WO2006109284A1

WO2006109284A1 - Use of filamentous hemagglutinin for the treatment of cognitive dysfunction and neurodegenerative disorders

Info

Publication number: WO2006109284A1
Application number: PCT/IE2006/000031
Authority: WO
Inventors: Kingston Mills; Marina Lynch; Brian Keogh; Ceire Costelloe
Original assignee: The Provost Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin
Priority date: 2005-04-14
Filing date: 2006-04-18
Publication date: 2006-10-19
Also published as: GB0507552D0

Abstract

Described is the novel finding that the administration of filamentous haemagglutinin (FHA) results in the maintenance of long term potentiation (LTP) . Moreover, the administration of FHA, through the modulation of inflammatory cytokine levels in the brain, can prevent the loss, and in some cases, can induce reversion to, normal levels of LTP in the brain. Further, where cognitive function may be impaired or diminished, for example in an aged animal or individual, FHA can be administered to reverse that impairment.

Description

"Methods and Compounds for the Treatment of Cognitive Dysfunction"

Field of the Invention The present invention relates to the treatment of cognitive dysfunction using filamentous haemagglutinin (FHA) . More specifically the present invention provides for the use of FHA or a fragment, derivative or variant thereof in the treatment or prophylaxis of cognitive dysfunction. The present invention has particular utility when the compounds of the present invention are administered to aged samples, most specifically humans, such that retention of long term potentiation results .

Background to the Invention

Inflammatory changes in the brain exert a negative impact on cognitive function. In animal studies, such changes have been associated with impairment in hippocampal dependent learning paradigms and also in long term potentiation (LTP) , which is a putative biological substrate for learning and/or memory. It is well documented that LTP is impaired in the hippocampus of the aged animal . Among the changes which contribute to this impairment is an increased hippocampal concentration of the pro-inflammatory cytokine interleukin-1 beta (IL-lbeta) , and increased IL-lbeta induced signaling.

Filamentous haemagglutinin (FHA) Bordetella pertussis causes a protracted and severe disease, which is often complicated by secondary infection and pneumonia, and can have a lethal outcome in young children. Recovery from infection is associated with the development of B. pertussis- specific ThI cells and these cells play a critical role in clearance of the bacteria from the respiratory tract. However, antigen-specific ThI responses in the lung and local lymph nodes, are severely suppressed during the acute phase of infection. B. pertussis has evolved a number of strategies to circumvent protective immune responses .

The virulence factor, filamentous haemagglutinin (FHA) from B. pertussis, is capable of inhibiting

LPS-driven IL-12 production by macrophages, IL-12 and IFN-γ production in a murine model of septic shock (3) and ThI responses to an unrelated pathogen, influenza virus, when administered simultaneously to the respiratory tract (4) . FHA is considered to function primarily as an adhesin, mediating^" binding^" όf^~B.^~"per^~tussis^~~iό ^"the: β2- integrins (CR3, CDllb/CDl8, αMβ2) via binding to leukocyte response integrin (αvβ3, CD61) and the integrin-associated protein (CD47) complex (5) . FHA may also contribute to suppressed ThI responses during acute infection with B. pertussis by the induction of T cells with regulatory activity, as a result of its interaction with cells of the innate immune system.

FHA interacts directly with dendritic cells (DCs) to induce IL-IO production and, inhibit LPS-induced IL- 12 and inflammatory chemokine production (6) . The dendritic cells generated following interaction with FHA selectively stimulate the induction of TrI cells from naϊve T cells .

A method of preventing or treating cognitive dysfunction in-vivo would have valuable potential for the treatment of age related changes and also neurodegenerative conditions and diseases.

Summary of the Invention

The present inventors have now surprisingly found that the administration of filamentous haemagglutinin (FHA) results in the maintenance of long term potentiation (LTP) . Further, the administration of FHA, through the modulation of inflammatory cytokine levels in the brain, can prevent the loss, and in some cases, can induce reversion to, normal levels of LTP in the brain.

Further, where cognitive function may be impaired or diminished, for example in an aged animal or individual, FHA can be administered to reverse that impairment .

