WO1999038529A1

WO1999038529A1 - Vaccine composition comprising milled lyophilisate of antigenic whole cells

Info

Publication number: WO1999038529A1
Application number: PCT/GB1999/000287
Authority: WO
Inventors: Roderick Peter Hafner
Original assignee: Provalis Uk Limited
Priority date: 1998-01-28
Filing date: 1999-01-28
Publication date: 1999-08-05
Also published as: GB9801870D0; AU2289799A

Abstract

A vaccine composition for the prevention of bacterial or fungal infections of mucosal surfaces comprises a lyophilisate of antigenic whole cells milled to a particle size of from about 20 to 350νm. The vaccine may contain killed organisms such as Haemophilus influenzae or Pseudomonas aeruginosa and is useful, for example, for preventing the colonisation by these organisms of patients suffering from chronic lung diseases or, in previously colonised patients, for preventing the occurrence of acute infection of the respiratory tract.

Description

VACCINE COMPOSITION COMPRISING MILLED LYOPHILISATE OF ANTIGENIC WHOLE CELLS

The present invention relates to a vaccine composition and, in particular, to a vaccine composition which elicits a mucosal immune response and protects against infections of mucosal membranes.

Mucosal membranes, such as those lining the gastrointestinal tract, respiratory tract, the urogen al tract, the eye and the mammary gland, are protected by the common mucosal immune system, which is characterised by the sub-epithelial distribution of both diffuse and aggregated collections of lyπφhoid tissue. Induction of mucosal immune responses for orally dosed products occurs primarily within the Peyer's patches of die gastrointestinal tract with the predominant flow of effector cells from the gut to other mucosal sites via the intestinal lymphatics, mesenteric lymph nodes and the blood.

The dome of the Peyer's patch is covered by specialised membranous epithelial cells called microfold, or M, cells, which are capable of sampling antigen from the gastrointestinal tract and transporting the sample antigen without degradation to the underlying lymphoid tissue. Within the Peyer's patch, there are distinct B cell and T cell areas. The B cells contain germinal centres which give rise primarily to precursor IgM and IgG B cells. Adjacent to the B cell follicles there are defined T cell areas which contain mature T cells and subsets.

Intestinally derived B and T effector cells traffic preferentially from the Peyer's patch to sites within the mucosal immune system, such as the gut itself, the airways, the mammary glands, urogenital tract and eyes and ears.

The nature of the immune response and its efficacy in controlling or preventing 99/38529

infection depend upon the nature of the immunogen (vaccine) and the invading pathogen. Examples of the responses that may be elicited are given below.

The clonal expansion and differentiation of antigen-stimulated B cells into IgM, IgA and IgG plasma cells leads to secretion of specific antibody (serosal and mucosal).

IgA antibody produced by IgA plasma cells is transported across the epithelial membrane into the mucosal fluid by a specialised transport system. However, some of this IgA antibody, which is produced in the sub-epithelial tissue, returns to the blood system by the regional lymphatics.

IgA in the mucosal immune system is distinct from IgA systemically in that it consists of two IgA molecules linked by a special connecting protein. It also has attached to it a protein called a secretory component, which protects the molecule against degradation in the hostile environment found at mucosal sites. This unique molecule is called secretory IgA (slgA). An important property of slgA is that it does not activate the classical complement pathways. This is important because activation of complement results in the induction of significant inflammatory mechanisms. The principle mechanism by which slgA protects the mucosal linings is by the inhibition of attachment of pathogens to the mucosal epithelial cells, thereby controlling the initial colonisation. This function prevents the establishment f infection at the target site. IgA probably has a lesser role in modulating established colonisation. IgG, whether derived from the serum, or mucosal, may also fulfil a protective function.

Antigen-stimulated CD4 positive T_H1 cells from the Peyer's patch may migrate from the gut-associated lymphoid tissue to, for example, the bronchus lumen. Following reactivation at this site due to exposure to the specific antigen of the pathogen, 99/38529 1

secreted cytokines may initiate (or contribute to) a sequence of events involving activated alveolar macrophages leading ultimately to increased polymorphonuclear neutrophil recruitment and activation. Neutrophils are able to phagocytose pathogens and inactivate them.

