure and hypertension. Tolerance is mediated by the oxidative actions of superoxide, peroxynitrite and protein kinase C; uric acid is postulated to protect against this (Abou-Mohamed, G. et al. (2004) Journal of Pharmacology and Experimental Therapeutics 308: 289-99). Nitric oxide (NO) has been shown to modulate the production of uric acid in humans via its influence on xanthine oxidase activity, and a cyclic and repeating relationship between NO and uric acid levels has been suggested (Lee, Y. J. et al. (2003) Metabolism-Clinical and Experimental 52: 1448-53). An elevated level of uric acid, or serum uric acid, has been linked with several disease conditions and metabolic disorders, either pathogenically or as a prognostic indicator. Examples include hyperuricemia (an abnormally high accumulation of uric acid in the blood; Martin, E.A. (ed.) (1998) The Oxford Concise Colour Medical

Dictionary, 2nd edition. Oxford University Press, Oxford, UK), gout (Terkeltaub, R.A.

(2003) New England Journal of Medicine 349: 1647-55), formation of uric acid stones in the kidney (Abate, N. et al. (2004) Kidney International 65: 386-92), the metabolic syndrome (Denzer, C. et al. (2003) Journal ofPediatric Endocrinology & Metabolism 16: 1225-32), tumour lysis syndrome (Jeha, S. (2001) Seminars in Hematology 38(suppl. 10): 4-8; Cairo, M.S. et al. (2002) Blood 100: 4670), stroke and ischemia (Chamorro, A. & Planas, A.M.

(2004) Stroke 35: E11-E12; Weir, C.J. et al. (2004) Stroke 35: E12; Kanellis, J. & Johnson, R.J. (2003) Stroke 34: 1956-7), cognitive disorder (Rinaldi, P. et al. (2003) Neurobiology of Aging 24: 915-9; Anderson, R.F. & Harris, T.A. (2003) Eree Radical Research 37: 1131-6), hypertension and obesity (Feig, D.I. & Johnson, R.J. (2003) Hypertension 42: 247-52; Masuo, K. et al. (2002) Hypertension 40: 36; Masuo, K. et al. (2003) Hypertension 42: 474-80), hypeiprolactinemia (Yavuz, D. et al. (2003) European Journal of Endocrinology 149: 187-93), osteoarthritis (Fickert, S. et al. (2003) Aktuelle Rheumatologie 28: 302-7), eclampsia (Ben Salem, F. et al. (2003) Annales Francoises D Anesthesie Et De Reanimation 22: 865-9), and a range of glomerulopathies (Deepa, P.R. & Varalakshmi, P. (2003) European Journal of Pharmacology 478: 199-205). Conversely, lowered serum uric acid level can be pathogenic (and prognostic), as in multiple sclerosis (Hooper, D.C. et al. (1998) PNAS USA 95: 675-80) and the precursor condition optic neuritis (Spitsin, S. et al. (2001) Multiple Sclerosis 7: 313-9). Therapeutic administration of uric acid in humans may boost anitoxidant capacity and aid in control of multiple sclerosis (Mousavizadeh, K. et al. (2003) Trends in Pharmacological Sciences 24: 563-4). Gout is characterised by the accumulation of monosodium urate crystals in the peripheral joints, leading to neutrophil activation and subsequent inflammation of the joint. Monosodium urate crystals are thought to activate neutrophils via a receptor- mediated process, involving in particular the Fc-receptor CD 16 and tyrosine kinase- dependent signal transduction (Desaulniers, P. et al. (2001) Journal of Leukocyte Biology 70: 659-68). There is evidence that inosine can modulate immune reactions, whilst a number of authors claim that it is anti-inflammatory [for example Marton, A. et al.,(2001) Int. J. Mol. Med. 8:617-21); Liaudet, L. et al. (2002) Annals of Surgery 235:568-78)], one recent paper [Idzko, M et al. (2004) J. Cellular Physiol. 199:149-56] claims inosine could stimulate chemotaxis, calcium signalling and actin polymerisation in dendritic cells. Vaccines have for many years included substances that have a direct or indirect stimulatory effect on the immune system, termed "adjuvants", such that the magnitude or quality of the immune response is altered or augmented. General information about the use of adjuvants is provided in Powell, M.F. & Newman, M.J. (eds.) (1995) Vaccine Design - The Subunit and Adjuvant Approach. Plenum Press, New York and London. Shi et al. (2003, Nature 425: 516-21) recently showed that uric acid can act as an adjuvant for a protein vaccine antigen. Uric acid purified from the cytosol of ultraviolet light-damaged BALB/c 3T3 cells could boost cytotoxic T-lymphocyte (CTL) killing responses in splenocytes from mice primed with particulate HIV gpl20 antigen. Commercially obtained pure uric acid had a similar effect, and was also able to boost killing in CTLs from mice primed against particulate ovalbumin. Uric acid was shown by Shi et al. to trigger increased expression of costimulatory molecules in dendritic cells, including CD86 and CD80 (also known as B7.1 and B7.2, the ligands for essential CD28 and CTLA-4 receptor-mediated activatory co-stimulation of T-cells). The concentrations at which dendritic cells were stimulated corresponded to the point at which uric acid would reach saturation, and crystallisation to form monosodium urate crystals would occur. Indeed, preformed monosodium urate crystals were shown to be highly stimulatory. The International Patent Application, publication number WO2004/100984 recently published covering the use of "Endogenous Adjuvant Molecules", for example monosodium urate crystals, uric acid and xanthine, as adjuvants for vaccines. This application published after the first filing of the present application. The adjuvants described therein may be used with any antigen, including proteins, peptides, lipids, carbohydrates and polynucleotides. Adjuvants for protein or polysaccharide vaccines, or in general those vaccines which comprise the antigen itself, have been known and developed since the 1920s. In contrast, polynucleotide vaccines where the vaccine comprises a polynucleotide that encodes the antigen and facilitates antigen production in the host cells of the vaccinee, are themselves a relatively recent development. Necessarily therefore, less is known about polynucleotide vaccine adjuvants. The adjuvant strategy for polynucleotide vaccines often involves the co-expression of immune modifiers, such as cytokines, together with the antigen. Recently described polynucleotide vaccine adjuvants include small molecules such as tucerasol (WO 00/12121), imidazoquinoline amines (WO 02/24225, WO 03/077944) and inducible nitric oxide synthase (iNOS) inhibitors (WO 03/030935). Summary of the Invention The present invention provides novel immunogenic compositions or vaccines comprising (a) a polynucleotide component that encodes an antigen against which it is desired to generate an immune response, and (b) an adjuvant component comprising an immune stimulatory quantity of an intermediate or final product of purine catabolism, or a derivative thereof. In the context of the present invention the polynucleotide component encoding the immunogenic compositions or vaccine antigen is any polynucleotide or vector that is capable of directing expression of the said antigen in the cells of the host vaccinee. The vector may be a live or attenuated viral or bacterial vector which delivers the foreign sequence that encodes the vaccine antigen. In one aspect of the present invention the immunogenic compositions or vaccines comprise a polynucleotide vector which is a DNA plasmid vector. In this aspect of the present invention the plasmid vector may be delivered to the vaccinee in liquid form, or in the form of dense micro-beads suitable for ballistic delivery into the skin, or formulated on the surface of dense micro-beads suitable for ballistic delivery into the skin, or coated onto microneedles. In one embodiment of the present invention the intermediate or final product of purine catabolism that forms the adjuvant composition is uric acid. In other embodiments of the present invention the intermediate or final products of purine catabolism may be selected from inosine, hypoxanthine or xanthine, or salts, solvates or physiologically active derivatives thereof. In another embodiment of the present invention, the adjuvant component comprises a combination of two or more intermediates or final products of purine catabolism. Accordingly, when two components are present, the adjuvant compositions may comprise a combination of uric acid and inosine, uric acid and hypoxanthine, or uric acid and xanthine. In one embodiment of the present invention the adjuvant is a salt of uric acid, such as the monosodium salt. The physical presentation of the uric acid, or other product of purine catabolism, in the polynucleotide vaccine of the present invention depends upon the form of the vaccine to be administered. For example, the uric acid, or other product of purine catabolism, may be in solution or in crystalline form. In one important aspect of the present invention the uric acid may be in the form of a crystal or in the form of a crystal formed of one of its salts, for example the adjuvants of the present invention may be a crystal form of the monosodium salt of uric acid. In other embodiments of the present invention the immunogenic compositions or vaccines of the present invention may be in solid form, such that the polynucleotide may be in a "dry" form and co-formulated with the uric acid. For example, the polynucleotide antigen and the uric acid, or other product of purine catabolism, may be in dry solid solution within a solid, or glassy, matrix. In such an embodiment the solid matrix may be a carbohydrate, or sugar, in solid form. In one form of the present invention the polynucleotide and uric acid in its crystalline form, or crystals formed of uric acid salt, are provided on the surface of microbeads suitable for ballistic delivery into the epidermis In a related aspect of the present invention the solid immunogenic composition or vaccine formulation may comprise a protein antigen and uric acid. In this context the solid vaccine may comprise the antigen and uric acid, or salt thereof, in a solid matrix such as a sugar. For example, in a lyophilised vaccine formulation. Further provided in this related aspect of the present invention is a method of stabilising a protein in its dry state, such as in its lyophilised form, by co-formulating said protein with uric acid, and optionally further comprising a stabilising sugar. This stabilised formulation and method has the additional advantage of enhancing the immune responses raised by the antigen. Also forming an aspect of the present invention is an immunogenic composition or vaccine formulation comprising a polynucleotide which encodes an antigen, uric acid or salt thereof (or other breakdown product of a purine) in a dry form wherein the polynucleotide is stabilised. In one embodiment this polynucleotide formulation may be lyophilised, optionally in the presence of a sugar. The uric acid, or other breakdown product of purine, is either in a crystalline form before administration to the patient, otherwise the crystals may be caused to form in the body of the vaccinee after administration of the vaccine. In this later context, the dose of the uric acid, or other product of purine catabolism, in the vaccines of the present invention is sufficient to enhance the immune response against the antigen, and in one embodiment is sufficiently high in concentration that crystallisation of uric acid occurs, to any appreciable extent, after administration. The vaccines of the present invention are particularly adapted, by the formulation with the adjuvants described herein, to the provision of highly potent immune responses, including cell mediated immune responses. In addition, the immunogenic compositions or vaccines of the present invention are also highly stable compositions, in that the stability of the polynucleotides in the vaccine is enhanced by the presence of uric acid, or other product of purine catabolism. An additional advantage of the present invention is the provision of a vaccine/adjuvant composition that does not have the toxicity issues associated with the persistence of potentially toxic adjuvants in the body of the vaccinee.