According to a first aspect of the present invention there is provided a method for the prophylaxis and/or treatment of cognitive dysfunction, the method comprising the step of administering an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof to an individual in need of such treatment .

In a further aspect of the invention, there is provided the use of filamentous haemagglutinin (FHA) in the preparation of a medicament for the prophylaxis and/or treatment of cognitive dysfunction.

In a preferred embodiment, the administration of the FHA containing agent results in the up-regulation of anti-inflammatory cytokines, the down-regulation of pro-inflammatory cytokines, or preferably both. Preferably this upregulation is in the hippocampus, most preferably this upregualtion of cytokine profile is in the microglial cells.

Typically the anti-inflammatory cytokines which are upregulated are IL-4 and IL-IO. The proinflammatory cytokines which are down-regulated are typically IL-lbeta, IL-17 and TNF-alpha. In one embodiment the modulation of pro-inflammatory and anti-inflammatory cytokine levels is in the hippocampus . In a further embodiment the modulation of the cytokine profile will be seen in the microglial cells .

A yet further aspect of the present invention provides for a pharmaceutical composition comprising an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof along with a pharmaceutically acceptable carrier or diluent.

The inventors have surprisingly found that FHA can be administered parenterally and still result in an effective modulation of both pro-inflammatory and anti-inflammatory cytokine levels in the hippocampus. Hence, in this aspect of the invention, parenteral administration is suitable for allowing the active agent, or the effector molecule (s) thereof, to cross the blood brain barrier and mediate an effect in the cells of the hippocampus . Thus , in a preferred embodiment the FHA containing agent is administered parenterally.

Accordingly a further aspect of the invention provides for the use of filamentous haemagglutinin (FHA) in the preparation of a medicament for the prophylaxis and/or treatment of cognitive dysfunction, wherein the medicament is administered parentally to an individual who requires such treatment . Typically a suitable dose may result in FHA or a derivative, fragment, variant, mutant or recombinant version thereof being administered at a dose range of between 0. Olμg/patient up to 50mg/patient .

In one embodiment the filamentous haemagglutinin (FHA) is the wild-type FHA molecule, for example as derived from Bordetθlla pertussis or Bordetella bronchisepetica or Bordetella parapertussis. However the term FHA also encompasses related molecules from other bacteria. Related molecules may include proteins from other bacteria with sequences homologous to those in FHA. "FHA" also encompasses, fragments, analogues and derivatives of FHA, including synthetic (e.g., recombinant) forms of FHA. Such synthetic molecules may be altered, removed or purified from its naturally occurring state through human intervention.

Where the form of the FHA molecule provided is not the naturally occurring, full length form, then preferably the amino acid sequence has at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, or 90%, 95%, 98% or 99% homology to amino acid sequence of the full length naturally occurring ^vwild type' form.

A derivative of FHA as disclosed herein may be, in certain embodiments, the same length or shorter than the wild type FHA peptide. In other embodiments, the peptide sequence or a variant thereof may comprise a larger peptide. As is well understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for 'conservative variation' , such as substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as lysine or glutamic acid for aspartic acid, or glutamine for asparagine.

Analogues of, and for use in, the invention as defined herein means a peptide modified by varying the amino acid sequence e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.

Such analogues of the amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids, while providing a peptide capable of inducing an alteration in the cytokine profile of a neural cell, and in particular a cell of the hippocampus .

Analogues also include derivatives of FHA, including FHA being linked to a coupling partner, e.g. a label, a drug, a toxin and/or a carrier or transport molecule.

In another embodiment, the term FHA also encompasses non-peptide mimetics of FHA, which may be used in the invention. Such mimetics of FHA may be prepared, either wholly or partly, by chemical synthesis .

In one embodiment, non-peptide mimetics of FHA can be used in the performance of the present invention. Such mimetics of FHA may be prepared, either wholly or partly, by chemical synthesis. Generation of the mimetics in this way can be performed by methods which are well known to the person skilled in the art.