Thus, in order to provide protection against mucosal infections, a vaccine composition must contain an active agent which is capable of being absorbed from the gastrointestinal tract by the M cells of the Peyer's patches and transported into the lymphatic system where it can elicit an immune response.

Vaccines which protect against infections of mucosal surfaces are known, for example from EP-A-0225329, which describes a non-adjuvanted composition comprising killed bacteria. However, in order to achieve the maximum response from this type of vaccine, the killed bacterial cells should be released close to die Peyer's patches so that proteolytic damage to the surface antigens of the killed cells is minimised. Transit time through the human small intestine is typically several hours and the Peyer's patches are distributed evenly along it and this means that the vaccine should ideally be formulated for sustained release. Formulation of a solid dosage product requires particles of the active material to be granulated to approximately equal size to that of the tablet excipients so as to allow good blending characteristics and even distribution of the active material throughout the product. The rate of release of particles from such a granulated material can then be controlled by the use of appropriate polymers. However, the stresses involved in the granulation process are not compatible with maintaining immunogenicity of the vaccine materials.

The present inventors have surprisingly found that a vaccine composition comprising a milled lyophilisate of antigenic whole cells can achieve sustained release of the cells over the length of the small intestine without the problems associated with granulation.

Therefore, in a first aspect of the present invention, there is provided a vaccine composition comprising a lyophilisate of antigenic whole cells milled to a particle size of from about 20 to 350μm.

It is somewhat surprising that the composition of the invention is effective as a vaccine because it would have been expected that particles of from about 20 to 350μm would be too large to be taken up into the Peyer's patches. However, the effectiveness of the vaccine appears to arise from the fact that, under the conditions which prevail in the gastrointestinal tract, individual cells gradually peel from the surface of the milled particles. It is these cells which are taken up into the Peyer's patches and which trigger the immune response. The peeling of cells from the surface of the lyophilised particles has been observed experimentally when the milled lyophilisate is dispersed in a buffer which simulates the conditions found in the gastrointestinal tract.

The vaccine composition is particularly advantageous because the gradual peeling off of the cells from the particles is, in effect, sustained release of individual cells along the length of the small intestine.

Thus the present invention has all the advantages of a sustained release vaccine product without die risk of destroying the antigenicity of the antigenic whole cells.

In the context of the present invention, the term "vaccine" refers to a composition for pharmaceutical or veterinary use which either prevents colonisation by an organism expressing the antigen contained in the antigenic whole cells or, in subjects 5

in which colonisation has already occurred, reduces acute exacerbation of infection by the orgamsm.

Whilst the particles may be milled to a size of from about 20 to 350 μm, it is preferred that the particles are within the range of about 20 to 250 μm. The best results are achieved when the particles are up to 150 μm in size.

The antigenic whole cells may be killed bacterial or yeast cells and the particular species of cell used will, of course, depend upon die condition against which the patient is being immunised. Examples of suitable species include Haemophilus influenzae, Pseudomonas species (such as P. aeruginosa and P. cepaceia), E. coli (both enteropathogenic and enterotoxigenic), Streptococcus species (such as S. pneumoniae, S. mitis, S. mutans, S. viridans and S. pyogenes), Staphylococcus aureus, Mycobacterium tuberculosis (such as Bacille Calmette-Guerin, BCG), Corynebacterium parvum, Candida species (such as C. albicans, C. cruzzi and C. glabrata), other causative agents of genitourinary tract infections, for example Chlamydia trachomatis and Neisseria gonnorhoea, Helicobacter pylori, bacteria known to cause respiratory exacerbations in diseased populations, for example Diplococcus pneumoniae, Klebsiella pneumoniae, Klebsiella ozaenae and Branhamella catarrhalis, bacteria known to cause meningitis, for example Neisseria meningitidis, bacteria which cause whooping cough, cholera, diphtheria and typhoid and organisms related to any of the above. Other suitable species will be familiar to those skilled in the art.

