WO2004108154A1 - Prevention of pulmonary immunopathology using mutant bacterial toxins - Google Patents

Abstract

The LT-K63 mutant of E.coli heat-labile toxin has been found to lead to protection against the pathological consequences of infection with unrelated organisms, particularly respiratory pathogens, including viruses. Alteration of the lung microenvironment by the protein may provide generic protection against a number of infectious diseases. The invention provides a method for protecting a patient against immunopathology, comprising the step of administering a detoxified mutant of an ADP-ribosyltransferase to the patient.

Description

PREVENTION OF PULMONARY IMMUNOPATHOLOGY USING MUTANT BACTERIAL TOXINS

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of immunopathology, particularly in the lungs, and its prevention.

BACKGROUND ART

Most pathogens enter and infect their host across a mucosal surface, in particular the gastrointestinal or respiratory tract. The mucosal associated lymphoid tissue (MALT) constantly encounters antigenic material from the environment and must therefore respond to potentially harmful microorganisms.

On encountering some microorganisms, however, the host's immune response can be inappropriate, and may even have pathological consequences. For example, T cells play an important role in control of infection by respiratory syncytial virus (RSV), but they are also involved in the immunopathogenesis of RSV disease. Mice primed with the attachment protein G of RSV and then challenged with live RSV develop pulmonary eosinophilia, weight loss and cachexia [1]. The eosinophilia is driven by an oligoclonal subset of CD4⁺ T cells secreting IL-4 and IL-5, while weight loss results from the excessive inflammatory infiltrate and the production of TNF [2].

Thus antiviral T cells are involved in an immunopathologic paradox where they are involved both in eliminating virus and in causing enhanced disease.

Various strategies have been investigated to decrease the vigour of the immune response. Depletion of CD4+ T cells abolishes Th2-driven lung eosinophilia [3]. Induction of CD8+ T cells during RSV infection greatly reduces eosinophilia by modifying the CD4+ Th2 phenotype through the production of IFN-γ. Administration of IL-12 during G-primed RSV infection also reduces eosinophilia and augments the moderation of pulmonary immunopathology via IFN-γ producing CD8+ T cells [4,5]. In addition, G-protein induced eosinophilia can be reduced by incorporating an immunodominant CTL epitope into immunological priming [6]. Immunomodulation in the G-RSV mouse model of RSV infection thus involves a balance between the production of Thl and Th2 cytokines by CD4+ and CD8+ T cells.

It has also been found that previous influenza virus infection of the lung reduces the pulmonary eosinophilia and illness caused by G-RSV. Moreover, this effect is long-lasting and transferable to syngeneic mice [7]. The reduction in RSV-induced lung immunopathology was associated with the recruitment and bystander activation of influenza-specific CD8+ T cells displaying intracellular IFN-γ production and cell surface markers of activation following RSV challenge.

It is an object of the invention to provide further and improved ways of reducing immunopathology, particularly in the lung, and more particularly for protecting against pulmonary eosinophilia. DISCLOSURE OF THE INVENTION

To demonstrate the pivotal role played by CD8+ T cells in reducing pulmonary eosinophilia, mice were immunised with the immunodominant CD8 epitope from the second matrix (M2) protein of RSV, prior to G protein priming and RSV challenge. The 'K63' mutant [8] of E.coli heat labile toxin ('LT') was used as an adjuvant [9]. This regime abolished pulmonary eosinophilia and weight loss. Surprisingly, it has now been found that control groups receiving LT-K63 alone benefited from an equivalent protective effect, but this effect was not recognised in reference 9.

Thus the invention provides a method for protecting a patient against immunopathology, comprising the step of administering a detoxified mutant of an ADP-ribosyltransferase to said patient. The invention also provides a detoxified mutant of an ADP-ribosyltransferase for use in protecting a patient from immunopathology. The invention also provides the use of a detoxified mutant of an ADP-ribosyltransferase in the manufacture of a medicament for protecting against immunopathology.

The invention provides a method for modifying the pulmonary phenotype of a patient, comprising the step of administering a detoxified mutant of an ADP-ribosyltransferase to said patient. The invention also provides a detoxified mutant of an ADP-ribosyltransferase for use in modifying the pulmonary phenotype of a patient. The invention also provides the use of a detoxified mutant of an ADP-ribosyltransferase in the manufacture of a medicament for modifying the pulmonary phenotype of a patient. In particular, the pulmonary phenotype may be modified to be biased towards a Thl -type cytokine environment and away from a Th2-type cytokine environment. The invention also provides a composition for preventing and/or treating the effects of viral respiratory infection(s), wherein the composition consists essentially of a detoxified mutant of an ADP-ribosyltransferase. The composition is preferably adapted for mucosal administration.

The immunopathology

The invention is used to protect a patient against immunopathology, and in particular against immunopathological side-effects resulting from an infection or from an antigen.

The immunopathology will generally be a pathogen-induced immunopathology. The pathogen will typically be a virus (e.g. a paramyxovirus, a pneumovirus, or a coronavirus) or a fungus (e.g. a yeast), but may also be a bacterium. The invention is particularly suited for protecting against immunopathology arising from infection by respiratory pathogens (particularly respiratory viruses). The immunopathology may arise from infection by respiratory syncytial virus, Cryptococcus neoformans (any of serotypes A, B, C or D), influenza virus (any type, including A and B), rotavirus, SARS coronavirus, Salmonella typhimurium, Citrobacter rodentium, and/or Mycobacteria (in particular M.tuberculosis and BCG). The ability of a bacterial antigen to modulate damaging immune responses subsequently raised against a viral or eukaryotic antigen is particularly surprising. Alternatively, the invention may be used to protect against vaccine-induced immunopathology. Vaccination with the attachment protein G of RSV results in lung immunopathology after intranasal RSV challenge. The invention can be used prior to vaccination to prevent such immunisation-related side effects, as well as preventing pathogen-induced immunopathology in patients who have previously received a vaccine.

The invention may also be used to protect against an allergen-induced immunopathology and against immunopathology resulting from the particulate nature of an antigen. For example, the invention can be used to protect against asthma, which is characterised by elevated lung eosinophils. The use of a toxin which shifts immune responses towards Thl and away from Th2 (e.g. LT-K63 [10]) is particularly beneficial in the case of allergic reactions. Mutant toxins with little or no enzymatic activity are thus preferred, as wild-type toxins can inhibit dendritic cell IL-12 production and impede IL-12 receptor expression on activated T cells [11].

The immunopathology may affect any organ or tissue of the body, but the invention is particularly suited for protecting against immunopathology of the lung, such as pulmonary eosinophilia. The invention is also suitable for protecting against immunopathological weight loss. Other immunopathological symptoms which can be prevented include atopic disorders, cachexia, malaise, immobility, cyanosis, ruffled fur, rhinitis, coryzea and fever.

The invention is particularly useful for enhancing the production of pathogen-specific mucosal IgA and in increasing the efficiency of antigen presentation. There may be alterations in antigen presenting cells, in chemokine receptor profiles and/or in adhesion molecule expression profiles.