Description of Drawings FIG. 1, Effect of vaccination with and without uric acid on mean tumour size. FIG 2. Effect of various parameters on tumour regression after vaccination with the vaccines of the present invention. A) uric acid concentration, B) precipitation time, C) time of admimstration of the uric acid after vaccination, D) injection site. FIG 3. Protocol timeline. FIG 4. Day 11 ELISPOT data. FIG 5. Day 14 ELISPOT data. FIG 6. Intracellular cytokine staining data. FIG 7. Tetramer staining data

Detailed Description of the Invention The present invention provides novel polynucleotide immunogenic compositions or vaccines comprising (a) a polynucleotide component that encodes an antigen against which it is desired to generate an immune response, and (b) an adjuvant composition comprising an immune stimulatory quantity of an intermediate or final product of purine catabolism, or a derivative thereof. The compositions of the present invention may be immunogenic compositions in that they are, after administration to a mammal, capable of generating an immune response, such as an antibody response or generation of T-cells that proliferate or secrete cytokines after stimulation with antigen, in that mammal which is specific for the antigen encoded by the polynucleotide component. Alternatively, the compositions of the present invention may be vaccine compositions, in that they are capable, after administration to a mammal, of generating an immime response in said mammal which is sufficient to afford a degree of protection against an infection or disease (prophylaxis), or ameliorate the symptoms of or eradicate an existing infection or disease. Elements of the present text which refer to vaccines or immunogenic compositions may be interchanged accordingly. The polynucleotide elements forming part of the vaccines of the present invention are vectors which, when administered to a vaccinee in an appropriate form, drive expression of an antigen in the cells of the vaccinee, thereby generating an immune response against the antigen. The vectors or polynucleotide elements of the vaccines of the present invention, which encode the antigen against which it is desired to generate an immune response, are operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence. The vectors may be, for example, plasmids, artificial chromosomes, live or attenuated bacterial, viral or phage vectors. Promoters and other expression regulation signals that form part of the polynucleotide vectors may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, and the β-actin promoter. Viral promoters such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, rous sarcoma virus LTR promoter, adenovirus promoter, or a HPN promoter, particularly the HPN upstream regulatory region (URR) may also be used. All these promoters are well described and readily available in the art. Examples of suitable viral vectors include herpes simplex viral vectors, vaccinia or alpha- virus vectors and retroviruses, including lentiviruses, human and simian adenoviruses and adeno-associated viruses. In an important aspect of the present invention the polynucleotide is in the form of a DΝA plasmid vector comprising covalently closed circular DΝA, in a super-coiled or open circular form, comprising an expression cassette having a promoter region and a coding region. The coding region encodes an antigen which, once expressed in the host cells of the vaccinee, generates an immune response. Optionally the coding region, or an additional coding region, may encode for an immunostimulatory cytokine such as IL-2, GM-CSF or lFΝ-γ. In one aspect of the present invention, there is provided a vaccine composition comprising a compound of Formula (I):

