WO2010032139A1 - Bacterial and fungal vaccines for the treatment of asthma - Google Patents

Abstract

Provided are methods for the treatment of asthma in a patient. The methods comprise administering to the patient an effective amount of a bacterial or fungal vaccine. In certain aspects, the vaccine is a monobacterial vaccine. The vaccines of the present application are particularly useful for treating intrinsic asthma.

Description

BACTERIAL AND FUNGAL VACCINES FOR THE TREATMENT OF ASTHMA

TECHNICAL FIELD

The present application relates to bacterial and fungal vaccines for the treatment of asthma.

BACKGROUND

Asthma is a chronic inflammatory condition of the airways characterized 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. Extrinsic asthma is also referred to as "allergic" asthma, whereas intrinsic asthma is also referred to as "non- allergic" or "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 ah, 2006, Respirology 11 :54-61). 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-typeable Haemophilus influenzae (NTHi) is the most common pathogenic bacteria associated with chronic bronchitis (CB) (Sethi and Murphy, 2001, Clin. Microbiol. Rev. 14:336-363). NTHi can be found in the upper airways (e.g., nose, middle ear, throat and sinuses) of healthy patients and patients with CB (Sethi and Murphy, 2001, Clin. Microbiol. Rev. 14:336-363) as well as several locations of the respiratory tract, including the lumen, adhering to mucosal epithelial cells in the interstitium of the submucosa (Moller et ah, 1998, Am. J. Respir. Crit. Care Med. 157:950-56). Studies of non-obstructive and obstructive CB have observed that a large proportion of patients have persistent infection with NTHi (Murphy et ah, 2004, Am. J. Respir. Crit. Care Med. 170:266-72). Bronchitis is an inflammation of the bronchi (medium-size airways) in the lungs. CB is not necessarily caused by infection and is generally part of a syndrome called chronic obstructive pulmonary disease (COPD); it is defined clinically as a persistent cough that produces sputum (phlegm) and mucus, for at least three months in two consecutive years.

In contrast, asthma is a typically chronic condition involving the respiratory system in which the airways occasionally constrict, become inflamed, and are lined with excessive amounts of mucus, often in response to one or more triggers. These episodes may be triggered by such things as exposure to an environmental stimulant such as an allergen, environmental tobacco smoke, cold or warm air, perfume, pet dander, moist air, exercise or exertion, or emotional stress. In children, the most common triggers are viral illnesses such as those that cause the common cold. This airway narrowing causes symptoms such as wheezing, shortness of breath, chest tightness, and coughing. The airway constriction responds to bronchodilators. Between episodes, most patients feel well but can have mild symptoms and they may remain short of breath after exercise for longer periods of time than the unaffected individual. The symptoms of asthma, which can range from mild to life threatening, can usually be controlled with a combination of drugs and environmental changes. 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 et ah, 1990, Allergy 45: 10-17). 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 et ah, 1994, Agents Actions 41, Special Conference Issue 1994:C22-C23). Several studies have also reported specific IgE antibodies produced in response to respiratory infection by fungi (e.g., Aspergillus) and viruses (e.g., respiratory syncytial virus, parainfluenza virus (Welliver et al, 1982, J. Pediatrics 101:889-96)) and bacteria (S. pneumoniae (Kjaergard et al, 1996, APMIS 104:61-67; Tee and Pepys, 1982, Clin. Allergy 12:439-50; Pauwels et al, 1980, Allergy 157:665-9), 5". aureus (Rhode et al, 2004, Respir. Med. 98:858-64; Tee and Pepys, 1982, Clin. Allergy 12:439-50), Pseudomonas aeruginosa (Shen et al, 1981, Infect. Immun. 32:967-68), and Mycoplasma pneumoniae (Seggev et al, 1996, Ann. Allergy Asthma Immunol. 77:67-73). IgE antibodies specific for NTHi have also been identified in the serum of patients with CB (Kjaergard et al, 1996, APMIS 104; 61-67; Tee and Pepys, 1982, Clin. Allergy 12:439-50) and cystic fibrosis (Tee and Pepys, 1982, Clin. Allergy 12:439-50).

In a study of patients with bronchial asthma, IgE antibodies to NTHi were found in 29% of patients. 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 et al, 1980, Allergy 157:665-9). While it has been hypothesized that bacterial infections may play a role in the induction and exacerbation of extrinsic 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 World Health Organization (WHO) recommendations that bacterial vaccines have no role in modern asthma treatment.

Despite massive amounts of research focused on therapeutic asthma intervention and treatment, the condition remains a major, costly and growing problem in modern Westernized societies.

Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present application. SUMMARY

Broadly stated, the present application stems from the recognition by the applicants that microorganisms that can colonize the airways can act as a trigger for severe asthma as a result of persistent colonization and/or recurrent exposure the microorganism.

Hence, in an aspect of the present application there is provided a method for treatment of asthma in a patient, comprising, administering to the patient an effective amount of a vaccine that elicits an immune response against microorganisms that colonize the airways. In one embodiment, the vaccine will be an oral vaccine against one or more airway microorganisms. In certain embodiments, the vaccine is an oral vaccine of killed microorganisms, such as killed bacteria and/or killed fungi.

The present application is illustrated in Examples 1-3 (below) by way of a vaccine against an exemplary organism, non-typeable Haemophilus influenzae (NTHi). However, without being bound by theory, it is applicants' belief that vaccines targeting other respiratory microorganisms will also yield beneficial results in the treatment of asthma. For example, vaccines of Pseudomonas aeruginosa result in protective immune responses against respiratory infections and in bacterial clearance from the lungs in animal models (see, e.g., Cripps et ah, 1994, Infection and Immunity 62(4): 1427-1436; Dunkley et ah, 1994, Immunology 83:362-69) and are also immunogenic in humans (see Cripps et ah, 2006, Infection and Immunity 74(2):968-974).