In preferred embodiments the fragment, analogue or mimetic of FHA defines FHA activity in that it can maintain long term potentiation, up-regulate the production of anti-inflammatory cytokines and down- regulate pro-inflammatory cytokines.

According to a further aspect of the present invention, there is provided the use of filamentous haemagglutinin (FHA) in the preparation of a medicament for the treatment and/or prophylaxis of cognitive dysfunction.

As defined above, an embodiment of this aspect of the invention extends to the use of a derivative or mutant or fragment or variant or peptide of FHA, and further to synthetic or recombinant forms.

In a preferred embodiment the FHA containing agent is administered parenterally. Typically a suitable dose may result in FHA or a derivative, fragment, variant, mutant or recombinant version thereof being administered at a dose range of between 0. Olμg/patient up to 50mg/patient .

The inventors of the present invention have also realised the utility of the present invention in the prevention and/or treatment of neurological conditions and diseases.

Accordingly, in further aspect of the present invention, there is provided a method for the treatment and/or prophylaxis of a neurodegenerative disease or condition, the method comprising the step of administering an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof.

In a further aspect of the invention, there is provided the use of filamentous haemagglutinin (FHA) in the preparation of a medicament for the prophylaxis and/or treatment of a neurodegenerative disease.

Typically the neurodegenerative disease or condition is selected from the group consisting of: Alzheimer's disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

In a preferred embodiment, the administration of the FHA containing agent results in the up-reguϊ^~ation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines . Preferably this modification of the cytokine profiles will be in the hippocampus, most preferably this modification of cytokine profile will be present in the microglial cells .

Typically the anti-inflammatory cytokines which are upregulated are IL-4 and IL-IO. The pro- inflammatory cytokines which are down-regulated are typically IL-lbeta and TNF-alpha. Preferably this downregulation will be in the hippocampus, most preferably this downregualtion of cytokine profile will be present in the microglial cells .

In a further embodiment of this aspect of the invention, FHA can be co-administered with a self or foreign antigen in order to treat a specific neurological disease or condition. Preferably the antigen is MOG or myelin basic protein or PLP where

FHA is being co-administered in order to treat Multiple Sclerosis. Further, the antigen may be beta amyloid peptide (s) or amyloid precursor protein (APP) where the condition is Alzheimer's disease.

In such embodiments, the antigen may be administed simultaneously, sequentially or separately from the FHA.

According to a further aspect of the present invention there is provided the use of filamentous haemagglutinin (FHA) in the treatment and/or prophylaxis of a neurological disease or condition.

Typically the neurodegenerative disease or condition is selected from the group consisting of:

Alzheimer's disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

In one embodiment of this aspect of the invention, FHA can be co-administered with a self or foreign antigen in order to treat a specific neurological disease or condition. Where the condition to be treated is Multiple sclerosis, preferably the antigen is MOG or myelin basic protein or PLP. Where the condition is Alzheimer's disease, then typically the antigen is beta amyloid peptide (s) or amyloid precursor protein (APP) .

In a preferred embodiment, the co-administration of the self antigen along with the FHA containing agent results in the up-regulation of anti-inflammatory cytokines and the down-regulation of proinflammatory cytokines . Preferably this modulation of cytokine levels occurs in the cells of the brain.

Typically the anti-inflammatory cytokines which are upregulated are IL-4 and IL-IO. The proinflammatory cytokines which are down-regulated are typically IL-lbeta, and TNF-alpha. Treatment

The term 'treatment' is used herein to refer to any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment) . Treatment may include curative, alleviation or prophylactic effects .

Administration

FHA, or a variant, analogue or fragment thereof for use in the present invention may be administered alone but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.

FHA, or a variant, analogue or fragment thereof for use in the present invention may be administered to a patient in need of treatment via any suitable route. The precise dose will depend upon a number of factors, including the precise nature of the form of FHA to be administered.

Although the preferred route of administration is parenterally (including subcutaneous, intramuscular, intravenous), some further suitable routes of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual), vaginal, intradermal, intrathecal and epidural) administration or administration via oral or nasal inhalation, by means of, for example a nebuliser or inhaler.

