AU2003225341A1 - Method of prophylaxis of infection - Google Patents

Description

WO 03/097094 PCT/AU03/00616 1 METHOD OF PROPHYLAXIS OF INFECTION This patent relates to the prevention in humans and other mammals of symptoms arising from the presence of air-borne pathogenic bacteria. Such 5 bacteria include Yersinia spp, Mycobacterium spp, Brucella spp, Bacillus anthracis, Chlamydia pneumoniae, Coxiella bumrnetii and Legionella pneumophila. Background 10 Kollberg (WO9841235) teaches the use of avian polyclonal antibodies against the bacteria Pseudomonas aeruginosa to treat respiratory tract infections caused by this bacteria. The antibody was applied topically to the respiratory tract of children suffering cystic fibrosis. The bacteria was present prior to 15 treatment commencing. The beneficial effect of the antibody was measured by isolating bacteria from the sputum. Prolonged treatment was associated with reduced bacterial counts. Pseudomonas aeruginosa does not survive within phagocytic cells. Kollberg did not demonstrate sustained prophylaxis since the antibodies were always applied after the bacteria were present. 20 Ramisse et al (Journal of Infectious Disease, 1996, May 173(5) 1123-8) teaches the use of polyclonal antibodies against Steptococcus pneumoniae to treat respiratory infections in mice caused by this bacteria. The antibody and its fragment were applied either intravenously or intranasally after the mice were 25 infected with the bacteria. Streptococcus pneumoniae does not survive within phagocytic cells. Sustained prophylaxis associated with the use of antibodies was not suggested. Cheng et al (Infectious Immunity 2001, April; 69(4): 2302-8) teaches the use of 30 polyclonal antibodies against group B Streptococcus bacteria to increase opsonisation and phagocytosis of this bacteria. Opsonisation is the process where an antibody bound to a bacteria stimulates the activity of phagocytic cells in the locality. This was measured by increased killing of the bacteria by macrophages in cell culture. The increase of phagocytosis is presumably WO 03/097094 PCT/AU03/00616 2 beneficial in vivo however no in vivo experiments were reported. Streptococcus bacteria do not survive in phagocytic cells. Sustained prophylaxis was not demonstrated. 5 De Hennezel et al (2000, Antimicrobial Agents and Chemotherapy, 45(1):316 teaches the use of polyclonal antibodies against Steptococcus pneumoniae to treat infections in mice caused by this bacteria. The antibody was delivered intranasally and parenterally. The beneficial effect was measured by recording lethality and counting bacteria in lung homogenates. Steptococcus pneumoniae 10 does not survive in phagocytic cells. De Hennezel et al did not demonstrate sustained prophylaxis since the antibodies were applied after the bacteria were present. Collins (US 4,994,269) teaches the use of a monoclonal antibody against 15 Pseudomonas aeruginosa to prevent and treat respiratory disease caused by that bacteria. The intranasal use of polyclonal and monoclonal antibodies is described and the beneficial effect was recorded by measuring lethality and counting bacteria in lung homogenates. Pseudomonas aeruginosa does not survive in phagocytic cells. Collins gave the antibody preparations 20 minutes 20 before challenge with the bacteria - this is does not suggest a sustained prophylactic function because it would require dosing to happen very frequently to provide a clinically useful effect. Eyles et al (Vaccine 1998 Apr; 16(7):698-707) teaches the use of an active 25 intranasal vaccine made of bacterial proteins to stimulate the body's own immune system and to prevent disease caused by the respiratory form of Yersinia pestis. Yersinia pestis does survive within phagocytic cells. Particular success was seen when cholera toxin B subunit was used as a strong mucosal adjuvant to stimulate an immune response in the mice. The preparation in the 30 above treatment (vaccination) does not comprise antibodies. Di Genaro et al (1998, Microbiological Immunology; 42(11):781) teaches the use of an active intranasal vaccine made of bacterial proteins to stimulate the body's own defence system and to prevent disease caused by a respiratory WO 03/097094 PCT/AU03/00616 3 form of Yersinia enterocolitica in mice. Yersinia does survive within phagocytic cells. The preparation in the above treatment (vaccination) does not comprise antibodies. 5 For bacteria that survive in phagocytic cells (eg Yersinia) the literature does not teach that the topical application of antibodies would result in a useful clinical result. The following concepts also teach away from the topical application of antibodies in managing respiratory disease caused by bacteria that survive in phagocytic cells: 10 Antibodies which bind with bacteria increase the phagocytosis of those bacteria by immune cells such as macrophages. This process (also known as Fc receptor-mediated phagocytosis) is well described in many medical texts and is known as 'opsonisation' (Huber et al, Journal of Immunology, 2001 Jun 15; 15 166(12): 7381-8). A consequence of opsonisation is as follows: for bacteria which survive within phagocytes, antibody treatments will be counter-productive because the antibodies will encourage the bacteria to enter a safe haven (the phagocytes). 20 The known vaccines for protection of mammals against air-borne bacteria that cause respiratory must be given at least weeks before possible exposure to the pathogen of concern to allow the immune system of the body to respond to the vaccine. 25 Summary We have made the surprising discovery that sustained prophylaxis against bacterial infection in the mammalian respiratory tract can be achieved by inhalation of binding proteins. By sustained prophylaxis we mean a clinically 30 useful period of prophylaxis of at least one hour and preferably at least 3 hours. Accordingly we provide a method of prophylaxis of bacterial infection of the respiratory tract of a patient, the method comprising inhalation by the patient of binding proteins.

