WO2012104647A1

WO2012104647A1 - Vaccine compositions

Info

Publication number: WO2012104647A1
Application number: PCT/GB2012/050226
Authority: WO
Inventors: Eric Edward Worrall
Original assignee: Anhydro Biologicals Limited
Priority date: 2011-02-04
Filing date: 2012-02-03
Publication date: 2012-08-09
Also published as: GB201101950D0

Abstract

Vaccine compositions comprise (a) an inactivated antigenic component derived from a virus seiected from Orthomyxovirus and Paramyxovirus; and (b) a sialidase-Gontaining, proteolytic enzyme-detoxified bacteria! exotoxin. Preferably, the compositions also comprise hyaluron!dase. The vaccine compositions have appiication in the treatment of, or prevention of, viral infections caused by a virus seiected from Orthomyxovirus and Paramyxovirus. The compositions are particularly useful for application to birds, especiaiiy chickens and other poultry, in the treatment of, or prevention of, influenza type A subtype H5N1 and Newcastle disease infections.

Description

S

VACCINE CO PQSmONS

The present invention relates to vaccine compositions useful for eliciting an immune response in subjects which is protective against a virus selected from Orthomyxovirus and Paramyxovirus.

Influenza, including the subfamily of Influenza A, B and C, is an infectious disease of mammals and birds caused by RNA viruses of the family Orthomyxoviridae. Typically, it is transmitted from infected animals by coughs and sneezes, creating aerosols containing the virus, and from birds through their droppings. The disease can also be transmitted through contact with bod fluids of infected subjects and with surfaces which have been contaminated with these.

The closely related family Paramyxoviridae includes:

Genus Aval a virus type species Newcastle disease virus;

Genus Henipavirus type species Hendravirus and Nipavirus;

Genus Morbiilivirus type species Measles virus, Rinderpest virus, Canine distemper virus, phocene distemper virus and Peste des Refits Ruminants virus;

Genus Rubaiavirus type species Mumps virus; and

Human Parainfluenza virus type 1 and 3.

There are other, minor, viruses in the Paramyxovirus family but those stated above are a major cause of disease in humans and animals which may be targets for therapeutic and immu_.noprophylaxis.

The term "avian influenza virus" is usually understood to mean an influenza type A virus since wild birds are the naturai hosts of influenza A virus. Some type A strains are called low pathogenic since such strains (LPAl viruses) are generally of low virulence although they can serve as progenitors of highly pathogenic strains (HPAI viruses). Highly pathogenic strains of Influenza A subtype H5N1 are endemic in birds in south east Asi and are believed to represent a long term pandemic threat. These strains are highly contagious between domestic poultry, such as chickens and turkeys. Outbreaks of the disease in poultry farms, where birds are often intensively reared in close contact with one another, have resulted in bird mortality rates of 100%.

Influenza viruses have two main antigenic glycoproteins at their surfaces: haemagglutinin (HA) and neuraminidase (NA) (also known as sialidase). Primary functions of HA are to bind to the sialic acid receptor sites on the cell membranes of the upper respiratory tract and on the surface of erythrocytes and to facilitate the entry of the viral genome into target cells in the host, NA has functions which aid and promote the release of progeny viruses from infected cells and also plays a role in promoting the entry of the virus into a host cell.

The pharmaceutical industry has produced neuraminidase inhibitors for combating influenza infection. These act by preventing or inhibiting the viral neuraminidase from performing its function. Examples of such neuraminidase inhibitors include Zanamivir and Ose!tamivi ("Tamifiu") which act by blocking the activity of the viral neuraminidase in releasing progeny virus particles from infected cells. Unfortunately, drug therapy using such compounds is often ineffective after the disease has been clinically recognised in a subject since the virus is, by this time, already well established. The use of such compounds prophyiacticaily i the treatment of poultry is usually too expensive for the poultry farmers, particularly those in the areas of the world where H5N1 is endemic.