Alternatively, the antigenic whole cells may be of an organism which has been genetically modified so as to be antigemc. Organisms which may be modified in this way include Lactococcus lactis which may be genetically modified to express antigenic elements. Similarly, antigenic whole cells may be recombinant organisms. 6

The genetically modified or recombinant organisms may include antigens from one or more of the species mentioned above.

A further type of antigenic whole cell which may be useful in the present invention is a bacterial or eukaryotic cell onto which a virus has been adsorbed.

The composition of the invention may contain cells of a single species or a mixture of species.

Since all mucosal surfaces are protected by the common mucosal immune system, the vaccine of the present invention may be used to protect against bacterial, viral or fungal infections of any mucosal surface, for example the surfaces lining the gastrointestinal tract (including the buccal cavity), the middle ear, the eustachian tubes, respiratory tract (including the nose and the nasopharynx), the urogenital tract, the eye and the mammary gland.

Killed whole cell vaccines have, in the past, been particularly useful for the prophylaxis of infections of the lungs and respiratory tract and the vaccine of the present invention is also of particular use in this field.

The vaccine of the present invention has a dual use and thus, for example, in a patient with a chronic lung disease such as cystic fibrosis, chronic bronchitis or bronchiectasis, a vaccine composition of the invention containing whole killed cells of Haemophilus influenzae or Pseudomonas aeruginosa or genetically modified or recombinant cells containing antigens from these organisms can prevent colonisation of the patient with Haemophilus influenzae or Pseudomonas aeruginosa.

In patients with chronic lung diseases who are already colonised with these organisms, the composition may be used to prevent acute infection of the patients by Haemophilus influenzae or Pseudomonas aeruginosa, although it will not necessarily clear the colonising organism. Acute infection by these organisms considerably worsens the prognosis of patients with severe lung disease.

This dual use of the vaccine also applies to the infection of other mucosal surfaces. Thus, the vaccine can either be used to prevent initial colonisation by an infecting organism or to reduce the incidence of acute infections in a patient already colonised by the orgamsm.

Vaccine compositions according to the invention may be used to prevent bacterial conjunctivitis, oral or vaginal thrush, which is caused by infection with Candida species, infection of the gastrointestinal tract with Helicobacter pylori, which can lead to inflammation and ulceration of the lining of the gastrointestinal tract, otitis media, meningitis, tonsillitis, pharyngitis, tuberculosis, secondary bacterial infections for example those occurring after a cold, diarrhoea caused, for example, by organisms such as E. coli, S. typhi and V. cholerae, pneumonia, conditions caused by Corynebacterium parvum and other sexually transmitted diseases.

The vaccine composition may contain an adjuvant but, in many cases, it is preferable not to include one. Adjuvants are well known to those skilled in the art of vaccine formulation and it would be a relatively simple matter to choose one which is suitable. In some cases, the killed cells in the vaccine may elicit a sufficient antigenic response so as not to require an adjuvant.

However, even species such as Haemophilus influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Helicobacter pylori, which do not elicit such a strong immune response may be 99/38529 ₈

included in the formulation without an additional adjuvant.

Compositions in which the antigenic whole cells are genetically modified or recombinant organisms or which include viruses adsorbed onto cells may, in some cases require an adjuvant. Those skilled in the art can easily determine whether or not an adjuvant is necessary using known methods and without undue experimentation.

The vaccine composition of the present invention is intended for oral administration and may be formulated in any suitable manner. For example, the milled lyophilisate may be encapsulated in hard or soft capsules or may be mixed with an excipient and tabletted. Sugars are especially suitable tabletting excipients and examples include lactose, sucrose, mannitol and sorbitol. However, other known tabletting excipients such as microcrystalline cellulose or dicalcium phosphate may be included in addition to or instead of sugars. In the presence of suitable excipients, the particle size of the milled lyophilisate is maintained in tabletting, indicating that the material retains substantial strength. Alternatively, the lyophilisate may be supplied as a powder which may be packaged in single-dose sachets.