In preferred embodiments, the composition is deliverable as an injectable composition, is administered orally, or is administered to the lungs as an aerosol via the oral or nasal inhalation routes .

For administration via the oral or nasal inhalation routes, preferably the active ingredient will be in a suitable pharmaceutical formulation which may be delivered using a mechanical form including, but not restricted to an inhaler or nebuliser.

Further, where the oral or nasal inhalation routes are used, administration is by a SPAG (small particulate aerosol generator) may be used.

For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, Lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (US Patent No. 3, 773, 919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl- methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981, and Langer, Chem. Tech. 12:98-105, 1982).

Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A. R. , Lippincott Williams & Wilkins; 20th edition (December 15, 2000) ISBN 0-

912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H^~.C.^~et aϊ7 7^fcl1 Edition ISBN 0-683305-72-7 the entire disclosures of which are herein incorporated by reference.

Pharmaceutical Compositions As described above, the present invention extends to a pharmaceutical composition for the treatment of cognitive dysfunction and further to the treatment of a neurological disease, such as those listed hereinbefore, wherein the composition comprises at least FHA, or a derivative, fragment, variant, mutant or recombinant version thereof.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention may comprise, in addition to active ingredient (i.e. FHA), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art.

Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous , intranasal or via oral or nasal inhalation.

The formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze dried powder. Dose

The composition is preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual .

The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of the neurological condition which is being treated or the state of cognitive dysfunction and diminishment of long term potentiation of the individual .

Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners, physicians or other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners .

The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.

For example, in one embodiment, a suitable dose may result in FHA or a derivative, fragment, variant, mutant or recombinant version thereof being administered at a dose range of between 0. Olμg/patient up to 50mg/patient .

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers .

"Polypeptide, peptide and protein" are used interchangeably herein to refer to a polymer of amino acid residues. Polypeptides can be modified by forms of post translational modification such as the addition of carbohydrate residues or the addition of a phosphate. The terms "polypeptide, peptides and proteins" include such modified proteins .

A "subject" includes any one or more different types of animal, including humans. Preferably the term means humans . The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention, and further, with reference to the figures .

Brief description of the figures

Figure 1, graphs A (electrophysiological read out) and B (bar chart) show that FHA reverses the age-related cognitive impairment in rats, as determined by its effect on long term potentiation (LTP) in the hippocampus,

Figure 2, graphs A and B, illustrate that systemic injection of FHA suppresses LPS- induced TNF-α production and enhances IL-IO production by peritoneal macrophages from aged rats,

Figure 3 illustrates that systemic injection of FHA reverses age-related enhancement of LPS- induced IL-lβ production by spleen cells from aged rats,

Figure 4 illustrates that systemic injection of FHA reverses age-related enhancement of phytohaemagglutinin-induced iFN-γ production by spleen cells from aged rats

(phytohaemagglutinin (PHA) is a polyclonal T- cell activator which is used in place of FHA) . Figure 5 shows 2 bar charts, A and B showing that IL-lbeta and IL-4 cytokine profiles following systemic injection of FHA, with FHA being shown to enhance anti-inflammatory cytokines in the hippocampus of aged rats, and

Figure 6 illustrates that FHA stimulates IL-IO production from rat microglial cells .

EXAMPLES

Example 1 - Effect of FHA on age-related cognitive impairment in rats

Materials and Methods :

Male Wistar rats (Charles River Laboratories, UK) of mean age 4 months (young; Y) or 22 months (aged; A) were injected subcutaneousIy with FHA (10 μg/rat) or PBS (Control) 3 times at 2 week intervals. 24 hours after the last injection rats were anaesthetised by intraperitoneal injection of urethane (1.5 g/kg i.p.) . In the case of aged rats, 75% of the urethane dose was administered initially and topped up if necessary to induce an adequate level of anaesthesia, which was identified as the absence of a pedal reflex.

Rats were placed in a head holder in a stereotaxic frame, and a window of skull was removed to allow placement of a bipolar stimulating electrode in the perforant path (4.4 mm lateral to lambda) and a unipolar recording electrode in the dorsal cell body region of the dentate gyrus (2.5 mm lateral and 3.9 mm posterior to Bregma) .