WO 03/097094 PCT/AU03/00616 4 The invention further provides the use of binding proteins in preparation. of a medicament for prophylaxis of bacterial infection of the respiratory tract by administration of the medicament to the respiratory tract. 5 In a further aspect the invention provides a composition for prophylaxis of bacterial infection of the respiratory tract, the composition comprising binding proteins and a propellant for delivering the composition as an aerosol for inhalation. 10 In yet a further embodiment the invention provides an inhaler product for prophylaxis of bacterial infection of the respiratory tract comprising a pressurised container enclosing a mixture of a binding protein composition and a propellant, preferably an HFC propellant. 15 Detailed Description In one preferment the bacteria are bacteria which survive inside phagocytes and the binding proteins are directed against such bacteria, for example 20 Yersinia spp, Mycobacterium spp, Brucella spp, Bacillus anthracis, Legionella pneumophila, Cosciella bumetii and Chlamydia pneumoniae. It is preferred that the binding proteins comprise antibodies or antibody fragments directed against the bacteria. Examples of antibodies and antibody 25 fragments include polyclonal antibodies, monoclonal antibodies, F(ab) fragments, F(ab)2 fragments, antibody tip fragments, chimeric and humanised antibodies and fragments, and recombinant antibodies and fragments. It is preferred that the antibody preparations are affinity purified. Preferably the 30 binding proteins are formulated in conjunction with other agents which protect the function of the antibodies in hostile environments. Examples of such agents are mammalian colostrum and colostrum extracts which have been described in International Patent Application No. PCT/AU03/00348, the contents of which are incorporated by reference.

WO 03/097094 PCT/AU03/00616 5 In one preferment the antibodies are taken from bovine colostrum or the yolk of a bird egg. Example, antibodies from hyperimmune colostrum (or hyperimmune egg yolk) may be used. It will be understood by those skilled in the art having 5 regard to the above disclosure that suitable binding protein may be prepared as hyperimmune colostrum (or egg yolk) by immunizing a mammal (or bird) with antigen derived from the pathogen. The method described in this patent gives immediate sustained protection (for 10 at least 1 hour) making it suitable for use immediately before entering a high risk area. In one embodiment the invention provides an inhaler composition comprising a mixture of binding protein or proteins and a propellant. The composition may 15 further comprise excipients in addition to the carrier. The propellant may be a fluorocarbon propellant such as a CFC, HCFC or HFC. Hydrofluorocarbon (HFC) propellants are particularly preferred. Examples of suitable hydrofluorocarbon propellants include HFC-134a and HFC-227. 20 The carrier for the binding protein is preferably essentially free of water. The carrier may be a finely divided particulate material or a liquid. In many cases the binding protein is moisture sensitive. The composition of the binding protein may be a solution or dispersion in the propellant and the composition may 25 include further solvents such as lower alkanols (eg ethanol) glycerol, lower alkylene glycols or mixture. The inhaler device of the invention preferably includes a container which maintains the formulation under pressure and is impermeable to the ingress of 30 moisture. The inhaler device may be a single use inhaler or multidose inhaler. A multidose inhaler may be provided with a drug metering valve. The inhaler may contain a moisture absorbing material.