(I) or a salt, solvate, or physiologically functional derivative thereof, and a polynucleotide which encodes an antigen against which it is desired to generate an immune response. As used herein, the term "physiologically functional derivative" refers to any pharmaceutically acceptable derivative of an adjuvant of the present invention (formed, for example, by addition of alkyl, alkenyl, alkynyl, aryl or polysaccharide groups to oxidised nitrogen atoms of the purine skeleton of uric acid or intermediate of the purine catabolism pathway), which upon administration to a mammal is itself capable of enhancing the immune response against the antigen encoded by the polynucleotide, or is capable of indirectly doing so through the action of a breakdown product formed from the derivative in situ after administration to the body. Such derivatives are clear to those skilled in the art, without undue experimentation, and with reference to the teaching of Burger's Medicinal Chemistry And Drug Discovery, 5 Edition, Nol 1: Principles and Practice, which is incorporated herein by reference to the extent that it teaches physiologically functional derivatives. As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of formula (I) or a salt or physiologically functional derivative thereof) and a solvent. Such solvents for the purpose of the invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid. In one embodiment the solvate is boric acid. Typically, the salts of the present invention are pharmaceutically acceptable salts. Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention. Salts of the compounds of the present invention may comprise salts derived from a nitrogen on a substituent in the compound of formula (I). Representative salts include the following salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, Ν-methylglucamine, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium and valerate. Suitable salts of uric acid, or derivatives thereof, include most metals including sodium, potassium, lithium, calcium, magnesium, zinc. Ammonium salts are also known and a guanidinium salt. These salts may be solvated with water. Salts of acids with uric acid may be used if basic groups are attached to the uric acid molecule. Uric acid may be manufactured according to a method of preparation given in The Merck Index: H

It is possible for the vaccination methods and compositions according to the present application to be adapted for protection or treatment of mammals against a variety of disease states such as, for example, viral, bacterial or parasitic infections, cancer, allergies and autoimmune disorders.

In one embodiment of the present invention the derivative of uric acid may be described as formula II

wherein, Rl can be hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, aryl; R2 can be hydrogen, alkyl, cycloalkyl, heteroalkyl; R3 can be hydrogen, alkyl, heteroalkyl, cycloalkyl, aralkyl, sugars (ribose etc); R4 can be hydrogen, alkyl, aryl, heteroalkyl, alkenyl. The polynucleotide sequences referred to in this application, which are to be expressed within a mammalian system in order to induce an antigenic response, may encode for an entire protein, or merely a shorter peptide sequence that is capable of initiating an antigenic response. For example, the antigens which may be used in the vaccines or immunogenic compositions may be surface exposed antigens derived from viral or bacterial pathogens. Throughout this specification and the appended claims, the phrase "antigenic peptide" or "immunogen" is intended to encompass all peptide or protein sequences which are capable of inducing an immune response within the animal concerned. In one embodiment, however, the polynucleotide sequence will encode for a full protein that is associated with the disease state, as the expression of full proteins within the animal system is more likely to mimic natural antigen presentation, and thereby evoke a full immune response. Antigens which are capable of eliciting an immune response against a human pathogen include those in which the antigen or antigenic composition is derived from any of a range of viral, bacterial, parasitic and yeast sources. Viral antigen sources include: HIN-1 (such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vif, vpr, vpu, rev); human herpes viruses (such as gH, gL gM gB gC gK gE or gD or derivatives thereof, or Immediate Early proteins such as ICP27 , ICP 47, IC P 4, ICP36 from HSN1 or HSN2); cytomegalo virus, especially human (such as gB or derivatives thereof); Epstein Barr virus (such as gp350 or derivatives thereof); Varicella Zoster Virus (such as gpl, II, III and IE63); hepatitis viruses such as hepatitis B virus (for example hepatitis B surface antigen or hepatitis core antigen or pol) or hepatitis C and hepatitis E virus antigens; and other viral pathogens such as the paramyxoviruses, including Respiratory Syncytial virus (such as F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18, and the anitgens LI, L2, El, E2, E3, E4, E5, E6, E7), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) and Influenza virus cells (such as HA, ΝP, ΝA, or M proteins, or combinations thereof). Bacterial sources include: Neisseria spp. such as N. gonorrhea and N. meningitidis (e.g. transferrin-binding proteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes (for example M proteins or fragments thereof, or C5A protease); S. agalactiae, S. mutans; H. ducreyi; Moraxella spp. such as M. catarrhalis (also known as Branhamella catarrhalis; antigens include high and low molecular weight adhesins and invasins); Bordetella spp., including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85A, 85B or 85C, MPT 44, MPT59, MPT45,

HSP10.HSP65, HSP70, HSP 75, HSP90, PPD 19kDa [Rv3763], PPD 38kDa [Rv0934]), M. bovis, M. leprae, M. avium, M. paratuberculosis and M. smegmatis; Legionella spp., including L. pneumophila; Escherichia spp., including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coli and enteropathogenic E. coli (for example shiga toxin-like toxin or derivatives thereof); Vibrio spp., including V. cholera (for example cholera toxin or derivatives thereof); Shigella spp., including S. sonnei, S. dysenteriae and S.flexnerii; Yersinia spp., including Y. enterocolitica (for example a Yop protein), Y. pestis and Y. pseudotuberculosis; Campylobacter spp., including C. jejuni (for example toxins, adhesins and invasins) and C. coli; Salmonella spp., including S. typhi, S. paratyphi, S. choleraesuis and S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp., including H pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp., including P. aeruginosa; Staphylococcus spp., including S. aureus and S. epidermidis; Enterococcus spp., including E. faecalis and E. faecium; Clostridium spp., including C. tetani (for example tetanus toxin and derivatives thereof), C. botulinum (for example botulinum toxin and derivatives thereof), and C. difficile (for example clostridium toxins A or B and derivatives thereof); Bacillus spp., including R. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB), and B. hermsii; Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp., including R. rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), and C. psittaci; Leptospira spp., including L. interrogans; and Treponema spp., including T. pallidum (for example the rare outer membrane proteins), T. denticola, and T. hyodysenteriae. Parasitic sources include: Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis; and Schisostoma spp., including S. mansoni.Yeast sources include: Candida spp., including C. albicans; and Cryptococcus spp., including C. neoformans.

Other group of specific antigens for tuberculosis are, for example, Rv2557, Rv2558, RPFs: Rv0837c, Rvl884c, Rv2389c, Rv2450, Rvl009, aceA (Rv0467), PstSl, (Rv0932), SodA (Rv3846), Rv2031c 16kDal., Tb Ral2, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPN, MTI, MSL, mTTC2 and hTCCl (WO 99/51748). Proteins for M tuberculosis also include fusion proteins and variants thereof in which at least two, or at least three, polypeptides of M. tuberculosis are fused into a larger protein. Some specific fusions include Ral2-TbH9-Ra35, Erdl4-DPN-MTI, DPN-MTI-MSL, Erdl4-DPN-MTI-MSL- mTCC2, Erdl4-DPN-MTI-MSL, DPN-MTI-MSL-mTCC2, and TbH9-DPN-MTI (WO 99/51748).