Test shocks were given at 30 second intervals for 10 minutes before and 40 minutes after tetanic stimulation (three trains of stimuli; 250 Hz for 200 ms; 30 second intertrain interval) . This protocol induces saturated LTP in perforant path- granule cell synapses in anaesthetised rats and has been shown to induce LTP in some, but not all, aged rats .

Results : The results (as shown in Figure 1, graphs A and B) show the mean data obtained from six animals in each treatment group over the 50-min experimental period.

In particular, the results show that delivery of high frequency stimulation to the perforant path of young rats resulted in an immediate and sustained increase in mean percentage change in the slope of the population excitatory post synaptic potential (EPSP); this effect was not observed in aged rats.

However, pre-treatment with 3 doses of FHA at 2 week intervals prior to assessment of LTP reverses the defect in aged rats which were now able to sustain LTP in a manner indistinguishable from the young rats (Figure IA) . Treatment with FHA had no effect in young rats . Analysis of the data in the last 5 min of the experiment (Figure IB) indicated that the mean percentage increases in EPSP slope, compared with the value in the 5 minutes prior to tetanic stimulation, was significantly (***, p<0.001 by Anova) less in aged compared with young rats.

Treatment with FHA significantly enhanced the mean EPSP slope (+++, p<0.001 untreated versus FHA- treated aged) ; there was no significant difference in mean EPSP between FHA-treated aged and un-treated young rats. FHA treatment also significantly enhanced the mean EPSP in young rats .

Example 2 - Effect of FHA on cytokine production in LPS-induced peritoneal macrophages in aged rats

Materials and Methods : At the end of the experiment described in Figure 1

(i.e at 40 minute time mark), rats were killed by decapitation. Peritoneal exudate cells were collected by flushing the peritoneal cavity twice with 20ml of ice cold PBS and removing the PBS with a syringe.

Cells were counted and incubated (lxioVml) with LPS (1, 10 or 100 μg /ml) or medium only. Supernatant was removed 24 hours later and TNF-α, and IL-10 concentrations were assayed by ELISA.

Results : The results, as shown in Figure 2, graphs A and B, show that peritoneal exudate cells (predominantly macrophages) from aged rats have enhanced LPS- induced TNF-α production, which is reduced to the level of young rats by treatment with FHA. In contrast, treatment with FHA enhances LPS-induced IL-IO production by peritoneal exudate cells from aged rats .

Example 3 - Effect of FHA on age related enhancement of LPS-induced Il-lbeta production by spleen cells from aged rats

Materials and Methods : At the end of the experiment described in Figure 1, rats were killed by decapitation. Spleens were removed and a spleen cell suspension prepared and incubated (1 xlO⁶/ml) with medium or LPS (1, 10 or 100 μg /ml) . Supernatant was removed 24 hours later and IL-Iβ concentrations were assayed by ELISA.

Results :

The results, as illustrated in Figure 3, show that spleen cells from aged rats have enhanced LPS- induced IL-lβ production, which is reduced by treatment with FHA.

Example 4 - Effect of FHA on phytohaemagglutinin induced IFN-gamma production by spleen cells of aged rats

Materials and Methods : At the end of the experiment described in Figure 1, rats were killed by decapitation. Spleens were removed and a spleen cell suspension prepared and incubated (1 xlO⁶/ml) with medium or phytohaemagglutinin (PHA) (1, 10 or 100 μg /ml) . PHA is a polyclonal activator of T-cells. It is used in this experiment in place of FHA. Supernatant was removed 24 hours later and IFN-γ concentrations were assayed by ELISA.

Results :

The results, as shown in Figure 4, show that spleen cells from aged rats have enhanced PHA-induced IFN-γ production, which is reduced to the levels observed with cells from young rats by treatment with FHA.