Efficacy can be monitored by standard assays for assessing immunopathology. In the lung, for instance, lung function and cellular infiltration can be assayed by selective lung lavage.

The invention may be used to prevent the future possibility of immunopathology, or to treat existing immunopathology.

The patient

The patient may be any mammal, and is preferably a human. The patient may be an adult or a child.

The patient may be immunocompromised. The use of live or attenuated vaccines is contraindicated in hosts with impaired immune function, as they may not mount an efficient immune response and the vaccine pathogen itself may disseminate or persist. The invention does not suffer from these problems and can give rise to generic protection against a number of unrelated pathogens.

The detoxified mutant of an ADP-ribosyltransferase

ADP-ribosylating bacterial exotoxins are widely known. Examples include diphtheria toxin (Corynebacterium diphtheriae), exotoxin A (Pseudomonas aeruginosά), cholera toxin (CT; Vibrio cholerae), heat-labile enterotoxin (LT; E.coli) and pertussis toxin (PT). Further examples are disclosed in references 12 & 13.

In their native form, the toxins catalyse the transfer of an ADP-ribose unit from NAD⁺ to a target protein. CT, for instance, transfers ADP-ribose to a specific arginine side chain of the α subunit of Gs, which blocks the ability of G_s to hydrolyse GTP to GDP. This locks the protein in its 'active' form, so adenylate cyclase activity is permanently activated. Cellular cAMP levels rise, leading to the active transport of ions from the cell and the loss of water into the gut [14].

The toxins are typically divided into two functionally distinct domains - A and B. The A subunit is responsible for the toxic enzymatic activity, whereas the B subunit is responsible for cellular binding. The subunits might be domains on the same polypeptide chain, or might be separate polypeptide chains. The subunits may themselves be oligomers e.g. the A subunit of CT consists of A_\ and A which are linked by a disulphide bond, and its B subunit is a homopentamer. Typically, initial contact with a target cell is mediated by the B subunit and then subunit A alone enters the cell. The A and/or B subunits of the toxins can be used with the present invention, as appropriate. The toxins are typically immunogenic, and have been proposed for use in subunit vaccines. As well as their immunogenic properties, the toxins have been used as adjuvants. Parenteral adjuvanticity was first observed in 1972 [15] and mucosal adjuvanticity in 1984 [16].

One problem, however, is that the proteins retain their toxic activity in the vaccines. To avoid this problem, site-directed mutagenesis of key active site residues has been used to remove toxic enzymatic activity whilst retaining immunogenicity [e.g. refs. 17 (CT and LT), 18 (PT), 19 etc.]. For example, acellular whooping cough vaccines include a form of pertussis toxin with two amino acid substitutions (Arg⁹→Lys and Glu¹²⁹→Gly; 'PT-9K/129G' [20,21]). An alternative method of detoxification is chemical treatment e.g. with glutaraldehyde. The invention utilises ADP-ribosyl- transferases (but preferably not PT) which have been detoxified, preferably by mutation. The degree of detoxification in mutant toxins used with the invention can vary according to needs. In a typical situation toxicity should be reduced at least 100-fold compared to the wild-type toxin (e.g. 1000-fold, 10000-fold or more) for maximum safety. Where a patient is suffering from severe immunopathology, however, residual toxicity in the toxin may be tolerable. Residual toxicity can be measured by any suitable assay e.g. in mouse cells, in CHO cells, by evaluation of the morphological changes induced in Yl cells, or preferably by the rabbit ileal loop assay.

The toxin can preferably retain the ability to bind GM1 ganglioside receptors.

The toxin used according to the invention is preferably a bacterial toxin. The toxin is preferably CT or LT. Suitable mutants of CT and LT are disclosed in refs. 17 & 22.

In general, LT is a preferred toxin for use with the invention. Two LT mutants of particular interest are LT-K63 (with a Ser⁶³— Lys substitution) and LT-R72 (with an Ala⁷²→Arg substitution), both of which have been evaluated clinically in humans [8]. These substitutions may be combined with one or more further mutations within LT. Corresponding mutants of CT are also known.

Reference 23 discloses mutations in the CTA subunit at Arg-7, Asp-9, Arg-11, His-44, His-70 and Glu-112. Reference 17 discloses detoxified CT and LT proteins having substitutions at one or more of amino acids Val-53, Ser-63, Val-97, Tyr-104 or Pro-106. Reference 24 discloses an LT mutant with a Lys-7 mutation (LT-K7). Reference 25 discloses an LT mutant where arginine at position 192 is substituted with glycine (mLT R192G). These mutations may be combined e.g. 2, 3, 4 or more different mutations.

Further toxins that can be used include diphtheria toxin (DT), Pseudomonas endotoxin A, Pseudomonas exotoxin S, B.cereus exoenzyme, B.sphaericus toxin, C.botulinum C2 and C3 toxins, C.limosum exoenzyme, as well as toxins from C.perfringens, C.spiriforma and C. difficile, Staphylococcus aureus EDIN, etc. Most ADP-ribosylating bacterial toxins are organized as an A:B multimer, wherein the A subunit contains the ADP-ribosyltransferase activity, and the B subunit acts as the binding moiety. New toxins are being identified in new genomes, and mutants of these toxins have been identified [12,13,26]. The production of mutants of new and known toxins is now straightforward and, although the data in the examples were obtained with LT-K63, the skilled person can easily screen further mutant toxins for their ability to reduce immunopathology.

The residue numbering of mutant toxins is relative to the native sequence e.g. residue 63 need not be the 63rd residue of the toxin used according to the invention, but is in the position which corresponds to residue 63 of the native sequence. Residue numbering for CT and LT can be found in reference 27.

Standard alignment techniques can be used to determine the position of any given amino acid according to the native numbering.

As well as using detoxified forms of toxins, the invention can use native forms of toxins in situations where the toxicity of the native toxin is either not exhibited under the circumstances of use (e.g. the toxin is administered by a route where it cannot exert any toxic effect) or is not of concern (e.g. in a patient where the severity of disease is so great that a risk/benefit assessment shows that the toxin should be administered despite of its toxicity).

Administration of the detoxified mutant The detoxified mutant can be delivered to various parts of the body by various routes. In general, however, it will be delivered to the cells, tissues and/or organs which are to be protected against immunopathology. If the lung is to be protected, for example, the mutant toxin will be delivered to the lung. Administration need not be directly to the lung, as material can be transported to the lung by inhalation e.g. by oral or intranasal administration of an aerosol. Intranasal administration is preferred. Of the various mucosal delivery options available, this route is the most practical as it offers easy access with relatively simple devices that have already been mass produced. Alternative routes for mucosal delivery of the vaccine are oral, intragastric, pulmonary, intestinal, rectal, ocular, respiratory and vaginal routes. The dose(s) suitable for protecting against immunopathological effects will vary according to host organism, target tissue, health and physical condition of the patient, age, the capacity of the individual's immune system to mount T cell responses, the degree of protection desired, the formulation of the administered composition, the treating doctor's assessment of the medical situation, and other relevant factors. The dose can fall in a relatively broad range, which can be determined through routine trials. In mice, for instance, it has easily been established that a dose of lμg is moderately effective in preventing RSV-induced pulmonary eosinophilia, and 5μg is wholly effective. The dose may be given in a single administration or as part of a series.

