WO2008109957A1

WO2008109957A1 - Method for determining suitability of treatment for asthma or chronic airways disease

Info

Publication number: WO2008109957A1
Application number: PCT/AU2008/000359
Authority: WO
Inventors: Margaret Dunkley; Robert Clancy
Original assignee: Hunter Immunology Limited
Priority date: 2007-03-15
Filing date: 2008-03-14
Publication date: 2008-09-18

Abstract

There is provided method for determining suitability of administration of a Non-typable Haemophilus influenzae (NTHi) vaccine to an individual for treatment or prophylaxis of asthma or chronic airways disease. The method involves evaluating whether the individual has one or more indicators selected from the group consisting of an elevated neutrophil level and at least one parameter indicative of exposure to NTHi. One or more embodiments find particular application in determining the suitability of the vaccine for prophylaxis or treatment of intrinsic asthma.

Description

METHOD FOR DETERMINING SUITABILITY OF TREATMENT FOR ASTHMA OR CHRONIC AIRWAYS DISEASE

FIELD OF THE INVENTION

The invention relates to a method for determining the suitability of administering a Non-typable Haemophilus influenzae (NTHi) vaccine to an individual for treatment or prophylaxis of asthma or chronic airways disease.

BACKGROUND OF THE INVENTION

Asthma is a chronic inflammatory condition of the airways characterised by reversible airway obstruction, and has traditionally been classified as extrinsic (due to allergic reaction to inhaled allergens such as pollens and house dust mite) or intrinsic (not due to classical allergy), the mechanism for which is unknown. This latter form of asthma has been called "idiopathic" asthma

In a recently reported study based on diagnosed asthma subjects, asthma was classified based on differences in eosinophil and neutrophil counts in sputum (Simpson et al, 2006). The subjects in the study were divided into different asthma subtypes based on the presence of these cell types compared to healthy control subjects. Several asthma sub- types were identified including neutrophilic asthma (> 61% neutrophils) and eosinophilic asthma (>1.01% eosinophils). The neutrophilic asthma group comprised approximately 20% of the overall number of asthmatics. The study further reported persistent neutrophilia in the majority of these subjects over both short term (4 week) and long term (mean 5.3 years) intervals between sampling despite no subject reporting respiratory tract infection during the month prior to assessment. While subjects with asthma were found to have higher levels of intracellular bacteria and macrophages than healthy controls, no significant differences were found between neutrophilic asthmatics and the other asthma groups. Indeed, the levels of bacteria found were stated to be less than that consistent with acute bacterial infections, and the report concluded there was no evidence of bacterial infection to explain the inflammatory process of neutrophilic asthma.

Non-typable Haemophilus influenzae (NTHi) is the most common pathogenic bacteria associated with chronic bronchitis (CB) (Sethi, 2001). NTHi can be found in the upper airways (eg., nose, middle ear, throat and sinuses) of healthy individuals and patients with CB (Sethi, 2001) as well as several locations of the respiratory tract, including the lumen, adhering to mucosal epithelial cells in the interstitium of the submucosa (Moller, 1998). Studies of non-obstructive and obstructive CB have observed that a large proportion of patients have persistent infection with NTHi (Murphy, 2004).

Both NTHi and Staphylococcus aureus have previously been shown to induce non-IgE-mediated and enhanced IgE-mediated histamine release from mast cells obtained by broncheoalveolar lavage from the airways of patients with CB . In the case of NTHi, it has been reported that exotoxin may be responsible for the enhancement of IgE-mediated histamine release (Clementsen, 1990). Immune cells isolated from patients with CB during acute exacerbations have been shown to be both sensitized and hyperactive to the patient's own bacteria (Norn, 1994). Several studies have also reported specific IgE antibodies produced in response to respiratory infection by fungi (Aspergillus) and viruses (eg., respiratory syncytial virus, parainfluenza virus, [Welliverl982]) and bacteria, (S. pneumoniae [Kjaergard, 1996; Tee, 1982; Pauwels,