In one embodiment, specific antigens for Chlamydia include, for example, the High Molecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP 366 412), and putative membrane proteins (Pmps). Other Chlamydia antigens of the vaccine formulation can be selected from the group described in WO 99/28475. In another embodiment bacterial antigens derived from Streptococcus spp., including S. pneumoniae (e.g. PsaA, PspA, streptolysin, choline-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins, J.B. et al. (1998) Microbial Pathogenesis 25: 337-42), and mutant detoxified derivatives thereof (WO 90/06951; WO 99/03884), PhtD, PhtA, PhtB, PhtE and CbpA . Other antigens derived from Haemophilus spp., include H. influenzae type B (for example PRP and conjugates thereof), non typeable H influenzae (for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides (US 5,843,464) or multiple copy variants or fusion proteins thereof). The antigens that may be used in the present invention may further comprise antigens derived from parasites that cause malaria. For example, antigens from Plasmodium falciparum include RTS,S and TRAP. RTS is a hybrid protein comprising substantially all the C-terminal portion of the circumsporozoite (CS) protein of P. falciparum linked via four amino acids of the preS2 portion of hepatitis B surface antigen to the surface (S) antigen of hepatitis B virus. Its full structure is disclosed in the International Patent Application No. PCT/EP92/02591, published under Number WO 93/10152 claiming priority from UK patent application No. 9124390.7. Other plasmodia antigens that are likely candidates to be components of a multistage malaria vaccine are P. falciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMPl, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and their analogues in other Plasmodium spp.. An embodiment of the present invention is a malaria vaccine wherein the antigenic preparation comprises a combination of the RTS,S and MSP-1 antigens. The invention contemplates the use of an anti-tumour antigen and may be useful for the immunotherapeutic treatment of cancers. For example, tumour rejection antigens such as those for prostrate, breast, colorectal, lung, pancreatic, renal or melanoma cancers. Exemplary antigens include MAGE 1, MAGE 3 and MAGE 4, or other MAGE antigens such as disclosed in WO99/40188, PRAME, BAGE, Lage (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or GAGE (Robbins, P.F. & Kawakami, Y. (1996) Current Opinion in Immunology 8: 628-36; Nan den Eynde, BJ.& Boon, T. (1997) International Journal of ^'Clinical and Laboratory Research 27: 81-6. Coneale, P. et al. (1997) Journal of the National Cancer Institute 89: 293-300). Indeed these antigens are expressed in a wide range of tumour types including melanoma, lung carcinoma, sarcoma and bladder carcinoma. MAGE antigens for use in the present invention may be expressed as a fusion protein with an expression enhancer or an immunological fusion partner. In particular,the MAGE protein may be fused to Protein D from Haemophilus influenzae B. In particular, the fusion partner may comprise the first one third of Protein D. Such constructs are disclosed in WO 99/40188. Other examples of fusion proteins that may contain cancer specific epitopes include bcr / abl fusion proteins. In one embodiment, prostate antigens are utilised, such as Prostate Specific Antigen (PSA), PAP, PSCA (Reiter, R.E. et al. (1998) PNAS USA 95: 1735 -40), PSMA or the antigen known as Prostase. Prostase is a prostate-specific serine protease (trypsin-like), and has been described by Nelson, P.S. et al. (1999; PNAS USA 96: 3114- 9). The nucleotide sequence and deduced polypeptide sequence of the mature protein, and homologues, are disclosed in (PNAS USA (1999) 96: 3114-9) and in International Patent Applications WO 98/12302 (and also the corresponding granted patent US 5,955,306), WO 98/20117 (and also the corresponding granted patents US 5,840,871 and US 5,786,148) (prostate-specific kallikrein) and WO 00/04149 (P703P). The present invention provides antigens comprising prostase protein fusions based on prostase protein and fragments and homologues thereof ("derivatives"). Such derivatives are suitable for use in therapeutic vaccine formulations that are suitable for the treatment of prostate tumours. Typically the fragment will contain at least 20, or at least 50, or at least 100, contiguous amino acids as disclosed in the above referenced patent and patent applications. A further prostate antigen for use in the present invention is known as P501S, sequence ID No. 113 of WO98/37814. -mmunogenic fragments and portions encoded by the gene thereof comprising at least 20, at least 50, or in another embodiment at least 100, contiguous amino acids as disclosed in the above referenced patent application, are contemplated. A particular fragment is PS 108 (WO 98/50567). Other prostate specific antigens are known from WO98/37418, and WO/004149. Another is STEAP (Hubert, R.S. et al. (1999) PNAS USA 96: 14523-8). Other tumoxir associated antigens useful in the context of the present invention include: Plu-1 (Lu, P.J. et al. (1999) Journal of Biological Chemistry 274: 15633-45), HASH -1, HasH-2, Cripto (Salomon, D.S. et al. (1999) Bioessays 21: 61 -70; US patent 5654140), and Criptin (US patent 5 981 215). Additionally, antigens particularly relevant for vaccines in the therapy of cancer also comprise tyrosinase and survivin. The present invention is also useful in combination with breast cancer antigens such as Muc-1, Muc-2, EpCAM, HER 2 / Neu, mammaglobin (US patent 5668267) or those disclosed in WO 00/52165, WO99/33869, WO99/19479, WO 98/45328. HER / 2 neu antigens are disclosed, ter alia, in US patent 5,801,005. hi one embodiment the HER / 2 neu comprises the entire extracellular domain (comprising approximately amino acids 1-645), or fragments thereof, and at least an immunogenic portion of or the entire intracellular domain (approximately the 580 C-terminal amino acids), hi particular, the intracellular portion should comprise the phosphorylation domain or fragments thereof. Such constructs are disclosed in WO00/44899. One specific embodiment is known as ECD PD; a second is known as ECD ΔPD (see WO/00/44899). The HER / 2 neu as used herein can be derived from rat, mouse or human. The antigens may also be associated with tumour-support mechanisms (e.g. angiogenesis, tumour invasion), for example tie 2. Vaccines of the present invention may also be used for the prophylaxis or therapy of chronic disorders in addition to allergy, cancer or infectious diseases. Such chronic disorders are diseases such as asthma, atherosclerosis, and Alzheimer's and other auto-immune disorders. Vaccines for use as a contraceptive may also be considered. Antigens relevant for the prophylaxis and the therapy of patients susceptible to or suffering from Alzheimer's neurodegenerative disease are, in particular, the N- terminal 39 -43 amino acid fragment of the β-amyloid precursor protein and smaller fragments). This antigen is disclosed in the International Patent Application No. WO 99/27944 (Athena Neurosciences). Potential self-antigens that could be included as vaccines for auto-immxme disorders or as a contraceptive vaccine include: cytokines, hormones, growth factors or extracellular proteins, such as a 4-helical cytokine, like IL13. Cytokines include, for example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL20, IL21, TNF, TGF, GM-CSF, MCSF and OSM. 4-helical cytokines include IL2, IL3, IL4, IL5, IL13, GM-CSF and MCSF. Hormones include, for example, luteinising hormone (LH), follicle stimulating hormone (FSH), chorionic gonadotropin (CG), VGF, GHrelin, agouti, agouti related protein and neuropeptide Y. The vaccines of the present invention are particularly suited for the immunotherapeutic treatment of diseases, such as chronic conditions and cancers, but also for the therapy of persistent infections. Accordingly the vaccines of the present invention are particularly suitable for the immunotherapy of infectious diseases, such as tuberculosis (TB), HIV infections such as AIDS, and hepatitis B (HepB) virus infections. In the context of the present invention the immunogenic compositions or vaccines are administered to a vaccinee. By this it is intended that the vaccinee is a mammal, and in one embodiment a human, to whom the vaccines or immunogenic compositions have been administered. The polynucleotide of the vaccines and the adjuvants of the present invention may be administered simultaneously or separately. For instance the polynucleotide and the adjuvant may be co-formulated in a single composition, or alternatively may be separately formulated in distinct compositions. In the latter instance the at least two compositions are administered in functional cooperation, and may be administered at substantially the same time, or alternatively be administered at different time points separated by, in different embodiments, within 30 minutes to 1 hour apart, or within 1 and 2 hours apart, or within 12-36 hours apart, such as 24 hours apart; or the two compositions may, substantially, be administered the next following day. When the at least two compositions are administered on different occasions, the polynucleotide may be administered before the adjuvant. The vaccine compositions may further be provided in a single composition, comprising both the polynucleotide and the adjuvant, wherein the adjuvant composition is in a delayed release formulation which allows the release of the adjuvant composition at the site of injection within 30 minutes to 1 hour after administration, or within 1 and 2 hours after admimstration, or within 12-36 hours after administration, such as 24 hours after administration. Accordingly, there is provided a kit comprising two compositions, the polynucleotide containing composition and the adjuvant containing composition, for separate administration. In this context the separate admimstration may be separated by administration site or time, or both. There is provided by the present invention a method of raising an immune response in an individual against an antigen, comprising administering to that individual a polynucleotide composition encoding the antigen, followed by administering to said individual an adjuvant composition comprising a product of purine catabolism, such as uric acid, or salt thereof. In one embodiment the adjuvant composition is administered within 12 to 36 hours after the administration of the polynucleotide. hi other embodiments the polynucleotide is a DNA plasmid vector, or alternatively in the form of a viral vector, such as a simian adenovirus vector. Also provided by the present invention is a method of therapeutically treating a patient having a tumour, comprising administering to that patient a polynucleotide composition encoding a tumour associated antigen, followed by administering to said patient an adjuvant composition comprising a product of purine catabolism, such as uric acid, or salt thereof. In one embodiment the adjuvant composition is administered within 12 to 36 hours after the administration of the polynucleotide. In other embodiments the polynucleotide is a DNA plasmid vector, or alternatively in the form of a viral vector, such as a simian adenovirus vector. In one embodiment plasmids of the vaccines are prevented from replicating within the mammalian vaccinee and integrating within the chromosomal DNA of the host, as such the plasmid may be produced without an origin of replication that is functional in eukaryotic cells. The immunogen component comprising a vector which comprises the nucleotide sequence encoding an antigenic peptide can be administered in a variety of manners. It is possible for the vector to be administered in a naked form (that is, as a naked nucleotide sequence not in association with liposomal formulations, with viral vectors or transfection facilitating proteins) suspended in an appropriate medium, for example a buffered saline solution such as PBS, and then injected intramuscularly, subcutaneously, intraperitonally or intravenously (Brohm, et al. (1998) Vaccine 16ι 949-54, the disclosure of which is included herein in its entirety by way of reference). It is additionally possible for the vectors to be encapsulated by, for example, liposomes or within polylactide co-glycolide (PLG) particles for administration via the oral, nasal or pulmonary routes in addition to the routes detailed above. It is also possible, according to one embodiment of the invention, for intradermal administration of the immunogen component, for example via use of gene-gun (particularly particle bombardment) administration techniques. Such techniques may involve coating of the immunogen component on to dense micro-beads, such as gold beads, which are then administered under high pressure into the epidermis, such as, for example, as described in Haynes, J.R. et al. (1996; Joxraial of Biotechnology 44: 37-42). In this context the adjuvant component may be co-formulated on the dense microbeads, or on separate populations of microbeads, or alternatively the polynucleotide vaccine may be administered ballistically on microbeads and the adjuvant administered separately via systemic or local delivery, possibly at the site of polynucleotide delivery by intradermal or subcutaneous injection. In an alternative embodiment of the present invention, there is provided a patch comprising a plurality of needles, being in the range of 30-1000 micrometers in length, the external surface of which is coated with a solid reservoir medixim. The solid reservoir medium in this context would comprise the vaccines of the present invention in solid form. Microneedles of this form are described in WO 02/07813 and WO