The mutant ADP-ribosyltransferase may be administered to the patient without co-administration of an antigen from the pathogen which induces the immunopathology in question. For instance, to prevent RSV-induced immunopathology the mutant ADP-ribosyltransferase may, surprisingly, be administered without an RSV antigen. Compositions for use with the invention may thus contain the mutant toxin as essentially the only antigenic component.

Compositions and medicaments for use with the invention

Compositions used according to the invention will generally have a pH between 6 and 8, preferably about 7. pH may be maintained by the use of a buffer. The compositions may be sterile and/or pyrogen-free.

Compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition). The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. Compositions are preferably adapted for intranasal administration, such as by nasal spray, nasal drops, gel or powder [e.g. refs. 28 & 29].

Further components of the composition

The composition of the invention will typically, in addition to the components mentioned above, comprise one or more 'pharmaceutically acceptable carriers', which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The compositions may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. A thorough discussion of pharmaceutically acceptable excipients is available in reference 30.

As toxoids such as LT are already adjuvantitious, it is not in general necessary to administer them with further adjuvants. To improve efficacy (e.g. to prolong half-life, or to modulate immune helper sub-populations), however, compositions of the invention may be administered in conjunction with other immunoregulatory agents. In particular, compositions will usually include an adjuvant. Preferred further adjuvants include, but are not limited to, one or more of the following set forth below:

A. Mineral Containing Compositions Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphoshpates, orthophosphates), sulphates, etc. (e.g. see chapters 8 & 9 of Vaccine design: the subunit and adjuvant approach (1995) Powell & Newman. ISBN 0-306-44867-X.), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt. See WO00/23105.

B. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). See WO90/14837. See also, Frey et al., "Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non- adjuvanted influenza vaccine in non-elderly adults", Vaccine (2003) 21:4234-4237.

Particularly preferred adjuvants for use in the compositions are submicron oil-inwater emulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80 ™ (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85™ (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L- alanine-2-( -2'-dipalmitoyl-OT-glycero-3-huydroxyphosphophoryloxy)-ethylamine (MTP-PE), for example, the submicron oil-in-water emulsion known as "MF59" (International Publication No. WO 90/14837; US Patent Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties; and Ott et al., "MF59 — Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines" in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M.F. and Newman, MJ. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene (e.g., 4.3%), 0.25-0.5%) w/v Tween 80™, and 0.5% w/v Span 85™ and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA). For example, MTP-PE may be present in an amount of about 0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100 μg/dose. As used herein, the term "MF59-0" refers to the above submicron oil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes a formulation that contains MTP-PE. For instance, "MF59-100" contains 100 μg MTP-PE per dose, and so on. MF69, another submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25%) w/v Tween 80™, and 0.75% w/v Span 85™ and optionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 0.4% Tween 80™, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion. MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 μg MTP-PE per dose. Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO 90114837 and US Patent Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention.

C. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.

Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP- LC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in US Patent No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO 96/33739). Combinations of saponins and cholesterols can be used to form unique particles called

Immunostimulating Complexs (ISCOMs). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP 0 109 942, WO 96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid of additional detergent. See WO00/07621. A review of the development of saponin based adjuvants can be found at Barr, et al, "ISCOMs and other saponin based adjuvants", Advanced Drug Delivery Reviews (1998) 32:247-271. See also Sjolander, et al., "Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines", Advanced Drug Delivery Reviews (1998) 32:321-338.

D. Bacterial or Microbial Derivatives Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as:

(DI) Non-toxic derivatives of enterobacterial lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. See Johnson et al. (1999) BioorgMed Chem Lett 9:2273-2278.

(D2) Lipid A Derivatives Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Meraldi et al, "OM-174, a New Adjuvant with a Potential for Human Use, Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242- 310 from the circumsporozoite protein of Plasmodium berghei", Vaccine (2003) 21:2485-2491; and Pajak, et al, "The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo", Vaccine (2003) 21:836-842.

(D3) Immunostimulatory oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine may be replaced with an analog such as 2'-deoxy-7-deazaguanosine. See Kandimalla, et al, "Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles", Nucleic Acids Research (2003) 31(9): 2393-2400;

WO 02/26757 and WO 99/62923 for examples of possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, "CpG motifs: the active ingredient in bacterial extracts?", Nature Medicine (2003) 9(7): 831-835; McCluskie, et al, "Parenteral and mucosal prime- boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA", FEMS Immunology and Medical Microbiology (2002) 32:179-185; WO 98/40100; US Patent No. 6,207,646; US Patent No. 6,239,116 and US Patent No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. See Kandimalla, et al, "Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs", Biochemical Society Transactions (2003) 3_i (part 3): 654-658. The CpG sequence may be specific for inducing a Thl immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al, "CpG-A-Induced Monocyte IFN-gamma-Inducible Protein- 10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha", J. Immunol. (2003) 170(8):4061-4068; Krieg, "From A to Z on CpG", TRENDS in Immunology (2002) 23(2): 64-65 and WO 01/95935. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, Kandimalla, et al, "Secondary structures in CpG oligonucleotides affect immunostimulatory activity", BBRC (2003) 306:948-953; Kandimalla, et al, "Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic GpG DNAs", Biochemical Society Transactions (2003) 31 (part 3):664-658; Bhagat et al, "CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents" BBRC (2003) 300:853-861 and WO 03/035836.

(D4) ADP-ribosylating toxins and detoxified derivatives thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin "LT), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211 and as parenteral adjuvants in WO 98/42375. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP- ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al, "The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin", Infection and Immunity (2002) 70(6):3012- 3019; Pizza, et al, "Mucosal vaccines: non-toxic derivatives of LT and CT as mucosal adjuvants", Vaccine (2001) 19:2534-2541; Pizza, et al, "LTK63 and LTR72, two mucosal adjuvants ready for clinical trials" Int. J. Med. Microbiol (2000) 290(4-5):455-461; Scharton-Kersten et al, "Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits and Unrelated Adjuvants", Infection and Immunity (2000) 68(9):5306-5313; Ryan et al, "Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Thl and Th2 Cells" Infection and Immunity (1999) 67(12):6270-6280; Partidos et al, "Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides", Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al, "Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines", Vaccines (2003) 2(2):285-293; and Pine et al, (2002) "Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63)" J. Control Release (2002) 85(l-3):263-270. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al, Mol. Microbiol (1995) 15(6):1165-1167, specifically incorporated herein by reference in its entirety.

E. Human Immunomodulators Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention. E.g., WO99/27960.

G. Microparticles Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ~100nm to ~150μm in diameter, more preferably ~200nm to ~30μm in diameter, and most preferably ~500nm to ~10μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes

Examples of liposome formulations suitable for use as adjuvants are described in US Patent No.