WO 03/097094 PCT/AU03/00616 6 In an alternative embodiment the composition may comprise a finely divided solid composition comprising the binding protein and a liquefied gas. The liquefied gas is preferably an inert gas such as nitrogen or a noble element. An example of a suitable method of formulation in liquid gas is described by 5 Meekka et al in US patent No. 6378518. In yet another embodiment the binding protein may be in finely divided form comprising particles of for example from 20 nm to 100 microns and preferably 50 nm to 10 microns. The finely divided composition may include anticaking 10 agent. The inhaler device of the invention may include a compartment containing a powder, a passage for providing an air-stream by inhalation of the user and means for releasing the binding protein powder formulation into the air stream. 15 The inhaler device may comprise a multiplicity of chambers. In one embodiment for example the device includes one chamber containing a binding protein in dry finely divided form and a second chamber containing a propellant or carrier. The device comprises means for providing mixing of the contents of 20 the first and second chambers. For example, the chambers may be separated by a frangible wall which in operation is breached to provide mixing of the finely divided dry binding protein and the carrier or propellant. Such an arrangement may be particularly useful where the binding protein has a limited shelf life in the carrier or propellant. In this embodiment the inhaler may include means for 25 rupturing the frangible wall. The frangible wall when used may be ruptured by a variety of means such as a piercing plunger or by relative rotation of the different section of an inhaler each provided with one of said chambers. In a further embodiment of the inhaler of the invention the composition of the 30 binding protein includes a liquid carrier which may be premixed with the binding protein or mixed therewith prior to inhalation and the inhaler comprises a spray nozzle and mechanical means for providing delivery of liquid binding protein mixture to the spray nozzle to form an aerosol.

WO 03/097094 PCT/AU03/00616 7 The dose of binding protein required to provide prophylaxis of infection will depend on the particular bacteria and the risk and of exposure. Typically the dose will be in the range of from 0.1 to 100 milligrams per kilogram of bodyweight of the individual in whom infection is to be prevented. More 5 preferably the dose will be in the range of from 0.8 to 80 mg/kg. The subject to be treated may be a human or lower animal subject. The invention will now be described with reference to the following examples. It 10 is to be understood that the examples are provided by way of illustration of the invention and that they are in no way limiting to the scope of the invention. Example 1 15 The results of Example 1 are discussed with reference to the attached drawings. In the drawings: Figure 1(a) is a chart comprising the effect of prophylactic treatment in accordance with the invention with controls; and 20 Figure 1 (b) examines the statistical significance of the results illustrated in Figure 1(a). Pernasal administration of specific antibody provides sustained immuno 25 protection of mice against respiratory challenge with a intracellular bacterial pathogen (Yersinia enterocolitica) when given before the bacterial challenge. In this example Yersinia enterocolitica serves as a model for Yersinia pestis. 30 Introduction The aim of this study was to determine if pernasally administered polyclonal antibodies obtained could provide sustained protection when challenged with an inhaled bacterial pathogen.