The vaccine composition of the present invention may be used in a method of preventing the colonisation of mucosal surfaces by an infecting organism, the method comprising immunising a patient at risk of such infection with a vaccine composition comprising a lyophilisate of antigemc whole cells milled to a particle size of from about 20 to 350μm.

The composition is also of use in a method for the prevention of acute infections by an organism in an individual already colonised by that orgamsm, the method comprising immunising a patient at risk of such infection with a vaccine composition 9/38529 _

comprising a lyophilisate of antigenic whole cells milled to a particle size of from about 20 to 350μm.

In a further aspect, the present invention provides the use of a lyophilisate of antigenic whole cells milled to a particle size of from about 20 to 350μm in the preparation of a vaccine composition for preventing colonisation of mucosal surfaces by an infecting organism expressing an antigen which is the same as the antigen of the lyophilisate or for preventing acute infection by the organism in a previously colonised patient.

The vaccine composition of the present invention is relatively simple to prepare and the method of preparation comprises the steps of:

i. preparing a suitable quantity of cells of the required organism by fermentation; ii. killing the organism by a method which enables antigenicity to be maintained; iii. washing and harvesting the killed cells by any suitable method; iv. lyophilising the harvested killed cells; and ii. milling the cake of killed cells to a particle size of about 20 to 350 μm.

Further optional steps include filling the milled lyophilisate into capsules or mixing the milled lyophilisate with a suitable excipient and tabletting.

Suitable methods of killing the cells include treatment with formaldehyde and the killed cells may be harvested by centrifugation.

The invention will now be described in greater detail, by way of example only, with reference to the following examples and to the Figures in which: 99/38529 JQ

FIGURE 1 is a plot showing the number of live bacteria recovered from bronchoalveolar lavage fluid in non immunised rats and in rats immunised with either a freshly prepared suspension of formalin-killed P. aeruginosa or with a milled lyophilisate of P. aeruginosa and subsequently challenged with live bacteria.

FIGURE 2 is a plot showing the number of live bacteria recovered from lung homogenate in non immunised rats and in rats immunised with either a freshly prepared suspension of formalin killed P. aeruginosa or with a milled lyophilisate of P. aeruginosa and subsequently challenged with live bacteria.

FIGURE 3 is a plot showing the in vitro proliferative response to l.Oμg of P. aeruginosa antigen for non immunised rats and rats immunised with either a freshly prepared suspension of formalin killed P. aeruginosa or with a milled lyophilisate of P. aeruginosa and subsequently challenged with live bacteria.

FIGURE 4 is a plot showing the in vitro proliferative response to l.Oμg concanavalin A for non immunised rats and rats immunised with either a freshly prepared suspension of formalin killed P. aeruginosa or with a milled lyophilisate of P. aeruginosa and subsequently challenged with live bacteria.

FIGURE 5 is a plot showing the extent of P. aeruginosa-speciRc antibody response in the serum of non immunised rats and rats immunised with either a freshly prepared suspension of formalin killed P. aeruginosa or with a milled lyophilisate of P. aeruginosa and subsequently challenged with live bacteria.

FIGURE 6 is a plot showing the extent of P. aeruginosa-specific antibody response in bronchoalveolar lavage fluid of non immunised rats and rats immunised with either a freshly prepared suspension of formalin killed P. aeruginosa or witii a milled lyophilisate of P. aeruginosa and subsequently challenged with live bacteria.

FIGURE 7 shows milled lyophilisate particles immediately after dispersion in deionised water and with a magmfication of x50.

FIGURE 8 is a close up (magnification x 1000) of the edge of one of the particles of Figure 7 and shows individual cells gradually being released from the surface of the particle.

FIGURE 9 is a similar view to Figure 8 (magnification x 1000) but taken two to three hours after dispersion of the lyophilisate particles in the deionised water.

FIGURE 10 is a plot showing the particles size profile of the lyophilisate when first dispersed in deionised water.