6,090,406, US Patent No. 5,916,588, and EP 0 626 169. I. Polyoxy ethylene ether and Poly oxy ethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxy ethylene ethers and polyoxyethylene esters. WO99/52549. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WOO 1/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152).

Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene- 4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. J Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al, "Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions", Biomaterials (1998) 19(1- 3): 109-115 and Payne et al, "Protein Release from Polyphosphazene Matrices", Adv. Drug. Delivery Review (1998) 31(3):185-196. K. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l '-2'-dipalmitoyl-.s'«- glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE). L. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues, described further in Stanley, "Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential" Clin Exp Dermatol (2002) 27(7):571-577 and Jones, "Resiquimod 3M", Curr Opin Investig Drugs (2003) 4(2):214-218. M. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA- phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi). VLPs are discussed further in WO 03/024480, WO 03/024481, and Niikura et al, "Chimeric Recombinant Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle Presenting Foreign Epitopes", Virology (2002) 293:273-280; Lenz et al, "Papillomarivurs-Like Particles Induce Acute Activation of Dendritic Cells", Journal of Immunology (2001) 5246-5355; Pinto, et al, "Cellular Immune Responses to Human Papillomavirus (HPV)-16 LI Healthy Volunteers Immunized with Recombinant HPV-16 LI Virus-Like Particles", Journal of Infectious Diseases (2003) 188:327-338: and Gerber et al, "Human Papillomavrisu Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Entertoxin Mutant R192G or CpG", Journal of Virology (2001) 75(10):4752-4760. Virosomes are discussed further in, for example, Gluck et al, "New Technology Platforms in the Development of Vaccines for the Future", Vaccine (2002) 20:B10 -B16.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention:

(1) a saponin and an oil-in-water emulsion (WO99/11241);

(2) a saponin (e.g., QS21) + a non-toxic LPS derivative (e.g., 3dMPL) (see WO 94/00153);

(3) a saponin (e.g., QS21) + a non-toxic LPS derivative (e.g., 3dMPL) + a cholesterol;

(4) a saponin (e.g. QS21) + 3dMPL + IL-12 (optionally + a sterol) (WO98/57659); (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (See

European patent applications 0835318, 0735898 and 0761231);

(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-

MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid

A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS

(Detox™); and

(8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dPML).

Aluminium salts (aluminium hydroxide in particular) and MF59 are preferred adjuvants for parenteral immunisation. Mutant bacterial toxins are preferred mucosal adjuvants.

Further antigens

Although toxoids such as LT can be used on their own to protect against immunopathological side- effects in general, other antigen(s) may be co-administered, with the toxoid enhancing the immune response raised against the other antigen(s). Further antigens for use with the invention include:

- an outer-membrane vesicle (OMV) preparation from N.meningitidis serogroup B, such as those disclosed in refs. 31, 32, 33, 34, etc.

- a saccharide antigen from N.meningitidis serogroup A, C, W135 and/or Y, such as the oligosaccharide disclosed in ref. 35 from serogroup C [see also ref. 36] or the oligosaccharides of ref. 37. - antigens from Helicobacter pylori such as CagA [38 to 41], VacA [42, 43], NAP [44, 45, 46], HopX [e.g. 47], HopY [e.g. 47] and/or urease.

- a saccharide antigen from Streptococcus pneumoniae [e.g. 48, 49, 50].

- an antigen from hepatitis A virus, such as inactivated virus [e.g. 51, 52]. - an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 52, 53].

- an antigen from hepatitis C virus [e.g. 54].

- a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 55] e.g. the CRMι₉₇ mutant [e.g. 56].

- a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 76]. - an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 57 & 58].

- a saccharide antigen from Haemophilus influenzae B [e.g. 36].

- polio antigen(s) [e.g. 59, 60] such as OPV or, preferably, IPV. - an antigen from N.gonorrhoeae [e.g. 61, 62, 63, 64].

- an antigen from Chlamydia pneumoniae [e.g. refs. 65 to 71].

- an antigen from Chlamydia trachomatis [e.g. 72].

- an antigen from Porphyromonas gingivalis [e.g. 73].

- rabies antigen(s) [e.g. 74] such as lyophilised inactivated virus [e.g. 75, RabAvert™]. - measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 76].

- influenza antigen(s) [e.g. chapter 19 of ref. 76], such as the haemagglutinin and/or neuraminidase surface proteins.

- antigen(s) from a paramyxovirus such as respiratory syncytial virus (RSV [77, 78]) and/or parainfluenza virus (PIV3 [79]). - an antigen from Moraxella catarrhalis [e.g. 80].

- an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 81, 82, 83].

- an antigen from Staphylococcus aureus [e.g. 84].

- an antigen from Bacillus anthracis [e.g. 85, 86, 87].

- an antigen from a virus in the flaviviridae family (genus flavivirus), such as from yellow fever virus, Japanese encephalitis virus, four serotypes of Dengue viruses, tick-borne encephalitis virus, West Nile virus.

- an antigen from the SARS coronavirus.

- a pestivirus antigen, such as from classical porcine fever virus, bovine viral diarrhoea virus, and/or border disease virus. - a parvovirus antigen e.g. from parvovirus B19.

- a prion protein (e.g. the CJD prion protein)

- an amyloid protein, such as a beta peptide [88] - a cancer antigen, such as those listed in Table 1 of ref. 89 or in tables 3 & 4 of ref. 90.

The composition may comprise one or more of these further antigens.

Preferred further antigens are those from respiratory-related pathogens (e.g. from RSV, from SARS-CoV, from influenza virus). Further preferred antigens are those that are used during pediatric immunisation (e.g. diphtheria, tetanus, pertussis, pneumococcus, Hib, HBV, poliovirus).

Where a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier protein in order to enhance immunogenicity [e.g. refs. 91 to 100]. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRMι₉₇ diphtheria toxoid is particularly preferred [101]. Other carrier polypeptides include the N.meningitidis outer membrane protein [102], synthetic peptides [103, 104], heat shock proteins [105, 106], pertussis proteins [107, 108], protein D from H.influenzae [109], cytokines [110], lymphokines [110], hormones [110], growth factors [110], toxin A or B from C. difficile [111], iron-uptake proteins [112] etc. Where a mixture comprises capsular saccharides from both serogroups A and C, it is preferred that the ratio (w/w) of MenA saccharide :MenC saccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Saccharides from different serogroups oϊ N.meningitidis may be conjugated to the same or different carrier proteins. Any suitable conjugation reaction can be used, with any suitable linker where necessary.

Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [58]).

Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens.

Antigens in the composition will typically be present at a concentration of at least lμg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

As an alternative to using protein antigens in the composition of the invention, nucleic acid encoding the antigen may be used [e.g. refs. 113 to 121]. Protein components of the compositions of the invention may thus be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid) that encodes the protein.