WO 03/097094 PCT/AU03/00616 8 The mouse model used in this study involved pernasal inoculation with Yersinia enterocolitica, a pathogen that is able to colonise and cause serious pneumonia in the lungs of mice. Yersinia bacteria survive within phagocytic cells. 5 For this study, purified rabbit antibodies (whole IgG or F(ab') 2 antibody fragments) directed against Y. enterocolitica and a variety of control materials were given pernasally to anaesthetised mice, either 3 hours before or 3 hours after the intranasal challenge with a suspension of 5 x 106 colony-forming units 10 (CFU) of living Y. enterocolitica bacteria. The extent of the protection against infection given by the antibodies and their fragments was determined from measurement of bacterial clearance 24 hours after infection. Experimental procedures 15 Bacteria A virulence plasmid - bearing strain of Y. enterocolitica 0:8 (strain 8081) was cultured in Tryptone Soy broth (Oxoid) for 24 hr at 280C. Cells were centrifuged and then washed several times following suspension in sterile PBS. Final 20 suspension was prepared to give 5 x 106 CFU in 50 pl of sterile PBS. The actual number of bacteria in the inoculum was determined by plating a sample onto Brain Heart Infusion plates (Oxoid) and counting the CFU after incubation for 24hr at 28 oC. 25 Mice Groups of five male Black Ten mice (6-8 week old) were kept in separate cages and were infected within a biosafety cabinet to minimise the airborne spread of Y. enterocolitica. Prior to the administration of antibodies and bacteria, mice 30 were anaesthetised briefly with inhaled Penthrane (methoxyflurane). Antibodies and bacteria were administered by placing 50 [d of the appropriate control solution, antibody preparation or bacterial suspension onto the nares of the anaesthetised mouse. The mouse inhaled the drop and the animal was then WO 03/097094 PCT/AU03/00616 9 allowed to recover. Animals were observed during the recovery period until the righting reflex returned. Treatment Groups 5 As detailed in Table 1, test mice received 100 [tg of either purified whole rabbit immunoglobulin (IgG) to Y. enterocolitica (0:8) or the F(ab') 2 fragment of the IgG. Antibodies were given pernasally (in 50 jl solution) to anaesthetised mice in each test group (10 mice per test group). Antibodies were given either 3 hours before or 3 hours after intranasal challenge with a suspension of 5 x 106 10 CFU of Y. enterocolitica serogroup. Control groups of mice (5 mice per control group) received either 50 pl PBS buffer, 100 gg of purified IgG from unimmunised rabbits (Non-specific IgG) or 100 jg of a pool of serum proteins from unimmunised rabbits, after removal of IgG using Protein A chromatography (Non-lgG proteins). An additional group of 5 control mice were 15 given whole IgG, without a bacterial challenge, to assess any histopathological effect of the antibody on the lungs. Table I - Treatment Groups No. of Timing of Group No. Test material Dose administration of test mice material -before or after infection 1. Test 10 Whole IgG 100 jg 3 hr before 2. Test 10 Whole IgG 100 jg 3 hr after 3. Test 10 F(ab') 2 Fragment 100 jig 3 hr before 4. Test 10 F(ab') 2 Fragment 100 jg 3 hr after 5. Control 5 Non specific IgG 100 ig 3 hr before 6. Control 5 Non-lgG protein 100 jg 3 hr before 7. Control 5 Buffer N/A 3 hr before 8. Control 5 Whole IgG 100 jg No Infection 20 Measurement of bacterial clearance from the lung WO 03/097094 PCT/AU03/00616 10 A comparison of bacterial clearance was made 24 hr after infection to determine the extent of the bacterial clearance induced by the test and control materials. After mice were killed using inhaled CO2, the cranial left lobe of the lung was 5 removed aseptically and placed in a preweighed bottle containing Iml of sterile PBS. The lobe was weighed and then homogenised in the PBS. Serial dilutions were plated on MacConkey agar to estimate counts of the recovered bacteria. Histopathological studies 10 The cranial right lobe of the lung was removed from each mouse, together with the heart, kidney, spleen and part of the liver and fixed in 10% formalin. These tissues were dehydrated in alcohol, embedded in paraffin and sections were stained in haematoxylin-eosin. Lung infection was identified by inflammatory cell 15 infiltration into alveoli, bronchiolitis and loss of normal histoarchitecture. In comparison, lung protection was evaluated by the presence of normal histoarchitecture. Statistics 20 Data from the estimates of recovered bacteria in the cranial lobe of the lungs were analysed using Student's 2-tailed t test. The estimates of CFU were transformed logarithmically to normalise their distribution and reduce the variance. A P value of <0.05 is considered statistically significant. 25 Results Bacterial clearance from the lung after passive immunisation As shown in the attached Figure la, bacterial clearance from the lungs of 30 treated mice (groups 1 and 3) was highly significant when compared to control mice (P<0.001 vs. group 6 and P<0.05 vs. group 5). These results indicate that pernasal administration, 3 hours before infection, of either the whole IgG of rabbit antibodies to Y enterocolitica or the F(ab') 2 fragment of the IgG, significantly reduced the numbers of Y. enterocolitica recovered from the lung.