03/061636, the contents of which are incorporated herein, and in particular the claims thereof are intended to be read, with the addition of uric acid, or other product of purine catabolism, in the context of this disclosure. The adjuvants and vaccines of the present invention may be administered via a variety of different administration routes, such as intramuscular, subcutaneous, intraperitoneal, intradermal, or topical routes. The adjuvant or polynucleotide components may be administered via the subcutaneous, intradermal or topical routes. In one embodiment, the administration of both components, the polynucleotide and adjuvant, is by the same route. In another embodiment, the polynucleotide is administered by ballistic delivery (gene gun) into the epidermis or dermis, and the adjuvant composition is delivered in the vicinity of the polynucleotide either topically or by intradermal or subcutaneous injection. The dose of admimstration of the adjuvant will also vary, but may, for example, range in a liquid form of the vaccine between about 5 μg per ml to about 5 mg per ml, and may be between 25 μg per ml to about 1 mg per ml, and may be between 50 to 500 μg per ml. In a liquid form between 0.5 and 1 ml of the vaccine may be administered to a human vaccinee. In a dry form of the vaccine a total mass of the adjuvant may also be in the range of 5 μg to about 5 mg per dose, and may be between 25 μg to about 1 mg per dose, and may be between 50 to 500 μg per dose. More specifically, a dosing schedule may be one where sufficient uric acid, or other product of purine catabolism, is administered to a vaccination site which results in high enough localised concentration such that crystallisation occurs and crystals of uric acid, or a salt thereof, can then mediate the adjuvant effect. When the adjuvant component is uric acid such a concentration is likely to be greater than or equal to 70 micrograms of uric acid per ml of solvent in a localised concentration at the site of administration. Administration of the adjuvant may be repeated with each subsequent or booster administration of the nucleotide sequence. The dose of the polynucleotide encoding the antigen will depend on the route of administration and will be readily determined by the man skilled in the art. Conventionally speaking for gene gun applications the dose will be between 0.5 and lOOμg per administration, and for intramuscular administration of "naked" DNA between 10 and 2000μg per administration. Uses of the word "comprising" in the disclosures throughout this specification are intended to be read in both senses of the word, in that in the context of each disclosure of elements the word "comprising" should be read in its inclusive sense, in that other elements may also be included, and also in its exclusive sense in that the disclosure is restricted to those elements, and therefore each word comprising may be substituted with the word "consisting". The present invention is exemplified by, and not limited to, the following examples. Example 1, Effect of Uric Acid on Solid E.G7-OVA Tumour Growth in vivo following DNA Vaccination by Gene Gun