Definitions

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The term "about" in relation to a numerical value x means, for example, x+10%. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the effect of treatments on (A) body weight, (B) eosinophil numbers, (C) CD4 cells in bronchoalveolar lavage, (D) CD8 cells in bronchoalveolar lavage, (E) CD4 cells in the lung, or (F) CD8 cells in the lung, after RSV infection. In Figures IB to IF the four groups of data were obtained from mice pre-treated with: (1) RSV peptide + LT-K63; (2) LT-K63 alone; or (3) PBS, and mice received either a primary infection (group 4) using the βgal-RSV vector or a normal RSV infection (groups 1, 2 & 3) using the rVV-G vector. Each point represents an individual mouse and is representative of 3 separate experiments.

Figure 2 shows the effect of LT-K63 dose (left-to-right in each graph: Og, 0.2μg, lμg, 5μg per dose) on (A) eosinophils, (B) neutrophils, (C) macrophages, (D) lymohpcytes, (E) total cell count and (F) total lymphocytes.

Figure 3 shows the TNF:IL-4 ratio in lungs (3A & 3B) or airways (3C & 3D). Figures 3A & 3C show CD4 cells, and Figures 3B & 3D show CD8 cells.

Figure 4 shows the effect of LT-K63 dose on RSV-specific recall responses. Figure 5 shows the effect of LT-K63 on (A) weight loss and (B) eosinophils, following intranasal vs. intraperitoneal administration

Figure 6 shows the effect of LT-K63 on (A) total lymphocytes, (B) macrophages, (C) CD4+ and CD8+ cells in the lung, (D) CD4+ and CD8+ cells in the airways, (E) total T cells in the lung, (F) total T cells in the airways, (G) CD44+ cells in the lung and (H) CD44+ cells in the airways. Figure 7 shows cell counts (xlO⁶) during C.neoformans infection. 7A & 7B show total cells, whereas 7C & 7D show only eosinophils. Cells were counted in lungs (7A & 7C) or airways (7B & 7D). The LT-treated group ('B') shows much lower cell numbers.

Figure 8 shows the change in weight over 7 days of influenza virus infection. Animals were pre-treated either with (8A) LT-K63 or (8B) PBS. Figure 9 shows total cells (♦) and lymphocytes (■) recovered from the lung after LT-K63 was given. Cell numbers are given xlO⁶.

Figure 10 shows % lymphocytes expressing CD4 (right axis; ■) and the total number xlO⁴ of CD4+ cells (left axis; ♦) following LT-K63 administration.

Figure 11 shows flow cytometric plots of CD4 (y axis) vs. TNF (x axis) at day 0 and day 10. Figure 12 shows % of lymphocytes co-expressing (12A) CD4 or (12B) CD8 in the TCR Vβ gene products shown on the x-axis. Open bars are PBS controls; filled bars are LT-K63-treated.

Figure 13 shows lung cellularity (xlO⁵).

Figure 14 shows the proliferative response of CD8+ T cells during influenza virus challenge, measured by a tritiated thymidine incorporation assay (values are counts per minute, xlO³). Cells were incubated with the immunodominant CD 8 epitope for RSV or influenza, LT-K63 itself or Con A. Open bars are PBS controls; open bars are LT-K63-pre-treated.

Figure 15 shows the IFN-γ/IL-5 ratios. Figure 15A shows results of intranasal administration at the indicated doses. Figure 15B shows results of 5μg intraperitoneal or intranasal doses. Figure 16 shows total number of CD4+ T cells (xlO³) on the left axis and % CD4+ cells on the right axis. Figure 16A shows IFNγ, and Figure 16B shows IL-5.

Figure 17 shows optical density of ELISA seven days after (A) RSV or (B) influenza virus infection.

Figure 18 shows lung tissue, formalin-fixed and embedded in paraffin at the peak of the inflammatory response (day 7 in A-D, and day 12 in E-F). Panels A, C and E show PBS control tissue; Panels B, D and F show tissue from LT-K63 -treated mice. The mice had been infected with G/RSV (A B), influenza virus (C/D) or C.neoformans (E/F). Figure 19 shows pathogen titres from the tissue: (19A) G/RSV CT value; (19B) influenza virus pfu; (19C) C.neoformans cfu.

Figure 20 shows cpm (xlO³) after a lμCi [³H]-thymidine assay.

Figure 21 shows % weight loss in mice after intranasal influenza virus infection. MODES FOR CARRYING OUT THE INVENTION

Weight loss induced by G protein

It is known that prior exposure of the lung to RSV prevents weight loss in mice further vaccinated with the G protein and re-challenged with RSV. To see if RSV-specific CD8+ T cells mediate this inhibition, an immunisation regime known to induce an efficient RSV-specific CTL response, capable of lysing RSV-specific targets [9] was used. BALB/c mice (8-10 weeks old) were intranasally immunised on three consecutive weeks with 24μg the immunodominant H-2k^d peptide (SYIGSINNI) from the second matrix protein (M2) of RSV together with LT-K63 (5μg) in 50μl PBS. One week after the final immunisation mice were scarified with 10⁶ pfu recombinant vaccinia virus ('rVV-G') expressing the G protein (or expressing a control protein β-gal) and challenged intranasally with whole RSV (strain A2) 2-3 weeks later (denoted as G-RSV).

Mice were weighed daily for 7 days following intranasal RSV challenge. Data were normalised to the group weight at challenge and are shown in Figure 1A as the mean +/- SEM. Treatment groups were RSVpep-G-RSV (closed triangles), LT-G-RSV (open triangles), PBS-G-RSV (closed diamonds) and βgal-RSV (open diamonds). As expected, mice undergoing a primary RSV infection (βgal-RSV) showed mild weight loss during the later stages of infection with recovery by day 7. Scarification with the G protein followed by intranasal RSV challenge (G-RSV) enhanced and accelerated this weight loss. Previous exposure of the lung to RSV peptide and LT-K63 prevented weight loss in G-RSV mice. Surprisingly, G-induced weight loss was also prevented by LT-K63 alone. There was a highly significant difference between mice previously immunised with LT-K63 either alone (p=**) or with RSV peptide (p=**) compared to G-RSV mice. The cellular composition of the bronchoalveolar lavage (BAL) fluid from the airways was analysed by haemotoxylin and eosin (H & E) staining of cytocentrifuge lung lavage preparations. BAL fluid from the airways, lung tissue and serum were obtained from mice as described previously [122]. In brief, lungs were inflated 6-times with 1.5ml Eagle's Minimum Essential Medium containing lOmM EDTA and kept on ice. 100 μl of this BAL fluid was cytocentrifuged onto glass slides, fixed in methanol and stained with haemotoxylin and eosin. The remainder was centrifuged, the supernatant decanted and the cell pellet re-suspended to lxl 0⁶ cells/ml in RPMI containing 10% FCS, 2 mM L-glutamine, 50μg/ml penicillin and 50μg/ml strepamyacin. Lung tissue was disrupted using 0.8μm filters to obtain single cell suspensions, the red blood cells lysed and the cell pellet re-suspended at lxlO⁶ cells/ml in R10F medium. A single lobe of lung tissue was frozen over liquid nitrogen and stored at -80°C until further use.