WO 03/097094 PCT/AU03/00616 11 This protection surprisingly occurred only when the antibodies were given 3hr before the bacterial challenge but not when given after the challenge. 5 From Figure 1 b which shows the structural significance of the results it is clear that treatments 1 and 3 (in which antibody was administered prior to challenge by the pathogen) are significantly better. Histopathological studies 10 There was no evidence of any histopathological effect of the pernasal administration of antibody (without a bacterial challenge) on the lungs of 5 control mice that were given whole IgG. 15 Example 2 Manufacture of hyperimmune serum against Yersinia spp. in rabbits. A virulence plasmid - bearing strain of Y. enterocolitica O:8 (strain 8081) was cultured in Tryptone Soy broth (Oxoid) for 24 hr at 280C. Bacteria were 20 harvested to yield 5 x 106 CFU in 50 il of sterile PBS. The number of bacteria in the sample was determined by plating a sample onto Brain Heart Infusion plates (Oxoid) and counting the CFU after incubation for 24hr at 28 oC. The bacteria were spun down, washed in PBS and then heat-killed by boiling. 25 The bacteria were again washed and aliquoted into doses corresponding to the following numbers of whole heat killed bacteria: 1x10 6 , 4x10 6 , 8x10 6 , 2x107, 4x107, lx1 08. These doses correspond to each vaccine dose in sequence. 1 ml of vaccine was injected into the thigh muscle of 3 rabbits twice a week for 3 30 weeks. 6 vaccinations were given to each rabbit. Blood was harvested from the ear vein of the rabbits. After harvesting, the blood was allowed to clot and the hyperimmune serum was removed, aliquoted and frozen at -200C.

WO 03/097094 PCT/AU03/00616 12 Example 3 Manufacture of F(Ab2) fragments against Yersinia spp. 5 Immunoglobulin was purified from rabbit antiserum to Y. enterocolitica (0:8), using Protein-A chromatography on a Millipore ProSep-A affinity column, with PBS pH 7.4 as the running buffer. Immunoglobulin was eluted with 0.1M Glycine / HCI pH 3.0 buffer, neutralised by addition of 1M Tris solution and then dialysed against PBS pH 7.4. Protein-A chromatography was also used to purify 10 non-specific immunoglobulin from unimmunised rabbit serum for use as "Non specific IgG" control. The F(ab') 2 fragment of the purified rabbit immunoglobulin to Y. enterocolitica (0:8) was prepared by digestion with immobilised pepsin (Pierce Chemical Co.) 15 to remove the crystallisable (Fc) region of the antibody molecule. Prior to digestion, purified immunoglobulin was dialysed against 20mM sodium acetate buffer at pH 4.5 and adjusted to a concentration of 10mg/ml. Immunoglobulin was then incubated with immobilised pepsin at 37 0 C for 4hrs. Digestion was stopped by centrifugation of the incubation mixture to remove the immobilised 20 pepsin and adjustment of the pH of the supernatant to 7.2. The F(ab') 2 fragment was purified from undigested IgG and whole Fc fragments by re-application over a ProSep-A affinity column. Results from the mice receiving the antibody fragments were used to indicate if passive immunity required intact immunoglobulin or only the antigen-binding portion. All antibody preparations 25 and control solutions were adjusted to a protein concentration of 2mg/mi in PBS before administration to mice. Example 4 Hyperimmune Colostrum Against Anthrax 30 Live anthrax vaccine (STERNE strain supplied by Fort Dodge Animal Health a division of Wyeth, located at Overland Park, Kansas City, Kansas, U.S.A.) was used to make hyperimmune bovine colostrum using the following method.