1980], S. aureus [Rhode, 2004; Tee, 1982], Pseudomonas aeruginosa [Shen, 1981], and Mycoplasma pneumoniae [Seggev, 1996]). IgE antibodies specific for NTHi have also been identified in the serum of patients with CB (Kjaergard, 1996; Tee 1982) and cystic fibrosis (Tee, 1982). In a study of patients with bronchial asthma, IgE antibodies to NTHi were found in 29%. Antibodies to NTHi and/or Streptococcus pneumoniae were also present in 22% of patients with no other IgE mediated hypersensitivity. However, higher levels of IgE bacterial antibodies were found in patients with demonstrable IgE antibodies to various inhalant antigens (suggesting an allergic phenotype) (Pauwels, 1980). While it has been hypothesised that bacterial infections may play a role in the induction and exacerbation of asthma, it has been considered that exacerbation of asthma is predominantly triggered by viral infection. Indeed, the clinical effect of bacterial vaccines in the treatment of asthma has been questioned leading to international (WHO) recommendations that bacterial vaccines have no role in modern asthma treatment. Similarly, chronic airways disease represents a major burden on health systems and improved and/or alternative treatments are required. Despite massive amounts of research being focused on therapeutic asthma and chronic airways disease intervention and treatment, these conditions remain a major, costly and growing problem in modern Westernised societies.

SUMMARY OF THE INVENTION

Broadly stated, the invention stems from the recognition by the inventors that Non-typable Haemophilus influenzae (NTHi) can act as a trigger for severe asthma as a result of persistent colonisation and/or recurrent exposure to NTHi. In particular, the present inventors have found that IgE antibody to NTHi is a highly significant mediator of asthma (often in a complex multi-factorial situation), and that administration of NTHi vaccine may also reduce asthma treatment needs and associated asthma medication. Moreover, the invention further relates to the observation that administration of NTHi vaccine can also reduce treatment needs in chronic airways disease. Hence, in an aspect of the invention there is provided a method for determining suitability of administration of an NTHi vaccine to an individual for treatment or prophylaxis of asthma or chronic airways disease, comprising evaluating whether the individual has one or more indicators selected from the group consisting of an elevated neutrophil level and at least one parameter indicative of exposure to NTHi. The indicator will generally be compared to corresponding reference data to determine whether the indicator is elevated or within a normal range. Elevated levels of one or more of the indicators is indicative that the individual is a suitable candidate for administration of the vaccine.

Typically, the parameter(s) exhibited by the individual indicative of exposure to NTHi will be selected from one or more of (i) antibody specific for NTHi, (ii) immune cells responsive to NTHi antigen, and (iii) NTHi infection. In at least one form, the NTHi specific antibody is IgE. The individual can have diagnosed asthma or chronic airways disease, or be an individual who is deemed at risk of asthma or chronic airways disease, such as a current or ex-smoker, an individual with recurrent airway infections, chronic cough and sputum (eg., as in chronic bronchitis), and/or intrinsic asthma. The vaccine can be any NTHi vaccine which induces an effective immune response against infection by the bacteria. Typically, the vaccine will be an oral vaccine against NTHi and more usually, an oral killed or inactivated (eg., attenuated) NTHi vaccine.

At least some embodiments of the invention have particular application in assessment of NTHi vaccines for the prophylaxis or treatment of neutrophilic asthma.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed anywhere before the priority date of this application.

The features and advantages of methods of the invention will become further apparent from the following detailed description of embodiments thereof.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 is a graph showing mean number of NTHi isolated in gargle of a placebo study group;

Figure 2 is a graph showing serum NTHi-specific IgG levels in the placebo group and a treatment group immunized with an oral killed NTHi vaccine; and

Figure 3 is a graph showing saliva NTHI-specific IgG levels in the placebo group and the treatment group immunized with an oral killed NTHi vaccine. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Asthma is defined clinically by wheeze, reversible airways obstruction and bronchial hyperactivity. The commonest cause of asthma is IgE mediated hypersensitivity to inhaled allergens resulting in the classification of asthma as "extrinsic" or "intrinsic". However, individuals with longstanding asthma can develop cough and sputum stemming from lung damage and associated recurring infection of the airways. There are also, for example, bronchitic individuals with longstanding cough and sputum who develop wheeze, and individuals with recurrent asthma and airways infection.