In an aspect of the present application there is provided an oral vaccine for treatment of asthma, the vaccine comprising one or more antigens that can elicit an immune response against one or more microorganisms that colonize the airways together with one or more physiologically acceptable carriers. In certain aspects, the vaccine comprises an adjuvant. In other aspects, the vaccine does not comprise an adjuvant.

In another aspect of the present application there is provided the use of at least one antigen for generating an immune response against microorganisms that colonize the airways for treatment of asthma in a patient. The one or more antigens can be in the form of a cell fraction, such as a membrane, cell wall or surface antigen preparation, from said microorganism and/or from an immunologically related microorganism.

In an embodiment, whole killed cells of the microorganism will be used in a vaccine or method for treatment of asthma as described herein.

The patient can have diagnosed asthma or be a patient whom is deemed at risk of asthma such as a current or ex-smoker, a patient with recurrent airway infections, chronic cough and sputum (e.g., as in chronic bronchitis), and/or intrinsic asthma. In at least one form, the patient will have one or more parameters indicative of exposure to the microorganism against which the immunization is targeted, such as an elevated neutrophil level, the presence of the microorganism in sputum or saliva, and/or antibodies specific for the microorganism. At least some embodiments of the present application have particular application in the treatment of neutrophilic asthma. In certain aspects of the present application, the patient does not have bronchitis. Advantageously, administration of a vaccine in accordance with one or more embodiments of the present application can lead to a reduction in IgE antibodies and/or a reduction in the symptoms or severity of the asthma (e.g., intrinsic or neutrophilic asthma) in the patient.

In certain aspects, the vaccine is a bacterial vaccine. In specific embodiments, bacteria being immunized against are one or more of the following species:

Staphylococcus aureus; Haemophilus influenzae; Streptococcus pneumoniae; Escherichia coli; Pseudomonas aeruginosa; Mycoplasma pneumoniae; Haemophilus parainfluenzae; β-Haemo lytic Streptococcus spp.; α-Haemo lytic Streptococcus spp.; Pseudomonas spp.;

Klebsiella pneumoniae; Serratia marcescens; Enterobacter cloacae; Chlamydia pneumoniae and Moraxella catarrhalis.

In other aspects, the vaccine is a fungal vaccine. In specific embodiments, fungi being immunized against are one or more of Candida albicans and Aspergillus fumigatus .

In yet other aspects, the vaccine is designed to immunize against both bacteria and fungi. The bacteria and fungi immunized against are one or more of the foregoing bacteria and fungi. In certain embodiments, the vaccine is a monobacterial vaccine comprising one or more strains of the same species of bacteria and/or one or more immunogenic fractions of said species of bacteria. In other embodiments, the vaccine is a monofungal vaccine comprising one or more strains of the same species of fungus and/or one or more immunogenic fractions of said species of fungus.

The present application further provides therapeutic regimens for asthma patients. In certain embodiments, the therapeutic regimens comprise (1) testing a patient, for example a patient who exhibits symptoms of asthma, for (a) an elevated neutrophil level, (b) the presence of a microorganism in sputum or saliva, and/or (c) antibodies specific for the microorganism and (2) administering a vaccine of the present application to a patient who tests positive for one, two or all three parameters. In certain embodiments, the vaccine administered comprises one or more microorganisms tested positive for and/or an immunogenic fraction of the one or more microorganisms tested positive for.

In certain embodiments the present application further provides a vaccine in the form of a tablet, said tablet having a core comprising a population of killed bacteria or cellular fraction thereof and an enteric coating surrounding said core.

In the vaccine of the present application the weight of said core is 400 mg to 500 mg.

In the vaccine of the present application killed bacteria or cellular fraction constitutes 7.5% to 15% of the weight of said core.

In the vaccine of the present application killed bacteria or cellular fraction constitutes approximately 10% of said core.

In the vaccine of the present application the subenteric coating results in a 2% to 3% of the weight of said core. In the vaccine of the present application, the enteric coating results in a 10% to

12% of the weight of the core.

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 application. 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 application as it existed anywhere before the priority date of this application.

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

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing mean number of non-typeable Haemophilus influenzae isolated in gargle of a placebo study group.

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

Figure 3 is a graph showing saliva non-typeable Haemophilus influenzae-spscific IgG levels in the placebo group and the treatment group immunized with an oral killed NTHi vaccine. Figure 4 shows the effect of vaccination of human subjects with HI- 164 on serum

IgG levels.

DETAILED DESCRIPTION

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, patients 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 patients with longstanding cough and sputum who develop wheeze, and patients with recurrent asthma and airways infection. Applicants have found that IgE antibody to an exemplary airways microorganism, non-typeable Haemophilus influenzae (NTHi), is a highly significant mediator of asthma (often in a complex multi-factorial situation). Without being limited by theory, it is believed by the applicants that the reduction of inhaled/colonizing microorganism in the lower airways reduces or essentially avoids the activation of mechanisms that trigger asthma. By reducing the induction of asthma, therapy with vaccines as described herein may also reduce asthma treatment needs and associated asthma medication.

Conventionally in asthma studies care has been taken to study discrete groups and generally, subjects with clearly defined asthma (e.g., classical extrinsic asthma) are separated from other groups (e.g., 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 Neutrophilic Recurrent acute Chronic extrinsic asthma wheezy bronchitis — airways

(allergic → Disease with asthma) Recurrent

(high sputum wheezy eosinophil 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 summarized as follows:

• The induction of IgE antibody to inhaled antigens (e.g., 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.