WO 03/097094 PCT/AU03/00616 13 Cows are immunised by a registered veterinarian with a Im injection of the STERNE strain vaccine as supplied into the muscle tissue on the side of the neck. Up to 5 injections are given at 2 weekly intervals during months 6 to 8.5 of gestation, ceasing 1 month before parturition. Test bleeds are taken from a 5 selection of the immunised cows and assayed to determine the level of specific antibodies. Results of these assays are used to determine if a satisfactory titre has been achieved. Hyperimmune colostrum was harvested and processed according to the method 10 of Example 2 of International Application No. PCT/AU03/00348. The following diagram shows the principles used to take colostrum and convert it to a processed form. 15 Process Waste Product Raw Colostrum 20 Separator Fat Microfilter Retentate: Cell 25 debris, bacteria Permeate Ultra filter [ Permeate: Water, lactose 30 Retentate High Protein final product 35 The raw colostrum is collected from dairy cows most preferably at the first milking after calving.. The colostrum is stored at 4oC on farm and then transported either for longer term storage at -20'C or sent directly to wet manufacturing. 40 WO 03/097094 PCT/AU03/00616 14 The raw colostrum is warmed to approximately 37 0 C and then skimmed with a rotary milk separator to remove fat. The resultant liquid may be pasteurised or microfiltered with a 7-10 micron ceramic filter system (to remove bacteria and debris. The liquid is then Ultrafiltered (for example in a Abcor 10m 2 Ultrafiltration 5 plant) to remove a majority of the water, lactose and electrolytes leaving a high protein concentrate. The resultant high protein concentrate is further processed preferably by lyophilization (freeze-drying) or spray-drying. The above method yield a processed bovine colostrum powder. This product is 10 suitable for inclusion in therapeutic goods. Example 5 Testing of hyperimmune colostrum for Anthrax binding capacity. 15 An Enzyme Linked Immuno-sorbent Assay was created using a recombinant protein form of Protective Antigen of Bacillus anthracis (PA), sourced from SAPHIRE LABS, an agent of AVANT Therapeutics of Massachusetts, U.S.A., part of the Collier Group. A test liquor was made by mixing the above bovine colostrum extract powder in 20 water to provide a 2% (by weight) aqueous mixture. This was tested alongside PBS (negative) and a standard mouse monoclonal antibody with a significant affinity for Protective Antigen (Bacillus anthracis) using the following assay method. Primary reaction time: 15 minutes. 25 Assay Method Method for testing colostrum from cows vaccinated with anthrax vaccine for anti-PA antibodies. (PA refers to protective antigen to B. anthracis). 1. One hundred microlitres (100 ul) of a 0.05M Carbonate-bicarbonate buffer containing lug of PA /ml was added to the wells of a 96-well 30 microtitre plate, and the plate was incubated overnight at room temperature. 2. The plate was washed (x3) with PBS/0.05% Tween 20.

WO 03/097094 PCT/AU03/00616 15 3. Three hundred microlitres (300 ul) of 3% skim milk in PBS/0.05% Tween 20 were added to each well containing PA to block any sites that did not bind PA. The plate was incubated at room temperature for 1 hr. 5 4. The plate was washed as above. 5. One hundred microlitres (100 ul) of a 1 in 500 dilution of a 20mg/mi solution of a colostrum sample was added to each well containing PA. 10 The plate was incubated at room temperature for 1 hr. 6. The plate was washed as above. 7. One hundred microlitres (100 ul) of a 1 in 2000 dilution of a goat anti 15 bovine alkaline phosphatase conjugate was added to each well, and the plate was incubated at room temperature for 1 hr. 8. The plate was washed (x3) with PBS/0.05% Tween 20 and once (xl) with distilled water. 20 9. One hundred microlitres (100 ul) of a p-nitrophenyl phosphate substrate was added to each well, and the plate incubated at room temperature for 1 hr.' 25 10. Finally, the plate was read at 405nm on an ELISA reader. The plate also contained a negative (same procedure as the test samples except that 100ul of 3% skim milk in PBS/0.05% Tween 20 was added instead of the colostrums sample) and positive control (same procedure as test samples 30 except 100ul of a 1 in 1000 dilution of a mouse anti-PA monoclonal antibody was added as the sample and a goat anti-mouse alkaline phosphatase conjugate was used instead of the anti-bovine conjugate. All dilutions were made in 3% skim milk in PBS/0.05% Tween 20.

WO 03/097094 PCT/AU03/00616 16 Results Assay results are shown in Table 2. Table 2 Sample Optical Density Bovine Colostrum Extract (raised against Bacillus anthracis) 3.7 Anti-PA monoclone (positive control) 4.2 Phosphate buffered saline (negative control) <0.1 5 The affinity for the bovine colostrum extract for PA is shown by the high optical density which is (a) similar to the positive control and (b) significantly greater than the negative control.