Conventionally in asthma studies care has been taken to study discrete groups and generally, subjects with clearly defined asthma (eg., classical extrinsic asthma) are separated from other groups (eg., those with smoking-related airways disease) leading to the studies being conducted on defined groups of asthmatics in isolation of other groups of asthmatics. However, this is an artificial categorization and rather, it is more realistic to view asthma as a spectrum of airways disease as illustrated in Scheme 1.

Classical extrinsic Neutrophilic Recurrent acute Chronic airways (allergic) asthma asthma wheezy bronchitis disease with (high sputum recurrent eosinophil count) wheezy bronchitis

Scheme 1: Spectrum of asthma disease

Various Different observations have been made with respect to these different clinical manifestations of asthma. In brief, these can be summarised as follows.

• The induction of IgE antibody to inhaled antigens (eg., pollens) gives rise to classical allergic asthma in which allergen-specific IgE binds to mast cells causing degranulation of the mast cells and releasing of mediators such as histamine that give rise to allergic symptoms. • Colonisation of damaged airways and intermittent viral infection can lead to neutrophil flux into the bronchus (acute bronchitis) (usually associated with wheeze - thought to follow "inflammation" of the bronchus).

• Smoking leading to lung damage can render the subject prone to infection of the airways.

However, many asthma subjects with clinically diagnosed asthma are 'mixed' with respect to these components and it is proposed that this spectrum of asthma disease can be reconciled by recognition that different pathogenic pathways can lead to asthma and that these pathways can co-exist. In particular, without being limited to theory, it is thought by the inventors that the dominant cause of wheeze in many asthmatics without demonstrable classical allergen hypersensitivity (eg., negative tests for IgE antibody to house dust, pollens and the like, and/or whom have elevated easinophil counts) is due to an IgE antibody mediated reaction to colonising and/or recurrent exposure NTHi in conjunction with the ability of NTHi to induce and activate neutrophils. Specifically, NTHi vaccine can reduce the load of NTHi to the small airways, and provide effective treatment for so-called "intrinsic asthma".

More broadly, benefit from NTHi vaccine can be derived by those individuals exhibiting one or more parameters indicative of exposure to NTHi such as elevated neutrophil levels (with or without elevated eosinophil levels), current NTHi infection as for instance indicated by NTHi in sputum or saliva and/or NTHi specific antibody, and those individuals with damaged airways such as arising from smoking (chronic obstructive pulmonary disease (COPD)) or chronic bronchitis (particularly those individuals with wheeze). It is recognised, for instance, that individuals with damaged airways are highly prone to infection/colonisation by NTHi and other bacterial pathogens. While damage to airways classically follows smoking, extrinsic asthma can also damage the airways (hence, later onset of cough and sputum associated with NTHi infection). Benefit may also occur in asthmatic individuals with combined mechanisms (eg., atopic subjects with IgE antibody to NTHi), and the treatment of asthma and asthma symptoms in general as a result of decrease or avoidance of induction of IgE production resulting from exposure to NTHi.

Chronic airways disease is to be taken to include chronic bronchitis, emphysema, chronic pulmonary obstructive disease (COPD). In particular, the invention relates to screening an individual to determine suitability of administration of NTHi vaccine to the individual for treatment or prophylaxis of asthma or asthma symptoms, or chronic airways disease (eg., wheezing, reversible airways obstruction). The screening will typically utilise a suitable biological sample from the individual. Any parameter indicative of exposure to NTHi can be evaluated such as antibody specific to NTHi, the presence of immune cells responsive to NTHi , and/or NTHi counts . The antibody specific to NTHi may for instance be selected from IgM, IgG, IgA and/or IgE. Measurement of antibody level can involve measurement of the total level of the antibody or one or more subclasses of antibody (eg., IgGl and/or IgG3).