Eosinophils in the BAL were enumerated by flow cytometry based on their size (forward scatter) and granularity (side scatter). The proportion of eosinophils was confirmed by their distinctive nuclear morphology and presence of acidophillic red granules. Neutrophils were identified by the presence of a multi-lobed nuclei and the absence of acidophilic granules.

The percentage of eosinophils in the BAL is shown in Figure IB. Mice experiencing a primary RSV infection (βgal-RSV; fourth column) did not develop pulmonary eosinophilia, whereas in those mice scarified with the G protein and challenged with RSV (third column) approximately 20% of cells from the airways were eosinophils. Previous exposure to RSV peptide and LT-K63 (first column on left-hand side) or to LT-K63 alone (second column) prevented eosinophilic inflammation during RSV challenge. Prior nasal administration of LT-K63 thus prevents G-induced weight loss and eosinophilia, even in the absence of RSV peptide.

Cells recovered by lavage (Figures IC and ID) and lung cell suspensions (Figures IE and IF) were surface stained for CD4 (Figures IC and IE) and CD8 (Figures ID and IF). Consistent with previous studies, during primary infection (column 4) CD8+ T cells dominated in the BAL whereas previous scarification with the G protein (column 3) enhanced CD4+ T cell accumulation (Figures IB and IC). Intranasal LT-K63 alone did not alter the proportion of CD4 or CD8 T cells in G-primed and RSV-challenged mice (column 2). Co-administration of LT-K63 with the RSV peptide significantly shifted the CD4:CD8 ratio in favour of CD8+ T cells (column 1). This skewing was evident in both the airways (IB and IC) and lung (IE and IF). Mucosal administration of peptide therefore successfully induced RSV specific CD8+ T cells, but these cells were not essential for the reduction in weight loss and eosinophilia in G-RSV mice. This effect was induced by the administration of LT-K63 alone.

LT-K63 dosage The effect of LT-K63 dose on its ability to abolish lung eosinophilia in mice was investigated. LT-K63 was intranasally administered to 8-10 week old balb/c mice every 7 days for 3 weeks at a dose of 0.2μg, lμg or 5μg. Control mice received PBS. One week later mice were scarified with a recombinant vaccinia virus construct expressing the G-protein of RSV and intranasally challenged with whole RSV 2-3 weeks later. Eosinophils were enumerated from cytocentrifuged lung lavage specimens as described above (Figure 2A). Neutrophils (2B), lymphocytes (2C) and macrophages (2D) were also enumerated in lavage cytospins by their nuclear morphology and cytoplasmic characteristics. Total live cells (2e) recovered after lavage were counted by trypan blue exclusion, and the total number of lymphocytes present (2F) was calculated by multiplying the percent lymphocytes by the total viable cell count. Results are shown in Figure 2, with the four groups in each graph from left to right having received Oμg (PBS only), 0.2μg, 2μg and 5μg of LT-K63. Prevention of eosinophilia was found to be directly related to dose.

Although 5μg LT-K63 prevented eosinophilia, lμg only reduced eosinophilia in 3/5 mice compared to the control PBS treated group (p=0.22) (Figure 2A). In addition to reducing eosinophilia, LT-K63 also caused a dose dependent increase in neutrophils and macrophages (Figures 2B & 2C) but not lymphocytes (Figure 2D) in the airways. Total cell recruitment into the airways was significantly (p=**) inhibited in mice previously given 5μg LT alone prior to G-RSV (Figure 2E). This translates into a reduction in the actual number of lymphocytes present (Figure 2F). Examining the lymphocyte population further, no significant alterations were noted in the proportion of CD4+ or CD8+ T cells. T cell cytokine profiles in the mice were analysed by intracellular staining and flow cytometry. TNF-expressing and IL4-expressing CD4 and CD 8 T cells in the lung or airways were enumerated by intracellular staining and flow cytometry. Figure 3 shows the ratio of TNF-expressing cells to IL4 expressing cells in the lungs (3A & 3B) or the airways (3C & 3D). Figures 3A & 3C show CD4 cells, and Figures 3B & 3D show CD8 cells. Administration of 5μg LT-K63 significantly increased the intracellular expression of TNF and reduced the expression of IL-4 in both CD4+ (p=0.043) and CD8+ (p=0.035) T cell subsets. This effect was particularly evident in cells extracted from the airways (3C & 3D).

The effect of dose on prevention of weight loss in the RSV infection model was also tested. Higher doses of LT-K63 were found to give greater reductions in RSV-induced weight loss (Figure 13).

LT-K63 enhances RSV-specific splenocyte proliferation

As shown above, LT-K63 decreases cell recruitment to the lung when administered prior to G-RSV, although those cells remaining produce high levels of intracellular TNF. To determine whether reduced cell recruitment moderates recall responses, splenocytes from mice which received different doses of LT-K63 followed by G-RSV were tested for proliferative responses to the immunodominant RSV G-protein peptide . Mice (Balb/C, 8-10 weeks old) received 0.2μg, lμg or 5μg LT-K63 (or PBS as a control) by intranasal administration every 7 days for 3 weeks. One week later mice were scarified with a recombinant vaccinia virus construct expressing the G-protein of RSV and intranasally challenged with whole RSV 2-3 weeks later. Spleens were removed from individual mice 7 days after RSV infection under sterile conditions and passed through a 0.8μm filter to produce a single cell suspension. After red blood cell lysis in 0.15M ammonium chloride, 1M potassium hydrogen carbonate and O.OlmM EDTA (pH 7.2), spleen cells were re-suspended in R10F media (2xl0⁶ cells/ml). 4xl0⁵ cells were added to each well of a round bottomed 96-well plate together with 25μg of purified G-protein peptide (AICKRIPNKKPGKKT; residues 184-198). Control wells were incubated with 25 μg of non-stimulatory G-protein peptide in which the major MHC class II I-E^d anchor region had been deleted (AICKRIPKNPGKKT). All samples were analysed in triplicate. After 48 hours at 37°C, 50μCi/ml of [³H]-thymidine was added for a further 24 hours. Cells were harvested, the incorporated [³H]-thymidine transferred to a filter and the beta-emissions read using a scintillation counter. The proliferation index was calculated by dividing the value obtained for the stimulatory peptide by the equivalent well containing control peptide. The results in Figure 4 represent the proliferative index of the mean +/- SEM of 5 duplicated samples.

Despite the reduction in cell recruitment to the lung, mice given 5μg LT-K63 prior to G-RSV exhibited the highest proliferative response. Lower doses of LT-K63 were not significantly different (p=**) from mice treated with PBS prior to G-RSV (Figure 4).

LT-K63 can thus enhance RSV-specific recall responses.

Route of administration

The above findings involved the lung microenvironment as the site of immune-education and subsequent effector function. For comparison, intraperitoneal administration was tested. LT-K63 (5μg) or PBS was intranasally or intraperitoneally administered to 8-10 week old BALB/c mice every 7 days for 3 weeks. One week later mice were scarified with a vaccinia virus construct expressing the G-protein of RSV and intranasally challenged with whole RSV 2-3 weeks later.