This experiment investigated the effect of uric acid admimstration on solid E.G7-OVA tumour growth in vivo following DNA vaccination by gene gxxn performed according to two different immunisation schedules.

1.1 Materials & Methods

1.1.1 Animals Female C57B1 6 mice (Charles River Ltd., Thanet, UK) were used throughout all experiments. All animals were shaved on the lower back immediately prior to tumour cell implantation, and on the abdomen immediately prior to initial gene gun application.

1.1.2 Tumour cells, implantation, and growth measurement The ovalbumin-transfected murine lymphoma line E.G7-OVA (Moore, M.W. et al. (1988) Cell 54: 777-85) was used in this experiment. 5 x 10⁶ live E.G7-OVA cells at nominal passage number 23 were injected subcutaneously into shaved skin of the lower back in 0.1 ml phosphate buffered saline. Date of implantation was designated day 0 ("dO"). Palpable tumours formed at the implant site were measured using callipers, and tumour size in mm² (representing the longest measured diameter multiplied by the diameter perpendicular to it) was recorded every two days up to the tumour end point for each animal. Tumour end points were defined as the points at which tumours either exceeded 250 mm² or became ulcerated (all such animals were euthanased for ethical reasons), or tumoxxrs regressed completely.

1.1.3 Gene gun DNA cartridge preparation Gold particles (mean diameter 2μm; Powderject/Chiron, Madison, WI, USA) were treated with 0.05 M spermidine (Sigma, St. Louis, MO, USA) and briefly sonicated, coated with plasmid DNA by precipitation with 0.9 M calcium chloride (American Pharmaceutical Partners Inc., Los Angeles, CA, USA), and then washed three times in dried absolute ethanol. Particles were transferred into a solution of polyvinyl pyrrolidone (Sigma) in dried absolute ethanol at 0.05 mg per ml. This solution was then coated onto the inside surface of tefzel tubing (Powderject) using custom- made tube turning apparatus (Powderject), and the tubing dried under nitrogen gas. Cartridges were cut from dried tubing. Samples from each batch of prepared cartridges were tested to ensure a DNA loading of 0.5 μg DNA per cartridge. Briefly, cartridge DNA was eluted by immersion of cartridges in DNAse/RNAse-free water and incubation at 37 °C for 30 min, followed by momentary centrifugation at 14,000 rpm. A supernatant sample (70 μl) was then transfened into a quartz capillary (Pharmacia Biotech), and the DNA concentration determined using a GeneQuant II DNA/RNA calculator (path length 10 mm, blanked against purified water; Pharmacia Biotech).

1.1.4 DNA vaccination Immunisation was achieved by particle mediated immunotherapeutic delivery (PMID) using the BioRad Helios gene gun device (BioRad, Hemel Hempstead, UK). Gene gun cartridges were prepared containing 0.5 μg DNA per cartridge of the ovalbumin-encoding plasmid pVacl.OVA(cyt) for therapeutic immunisation, or 0.5 μg DNA per cartridge of the pVacl empty vector for controls. Plasmid-coated gold particles were accelerated into shaved abdominal skin using helium gas at approximately 400 p.s.i.. Two cartridges were fired per immunisation date per mouse. Immunisation schedules are shown in Table 1.

1.1.5 Uric acid formulation and administration Purified uric acid crystals (Sigma) were dissolved in 0.1 M boric acid at 5 mg per ml and the pH adjusted to 8.5 using 1 M NaOH. The solution was heated to 55 °C, filtered through a sterile 0.22 micrometer filter, and allowed to stand at room temperature for 48 h. The resultant mixture of soluble and crystallised uric acid was then injected subcutaneously at the site of gene gun immunisationat a total concentration of 1 milligram per ml in 100 microlitres solution . Control animals were injected with borate solution prepared as above but without addition of uric acid.

1.2 Results All animals were immunised on either days 2 and 4 ("d2, d4") or on day 7

("d7") post-implantation with two gene-gun shots of 0.5 micrograms plasmid DNA each, and then injected subcutaneously at the immunisation site with either uric acid solution or borate (for controls) as shown in Table 1. Animals immunised on the d2, d4 schedule were injected following immunisation on d4; animals immunised on d7 only were injected on day 8 (d8). The experimental groups are sximmarised below:

Table 1: Experimental groups for experiment C02-1103

Txxmour size was recorded for each animal every two days up to tumour end points. Plotted growth curves showing mean tumour size over time for each group are presented in Figure 1. The control groups ("pVacl d2, d4 + borate d4" and "pVacl d7 + borate d8";

Fig 1) show typical uninliibited txxmour growth. Immunisation with pVacl.OVA(cyt) via the d2, d4 schedule delays tumour growth, and immxxnisation at d7 only has a similar effect. Admimstration of uric acid at d4 following immunisation at d2 and d4 appears to alter the kinetics of tumour growth. The administration of uric acid at d8 following a single immunisation at d7

(group 6; "pVacl.OVA(cyt) d7 + UA d8") has the greatest effect on tumour growth, with three of the six mice having tumours that regressed completely by day 31 post- implantation (no tumour re-growth was subsequently observed). Of the remaining mice in group 6, two had tumours which exceeded 250 mm² (and were therefore euthanased for ethical reasons), and one had a tumour which appeared to have anested growth but did not regress (this animal was euthanased on day 31 due to txxmour ulceration). Example 2, Variation of adjuvantsolution preparation and administration protocol, and its effect on solid E.G7-OVA tumour growth in vivo following DNA vaccination and adjuvant administration In this series of four experiments, aspects of the adjuvant solution preparation and administration protocols were varied, and the effect on solid E.G7-OVA tumour growth in vivo observed following DNA vaccination by gene gun and adjuvant administration.

2.1 Materials & Methods

2.1.1 Animals, cells and tumour implantation Animals and cells were sourced and prepared, and tumours implanted and measured, as described in Example 1 (sections 1.1.1 - 1.1.2).