The NTHi specific antibody measured can be total NTHi-specific antibody or antibody selected from one or more of IgA, IgM, IgG and IgE and subclasses thereof, such as IgGl and/or IgG3. Antibody level(s) can be measured in blood, serum, plasma, sputum and/or saliva samples. IgE antibody levels will generally be measured in sputum or saliva. The immune cells responsive to NTHi can be T lymphocytes (eg. ThI and/or Th2 cells), phagocytic cells, neutrophils, antigen presenting cells and for instance, mixtures of immune cells. Immune cell responsiveness can be conveniently determined by limiting dilution analysis or other form of conventional antigen induced proliferation assay utilising peripheral blood mononuclear cells (PBMCs), or discrete cell populations as may be obtained by fluorescent activated cell sorting (FACS) or other suitable immune cell isolation or purification protocol. NTHi vaccine antigen can be used to stimulate immune cell proliferation in such assays. Neutrophil levels can also be measured in saliva or sputum using any appropriate conventionally known assay including microscopic evaluation following cell staining. Similarly, any suitable method known in the art can be employed to determine NTHi counts/level of infection. NTHi infection can for instance be evidenced by NTHi in sputum or saliva.

Antibody levels, neutrophil levels, NTHi counts and other suitable indicators measured in accordance with embodiments of the invention can be compared with corresponding reference level(s) derived from classical extrinsic asthmatics (eg., exhibiting eosinophilia and/or airways hyper-responsiveness) for example, (eg., a non- asthmatic or chronic airways disease) control or reference group. Statistical methods for differentiating asthma groups are for instance, described in Simpson et al, 2006. A higher level of the measured indicator compared to the reference level will generally indicate that the individual is a suitable candidate for the administration of the NTHi vaccine.

Suitable immunoassays which can be utilized to measure antibody level include radioimmunoassays (RIA) or ELISA as outlined above (eg., see Handbook of

Experimental Immunology, Weir et ah, Vol. 1-4, Blackwell Scientific Publications 4^th Edition, 1986 and subsequent editions thereof). Such assays include those in which the target antibody is detected by direct binding with a labelled antibody, and those in which the target antibody is bound by first antibody, typically immobilized on a solid substrate (eg., a microtitre tissue culture plate formed from a suitable plastics material such as polystyrene or the like), and labelled with second antibody specific to the first antibody to form an antigen-first antibody- second antibody complex that is detected by a signal emitted by the label. Sandwich techniques in which the target antibody is immobilized by an antibody for presentation to a labelled second antibody specific for the target antibody are also well known. Antibodies used in such assays can be bound to a solid substrate covalently utilising any commonly used amide or ester linkers, or by adsorption.

The "label" used in any such assay can be any molecule which by its nature is capable of providing or causing the production of an analytically identifiable signal which allows the detection of the antibody complex. Such detection may be qualitative or quantitative. An antibody can, for instance, be labelled with a radioisotopes such as ³²P, ² I or ³ I, an enzyme, a fluorescent label, chemiluminescent molecule or an affinity label such as biotin, avidin, streptavidin and the like. An enzyme can be conjugated with an antibody by means of coupling agents such as gluteraldehyde, carbodiimides, or for instance, periodate although a wide variety of conjugation techniques exists. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase amongst others.

Detection utilising enzymes is achieved with the use of a suitable substrate for the selected enzyme. The substrate will generally be chosen for production upon hydrolysis of a detectable colour change. However, fluorogenic substrates can also be used which yield a fluorescent product rather than a chromogen. Suitable fluorescent labels include those capable of being conjugated to an antibody substantially without altering the binding capacity of the antibody, examples of which include fluorescein, phycoerythrin (PE) and rhodamine which emit light at a characteristic wavelength in the colour range following illumination with light at a different wavelength. Methods for labelling antibodies can be found in, for example, Current Protocols in Molecular Biology. Ausubel FM., John Wiley & Sons Inc. Optimal concentrations of antibodies, temperatures, incubation times and other assay conditions can be determined by reference to conventional assay methodology and the application of routine experimentation .

The NTHi vaccine will typically contain whole killed NTHi isolate(s) (eg., formalin-killed). However, soluble or particulate NTHi antigen comprising or consisting of outer cell membrane and/or surface antigens can be utilised as well, or instead of, whole killed organisms. In one or more forms, the outer cellular membrane fraction or membrane protein(s) of the selected NTHi isolate(s) can be utilised. For instance, NTHi OMP P6 is a highly conserved 16-kDa lipoprotein (Nelson, 1988) which is a target of human bactericidal antibody and induces protection both in humans and in animal models. In chronic pulmonary obstructive disease (CPOD), OMP P6 has been shown to evoke a lymphocyte proliferative response that is associated with relative protection from NTHi infection (Abe, 2002). Accordingly, OMP P6 or any other suitable outer membrane NTHi proteins polypeptides (eg., P2, P4 and P26) or antigenic fragments of such proteins or polypeptides can find application in the NTHi vaccine.