Claims (23)

1. A method for prophylaxis of infection of the respiratory tract of a subject by pathogenic airborne bacteria the method comprising administering to the subject by inhalation binding proteins directed against the bacteria. 5

2. A method according to claim 1 wherein the pathogenic bacteria is a bacteria which survives inside phagocytes and the binding proteins are directed against said bacteria which survives inside phagocytes. 10

3. A method according to claim 2 wherein the binding proteins comprise antibodies or antibody fragments directed against said bacteria which survives inside phagocytes.

4. A method according to claim 3 wherein the binding proteins are selected 15 from the group consisting of polyclonal antibodies, monoclonal antibodies, F(ab) fragments, F(ab') 2 fragments, antibody tip fragments, chimeric and humanised antibodies and fragments and recombinant antibodies and fragments.

5. A method according to claim 2 wherein the binding proteins comprise 20 antibodies which have been affinity purified.

6. A method according to claim 2 wherein the binding proteins are administered in conjunction with a protective agent selected from mammalian colostrum and extracts thereof. 25

7. A method according to claim 2 wherein the binding proteins comprise antibodies taken from colostrum of a mammal or the yolk or an egg.

8. A method according to claim 1 wherein the binding protein is prepared as 30 a hyperimmune protein against a bacteria which survives inside phagocytes wherein the hyperimmune protein is taken from hyperimmune colostrum or hyperimmune egg yolk. WO 03/097094 PCT/AU03/00616 18

9. A method according to claim 2 wherein the bacteria is selected from the group consisting of Yersinia spp., Mycobacterium spp., Brucella spp., Coxiella burnetil, Chlamydia pneumoniae, Bacillus anthracis and Legionella pneumophila. 5

10. A method according to claim 9 wherein the bacteria is selected from Yersinia spp and B. anthracis.

11. A method according to claim 2 wherein the binding proteins are 10 administered by inhalation as an aerosol.

12. A composition for inhalation as an aerosol for prophylaxis of bacterial infection by a bacteria which survives inside phagocytes the composition comprising binding proteins directed against said bacteria which survives inside 15 phagocytes.

13. A composition according to claim 12 comprising a propellant for delivering the composition as an aerosol for inhalation. 20

14. A composition according to claim 12 comprising a protective agent for the binding proteins selected from mammalian colostrum and extracts thereof.

15. A composition according to claim 13 wherein the propellant is a hydroflurocarbon propellant. 25

16. A composition according to claim 12 wherein the binding protein is prepared from hyperimmune colostrum or hyperimmune egg yolk wherein said hyperimmune colostrum or hyperimmune egg yolk was made using an antigen derived from a bacteria which survives inside phagocytes. 30

17. A composition according to claim 13 comprising a non-aqueous carrier selected from lower alkanols, glycerol, lower alkyleneglycols and mixtures thereof. WO 03/097094 PCT/AU03/00616 19

18. A composition according to claim 13 in the form of particles of size in the range of from 20 nm to 10 microns.

19. An inhaler product for prophylaxis of bacterial infection of the respiratory 5 tract by a bacteria which survives inside phagocytes the product comprising a chamber containing a composition comprising binding protein directed against said bacteria and a protective agent for the binding protein selected from colostrum and a means for providing an aerosol of the composition. 10

20. An inhaler product according to claim 19 wherein the composition comprising a binding protein is in the form of a powder and the inhaler product comprises a passage for providing an airstream by inhalation of the user and means for releasing the powder composition into the airstream to form an aerosol thereof. 15

21. An inhaler product according to claim 19 comprising a propellant and a valve for delivering an aerosol of the composition.

22. An inhaler product according to claim 21 comprising a multiplicity of 20 chambers including a first chamber comprising said composition and s second chamber containing a propellant and the inhaler further comprises means for providing mixing of the chambers prior to use.

23. An inhaler product according to claim 22 wherein the composition 25 comprises a liquid carrier or is mixed with a liquid carrier prior to inhalation and the inhaler comprises a spray nozzle and mechanical means for providing delivery of the liquid to the nozzle under pressure.