A vaccine composition comprising influenza type A virus antigen and a bacterial sialidase is disclosed in WO 2010/052492. The preparation of the bacterial sialidase described involves the detoxification of sialidase-containing exotoxin obtained from Clostridium petfringens using formalin, it has been found, however, that the formalin treatment damages the sialidase enzyme in the toxoid and, thus, reduces the effectiveness of the bacterial sialidase in the vaccine compositions disclosed in the document.

The aim of the present invention is to provide a cost-effective vaccine which has improved effectiveness and which can be used in large scale immunisation programmes for therapeutic or prophylactic treatment against an infection by a virus selected from Orthomyxovirus and Paramyxovirus. Accordingly, the present invention provides a vaccine composition comprising

(a) an inactivated antigenic component derived from a virus selected from Orthomyxovirus and Paramyxovirus; and

(b) a sialidase-containing, proteolytic enzyme-detoxified bacteria! exotoxin.

St has surprisingly been found that the incorporation into the vaccine composition of a sialidase-containing, proteolytic enzyme-detoxified bacterial exotoxin has the effect of increasing the potency of the vaccine. Although we do not wish to be bound by theory, it Is possible that the potentiating effect of this bacterial sialidase-containing detoxified exotoxin results from the selective removal of sialic acid from the ssaiated membrane glycolipids present on the surfaces of the host's epithelia by the sialidase thereby preventing any Orthomyxovirus or Paramyxovirus from binding at these sialic acid receptor sites. We have found that, unlike in the prior art, formalin-detoxification of bacterial, sialidase-containing exotoxin detoxification of the exotoxin using a proteolytic enzyme does not affect the potency of the sialidase. By blocking the entry of the virus into the vuinerable ceils of the host, more time is provided fo the host's immune response, elicited by the antigen in the vaccine composition, to act against the attacking virus. In addition, the siaildase will, itself, have an antigenic effect in the host and, thus, will elicit the production of anti-bacterial sialidase antibodies by the host's immune system which may have an effect against the viral neuraminidase.

Examples of suitable sources for the sialidase-containing bacterial exotoxin, which must be detoxified by a proteolytic enzyme according to the present invention, are Pseudomonas aeruginosa and Clostridium perfringens. Preferably, the bacterial sialidase s obtained from Clostridium perfringens and more preferably from Clostridium perfringens Type A, especially Type A strain 107, a high sialidase strain. The preparation of a purified sample of sialidase from Clostridiu ' perfringens A strain 107 is described herein in the following Examples. According to the method used herein, cultured bacterial cells were treated with a proteolytic enzyme to produce a CI perfingens toxoid containing sialidase. It is known that the non-toxoided form of the Clostridium perfringens alpha toxin is haemolytic and necrotic and is the cause of infectious necrotic enteritis in chickens. It is believed that the use of the Clostridium perfringens sialidase in the vaccine composition of the invention has the additional effect of improving resistance, in chickens to which the vaccine compositions is administered, to infectious necrotic enteritis.

The vaccine composition of the invention contains a sialidase- containing proteolytic enzyme-detoxified bacterial exotoxin. By the expression "detoxified bacteria! exotoxin" we mean a bacterfal exotoxin which has been weakened or modified such that it is no longer toxic but which still retains its antigenic functions. As used herein, the term "toxoid" means a detoxified bacteria! exotoxin. By the expression "proteolytic enzyme- detoxified" we mean that the weakening or modification of the exotoxin has been achieved by the action of a proteolytic enzyme on the exotoxin.

The action of the proteolytic enzyme on the exotoxin to detoxify the exotoxin does not adversely affect the sialidase activity of the exotoxin, Useful proteolytic enzymes in the present invention will typically be endopeptidases. According to an embodiment of the present invention, the endopeptidase will be a serine protease, a cysteine protease or an aspartic protease, preferably a serine protease or a cysteine protease or a combination thereof. Examples of suitable proteolytic enzymes include trypsin, papain, bromelain, ficin, Bacillus suhti!is protease, Bacillus iicheniformis protease and microbial rennet from Rhizomucor miehei. Preferably the proteolytic enzyme is trypsin or papain.