Soluble and/or particulate antigen can be prepared by disrupting killed or viable selected NTHi isolate(s). A fraction for use in the vaccine can then be prepared by centrifugation, filtration and/or other appropriate techniques known in the art. Any method which achieves the required level of cellular disruption can be employed including sonication or dissolution utilising appropriate surfactants and agitation, and combination of such techniques. When sonication is employed, the isolate can be subjected to a number of sonication steps in order to obtain the required degree of cellular disruption or generation of soluble and/or particulate matter of a specific size or size range. The NTHi vaccine itself may be a freeze -dried or lyophilised vaccine reconstituted utilising a physiologically acceptable buffer or fluid. The vaccine can also contain one or more anti-caking agents, preservatives such as thimerosal or which are otherwise suitable for the proposed mode of administration, stabilisers such as amino acids and sugar moieties, sweetening agents such sucrose, lactose or saccharin, surfactants, pH buffering agents and pH modifiers such sodium hydroxide, hydrochloric acid, monosodium phosphate and/or disodium phosphate, a pharmaceutically acceptable carrier such as physiologically saline, solvents and dispersion media and isotonic preparations. The vaccine can also comprise one or more adjuvants. Suitable adjuvants include for instance cholera toxin B subunits and conventionally known alum adjuvants. Typically, although not exclusively, the vaccine is non-adjuvanted.

Use of such ingredients and media in vaccines is well known in the art. Except insofar as any conventional media or agent is incompatible with the NTHi isolate(s) or antigens, or the proposed mode of administration, their use in NTHi vaccines is specifically encompassed. Supplementary active agents for boosting the immune response including for instance, probiotic microorganisms, fractions and biological products thereof, and appropriate cytokines, can also be included to the vaccine. Pharmaceutically acceptable carriers and combinations of ingredients useful in vaccine compositions of the present invention may for instance be found in handbooks and texts well known to the skilled addressee such as "Remington" The Science and Practice of Pharmacy (Mack Publishing Co., 1995)", the contents of which is incorporated herein in its entirety by reference. Specific examples of adjuvants include cholera toxin B subunit and conventionally known alum adjuvants.

The NTHi vaccine can be administered as a dry powder or in liquid form. Administration can for example be achieved by injection (eg. subcutaneous, or intravenous), orally such as by dosage unit form (eg tablet, capsule or dosed liquid form) instillation, or as a spray. Particularly suitable forms of NTHi vaccine that can be administered include enterically coated tablets, capsules and dragees.

The individual will normally be a human being although the vaccine may also be administered to any suitable mammalian asthma or chronic airways disease model. Studies illustrating the relationship between exposure to NTHi, asthma and chronic airways disease are described below. EXAMPLE 1 Subjects with chronic airways disease have high levels of IgE antibody to NTHi

A study was performed in which subjects with chronic obstructive pulmonary disease (COPD) and an age-matched control group were assessed for levels of total IgE and NTHi-specific IgE in saliva, serum and sputum. A physical examination and a comprehensive questionnaire which included data on sex, age, smoking habits, and respiratory symptoms were completed. Use of corticosteroids and antibiotics were recorded from all subjects. Lung function was assessed by spirometry. None of the healthy controls were active smokers or had a history of ever having smoked. All but one of the subjects in the COPD group exhibited wheeze. Wheeze was defined as a wheezing or whistling sound in the chest at any time. None of the subjects studied had a respiratory infection within the preceding month. All patients were clinically stable. Saliva and bloods samples were collected.

1.1 Methodology

1.1.1 Saliva

Whole paraffin stimulated saliva was collected for 10 mins in ice-chilled tubes by mild suction, clarified by centrifugation at 20,000 x g for 20 mins at 4⁰C and the clear supernatant was kept frozen at -70⁰C until analysed.

1.1.2 Serum

Ten millilitres of blood was collected by routine venipuncture and allowed to clot at room temperature, centrifuged at 5,000 x g at 4⁰C for 10 mins, and serum stored at -70⁰C until analysed.