FIGURE 11 is a plot which is similar to Figure 10 but which shows the particles size profile of the lyophilisate one hour after dispersion in deionised water.

O 99/38529 12

EXAMPLE 1 - Preparation of Tablets Containing Milled Lyophilised P. aeruginosa

Ingredient Quantity per tablet

Tablet Core

Milled lyophilisate of P. aeruginosa 15.6 mg

Lactose 223.15mg

Povidone lO.Omg

Magnesium stearate 1.25mg

Enteric Coat f20mg per tablet)

Polyvinyl acetate phthalate 60.048% w/w

Titanium dioxide 27.250% w/w

Diethyl phthalate 5.976% w/w

Stearic acid 5.976% w/w

Iron oxide yellow 0.750% w/w

A culture broth of P. aeruginosa is used to inoculate two 500ml flasks of Tryptone Soya Broth which is incubated aerobically at 37°C. Once the OD660,,,,, reaches a value greater than 4, two 500ml flasks are used to inoculate a fermenter containing 30L of Tryptone Soya Broth with a suitable quantity of Antifoam C™. The fermenter is controlled to maintain an oxygen tension of at least 40% and the cells grown until an OD660,,,, of 5-6 is reached. The seed fermenter is used to inoculate a fermenter containing 400L of Tryptone Soya Broth. The fermenter is controlled to maintain an oxygen tension of at least 40% and d e cells grown until an ODόόO_^, of 5-6 is reached. The cells are then killed by addition of formaldehyde to a final concentration of 1 % followed by incubation for 10-12 hours. The contents of the fermenter are then transferred to a holding tank and held for a further 6-8 hours before harvesting using a centrifuge. The resulting cell paste is frozen to -70°C and lyophilised using a shelf temperature of -20°C and a condenser temperature of -51°C and a pressure of 0.13mBar. When primary drying is complete and die vacuum stabilises, the secondary drying cycle is initiated by ramping the shelf temperature up by 5°C increments to a maximum of 15°C. 13

The lypophilisate is milled using a Fritsch™ variable speed rotor mill fitted with a 200 micron trapezoidal screen operating at 12000 rpm and a feed rate of 20g/min. The milled material is blended witii die tablet core excipients using a drum blender and compressed using a rotary tablet press fitted witii 9mm tooling. The resulting tablets are enteric coated in a perforated drum coater, applying the enteric coat components from an ethanol/water suspension.

EXAMPLE 2 - Preparation of Capsules Containing Milled Lyophilised C. albicans

Using the general method of Example 1, a milled lyophilisate of Candida albicans was prepared. 175-250mg of the lyophilisate was filled into white opaque size 1 gelatin capsules. The capsules were gelatin banded and enteric coated with the following coating composition.

Material Quantity per Capsule

Methacrylic acid copolymer 22mg

Diacetylated monoglycerides 3.35mg

Talc 8.8mg

Magnesium stearate 5.25mg

EXAMPLE 3 - Preparation of Capsules Containing a Milled Lyophilisate of Helicobacter pylori

Using the general method of Example 1, a milled lyophilisate of Helicobacter pylori was prepared. 5mg of the lyophilisate was mixed wim 100-250mg of lactose and O 99/38529 ₁₄

filled into white opaque size 1 gelatin capsules. The capsules were gelatin banded and enteric coated with the following coating composition.

Material Quantity per Capsule

Methacrylic acid copolymer 22mg

Diacetylated monoglycerides 3.35mg

Talc 8.8mg

Magnesium stearate 5.25mg

EXAMPLE 4 - Effectiveness of Composition Containing MiUed Lyophilisate of P. aeruginosa in Animal Model

In this example, the following abbreviations are used:

BAL Bronchoalveolar lavage fluid

CFU Colony forming units

ConA Concanavalin A

CPM Counts per minute LH Lung homogenate

ML Milled lyophilisate of P. aeruginosa

PBS Phosphate buffered saline

SE Standard error of die mean

SER Serum SI Stimulation index

SPF Specific pamogen free P. aeruginosa were grown overnight on a nutrient agar plate and harvested into PBS. The cell concentration was adjusted to 10¹⁰/mL by comparison of optical density at 405nm with a standard regression curve. An equal volume of 2% parafoπnaldehyde in PBS was added and the suspension incubated for 2h at 37°C. Following three washes with PBS, the live P. aeruginosa was resuspended in PBS to 10^,0mL. The actual concentration of live bacteria was checked by plating out 20μL of serial dilutions onto nutrient agar plates, incubation overnight at 37°C and then counting the CFU.