• Colonization 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 by theory, it is thought by the applicants that the dominant cause of wheeze in many asthmatics without demonstrable classical allergen hypersensitivity (e.g., negative tests for IgE antibody to house dust, pollens and the like, and/or whom have elevated eosinophil counts) is due to an IgE antibody mediated reaction to colonizing and/or recurrent exposure microorganisms in conjunction with the ability of the microorganism to induce and activate neutrophils. Specifically, microorganism-based vaccines can reduce the load of the corresponding microorganism to the small airways, and provide effective treatment for so-called "intrinsic asthma". In other aspects, and also without being limited by theory, it is thought that some patients who are amenable to treatment by the vaccines of the present applicaiton are allergic to bacteria and have IgE antibodies. It is also believed that in such individuals interactions may exist between bacteria and state of allergy to other allergens that contribute to the asthmatic state. Thus, in certain aspects, the vaccines of the present application are used to treat a patient who is an allergic asthmatic.

More broadly, benefit from the vaccines of the present application can be derived by those patients exhibiting one or more parameters indicative of exposure to a microorganism that is capable of colonizing the airways, such as elevated neutrophil levels (with or without elevated eosinophil levels) in salive, current infection with the microorganism as for instance indicated by the presence of the microorganism in sputum or saliva and/or antibodies specific to the microorganism, and those patients with damaged airways such as arising from smoking (chronic pulmonary obstructive disease (COPD)) or chronic bronchitis (particularly those patients with wheeze). It is recognized, for instance, that patients with damaged airways are highly prone to infection/colonization by pathogenic microorganisms. While damage to airways classically follows smoking, extrinsic asthma can also damage the airways (hence, later onset of cough and sputum associated with airways infection). Benefit may also occur in asthmatic patients with combined mechanisms (e.g., atopic subjects with IgE antibody to a microorganism that colonizes the airways), 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 a vaccine targeting the microorganism. Antibody levels can be measured in blood, serum, plasma, sputum or saliva samples using any suitable conventionally known assay protocol including, enzyme linked immunosorbent assay (ELISA) or other immunoassay. The antibody tested for can be selected from one or more of IgA, IgM, IgG and IgE, and subclasses thereof, such as IgGl and/or IgG3. Total IgE and/or IgE antibody specific to a microorganism that colonizes the airways will generally be measured in sputum or saliva sample. 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 microorganism counts/level of infection. Antibody levels, neutrophil levels and NTHi counts can be compared against corresponding reference level(s) derived from classical extrinsic asthmatics (e.g., exhibiting eosinophilic and/or hyper-responsiveness) or for example, a non-asthmatic control or other suitable reference group. Statistical Methods for differentiating asthma groups are described in, for instance, Simpson et ah, 2006, Respirology 11:54-61.

The patient to whom the vaccine is administered in accordance with the present application will normally be a human being although the vaccine may also be administered to any suitable mammalian asthma model.

The patient needs not have an infection with the microorganism against which an immune response is specifically elicited by the vaccine of the present application to benefit from these vaccines. Thus, in certain aspects, a patient to whom a vaccine of the present application is administered does is not infected with a microorganism present in a vaccine that is administered to said patient, or markers indicative of infection with the microorganism (e.g., antibodies specific to the microorganism). In other aspects, the patient is positive for infection with the microorganism or has markers indicative of an infection with the microorganism.

In certain embodiments, a vaccine of the present application is capable of eliciting a non-specific immune response against microorganism other than the microorganism contained in the vaccine, a patient may be positive for microorganisms other than the microorganism of the vaccine or has markers indicative of infection by microorganisms other than the microorganism(s) present in the vaccine. In specific embodiments, the microorganism not contained in the vaccine but with which the patient to whom the vaccine is administered is infected is one or more of: non-typeable Haemophilus influenzae, Staphylococcus aureus; a typeable strain of Haemophilus influenzae, such as serotype B; Streptococcus pneumoniae; Escherichia coli; Pseudomonas aeruginosa; Mycoplasma pneumoniae; Haemophilus parainfluenzae; β-Haemolytic Streptococcus spp.; α-Haemo lytic Streptococcus spp.; Pseudomonas spp.; Klebsiella pneumoniae; Serratia marcescens; Enterobacter cloacae; Chlamydia pneumoniae; and Moraxella catarrhalis.

In certain aspects, the patient to whom the vaccines of the present application are administered do not have COPD, do not have one or more symptoms of COPD such as emphesyma and wheezing, and/or do not have chronic bronchitis (whether or not associated with COPD).

According to the present application, treatment of asthma encompasses the treatment of patients already diagnosed as having any form of asthma at any clinical stage or manifestation; the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of asthma; preventing and/or reducing the severity of nighttime and/or daytime asthma attacks; improving lung capacity; preventing a reduction in lung capacity of asthmatic patients; preventing or limiting adverse exacerbations; preventing or limiting hospital admissions from asthma symptoms; and/or reducing or limiting the need for antibiotics, steroids, bronchodilators or other medications.