2.1.2 Gene gun DNA cartridge preparation and vaccination DNA cartridges were prepared and DNA vaccination by gene gun performed as described in Example 1 (sections 1.1.3 - 1.1.4). All animals were vaccinated by gene gun on day 7 post-implantation only.

2.1.3 Adjuvant formulation and administration Purified uric acid crystals (Sigma) were dissolved in 0.1 M boric acid at 1 mg per ml and the pH adjusted to 8.5 using 1 M NaOH. The solution was heated to 55 °C, filtered through a sterile 0.22 micrometer filter, and allowed to precipitate at room temperature for 48 h. The resultant mixture of soluble and crystalline uric acid was injected subcutaneously at the site of DNA vaccination 24 hours post-immunisation at a total of 1 milligram uric acid per ml in 100 microlitres solution. Single aspects which were a derivation from this protocol were varied in four experiments as shown in Table 2.

Table 2: Experiments Six animals per group, five or six groups per experiment

2.2 Results The mean tumour size per group was recorded at day 10 and day 20 post- implantation in each experiment. The percentage of tumours in each group that progressed (i.e. grew to a size greater than or equal to 250 mm and did not regress) was also recorded in each experiment. These data have been illustrated graphically in Figure 2. hi experiment A, the concentration of uric acid in the adjuvant solution was varied. An adjuvant effect was observed at concentrations greater than or equal to 0.10 milligrams uric acid per millilitre. Adjuvant effect was indicated by a decrease in mean tumour size at day 20 and by a decrease in the percentage of tumours that regressed. In experiment B, the precipitation time allowed during preparation of the adjuvant solution was varied. A precipitation time of at least 24 hours was required to illicit an adjuvant effect, and this was improved after 48 hours. There was no further improvement after 72 hours compared to 48 hours. The degree of crystallisation allowed to occur in the adjuvant solution seems, therefore, to influence the adjuvanticity of the solution independently of the prepared uric acid concentration. In experiment C, the time of adjuvant administration relative to DNA vaccination was varied. An adjuvant effect was observed when the uric acid solution was administered 24 hours post- vaccination. In experiment D, the site of adjuvant injection was varied, and compared to injections of control borate buffer prepared as described in section 2.1.3 but without addition of uric acid. Adjuvant effect was observed when the adjuvant solution was administered at the site of DNA vaccination, but not when it was injected at a remote site (the flank). There was some indication of adjuvant effect when the solution was injected at the tumour growth site, but may not be significant and has not been observed in a repeat experiment.

Example 3, Measurement of cytokine production following immunisation and administration of uric acid adjuvant solution against a growing tumour.

This experiment investigated levels of cytokine production following DNA vaccination by gene gun and administration of uric acid adjuvant solution against a growing solid E.G7-OVA tumour.

3.1 Materials & Methods

3.1.1 Animals Female C57B1/6 mice (Charles River Ltd., Thanet, UK) were shaved on the flank immediately prior to tumour cell implantation, and on the abdomen immediately prior to initial gene gun application.

3.1.2 Tumour cells 1 x 10⁶ live E.G7-OVA cells were injected subcutaneously into shaved skin of the flank in 0.1 ml phosphate buffered saline.

3.1.3 Gene gun DNA cartridge preparation and vaccination DNA cartridges were prepared and DNA vaccination by gene gun performed as described in Example 1 (sections 1.1.3 - 1.1.4). Animals were vaccinated by gene gun on days 5 and 7 post tumour-implantation with 0.5 μg of the plasmid pVacl.OVA(cyt), or with the empty vector pVacl for controls. 3.1.4 Adjuvant formulation and administration The uric acid adjuvant solution was prepared and admimstered as described in Example 2 (section 2.1.3). Animals were injected with uric acid adjuvant solution (or borate buffer for controls) 24 hours after each gene gun vaccination.

3.1.5 Detection of IFN-gamma and IL2 production in ex vivo splenocyte populations by ELISPOT assay Three animals per group were sacrificed and spleens excised on days 11 and 14 post tumour-implantation. Each spleen was processed to remove red blood cells and produce a single-cell suspension of splenocytes. Cells were loaded onto 96-well plates coated with IFN-gamma or IL2 capture antibodies (Pharmingen) at 4 x 10⁵ cells per well, and incubated for 24 hours with 50 μl per well of either SIINFEKL (300nM) or TEWT (lOμM) ovalbumin-derived peptides or with medium for controls. After 24 hours plates were washed using phosphate buffered saline, and then developed by incubation with 50 μl per well of biotin-conjugated IFN-gamma or IL2 detection antibody (Pharmingen), followed by streptavidin alkaline phosphatase (Caltag

Laboratories) and finally substrate from the Alkaline Phosphate Conjugate Substrate Kit (BioRad), with washing between each step. Spots representing cytokine- producing cells were counted using ELISPOT Robot software (Autoimmun Diagnostika). Background counts from cells incubated with medium only were subtracted from those stimulated with peptide.

3.1.6 Intracellular cytokine staining (ICS) 10 μl of anti-mouse CD28 antibody / anti-mouse CD49d antibody solution was added to 4 x 10 splenocytes and the mixture incubated for 10 minutes at room temperature. 1 ml of SIINFEKL peptide (300nM) was added followed by incubation at 37° C for 1 hour in a humidified atmosphere with 5% CO₂. Brefeldin A (20 μl) was added and the tubes placed in a waterbath for 6 hours at 37° C, before storage overnight at 4° C. Following overnight stimulation the cells were centrifuged and the supernatant discarded, then 10 μl of CD4p_ercp/CD8APc solution added and the cells incubated for 30 minutes at room temperature in the dark. The cells were washed resuspended in flow cytometry buffer. Fixative Reagent A (100 μl; Caltag) was added and the cells incubated for 15 minutes at room temperature in the dark. The cells were washed and resuspend again in flow cytometry buffer, then 100 μl IFN-Y_PE/IL-2_FITC added and incubated for 30 minutes at room temperature in the dark. Following a final wash and resuspension in flow cytometry buffer, cells were analysed for cytokine staining on a FACS Calibur flow cytometer (Becton Dickinson). Baseline responses were subtracted from responses detected following stimulation with peptide.

3.1.7 Tetramer staining ofCD8 T-cells Blood samples were collected from mice sacrificed at days 7 and 11 post tumour-implant into 100 μl sodium citrate. Blood or splenocytes (100 μl) were transfened to tubes with 5 μl of tetramer per tube (SIINFEKL-MHC complex labelled with PE fluorochrome; Beckman Coulter) and incubated for 20 minutes at room temperature. Five microlifres of CD8 CyChrome (2 mg/ml; BD Biosciences) was added and tubes incubated for 10 minutes at room temperature then washed in flow cytometry buffer. Splenocytes were resuspended in 250 μl of BD cellfix reagent (Becton Dickinson) then analysed on a FACSCalibur flow cytomoter. Blood samples were resuspended in 250 μl of lysis buffer (Beckman Coulter) diluted 1 part in 25 parts PBS, left at room temperature for 2 minutes, and then 250 μl of fixative (Beckman Coulter) added. Blood cells were washed and resuspended in flow cytometry buffer then analysed on a FACSCalibur flow cytometer.