P. aeruginosa were killed using formalin and a freshly prepared suspension of the killed bacteria was used as a "gold standard". The gold standard was compared for effectiveness in an animal model against a milled lyophilisate of P. aeruginosa prepared using the method described in Example 1.

Animal Model

Animals used were specific pathogen free (SPF) male Dark Agouti (DA) rats. Groups of five rats were immunised intra-duodenally with 0.5mL of either the gold standard or die milled lyophilised P. aeruginosa preparation suspended in PBS to 10¹⁰ organisms per mL. The dose of P. aeruginosa per rat was therefore 5 x 10⁹ organisms, equivalent to 3.9 mg of original lyophilisate. The immunisation was performed by anaesthetising the rats, opening the peritoneum to expose the intestine and injecting 0.5mL of the suspension of killed P. aeruginosa into the duodenum. The incision was closed and d e rat allowed to recover from anaesthesia. Another group of rats was left untreated and this was the non-immune control group.

Fourteen days after immunisation, rats were infected by intra-tracheal instillation of 5 x 10⁸ live bacterial in 50μL of PBS. Rats were killed 4 hours later by intraperitoneal overdose of sodium pentobarbitone. Blood was collected by heart- 99/38529 j₆

bleed and allowed to clot. Serum was separated and stored frozen at -20°C for subsequent antibody analysis. Lungs were lavaged wim 5 x 2 mL of PBS. Pooled BAL was analysed for number of live bacteria by plating out 20μL of serial 10-fold dilutions onto nutrient agar and counting CFU after overnight incubation at 37°C. Lavaged lungs were homogenised in 10 mL PBS and this homogenate was also assessed for number of CFU. Mesenteric lymph nodes were removed from the rats. A single cell suspension was prepared and after two washes the cells were suspended in supplemented RPMI medium at 2 x 10¹⁰ cells/mL. Triplicate cultures were set up containing 2 x 10⁵ cells/200μL/well in flat-bottom 96 well tissue culture plates. Cultures contained no antigen, P. aeruginosa antigen (Pa antigen) at 0. lμg/mL or

1.Oμg/mL or ConA at 2μg/mL. Pa antigen was prepared and treated as described by Dunkley et al in Advances in Mucosal Immunology, J. Mestecky et al Ed. (1995), Plenum Press, New York, pages 755-759. Plates were cultured for 4 days and wells were pulsed witti 0.5 μCi ³H-thymidine for the last 7 hours of culture. Wells were harvested onto glass fibre filter paper. Filters were placed in tubes with 4 mL of BCS scintillant and counted for 60 seconds in a β-counter to get a measurement of counts per minute. Stimulation index was assessed by dividing die CPM in die presence of antigen or ConA by the CPM in the absence of antigen.

Statistical Method - Comparison of Groups

The mean and SE for each parameter for each group was calculated. Immunised groups were compared wim the non-immune control group and groups immunised wim the milled lyophilisate were compared wim the group immunised wim the gold standard using an unpaired t test (Macintosh Systat). P values are shown in Table 1. P<0.05 is considered to be statistically significant. O 99/38529

17

Table 1 - Statistical Analysis

Comparison BAL LH SI SI SER SER SER BAL BAL BAL CFU CFU Pa 1.0 ConA IgG IgA IgM IgG IgA IgM

Non-immune 0.000 0.001 0.260 0.393 0.000 0.114 0.037 0.014 0.050 0.047 vs Gold Std

Non-immune 0.000 0.011 0.781 0.734 0.036 0.004 0.002 0.015 0.002 0.000 vs ML

Gold Std vs 0.432 0.943 0.348 0.430 0.067 0.924 0.174 0.059 0.004 0.811 ML

Results

Live bacteria recovered is shown in Figures 1 and 2 and mean bacterial clearance in Table 2. In BAL, the number of live bacteria in both immunised groups is significantly lower than in the non-immune group (Figure 1 and Table 2). The milled lyophilisate immunised group was not significantly different from the gold standard immunised group.