Mice were weighed daily following intranasal RSV challenge. Data were normalised to the group weight at challenge and shown as mean +/- SEM in Figure 5A. Confirming the previous findings, mice receiving 5μg LT-K63 intranasally (open triangles) were protected against weight loss. When administered intraperitoneally (open squares), however, LT-K63 had no protective effect. Similarly, intranasal (closed triangles) or intraperitoneal (closed squares) PBS had no protective effect.

Eosinophils were enumerated from cytocentrifuged lung lavage specimens, as described above. The only treatment to show protection against eosinophilia was intranasal administration of LT-K63 (Figure 5B, third column from left). The beneficial effects of LT-K63 therefore depend on the correct route of delivery, as intraperitoneal administration had no effect. The reduction in weight loss and eosinophilia was not related to the anaesthetic or the administration medium as the same volume of PBS had no beneficial effect.

Eosinophilia caused by Cryptococcus neoformans C.neoformans is a pathogenic yeast which causes cryptococcosis, often clinically manifested as meningoencephalistis. Primary infection of C57BL/6 mice with C.neoformans induces extensive lung eosinophilia, again driven by induction of Th2 cells [123]. Prior administration of LT-K63 was found to protect against eosinophilia induced by C.neoformans.

5μg LTK63 was administered intranasally to 8-12 week old female BALB/c mice. After 2 weeks, the mice were challenged with 2 x 10⁴ C.neoformans by the same route. Lung lavage and residual lung tissue were removed 12 days later. Total viable cell count was determined by trypan blue exclusion.

Eosinophils were enumerated by histological analysis of H and E stained cytocentrifuge preparations.

The proportion of eosinophils was confirmed by flow cytometry. As shown in Figure 7, animals pre-treated with the mutant toxin (group 'B') showed lower total cell counts (7A & 7B) and lower eosinophil counts (7C & 7D) in the lungs (7A & 7C) and airway (7B & 7D) during C.neoformans infection when compared to animals pre-treated with PBS alone (group 'A').

Weight loss caused by influenza virus

Infection with influenza virus may result in immunopathological symptoms, including weight loss due to excessive cellular infiltration and the production of inflammatory cytokines. Human influenza A virus infection typically causes tracheobronchitis and occasional fatal pneumonia and, in mice, it induces similar inflammation with infiltration of neutrophils and Thl -driven CD8+ T cells.

As LT-K63 activates lung APCs and stimulates a mild Thl cytokine environment, mice were treated with LT-K63 prior to influenza virus infection. 5μg LTK63 was administered intranasally to 8-12 week old female BALB/c mice. After 2 weeks, 50 HA units of influenza virus (strain X31) was applied by the same route. The weight of each mouse was recorded daily and the percent weight loss determined from the original body weight prior to infection. As shown in Figure 8, pre-treatment of animals with LT-K63 (8A) resulted in little or no weight loss over the 7 days following influenza virus infection when compared to animals pre-treated with PBS alone (8B).

LT-K63 modifies the lung microenvironment BALB/c mice were intranasally immunised with 5μg LT-K63 and lung compartments were sampled every other day over 14 days. The total number of lymphocytes was tracked after immunisation (Figure 6A; Figure 9) by flow cytometry using antibodies to CD4, CD8 and B cells to delineate the correct population. The mild increase in cellularity was mainly caused by lymphocytic expansion (Figure 9). Figure 6B shows the numbers of macrophages (open squares) and granulocytes (closed squares), enumerated by flow cytometry based on size and granularity. Total CD4+ (diamonds) and CD8+ (squares) cells were enumerated by multiplying the percent CD4+ and CD8+ positive cells by the number of live cells recovered from the lung (Figure 6C) or airways (6D). Although both CD4+ and CD8+ cells increased in number, the proportion of the two subsets was essentially constant throughout the analysis. Figures 6E (lung) and 6F (airways) show the number of T cells expressing high levels of CD44 over the time course. CD44 FACS analysis is shown on days 0 and 10 in the lung (Figure 6G) and the airways (Figure 6H).

Major alterations in the cellular content of the lung tissue were observed, as the number of lymphocytes, granulocytes and macrophages increased. Similar effects were evident in the airways, but the lymphocytic response was less pronounced. Flow cytometric analysis revealed that LT-K63 predominantly affected the CD4+ T cell population, which increased rapidly in number (6C) and activity (6E) in the lung and in the airways (6D and 6F). This effect was less pronounced in the airways compared to the lung and the difference between CD4+ and CD8+ T cells less evident.

The most striking effect exerted by LT-K63 was on the antigen presenting cell compartment. The proportion and total number of B cells increased here, as did their expression of markers of activation (MHC class II, CD40L and CD80; Figure 10). LT-K63 induced a Thl-type cytokine profile in the lung, with increased TNF (Figure 11) and IFN-γ, but not increased IL-4 or IL-5. This enhanced state of activation in T cells and APCs persisted for 14 days. Further analysis till day 28 showed that the lung had essentially returned to a pre-treatment condition. Furthermore, the CD4+ and CD8+ T cell clonality was analysed based on the proportion of cells expressing different Vβ TCRs. The CD4+ Vβ8.1/8.2 population and the CD8+ Vβ2 populations increased (Figure 12). These populations, receded with time however, and were back at pre-infection levels by day 14.

Thus nasal instillation of LT-K63 alone causes T cell recruitment and activation, and the detoxified toxin seems to mature the lung environment such that subsequent infections are handled better. This effect may be due to the maturation of antigen presenting cells in the lung, or to the minimal Thl infiltration to LT-K63 alone.

Histology

Histological (H and E) analysis of lung sections confirmed that prior LT-K63 treatment significantly reduced the inflammatory infiltrate during G/RSV (Figure 18B, vs. PBS control in Figure 18A), influenza virus (Figure 18C/D) and C.neoformans (Figure 18E/F) infections. Analysis of pathogen titre in lung homogenates showed that LT-K63 had no effect on RSV titres 7 days after infection (Figure 19A) but markedly reduced both influenza virus (Figure 19B) and C.neoformans (Figure 19C) titres. Moreover, influenza virus was undetectable on day 7 after infection. In addition, LT-K63 pre-treatment prevented uncontrolled pulmonary C.neoformans dissemination to the brain. T cell activation

LT-K63 was found to reduce Th2-driven eosinophilia and immunopathology during infection, while at the same time moderating illness and pathology to Thl -driven influenza infection. The reasons for this activity were investigated. The proportion of CD4+ and CD8+ T cells was unaltered in both the G/RSV and influenza virus infection models, but a greater proportion of T cells were activated. The LT-K63-induced increase in the activation of CD8+ T cells (8-fold) was greater than that observed for CD4+ T cells (2 -fold) in the G/RSV, influenza virus and C.neoformans lung infection models. Prior LT-K63 treatment enhanced the RSV-specific and influenza-specific CD8+ T cell proliferative responses following both RSV (Figure 4) and influenza virus infection (Figure 14).