1.1.3 Sputum sol.

In general, subjects were instructed to expectorate on arising and to keep samples refrigerated. Sputum samples were assessed for oropharyngeal contamination by microscopic examination according to the criteria described by Courcol et al (1985). Sputum sol was prepared from acceptable samples by centrifugation at 4⁰C for 60 mins at 30,000 x g and stored at -70 C until analysed. 1.1.4 Preparation of NTHi antigens

A zwittergent extract of NTHi OMP was prepared as described by Murphy et al (1988). P6, a highly conserved 16-kDa lipoprotein of NTHi, was purified using preparative polyacrylamide gel electrophoresis (PAGE) by the sodium dodecyl sulphate (SDS) method as described by Kyd et al (1994). Peparative SDS-PAGE for purification of P6 was performed using a Bio-Rad 491 Cell (BioRad, Hercules, CA). SDS-PAGE was carried out using the PHAST System (Pharmacia Piscataway, NJ) to analyse the OMP zwittergent and P6 fractions with 10-15% gradient gels. Low molecular weight standards (Pharmacia) were run on each gel. Gels were stained with coomassie blue and silver nitrate.

1.1.5 IgE Enzyme linked immunosorbant assay (ELISA)

Goat anti-human IgE (Tago, Inc. CA) at a concentration of 2.0 μg/ml was used for the measurement of total IgE in samples. IgE antibodies were measured by ELISA. Briefly, flat-bottomed 96-well ELISA plates (Immunoplate I; Polysorp, Nunc, Roskilde, Denmark) were coated overnight at 4 C with 100 μl of antigen at the appropriate concentration in sodium-bicarbonate buffer (pH 9.6) or sodium-bicarbonate buffer alone. The wells were washed three times with PBS pH 7.2 containing 0.05% (v/v) Tween 20 (PBS/Tween) and then 100 μl of 1% (w/v) BSA (Radioimmunoassay grade; Sigma, St Lois, MO) in PBS/T was added and left for 60 min at 37⁰C. The wells were washed with PBS/T and then 100 μl of sample diluted in 1% BSA/PBS/T were added to each well. The plates were incubated for an additional 60 min at 37⁰C, after which they were washed and 100 μl of biotinylated goat anti -human IgE (Tago, Inc. California, USA) diluted 1:1000 in 1% BSA/PBS/T was added and incubated for another 60 min at 37⁰C. After washing, 100 μl of peroxidase-conjugated streptavidin (Tago) diluted

1:40,000 in 1% BSA/PBS/T was added to each well and incubated for 45 min at 37⁰C. After washing, 100 μl of enzyme substrate 3,3',5,5'-tetramethyl-benzidine (Sigma) in substrate buffer was added to each well and incubated for 15 - 30 min at room temperature. The reaction was stopped with 100 μl of sulphuric acid (1.0 M) and absorbance was read at 490 nm on an ELISA plate reader. Standard curves were generated by running five two fold dilutions of goat anti-human IgE (2.0 μg/ml) (Bioclone, Australia) for the measurement of total IgE in samples and pooled serum from 10 chronic bronchitic subjects for the measurement of OMP IgE and P6 IgE in samples. Standard curves and samples were tested in duplicate. The absorbance of samples in carbonate buffer wells was subtracted from each antibody coated well to give the final result. The sensitivity range for total IgE was 0.15 - 2.43 ng/ml.

Checkerboard titrations were conducted to optimize all antibody concentrations and useful ranges for protein standard concentrations and sample dilutions.

1.2 Serum and sputum IgE levels in control and treatment groups Total IgE and NTHi IgE antibodies were measured by ELISA assay as described in Example 1.1.5. The patient profile is shown in Table Ia. The values obtained are shown in Table Ib (values presented represent the mean +/- SEM).