In LH, the number of live bacteria recovered in the gold standard and milled lyophilisate immunised groups is significantly lower than in the non immune group

(Figure 2 and Table 2). The groups immumsed with milled lyophilisate and gold standard are not significantly different.

In Table 2, % bacterial clearance is defined as:

(CFU non immune - CFU immunised) x 100 CFU non immune O 99/38529

18

Table 2 - % Bacterial Clearance

Group % Clearance BAL % Clearance LH

Non-immune 0 0

Gold Standard 93.7 68.1

Milled Lyophilisate 95.9 65.9

The in vitro proliferative response is shown in Figures 3 and 4. The response to 1.Oμg Pa antigen of immunised groups is not significantly different from that of the non-immune group and die test groups are not significantly different from the gold standard (Figure 3). For the ConA response, the immunised groups are not significantly different from me non-immune group or from each other (Figure 4).

The P. aeruginosa-specific antibody response is shown in Figures 5 and 6. Serum P. aeruginosa-specific IgG, IgA and IgM are significantly higher in the immumsed groups than the non-immune group (Figure 5). There is no significant difference between the group immunised with milled lyophilisate and die gold standard group. Figure 6 shows that BAL IgG, IgA and IgM are all sigmficantly higher in the immunised groups than in the non immune group (except for the gold standard which is close to significance, P=0.05). The BAL IgA of the Pa milled lyophilisate immunised group is sigmficantly higher than that of the gold standard immunised group. The BAL IgG and IgM of die milled lyophilisate immunised group are not significantly different from those of the gold standard.

Conclusion

The bacterial clearance assay is the most relevant assay to perform as it is an actual measure of protection against infection. The proliferation and antibody assays provide extra information about die immune status of the rats following immunisation.

In the bacterial clearance assay, there does not appear to be an appreciable difference between the gold standard and die milled lyophilisate. Both provide substantial bacterial clearance from the lung. This indicates diat the particle size of the milled lyophilisate does not, as might have been expected, prevent it from being absorbed by M cells in the gastrointestinal tract.

EXAMPLE 5 - Peeling of Cells from the Milled Particles

Milled lyophilisate particles were dispersed in distilled water and observed under a light microscope. Figure 7 shows die particles immediately after dispersion. Figure 8 shows a close up of the edge of one of die particles with individual bacterial cells gradually being released from the surface. After 2-3 hours, the large milled particles break up into smaller particles about 20-3 μm across, which continue to release individual cells (Figure 9)

The gradual release of smaller particles and cells can also be visualised using a Malvern Mastersizer X™. Figures 10 and 11 demonstrate me behaviour of a milled lyophilisate dispersed in deionised water. Figure 10 shows the particle size profile of the material when first dispersed in deionised water. The majority of the material is in the 100-200μm particle size range witii only a small quantity between 1 and lOμm. Figure 11 shows the same material after one hour. There has been a significant decrease in die material with a particle size of between 100 and 200μm and a noticeable increase in the material with a particle size of 1 to lOμm. Importantiy, a significant proportion of the material remains with a particle size of 100 to 200μm, providing a reservoir of material for ongoing release.

Claims

C A S

1. A vaccine composition comprising a lyophilisate of antigenic whole cells milled to a particle size of from about 20 to 350μm.

2. A vaccine composition as claimed in claim 1 , wherein the particle size is from about 20 to 250μm.

3. A vaccine composition as claimed in claim 2, wherein the particle size is from about 20 to 150μm.

4. A vaccine composition as claimed in any one of claims 1 to 3, wherein the antigenic whole cells are bacterial or yeast cells.