Advantageously, administration of a vaccine in accordance with one or more embodiments of the present application can lead to a reduction in IgE antibodies and/or a reduction in the symptoms or severity of the asthma (e.g., intrinsic or neutrophilic asthma) in the patient. The vaccine of the present application can be administered in accordance with any regimen suitable for generating an effective immune response against a microorganism infection. The vaccine of the present application can be administered as a single dose or, where desired or necessary, the initial dose can be followed by boosters at several days, several weeks, or several months or years following the initial dose. In an exemplary embodiment, a single dose of the vaccine can be administered once per year pre-winter. Optionally, one or more "booster" doses of the vaccine administered at an interval of a number of weeks or months may also be given. Alternatively, a number of doses of the vaccine may be administered over the course of a number of weeks in order to generate an effective immune response against infection and/or colonization by a microorganism that is capable of colonizing the airways.

Each dosage administered to a patient can consist of one unit dose (as described below), or more or less. The specific dosage mounts effective for therapeutic use will depend on, e.g., the immunogenic component of the vaccine (as described below), the weight and general state of health of the patient, the judgment of the prescribing physician, and the proposed mode of delivery and nature of the vaccine (e.g., capsule, powder, liquid, aerosol delivery, tablets, enterically coated tablets etc.).

The vaccines of the present application may be administered using any desired route of administration, including but not limited to, e.g., subcutaneous Iy, intravenously, intramuscularly or intradermally, although mucosal administration is preferred.

Mucosal routes of administration include, but are not limited to, oral, rectal and nasal administration. Preparations for mucosal administrations are suitable in various formulations as described below. The route of administration can be varied during a course of treatment. The vaccine utilized in a method of the present application will typically contain whole killed or inactivated (e.g., attenuated) microorganism isolate(s) (e.g., formalin- killed). However, soluble or particulate antigen comprising or consisting of outer cell membrane and/or surface antigens of the microorganism can be utilized as well, or instead of, whole killed organisms. Soluble and/or particulate antigen can be prepared by disrupting killed or viable selected microorganism 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 utilizing 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.

In specific embodiments, the immunogenic component are killed cells and/or an immunogenic fraction of one or more of the following species, or an immunogenically related microorganism that is capable of eliciting an immune response against one or more of the following species Staphylococcus aureus; Haemophilus influenzae;

Streptococcus pneumoniae; Escherichia coli; Pseudomonas aeruginosa; Mycoplasma pneumoniae; Haemophilus parainfluenzae; β-Haemolytic Streptococcus spp.; α- Haemolytic Streptococcus spp.; Pseudomonas spp.; Klebsiella pneumoniae; Serratia marcescens; Enterobacter cloacae; Chlamydia pneumoniae; Moraxella catarrhalis;

Candida albicans and Aspergillus fumigatus.

The lack of a beta-lactamase gene is an optional feature of the bacterial strains of the present application. Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillins, cephalosporins, cephamycins, ertapenems and carbapenems. Beta-lactam antibiotics are typically used to treat a broad spectrum of gram positive and gram-negative bacteria. Because beta- lactamase expression may result in antibiotic resistance, the presence of a beta-lactamase gene is generally not preferred during the manufacture and administration of killed bacteria in accordance with the present application. The lack of a beta-lactamase gene allows the organism to be controlled with beta-lactam antibiotics should the need arise during manufacturing or an adverse event in a patient.

In a specific embodiment, the immunogenic component comprises killed cells or a cellular fraction of Pseudomonas bacteria, such as Pseudomonas aeruginosa. A mucoid strain of Pseudomonas aeruginosa may advantageously be used in the vaccines of the present application; however, the use of non-mucoid strains is also contemplated. An exemplary mucoid strain is strain 385, which is a serotype 2, phage type 21/33/109/110X/1214 strain whose immunogenicity in humans has been demonstrated (see Cripps et ah, 2006, Infection and Immunity 74(2):968-974). Other members of the Pseudomonas aeruginosa group that may be used in a vaccine of the present application include, but are not limited to, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P.flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, and P. straminea.

In a specific embodiment, the immunogenic component comprises killed cells or a cellular fraction of Staphylococcus bacteria, such as Staphylococcus aureus. Other Staphylococcus species that may be used in a vaccine of the present application include, but are not limited to S. auricularis, S. epidermidis, and S. haemolyticus.

In another specific embodiment, the immunogenic component comprises killed cells or a cellular fraction of Streptococcus bacteria, such as Streptococcus pneumoniae.

Other Streptococcus species that may be used in a vaccine of the present application include, but are not limited to S. agalactiae, S. aginosus, S. mutans, S. oralis, S. per oris,

S. pyogenes, S. thermophilus, and S. viridans.

In another specific embodiment, the immunogenic component comprises killed cells or a cellular fraction oiMoraxella bacteria, such as Moraxella catarrhalis.

The non-typable H. influenzae isolate NTHi- 164 (Hunter Immunology Limited, Frenchs Forest, NSW 2086, Australia) is particularly suitable for use in vaccines for the treatment of asthma as described herein. NTHi- 164 was deposited with the National

Measurement Institute (NMI) in Melbourne, Australia on August 13, 2008 and assigned deposit no. V08/021002.

In one or more embodiments, the outer cellular membrane fraction or membrane protein(s) of the selected microorganism(s) will be utilized as the immunogenic component of a vaccine of the present application.