Fig 3: Protocol timeline dO d5 d6 d7 d8 d11 d14

tumour DNA adjuvant DNA adjuvant ELISPOT ELISPOT, ICS implant vaccination vaccination & tetramer

3.2 Results ELISPOT data from splenocytes collected at days 11 and 14 post tumour- implantation are shown in Fig. 4 and 5 respectively. The results of intracellular cytokine staining from splenocytes collected at day 14 are illustrated in Fig. 6. Tetramer staining from splenocyte and blood samples taken at day 14 is shown in Fig. 7. Splenocytes stimulated ex vivo with the CD8-recognised peptide SIINFEKL showed IFN-gamma and IL2 production at both days 11 (Fig. 4 a, b) and 14 (Fig. 5 a, b). There was an increase in cytokine production in the groups immunised with ova- encoding DNA compared with the controls immunised with empty vector. Cytokine production in empty vector-immunised groups may be attributable to CD8⁺ cells activated by tumour-derived antigen or the inflammatory effects of uric acid admimstration. Of the ovalbumin-immunised groups, IFN-gamma and IL2 production was increased in groups that received the uric acid adjuvant compared with those that received only borate buffer. Splenocytes stimulated with the CD4-recognised peptide TEWT (Fig. 4 c, d and

Fig. 5 c, d) showed little production of either cytokine, and there were only marginal increases in groups immunised with ova-encoding DNA compared with empty vector controls. Intracellular cytokine production at day 14 in CD8⁺ spleen-derived T-cells detected by ICS (Fig. 6) reflected the pattern that was detected by the ELISPOT assay. Groups immunised with ova-encoding DNA produced higher levels of intracellular cytokines (particularly IFN-gamma) than empty vector controls, and production appeared highest in the group that received the uric acid adjuvant. Tetramer staining of spleen and blood derived cells taken at day 14 (Fig. 7) revealed the percentage of all CD8⁺ T-cells that have a T-cell receptor specific for the ovalbumin-derived peptide sequence SIINFEKL. The group that was immunised with ovalbumin-encoding DNA and received the uric acid adjuvant had a higher percentage of CD8⁺ T-cells specific for the ovalbumin SIINFEKL epitope than the empty vector control or the immunised group that received only borate buffer.

3.3 Conclusions In the experiment presented here, cytokine production by immune cells is quantified ex vivo in tumour-bearing mice that have been immxxnised by DNA vaccination against a tumoxxr associated antigen with or without subsequent administration of the uric acid adjuvant. The SIINFEKL peptide, an ovalbumin-derived CD8-stimulatory sequence, was used to quantify ex vivo CD8⁺ cell populations for cytokine capture (ELISPOT) assays, while the ovalbumin-derived CD4-stimulatory peptide TEWT was used to quantify CD4⁺ cells. Administration of the uric acid adjuvant solution following DNA immunisation against ovalbumin stimulated intracellular production and extra-cellular release of IFN- gamma and IL2 by CD8⁺ cells in excess of that stimulated by admimstration of the buffer alone, or admimstration of the adjuvant following vaccination with an empty plasmid. There also appeared to be some expansion of CD8⁺ T-cells bearing an ovalbumin-specific TCR in both the spleen and the blood, above the levels stimulated by buffer or empty vaccination.

Claims

Claims

1. An immunogenic composition comprising a polynucleotide encoding an antigen, and an intermediate or final product of purine catabolism.

2. An immxxnogenic composition as claimed in claim 1, wherein the intermediate or final product of purine catabolism is uric acid.

3. An immunogenic composition as claimed in claim 2, wherein the uric acid is in a crystalline form.

4. An immxxnogenic composition as claimed in claim 2, wherein the uric acid is at sufficiently high concentration such that crystallisation of uric acid occurs after administration.

5. An immunogenic composition as claimed in any of claims 2 to 4, wherein the uric acid is in the form of a salt.

6. An immxmogenic composition as claimed in claim 5, wherein the salt of uric acid is a monosodium salt.

7. An immunogenic composition as claimed in claim 1, wherein the intermediate or final product of purine catabolism is selected from the group consisting of inosine, hypoxanthine and xanthine.

8. An immunogenic composition composition comprising a compound of Formula

(I):

(I) or a salt, solvate, or physiologically functional derivative thereof, and a polynucleotide that encodes an antigen against which it is desired to generate an immune response.

9. An immunogenic composition as claimed in any one of claims 1-8 wherein said immxmogenic composition is formulated such that the intermediate or final product of purine catabolism or compound of formula I, can be administered to the vaccinee between 12 and 36 hours after the admimstration of the polynucleotide encoding an antigen.

10. An immunogenic composition as claimed in any one of claims 1 to 9, wherein the polynucleotide is a DNA plasmid vector.

11. An immunogenic composition as claimed in claim 10, wherein the plasmid vector is in the form of dense micro-beads suitable for ballistic delivery into the skin.

12. An immxmogenic composition as claimed in claim 11, wherein the plasmid vector is formulated on the surface of dense micro-beads suitable for ballistic delivery into the skin.

13. An immxmogenic composition as claimed in claim 12, wherein the intermediate or final product of purine catabolism is co-formulated with the plasmid vector.

14. An immxmogenic composition as claimed in any one of claims 1 to 9, wherein the polynucleotide is in the form of an adenovirus vector.

15. A method of vaccinating an individual comprising admimstering to said individual a polynucleotide encoding an antigen and an intermediate or final product of purine catabolism.

16. A method as claimed in claim 15, wherein the polynucleotide encoding an antigen and the intermediate or final product of purine catabolism are present in separate compositions, and the composition comprising the intermediate or final product of purine catabolism is administered to said individual between 12 and 36 hours after the administration of the polynucleotide encoding an antigen.

17. A method as claimed in claim 15 or 16 wherein said intermediate or final product of purine catabolism is uric acid.

18. A kit comprising (a) a composition comprising a polynucleotide encoding an antigen, and (b) a composition comprising an intermediate or final product of purine catabolism, the kit optionally further comprising instructions to administer the two compositions simultaneously or separately.

19. A vaccine comprising the immxmogenic composition as claimed in any one of claims 1 to 14.

20. Use of an immunogenic composition as claimed in any one of claims 1 to 14, in the manufacture of a vaccine composition for the amelioration or treatment of a cancer or a txxmour.

21. A method of treating an individual suffering from cancer comprising administration to that individual of an immxxnogenic composition as claimed in any one of claims 1 to 14.