As T cells generally exert their effect on eosinophils by secretion of cytokines, T cell cytokine profiles were investigated by intracellular staining and flow cytometry in the G/RSV infection model. Generally, the higher the dose of LTK63, the lower the Thl and Th2 cytokines present during subsequent infection. For example, control mice had 17009 +/- 120 TNF producing CD4+ T cells at day 7 after RSV challenge whereas those given 5μg LT-K63 had only 7083 +/- 87 (p<0.05). Of the cytokines remaining in the 5 μg LTK63 treated group, intracellular TNF or IFN-γ dominated over IL-5 in both CD4+ (Figure 15 A; p=0.043) and CD8+ (p=0.035) T cell subsets. This effect was seen in mice given LT-K63 intranasally, but not intraperitoneally (Figure 15B). Treatment of mice with 5μg LT-K63 14 days prior to influenza infection also reduced the total number of CD4+ and CD 8+ T cells producing IFN-γ (Figure 16A) or IL-5 (Figure 16B). Despite reduced cytokine production, significant IgA was detected in nasal washes taken from mice infected with G/RSV (Figure 17A) or influenza virus (Figure 17B). In both cases prior intranasal LTK63 treatment enhanced recovered antigen specific IgA.

Antigen processing LT-K63 is shown above to stimulate a mild Thl environment in the lung, and to mature antigen presenting cells by increasing MHC class II, B7 co-stimulatory molecules and CD40. The outcome and immune phenotype of subsequent lung infections are altered beneficially. Though total T cells are reduced during pathogen challenge, the proportion of activated cells in those remaining is increased. This increase was investigated further. The ability of naϊve mouse alveolar cells (>95% macrophages by flow cytometry) to process and present whole ovalbumin to ovalbumin-specific T cells (DO 11.10 T cells) was analysed, and the proliferative responses were compared to OVA-specific peptide. Untreated lung macrophages efficiently induced proliferation of DO 11.10 T cells in the presence of ovalbumin or OVA peptide (Figure 20, left-hand side). In contrast, macrophages removed from LT-K63 primed mice proliferated well in response to peptide but not to whole ovalbumin (Figure 20, right-hand side). This difference suggests that presentation of peptides to antigen-specific T cells is not affected by LT-K63, but that processing of whole antigens is reduced.

The alteration of lung antigen presenting cells by LT-K63 may explain the difference in subsequent immune pathology to unrelated infections. To prove the impact of LT-K63 modulated APCs on subsequent infection, alveolar cells were removed by lavage (>95% pure macrophages) from RAG-/- mice (i.e. lacking B and T cells) that had been pre-treated or not with 5μg LT-K63 three weeks earlier. lxlO⁵ cells were transferred intranasally into immunocompetant littermates. Mice were then infected with influenza virus one day later and weight loss was evaluated. Transfer of LTK63, but not PBS treated lung cells reduced weight loss during subsequent influenza virus infection (Figure 21). These results imply that the influence of LTK63 is only partially dependent on T and B cells.

Conclusions

1. Prior intranasal administration of a modified heat-labile toxin from Escherichia coli (LT-K63) eliminates eosinophilic lung immunopathology during respiratory syncytial virus (RSV) infection and C.neoformans infection. 2. LT-K63 modifies T cell activation and cytokine expression and prevents RSV-induced and influenza virus-induced weight loss and lung eosinophilia, without inhibiting virus clearance. Treated mice still develop powerful antibody responses to each virus but lack the exuberant cellular inflammation characteristic of respiratory infections.

3. The number of activated CD4+ and CD8+ T cells increase in the lung and the airways following LT-K63 administration and RSV infection.

4. LT-K63 can enhance RSV-specific recall responses.

5. The dose of LT-K63 and the route of immunisation influence these effects.

6. LT-K63 provides generic protection against immunopathology caused by various lung pathogens.

7. These effects of LT-K63 may be due in large part to changes in the maturation of antigen presenting cells. Pre-treatment with LT-K63 significantly affects the APC compartment, where

MHC II, CD80 and CD40L expression are increased.

8. LT-K63 promotes a mild Thl -type cytokine environment in the lung, and may imprint this phenotype on the lung. This effect would be particularly useful in infants at risk from the development of asthma or viral-induced atopic disorders. 9. LT-K63 increases pathogen-specific nasal IgA.

The use of non-toxic proteins such as LT-K63 fulfils many of the criteria required for an effective protection strategy against respiratory infection. It induces appropriate immune responses to both viral and non-viral pathogens, and to both Thl- and Th2-driven disease or immunopathology. It effectively matures the APC compartment of the respiratory tract and induces potent CD8+ T cells and local IgA production. Excessive inflammation is eliminated without sacrificing effective pathogen clearance, and it is most effective at the site of pathogen entry and replication. In addition, the intranasal route of administration benefits from being non-invasive. Furthermore treatment with LT-K63 would also bypass the problem of antigenic shift and drift in surface-exposed pathogen proteins since the mechanism of protection is not restricted to one antigen. Overall LT-K63 primes the lung to cope with infection more efficiently, but not in the same way as a vaccine would be expected to work.

The ability of LT and mutant derivatives to modify dendritic cells, macrophages and B lymphoma cells in vitro has been described before [124-126], but these results go against the previous suggestion that these effects require at least partial retention of ADP ribosyltransferase activity. The discrepancy could be explained in various ways e.g. the present work has been performed in vivo.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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Claims

1. A method for protecting a patient against an immunopathology, comprising the step of administering a detoxified mutant of an ADP-ribosyltransferase to said patient.

2. A detoxified mutant of an ADP-ribosyltransferase, for use in protecting a patient from an immunopathology.

3. Use of a detoxified mutant of an ADP ribosyltransferase in the manufacture of a medicament for protecting against an immunopathology.

4. The method, mutant or use of any preceding claim, wherein the immunopathology is a side effect of an infection.

5. The method, mutant or use of any preceding claim, wherein the immunopathology is a pathogen- induced or allergen-induced immunopathology.

6. The method, mutant or use of claim 5, wherein the pathogen is a virus, a fungus or a bacterium.

7. The method, mutant or use of claim 6, wherein the pathogen is respiratory syncytial virus, influenza virus or Cryptococcus neoformans.

8. The method, mutant or use of any preceding claim, wherein the immunopathology affects the lung.

9. The method, mutant or use of claim 8, wherein the immunopathology is pulmonary eosinophilia.

10. The method, mutant or use of any preceding claim, wherein the mutant ADP ribosyltransferase is a mutant of cholera toxin, pertussis toxin, or heat-labile E.coli toxin.

11. The method, mutant or use of any preceding claim, wherein the mutant ADP ribosyltransferase is a heat-labile E.coli toxin with a mutation at Ser-63.

12. The method, mutant or use of any preceding claim, wherein the mutant ADP ribosyltransferase is delivered mucosally.

13. The method, mutant or use of claim 12, wherein the mutant ADP ribosyltransferase is delivered intranasally.

14. The method, mutant or use of any preceding claim, wherein the patient's pulmonary phenotype is biased towards a Thl -type cytokine environment.