In an exemplary embodiment, the microorganism is NTHi. Immunogenic proteins and peptides of NTHi have been described. In a specific embodiment, an NTHi outer membrane protein ("OMP") fraction or OMP protein is used as an immunogenic component of a vaccine of the present application. NTHi OMPs include OMP P6, a highly conserved 16-kDa lipoprotein (Nelson et ah, 1988, Infect. Immun. 56:128-134) that is a target of human bactericidal antibody and induces protection both in humans and in animal models. In chronic pulmonary obstructive disease (COPD), OMP P6 has been shown to evoke a lymphocyte proliferative response that is associated with relative protection from NTHi infection (Abe et ah, 2002, Am. J. Respir. Crit. Care Med. 165: 967-71). Accordingly, OMP P6 or any other suitable outer membrane NTHi proteins, polypeptides (e.g., P2, P4 and P26) or antigenic peptides of such proteins may suitable be used as the immunogenic components of the vaccines of the present application, either in isolated and purified form or as a component of a cellular fraction, such as an OMP fraction. In a certain aspect, the immunogenic protein or peptide is OMP P26 or an immunogenic fragment thereof. In another aspect, the immunogenic protein or peptide is OMP P2 or an immunogenic fragment thereof. In specific embodiments, the vaccine comprises (1) an OMP P26 protein or immunogenic fragment thereof that has at least 99% or at least 99.5% sequence identity with the OMP P26 protein of NTHi- 164, and/or (2) an OMP P2 protein or immunogenic fragment thereof that has at least 99% or at least 99.5% sequence identity with the OMP P2 protein of NTHi-164.

In another exemplary embodiment, the microorganism is Streptococcus pneumoniae. Immunogenic proteins and peptides of Streptococcus pneumoniae have been described (see, e.g., Zysk et al, 2000, Infect. Immun. 68(6): 3740-3743; U.S. Patent No. 6,689,369). Such proteins, including but not limited to PspA, SpsA, DnaK, NanA, AIiB and StrH, and immunogenic peptide fragments thereof, may be suitably included in a vaccine of the present application, either in isolated and purified form or as a component of a cellular fraction.

In another exemplary embodiment, the microorganism is Pseudomonas aeruginosa. Immunogenic proteins and peptides of Pseudomonas aeruginosa have been described (see, e.g., Brennan et al, 1999, Microbiology 145:211-220; WO/2001/002577). Such proteins, including but not limited to OMP F, and immunogenic peptide fragments thereof, may be suitably included in a vaccine of the present application, either in isolated and purified form or as a component of a cellular fraction. In another exemplary embodiment, the microorganism is Moraxella catarrhalis.

Immunogenic proteins and peptides of Moraxella catarrhalis have been described (see, e.g., Liu et al, 2007, Infect. Immun. 75(6):2818-2825; U.S. Patent No. 6,004,562; European Patent EP 1204752). Such proteins, including but not limited to OMP CD and OMP Bl, and immunogenic peptide fragments thereof, may be suitably included in a vaccine of the present application, either in isolated and purified form or as a component of a cellular fraction. In another exemplary embodiment, the microorganism is Candida albicans. Immunogenic proteins and peptides of Candida albicans have been described (see, e.g., Checkley et ah, 2002, Abstr. Intersci. Conf. Antimicrob. Agents Chemother. Intersci. Conf. Antimicrob. Agents Chemother. 2002 Sep 27-30; 42: abstract no. M-469; Eroles et ah, 1997, Microbiology 143 (2):313-320). Such proteins, including but not limited to CaNot5p, enolase, and hsp70, and immunogenic peptide fragments thereof, may be suitably included in a vaccine of the present application, either in isolated and purified form or as a component of a cellular fraction.

A vaccine of the present application will typically comprise the cells of the selected immunogenic component {i.e., microorganism isolate(s) and/or cellular fractions and/or isolated or purified proteins and/or peptides) in an amount of from about 0.1% to

100% w/w of the vaccine composition, more preferably in an amount of from 1% to 50% of w/w of the vaccine composition.

For whole cell killed vaccines, the unit dose will typically be in a range of about 10⁹ to about 10¹² killed cells, more preferably from about 10⁹ to about 10¹¹ killed cells, and most preferably about 10¹⁰ to about 10¹¹ killed cells.

For vaccines made of cellular fractions of the microorganism, the unit dose will be fractionated from about 10⁹ to about 10¹⁴ cells, more preferably fractionated from about 10¹⁰ to about 10¹³ cells, and most preferably fractionated about 10¹⁰ to about 10¹² cells. For vaccines containing isolated or purified proteins and peptide fragments, the unit dose is generally 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175- 200 mg or more.

The optimum dosage of the vaccine can be determined by administering different dosages to different groups of test mammals, prior to subsequently infecting the animals in each group with the microorganism, and determining the dosage level required to achieve satisfactory clearance of the pathogen.

A vaccine of the present application may also comprise one or more pharmaceutically acceptable carriers and/or adjuvants. Exemplary adjuvants that may be used are further detailed below. Typically, although not exclusively, the preferred oral vaccine formulation is non-adjuvanted. Actual methods for preparing the vaccine formulations of the present application will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980). The vaccines of the present application are generally provided in compositions with pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carriers" includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and preferably does not cause disruptive reactions with the subject's immune system. In general, the vaccines can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules (e.g., adapted for oral delivery), microbeads, microspheres, liposomes, suspensions, salves, lotions and the like. The vaccine itself can be a freeze-dried or lyophilized vaccine reconstituted utilizing 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, stabilizers 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 is advantageously presented for oral administration, for example in a lyophilized encapsulated or tabletted form. It is recognized that the immunogenic component of a vaccine of the present application, when administered orally, is preferably protected from digestion. This can be accomplished either by mixing or packaging the immunogenic component in an appropriately resistant carrier, such as a liposome, or within an enteric coating. The preparations may also be provided in controlled release or slow-release forms.

In an embodiment, the oral formulation is in the form of a capsule or tablet. Such capsules and tablets may be provided with an enteric coating comprising, for example, Eudragate "S" (Trade Mark), Eudragate "L" (Trade Mark), cellulose acetate, cellulose phthalate or hydroxypropylmethyl cellulose. Other carriers suitable for formulating capsules or tablets include binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Preparations for oral administration may be suitably formulated to give controlled release of the immunogenic component.