Table Ia: Subject profile

Table Ib: Total IgE, IgE to NTHi OMP, and IgE to NTHi P6 in subjects with recurrent airways infection and asthma

ND = Not detectable EU = Elisa Units

1.3 IgE levels and allergic airways disease

The relationship between IgE level and allergic respiratory disease in subjects was evaluated. The patient profile is shown in Table 2a. In brief, subjects with recurrent acute bronchitis with bronchospasm (most of whom smoke and have early chronic airways disease) were found to have high levels of IgE antibody irrespective of existence of allergic disease. Total IgE and NTHi specific IgE levels are shown in Table 2b (values presented represent the mean +/- SEM). Table 2a: Subject profile

Table 2b: Total IgE and IgE specific to NTHi antigens (OMP & P6)

ND = Not detectable EU = Elisa Units 1.4 Discussion

The results presented in Examples 1.2 and 1.3 show no significant difference in the level of total IgE in control and COPD groups in serum and saliva. However, a significant increase in IgE OMP antibody in the COPD group compared to control group (P<0.01) was found, and both IgE OMP antibody and IgE P6 antibody were detected in sputum. The results show that NTHi- specific IgE antibody is common in serum and secretions in individuals with chronic airways disease or asthma (wheeze).

Subjects that had mild to severe COPD and were treated with the oral vaccine in the active treatment group were found to have a 50% reduction in the usage of bronchodilator therapies. Moreover, eosinophil counts following the administration of a triple course of oral NTHi vaccine therapy were found to be significantly reduced in the active treatment group only. In conclusion, the oral NTHi therapy reduces the usage bronchodilator therapies in acute episodes and also reduces eosinophil counts which are associated with allergic reactions specific to NTHi.

EXAMPLE 2

A placebo-controlled double-blind clinical study was performed in which 64 subjects on the basis of having smoked at least 10 cigarettes per day for the past two years were recruited and allocated to oral NTHi therapy or placebo treatment groups in a double-blind study. Subjects were randomised into placebo and active groups and were given three courses of study medication at monthly intervals. Each course consisted of two tablets per day for three days. The active tablets each contained 45mg of formalin-killed NTHi (equivalent to 10 killed bacteria per active tablet). Blood, saliva, gargles, throat swabs, and nasal swabs (for microbiological assessment) were collected at seven fortnightly visits.

2.1 Detection of NTHi and measurement of NTHi-specific IgG

Surprisingly, measurements in the placebo -treated and vaccine -treated groups over the winter period detected NTHi in both groups indicating random exposure to the bacterium. Fig. 1 shows the mean level of NTHi in the gargles of the placebo group at each visit. NTHi-specific IgG was measured in serum and saliva by ELISA assay. Briefly, wells of 96- well Nunc Maxisorp plates were coated with H. influenzae 164 sonicate antigen preparation. After incubation overnight at 2-8°C the plates were washed and samples of serum or saliva at various dilutions were added. Following incubation at room temperature for 60 minutes, the plates were washed and horse-radish peroxidise- conjugated anti-human IgG antibody (Chemicon catalogue number API 12P) was added. After incubation for a further 60 minutes at room temperature the plates were washed and TMB substrate (Biomediq catalogue number 50-76-00) was added prior to an additional incubation for 10 minutes at room temperature and the reaction being stopped by addition of IM phosphoric acid. Absorbance was read on a BioRad microplate reader on dual wavelength mode with a primary filter of 450nm and reference filter of 655nm. A standard curve was used to determine the ELISA units in each sample.

Levels of NTHi-specific IgG in serum and saliva in the placebo group were higher and more variable than the levels in the vaccine-treated group (see Fig. 2 and Fig. 3). The inventors believe this because NTHi reaching the lower airways in the placebo group results in systemic production of IgG and that NTHi was essentially prevented from reaching the lower airways in the vaccine treated group. To test this, plots were prepared of relationship between the number of visits at which NTHi was detected in the gargle and the Log change in serum IgG between visits 1 and 6. Placebo and active subjects were grouped according to whether they had 0-1 visits or 2-4 visits where NTHi was found in the gargle. In the placebo group, positive increases in serum IgG were associated with increased number of NTHi detections. This was not found in the active treatment group. The difference between the placebo and active change in IgG was statistically significant (p=0.0186) indicating the serum IgG in the placebo group was indeed generated by NTHi as a result of the bacteria reaching the lower airways. Moreover, the more NTHi present in the placebo washings, the higher the IgG antibody level. This is also believed to apply to the appearance of salivary NTHi- specific IgG in the placebo group. 2.2 Discussion