5. A vaccine composition as claimed in claim 4, wherein the bacterial or yeast cells are of one or more of the species Haemophilus influenzae, Pseudomonas species (such as P. aeruginosa and P. cepaceia), E. coli (both enteropathogenic and enterotoxigenic), Streptococcus species (such as S. pneumoniae, S. mitis, S. mutans,

5. viridans and S. pyogenes), Staphylococcus aureus, Mycobacterium tuberculosis (such as Bacille Calmette-Guerin, BCG), Corynebacterium parvum, Candida species

(such as C. albicans, C. cruzzi and C. glabrata), other causative agents of genitourinary tract infections, for example Chlamydia trachomatis and Neisseria gonnorhoea, Helicobacter pylori, bacteria known to cause respiratory exacerbations in diseased populations, for example Diplococcus pneumoniae, Klebsiella pneumoniae, Klebsiella ozaenae and Branhamella catarrhalis, bacteria known to cause meningitis, for example Neisseria meningitidis, bacteria which cause whooping cough, cholera, diphdieria and typhoid and organisms related to any of die above.

6. A vaccine composition as claimed in claim 4, wherein the bacterial or yeast cells are cells of an orgamsm which has been genetically modified so as to be antigenic, for example Lactococcus lactis which has been genetically modified to express antigenic elements, or cells of a recombinant organism.

7. A vaccine composition as claimed in claim 6, wherein the genetically modified or recombinant organism includes antigens from one or more of the species defined in claim 5.

8. A vaccine composition as claimed in claim 4, wherein die bacterial or yeast cells are cells onto which a virus has been adsorbed.

9. A vaccine composition as claimed in any one of claims 1 to 8, which protects against infections of the surfaces lining the gastrointestinal tract (including me buccal cavity), the middle ear, the eustachian tubes, respiratory tract (including die nose and the nasopharynx), the urogenital tract, the eye and the mammary gland.

10. A vaccine composition as claimed in any one of claims 1 to 9 for the prevention of bacterial conjunctivitis, oral or vaginal thrush, which is caused by infection with Candida species, infection of the gastrointestinal tract with

Helicobacter pylori, which can lead to inflammation and ulceration of me lining of the gastrointestinal tract, otitis media, meningitis, tonsillitis, pharyngitis, tuberculosis, secondary bacterial infections for example those occurring after a cold, diarrhoea caused, for example, by organisms such as E. coli, S. typhi and V. cholerae, pneumonia, conditions caused by Corynebacterium parvum and omer sexually transmitted diseases.

11. A vaccine composition comprising whole killed cells of one or more of: Haemophilus influenzae, Pseudomonas aeruginosa or genetically modified or recombinant cells containing antigens from these organisms, wherein the cells are milled to a particle size of from about 20 to 350μm.

12. A vaccine composition as claimed in any one of claims 1 to 11, further including an adjuvant.

13. A vaccine composition as claimed in any one of claims 1 to 12, wherein the milled lyophilisate is encapsulated in hard or soft capsules or is mixed with an excipient and tabletted or is provided as a powder.

14. The use of a lyophilisate of antigenic whole cells milled to a particle size of from about 20 to 350μm in the preparation of a vaccine composition for preventing colonisation of mucosal surfaces by an infecting organism expressing an antigen which is the same as an antigen expressed by die lyophilisate or for preventing acute infection by the organism in a previously colonised patient.

15. A process for the preparation of a vaccine composition for preventing bacterial or fungal infections of mucosal surfaces or for preventing acute infections in patients colonised with a bacteria or fungus, the process comprising the steps of: i. preparing a suitable quantity of cells of the required organism by fermentation; ii. killing the organism by a method which enables antigenicity to be maintained; iii. washing and harvesting the killed cells by any suitable method; iv. lyophilising the harvested killed cells; and ϋ. milling the cake of killed cells to a particle size of about 20 to 350 μm.