These capsules and tablets may be used as such, or alternatively, the lyophilized material may be reconstituted prior to administration, e.g., as a suspension. In another embodiment, the oral vaccine may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid).

In order to protect the immunogenic component of the vaccine from gastric acidity, a sodium bicarbonate preparation may be advantageously administered before each administration of the vaccine.

In another embodiment, the vaccine may also be formulated for administration by inhalation or injection.

Except insofar as any conventional media or agent is incompatible with the immunogenic component of a vaccine of the present application, or the proposed mode of administration, their use the vaccines that can be employed in methods embodied by the present application 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 of the present application. A vaccine of the present application optionally comprises one or more adjuvants. Examples of suitable adjuvants are presented hereinbelow.

Suitable adjuvants include mineral salt adjuvants or mineral salt gel adjuvants. Such mineral salt and mineral salt gel adjuvants include, but are not limited to, aluminum hydroxide (ALHYDROGEL®, REHYDRAGEL®), aluminum phosphate gel, aluminum hydroxyphosphate (ADJU-PHOS®), and calcium phosphate. Other suitable adjuvants include immunostimulatory adjuvant. Such class of adjuvants include, but are not limited to, cytokines (e.g., interleukin-2, interleukin-7, interleukin-12, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ, interleukin-lβ (IL-lβ), and IL-I (3 peptide or Sclavo Peptide), cytokine-containing liposomes, triterpenoid glycosides or saponins (e.g., QuilA and QS-21, also sold under the trademark STIMULON^™, ISCOPREP^™), Muramyl Dipeptid (MDP) derivatives, such as N-acetyl-muramyl-L- threonyl-D-isoglutamine (Threonyl-MDP, sold under the trademark TERMURTIDE ), GMDP, N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine- 2- (l'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine, muramyl tripeptide phosphatidylethanolamine (MTP-PE), unmethylated CpG dinucleotides and oligonucleotides, such as bacterial DNA and fragments thereof, LPS, monophosphoryl Lipid A (3D-MLA sold under the trademark MPL®), and polyphosphazenes. Yet other suitable adjuvants include particulate adjuvants, including, but not limited to, emulsions, e.g., Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, squalene or squalane oil-in-water aduvant formulations, such as SAF and MF59, e.g., prepared with block-copolymers, such as L- 121 (poly oxypropylene/polyoxy ethylene) sold under the trademark PLURONIC® L-121, Liposomes, Virosomes, cochleates, and immune stimulating complex, which is sold under the trademark ISCOM. Yet other suitable adjuvants are microparticulate adjuvants such as, but not limited to, biodegradable and biocompatible polyesters, homo-and copolymers of lactic acid (PLA) and glycolic acid (PGA), poly (lactide-co-glycolides) (PLGA) microparticles, polymers that self-associate into particulates (poloxamer particles), soluble polymers (polyphosphazenes), and virus-like particles (VLPs) such as recombinant protein particulates, e.g., hepatitis B surface antigen (HbsAg). A preferred class of adjuvants are mucosal adjuvants, including but not limited to heat-labile enterotoxin from Escherichia coli (LT), cholera holotoxin (CT) and cholera Toxin B Subunit (CTB) from Vibrio cholerae, mutant toxins (e.g., LTK63 and LTR72), microparticles, and polymerized liposomes. In certain aspects, the adjuvant is an adjuvant that activates a ThI immune response. Preferably, the adjuvant does not activate a Th2 immune response, although adjuvants that activate a Th2 immune response are within the scope of the present application.

The vaccines of the present application can be formulated in any suitable manner. In general, the vaccines of the present application can be administered orally, nasally, nasopharyngeal^, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added carriers.

A vaccine of the present application can be administered as a capsule or tablet, 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 (e.g., tablet, capsule or dosed liquid form), or by inhalation.

In certain aspects, a vaccine of the present application is administered in a manner that allows the immunogenic component to reach a lymphoid tissue, more preferably a secondary lymphoid tissue, and most preferably a mucosa-associated lymphoid tissue. In certain embodiments, the mucosa-associated lymphoid tissue is BALT (bronchus- associated lymphoid tissue), NALT (nose-associated lymphoid tissue), LALT (larynx- associated lymphoid tissue), VALT (vascular-associated lymphoid tissue, or GALT (gut- associated lymphoid tissue). Most preferably, administered by way of an enterically coated tablet to allow delivery of the immunogenic component of the vaccine to Peyer's patches, which are aggregations of lymphoid tissue that are predominantly found in the lowest portion of the ileum, and other gut-associated lymphoid tissue (GALT) in the patient's gut.

Described below are combinatorial methods in which the vaccines of the present application can be utilized. The combinatorial methods of the present application involve the administration of at least two agents to a patient, the first of which is a vaccine targeting a microorganism according to the present application, and the second of which is a second therapeutic agent.

The combinatorial therapy methods of the present application can result in a greater than additive effect, providing therapeutic benefits where neither the vaccine nor second therapeutic agent administered in an amount that is alone effective for treatment of asthma.

In the present methods, the vaccine and the second therapeutic agent can be administered concurrently or successively. As used herein, the vaccine and the second therapeutic agent are said to be administered concurrently if they are administered to the patient on the same day, for example, simultaneously, or 1, 2, 3, 4, 5, 6, 7 or 8 hours apart. In contrast, the vaccine and the second therapeutic agent are said to be administered successively if they are administered to the patient on the different days, for example, the vaccine and the second therapeutic agent can be administered at a 1-day, 2- day or 3-day, one-week, 2-week or monthly intervals. In the methods of the present aplication, administration of the vaccine can precede or follow administration of the second therapeutic agent.