Example 5 - Effect of FHA administration on cytokine production in the hippocampus of aged rats

Materials and Methods:

At the end of the experiment described in Figure 1, rats were killed by decapitation and the brains rapidly removed. The hippocampus was dissected free from the whole brain; cross-chopped slices (350x350 μm) were prepared using a Mcllwain tissue chopper and stored in Krebs buffer containing CaCl₂ (1.13 mM) and 10% DMSO at -8O⁰C.

Hippocampal slices were thawed, and rinsed three times in ice-cold Krebs solution and homogenised in _ ice-cold. Krebs solution. _ .Prjotein._concentrat_ions_in__. homogenates were equalised and triplicate aliquots (100 μl) were used for quantification of IL-4 and IL-lβ concentrations by ELISA. Values were corrected for protein concentration in homogenate samples and values were expressed as pg/mg protein.

Results :

The data, as illustrated in Figure 5, graph A (IL- lbeta) and graph B (IL-4) show that aged rats have significant lower IL-4 concentrations and higher IL- lβ concentrations in the hippocampus. The deficit in IL-4 in the aged animals is reversed by treatment with FHA. Conversely IL-lβ concentrations in the hippocampus of aged rats is reduced following treatment with FHA.

Example 6 - Effect of FHA administration on IL-IO production in rat microglial cells

Materials and Methods : Glial cultures were prepared from whole brains of 1- day-old Wistar rats. Dissected brain tissue was roughly chopped and added to pre-warmed Dulbecco ' s modified Eagle's medium (DMEM; Gibco BRL) containing 10% foetal calf serum, penicillin (100 U/ml) and streptomycin (100 U/ml) . Tissue was triturated, the suspension was filtered through a sterile mesh filter (40 μm) , centrifuged at 2000xg for 3 min at 20⁰C and the pellet resuspended in warmed DMEM.

Resuspended cells were pipetted onto poly-L-lysine- coated. (.60.μg/mLL .cover, slips in_2_4-well__plates_at a. density of 0.25xl0⁶ cells and incubated for 1 h before addition of warmed DMEM. Cells were grown at 37°C in a humidified 5% CO₂: 95% air environment and media were changed every 3 days .

Neuronal cells cannot survive under these incubation conditions and the resultant population of cells was a mixed culture of microglia and astrocytes . Mixed glial cell cultures were treated with medium, LPS (1 μg/ml) , FHA (5μg/ml) or FHA and LPS. Supernatants were removed at 6 and 24 hours and assayed for IL-IO concentrations by ELISA

Results :

The results, as shown in Figure 6, show that FHA stimulates IL-IO production from mixed glial cells prepared from neonatal rats. Furthermore, when examined 6 hours after stimulation FHA synergises with LPS to further enhance IL-IO production (IL-IO is not detected with LPS or FHA stimulation alone but is observed with the combination) .

All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various

^{" "}modifications of ^"the-^"described modes ^~ of -carrying out' the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1. A method for the prophylaxis and/or treatment of cognitive dysfunction, the method comprising the step of administering an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof to an individual in need of such treatment .

2. The method of claim 1 wherein the administration of the FHA containing agent results in the up-regulation of cytokines which mediate an anti-inflammatory activity and the down-regulation of cytokines which mediate a pro-inflammatory effect.

3. The method of claim 2 wherein the up-regulation of the anti-inflammatory cytokines occurs in the hippocampus .

4. The method of claim 2 wherein the up-regulation of the anti-inflammatory cytokines is in the microglial cells .

5. The method of any of claims 2 to 4 wherein the anti-inflammatory cytokines that are upregulated are IL-4 and IL-IO.

6. The method of claim 2 wherein the pro- inflammatory cytokines which are down-regulated are IL-lbeta and TNF-alpha.

7. The method of any of claims 2, 5 or 6 wherein the up-regulation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines occurs in the hippocampus .

8. The method of any of claims 2, 5 or 6 wherein the up-regulation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines is in the microglial cells.

9. The method of any preceding claim wherein the FHA containing agent is administered parenterally.

10. The method of claim 9 wherein the FHA containing agent is administered at a dose range of between 0. Olμg/patient up to 50mg/patient .

11. The method of any preceding claim wherein the filamentous haemagglutinin (FHA) is derived from Bordetella pertussis or Bordetella bronchisepetica or Bordetella parapertussis .