Serum IgG antibody as a marker for the efficacy of the vaccine was measured. An apparent lack of an IgG response in the vaccine-treated group was found while the placebo treated group of patients showed an increase in serum IgG. Without being limited by theory, it is believed by the inventors that the increase in IgG observed in the placebo group is reflecting an immune response to infecting bacteria reaching the lower airways where uptake of the bacteria by antigen-presenting cells and transport to draining lymph nodes induces an anti-bacterial IgG response. In contrast, the lack of such a response in the vaccine -treated group indicates that the bacteria are being essentially prevented (by a mucosal vaccine -specific immune response) from reaching the lower airways. A comparison of the IgG response in subjects with NTHi detected in the upper airways at 0-1 or 2-4 visits also showed the increase in IgG in the placebo group but not in the active (vaccine) treatment group. This suggests that serum IgG measurement following oral vaccination with NTHi reflects exposure to infection and the degree to which this is prevented by mucosal immunization. The saliva IgG response reflected that seen in the serum.

Overall, this study demonstrates detection of NTHi in the upper respiratory tract of subjects in both the treatment and placebo groups, and that treatment with oral killed NTHi vaccine therapy led to a reduction of NTHi- specific IgG in serum and saliva in the treatment group indicating the vaccine was successful in limiting or preventing access of NTHi to the lower airways (ie., less allergen to initiate asthma).

Thus, only in the placebo group did NTHi access the lower airways as evidenced by stimulation of IgG antibody, and oral 'immunisation' with NTHi vaccine reduced NTHi allergen in the airways

EXAMPLE 3 Killed NTHi vaccine orally administered to subjects with mild, moderate or severe airway disease reduce usages of anti-asthma therapy

One hundred and forty human subjects with mild- to -moderate or moderate- to- severe airway disease were recruited into a double blind, placebo-controlled study to assess the effect of an oral killed non-typeable Haemophilus influenzae (NTHi) vaccine on number and severity of wheezy reversible airways obstruction, and usage of concomitant medication as well as the presence of NTHi and other bacteria in the airways.

A reduction in use of anti-asthma-type medication (bronchodilators, steroids etc) and reduced infection by NTHi was found in the treatment group compared to the control group. In particular, a specific reduction of NTHi within the airways of subjects with high IgE antibody levels (serum and secretions) to NTHi, and a reduction in asthma symptoms with a consequential reduction in the need for asthma medication was obtained.

Although the invention has been described with reference to particular examples, it will be appreciated by those skilled in the art that numerous variations and/or modifications may be made without departing from the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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Claims

1. A method for determining suitability of administration of a Non-typable Haemophilus influenzae (NTHi) vaccine to an individual for treatment or prophylaxis of asthma or chronic airways disease, comprising evaluating whether the individual has one or more indicators selected from the group consisting of an elevated neutrophil level and at least one parameter indicative of exposure to NTHi.

2. A method according to claim 1 wherein at least one parameter exhibited by the individual is selected from the group consisting of (i) antibody specific for NTHi, (ii) immune cells responsive to NTHi, and (iii) NTHi infection.

3. A method according to claim 2 comprising evaluating whether the individual exhibits antibody specific for NTHi.

4. A method according to claim 3 wherein the antibody is IgE antibody.

5. A method according to claim 1 comprising evaluating whether the individual has an elevated neutrophil level.

6. A method according to any one of claims 1 to 5 comprising comparing a level of the measured indicator with corresponding reference data to determine the suitability of administering the vaccine to the individual.

7. A method according to any one of claims 1 to 6 being a method for prophylaxis or treatment of asthma.

8. A method according to claim 7 wherein the asthma is intrinsic asthma.

9. A method according to claim 7 wherein the asthma is neutrophilic asthma.

10. A method according to any one of claims 1 to 6 being a method for prophylaxis or treatment of chronic airways disease.

11. A method according to claim 10 wherein the chronic airways disease is chronic pulmonary obstructive disease (COPD).

12. A method according to any one of claims 1 to 11 wherein the vaccine is an oral vaccine.

13. A method according to claim 12 wherein the vaccine is an oral killed vaccine.

14. A method according to claim 13 wherein the vaccine is contains one or more whole killed NTHi isolates.