As a non-limiting example, the vaccine and second therapeutic agent can be administered concurrently for a period of time, followed by a second period of time in which the administration of the vaccine and the second therapeutic agent is alternated.

Because of the potentially synergistic effects of administering a vaccine and a second therapeutic agent, such agents can be administered in amounts that, if one or both of the agents is administered alone, is/are not effective for treating asthma.

Patients with persistent asthma use a combination of long-term control medications and quick-relief medications, taken with a hand-held inhaler. Asthma symptoms triggered by airborne allergens, such as pollen or pet dander, are also treated with allergy medications.

Accordingly, suitable second therapeutic agents include long-term control medications, quick-relief medications, and allergy medications. Examples of long-term control medications include, but are not limited to, (1) inhaled corticosteroids such as fluticasone (e.g., Flovent Diskus™), budesonide (e.g.,

Pulmicort™), triamcinolone (e.g., Azmacort™), flunisolide (e.g., Aerobid™), and beclomethasone (e.g., Qvar™); (2) long-acting beta-2 agonists (LABAs) such as salmeterol (e.g., Serevent Diskus™) and formoterol (e.g., Foradil Aerolizer™ , Oxis™,

Performist™ and Brovana™); (3) long acting muscarinic antagonists such as tiotropium

(e.g., Spiriva™) and ipratropium (e.g., Atrovent™); (4) leukotriene modifiers such as montelukast (e.g., Singulair™), zafirlukast (e.g., Accolate™) and zileuton (e.g., Zyflo

CR™); (5) mast cell inhibitors such as cromolyn (e.g., Intal™) and nedocromil (e.g., Tilade™); and (7) theophylline.

Examples of quick-relief medications include (1) short-acting beta-2 agonists (SABAs) such as albuterol or albuterol sulfate (e.g., as sold under brand name Xopenex™ and Ventolin™), (2) short acting muscarinic antagonists, and (3) oral and intravenous corticosteroids such as prednisone, methylprednisolone, mometasone furoate (e.g., as sold under brand name Asmanex ) and ciclesonide (e.g., Aerobid /Alvesco ).

Examples of allergy medications include (1) immunotherapy, (2) anti-histamines

(e.g., CCllaarriittiinn™ aanndd z Zyrtec ) and (3) anti-IgE monoclonal antibodies, such as omalizumab (Xolair™).

Examples of mucolytics include, but are not limited to, Bronchitol™ (a mannitol inhaler) and Mucomyst™ (an acetylcysteine inhaler).

In certain aspects, the second therapeutic agent itself is a combination product, i.e., a product containing more than one active ingredient. Examples of suitable combination products include Symbicort™ (a combination of formoterol and budesonide); Combivent™ (a combination of atrovent and albuterol); Advair™ or Seretide™ (a combination of salmeterol and fluticasone); a combination of long acting beta-adrenoceptor agonist such as indacaterol with mometasone; and a combination of long acting beta-2 agonist such as formoterol with mometasone. 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 groups 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.

Whole paraffin stimulated saliva was collected for 10 minutes in ice-chilled tubes by mild suction, clarified by centrifugation at 20,000 x g for 20 minutes at 4°C and the clear supernatant was kept frozen at -70⁰C until analyzed.

1.1 Materials and Methods /././ Serum

Ten milliliters of blood was collected by routine venipuncture and allowed to clot at room temperature, centrifuged at 5,000 x g at 4°C for 10 minutes, and serum stored at - 70⁰C until analyzed.

1.1.2 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 ah, 1985, Eur.

J. Clin. Microbiol. 3: 122-25. Sputum sol was prepared from acceptable samples by centrifugation at 4°C for 60 mins at 30,000 x g and stored at -70⁰C until analyzed.

1.1.3 Preparation of NTHi antigens

A zwittergent extract of NTHi OMP was prepared as described by Murphy and Bartos, 1988, Infect. Immun. 56: 1084-89. 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 ah, 1994, Infect Immun 62:5652-58. Preparative SDS-PAGE for purification of P6 was performed using a Bio- Rad 491 Cell (Bio-Rad, Hercules, CA). SDS-PAGE was carried out using the PHAST System (Pharmacia Piscataway, NJ) to analyze 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,

1.1.4 IgE Enzyme linked immunosorbant assay (ELISA)

Goat anti-human IgE (T ago, Inc. CA) at a concentration of 2.0 ug/ml 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. Louis, MO) in PBS/T was added and left for 60 min at 37°C. 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 37°C, after which they were washed and 100 μl of biotinylated goat anti-human IgE (T ago, Inc. California, USA) diluted 1 : 1000 in 1% BSA/PBS/T was added and incubated for another 60 min at 37°C. After washing, 100 μl of peroxidase-conjugated streptavidin (T ago) diluted 1 :40,000 in 1% BSA/PBS/T was added to each well and incubated for 45 min at 37°C. 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 ELlSA plate reader. Standard curves were generated by running five two fold dilutions of goat anti-human IgE (2.0 ug/ml) (Bioclone, Australia) for the measurement of total IgE in samples and pooled serum from 10 chronic bronchitis 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 below. 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 who 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

ND=Not detectable EU=Elisa Units 1.4 Discussion

The results 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 patients with chronic airways disease and 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; CLINICAL BENEFITS OF A MONOBACTERIAL

VACCINE

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 randomized 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 45 mg of formalin- killed NTHi (equivalent to 10¹¹ killed bacteria per active tablet). Blood, saliva, gargles, throat swabs, and nasal swabs (for microbiological assessment) were collected at seven fortnightly visits.