12. The use of an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof in the preparation of a medicament for the treatment and/or prophylaxis of cognitive dysfunction.

13. The use of claim 12 wherein the FHA containing agent is administered parenterally.

14. The use of claim 13 wherein the FHA containing agent is administered at a dose range of between 0. Olμg/patient up to 50mg/patient .

15. A method for the treatment and/or prophylaxis of a neurodegenerative disease or condition, the method comprising the step of administering an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof.

16. The method of claim 15 wherein the neurodegenerative disease or condition is selected from the group consisting of: Alzheimer's disease (AD) , mild cognitive impairment (MCI) , multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

17. The method of claims 15 or 16 wherein the administration of the FHA containing agent results in the up-regulation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines.

18. The method of claim 17 wherein the up- regulation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines is in the hippocampus .

19. The method of claim 17 wherein the up- regulation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines is in the microglial cells.

20. The method of any of claims 17 to 19 wherein the anti-inflammatory cytokines which are up- regulated are IL-4 and IL-IO.

21. The method of any of claims 17 to 19 wherein the pro-inflammatory cytokines which are down- regulated are IL-lbeta and TNF-alpha.

22. The method of claim 21 wherein the downregulation is in the hippocampus.

23. The method of claim 21 wherein the downregualtion is in the microglial cells .

24. The method of any of claims 15 to 23 wherein the FHA containing agent is co-administered with a self or foreign antigen in order to treat a specific neurological disease or condition.

25. The method of claim 24 wherein the antigen is

MOG or myelin basic protein or PLP and the condition to be treated is Multiple Sclerosis.

26. The method of claim 24 wherein the antigen is beta amyloid peptide (s) or amyloid precursor protein (APP) and the condition to be treated is Alzheimer's disease.

27. The method of any of claims 15 to 26 wherein the FHA containing agent is administered parenterally.

28. The method of claim 27 wherein the FHA containing agent is administered at a dose range of between 0. Olμg/patient up to 50mg/patient .

29. The use of filamentous haemagglutinin (FHA) in the treatment and/or prophylaxis of a neurological disease or condition.

30. The use of claim 29 wherein the neurodegenerative disease or condition is selected from the group consisting of: Alzheimer's disease (AD) , mild cognitive impairment (MCI) , multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.

31. The use of claims 29 or 30 wherein FHA is coadministered with a self or foreign antigen in order to treat a specific neurological disease or condition.

32. The use of claim 31 wherein the condition to be treated is Multiple sclerosis and the antigen is MOG or myelin basic protein or PLP.

33. The use of claim 31 wherein the condition is Alzheimer's disease and the antigen is beta amyloid peptide (s) or amyloid precursor protein (APP) .

34. The use of any of claims 31 to 33 wherein the co-administration of the self antigen along with the FHA containing agent results in the up-regulation of anti-inflammatory cytokines and the down-regulation of pro-inflammatory cytokines.

35. The use of claim 34 wherein the up-regulation of anti-inflammatory cytokines and the down- regulation of pro-inflammatory cytokines occurs in the cells of the brain.

36. The use of claims 34 or 35 wherein the antiinflammatory cytokines which are upregulated are IL- 4 and IL-IO.

37. The use of claims 34 or 35 wherein the proinflammatory cytokines which are down-regulated are typically IL-lbeta, and TNF-alpha.

38. The use of any of claims 29 to 37 wherein the FHA containing agent is administered parenterally.

39. The use of claim 38 wherein the FHA containing agent is administered at a dose range of between

0. Olμg/patient up to 50mg/patient .

40. Use of filamentous haemagglutinin (FHA) in the preparation of a^~ medicament for the ^"prophylaxis and/or treatment of cognitive dysfunction, wherein the medicament is administered parentally to an individual who requires such treatment.

41. A pharmaceutical composition comprising an agent comprising filamentous haemagglutinin (FHA) or a derivative or mutant or fragment or variant or peptide thereof along with a pharmaceutically acceptable carrier or diluent.