1.5 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). Applicants believe this because NTHi reaching the lower airways in the placebo group results in systemic production of IgG and that 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.

1.6 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 applicants 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 2-6 visits also showed the increase in IgG in the placebo group but not in the active (vaccine) treatment group (Figure 4). 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 NTHi-specific 10 in serum and saliva in the treatment group indicating the vaccine was successful in limiting or preventing access of

NTHi to the lower airways (i.e., 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 'immunization' 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 REDUCED 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.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the present application.

Claims

WHAT IS CLAIMED IS:

1. A method of treating asthma, comprising administering to a patient in need thereof an effective amount of a vaccine, wherein the vaccine is (a) a monobacterial vaccine comprising a population of bacteria or a cellular fraction thereof, said bacteria selected from the genera Pseudomonas, Streptococcus, Staphylococcus and Moraxella or (b) a monofungal vaccine comprising a population of Candida or a cellular fraction thereof

2. The method of claim 1 wherein the vaccine is a monobacterial vaccine.

3. The method of claim 2 wherein said monobacterial vaccine comprises a population of Pseudomonas bacteria.

4. The method of claim 3 wherein said Pseudomonas bacteria belong to the Pseudomonas aeruginosa group.

5. The method of claim 4 wherein said Pseudomonas bacteria are P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea.

6. The method of claim 5 wherein said Pseudomonas bacteria are P. aeruginosa.

7. The method of claim 14 wherein said killed bacteria are killed Streptococcus bacteria.

8. The method of claim 7 wherein said Streptococcus bacteria are S. agalactiae, S. aginosus, S. mutans, S. oralis, S. peroris, S. pneumoniae, S. pyogenes, S. thermophilus, or 5^*. viridans.

9. The method of claim 8 wherein said Streptococcus bacteria are S. pneumoniae.

10. The method of claim 14 wherein said killed bacteria are killed

-31-

14572031 1 BUSINESS Staphylococcus bacteria.

11. The method of claim 10 wherein said Staphylococcus bacteria are S. aureus, S. auricularis, S. epidermidis , or S. haemolyticus.

12. The method of claim 2 wherein said killed bacteria are killed Moraxella bacteria.

13. The method of claim 12 wherein said Moraxella bacteria are Moraxella catarrhalis.

14. The method of any one of claims 1 to 13 wherein said bacteria are killed.

15. The method of claim 1 wherein the vaccine is a mono fungal vaccine.

16. The method of claim 15 wherein said mono fungal vaccine comprises a population of Candida.

17. The method of claim 16 wherein said Candida is Candida albicans.

18. The method of claim 16 wherein said Candida is killed.

19. The method of any one of claims 1 to 18, wherein said vaccine is an oral vaccine.

20. The method of claim 19 wherein said vaccine having a core comprising a population of killed bacteria or cellular fraction thereof and an enteric coating surrounding said core.

21. The method of claim 20 wherein said vaccine further comprises a subenteric coating between said core and said enteric coating.

22. The method of claim 21 wherein said vaccine further comprises a film coating as its outermost layer.

23. The method of any one of claims 20 to 22 wherein said core comprises lactose.

24. The method of any one of claims 20 to 23 wherein said core comprises cellulose or a cellulose derivative.

25. The method of claim 24 wherein said cellulose or cellulose derivative is croscarmellose sodium.

26. The method of any one of claims 20 to 25 wherein said core comprises a filler.

27. The method of claim 26 wherein said filler is magnesium stearate.

28. The method of any one of claims 20 to 27 wherein the weight of said core is 400 mg to 500 mg.

29. The method of any one of claims 20 to 28 wherein said killed bacteria or cellular fraction constitutes 7.5% to 15% of the weight of said core.

30. The method of claim 29 wherein said killed bacteria or cellular fraction constitutes approximately 10% of said core.

31. The method of any one of claims 21 to 30 wherein said subenteric coating results in a 2% to 3% of the weight of said core.

32. The method of claim 31 wherein said subenteric coating comprises Opadry II white.

33. The method of any one of claims 20 to 32 wherein said enteric coating results in a 10% to 12% of the weight of the core.

34. The method of any one of claims 20 to 33 wherein said enteric coating is an aqueous acrylic coating.

35. The method of claim 34 wherein said aqueous acrylic coating is Acryl- EZE Red.

36. The method of claim 22 wherein said film coating is purified water.

37. The method of any one of claims 2 to 36 wherein the asthma is intrinsic asthma.

38. The method of any one of claims 2 to 37 wherein the asthma is neutrophilic asthma.

39. The method of claim 38 wherein the asthma is characterized by an elevated neutrophil level in sputum or saliva.

40. The method of any one of claims 1 to 39 wherein said patient exhibits one or more parameters indicative of exposure to said bacteria or said Candida.

41. The method of claim 40 wherein said patient exhibits antibodies specific to said bacteria or said Candida.

42. The method of claim 41 wherein said antibodies are IgE antibodies.

43. The method of any one of claims 1 to 42 wherein said cellular fraction is a membrane preparation, a surface protein preparation, or a cell wall preparation.

44. The method of any one of claims 1 to 42 wherein said population of bacteria or Candida is a population of 10⁸ to 10¹² killed bacterial or Candida cells.

45. The method of claim 44 wherein said population of bacteria or Candida is a population of 10⁹ to 10¹¹ killed bacterial or Candida cells.