According to one embodiment, the virus is an Orthomyxovirus, preferably an influenza type A virus.

According to a particularly preferred embodiment, the virus is an influenza type A virus subtype H5N1.

According to a different embodiment, the virus is a virus of the Paramyxoviridae family, preferably a virus selected from:-

Genus Avulavirus type species Newcastle disease virus:

Genus Henipavirus type species Hendravirus and Nipavirus; Genus MorbiMvirus type species Measles virus, Rinderpest virus, Canine distemper virus, phocene distemper virus and Peste des Petits Ruminants virus;

Genus Rubalavirus type species Mumps virus; and

Human Parainfluenza virus type 1 and 3.

!n the vaccine composition of the invention, an inactivated antigenic component derived from the virus selected from Orthomyxovirus- and Paramyxovirus is used. The inactivated antigenic material may, according to one embodiment, comprise inactivated who!e virus particles. Alternatively, according to a different embodiment, the inactivated antigenic materia! may comprise disrupted virus (split virus) in which immunogenic protein, for example, 2 ion channel protein or glycoprotein, is retained. According to yet a further embodiment, the inactivated antigenic material comprises a purified viral membrane glycoprotein. A vaccine composition according to the invention may comprise one or more types of these antigenic materials.

The virus used to prepare the antigenic material used in the vaccine of the present invention will, of course, depend on the disease against which the recipient of the vaccine is to be immunized, in the case of vaccinating poultry against influenza type A subtype H5N1 , it will, of course, be necessary for the composition to comprise inactivated antigenic material derived from at least one strain of influenza A subtype H5N1. More preferably, the vaccine composition will comprise inactivated antigenic material from more than one strain of H5 1 in order to elicit broader protection in a subject against different strains of H5 1 in circulation.

Hya!uronidases are a famiiy of enzymes that are known to catalyse the hydrolysis of hyaluronic acid, which is a constituent of epithelia! tissue. According to a preferred embodiment, the vaccine composition of the invention also comprises hyaluronidase. It is believed that the presence, in the vaccine, of hyaluronidase will, because of its degradative effect on hyaluronic acid in the epithelium, increase tissue permeability and, thus, aid the delivery of the inactivated antigenic component of the vaccine to the body of the subject through epithelial tissue. As stated above, the preferred source of sialidase-containing bacterial exotoxin for use in the manufacture of the vaccine of the present invention is Clostridium petfringens, especially Clostridium petfringens Type A and most especially Clostridium petfringens Type A strain 107, Hyaiuronidase is produced by Clostridium petfringens Type A bacteria and, thus, is present in the sialidase-containing exotoxin derived from these bacteria.

The vaccine composition may comprise any one or more suitable carrier, e cipient, stabiliser, preservative, or other additive, as is conventional in the art. Since the main point of entry of the influenza virus into a host is via mucosal surfaces in the upper respiratory tract, it is a preferred embodiment of the present invention that the vaccine composition is adapted for mucosal administration and more preferably intranasal administration, InfranasaSiy administered vaccines are, of course, well known and the common general knowledge of the person skilled in the art teaches how vaccines for intranasal administration may be prepared.

if is also well known in the vaccine art to use an adjuvant in association with an antigenic component of a vaccine. Adjuvants are substances which enhance the immune response to an antigen in a subject. Vaccines adapted for mucosal administration, particularly those adapted for intranasal administration, have been prepared using chitosan, in addition to the antigenic materia!, to take advantage of its ability to increase transceiluiar and paracellu!ar transport across mucosal epithelium. Chitosan is the general name given to a class of cationic polysaccharides prepared by the deacetylation of chitin which is a natural biopolymer that occurs abundantly in the exosketetons of marine crustaceans. Chitosan is a linear cationic polyelectrolyte which is non-toxic, biodegradable and biocompatible and is a white or off-white amorphous translucent solid which is soluble in dilute organic acids. Chitosan, typically, is used in the form of a non-toxic acid addition salt, e.g. chitosan HGi, which is more soluble than chitosan. Such materials are available from Nova atrix.

The chitosan will typically be a deacetylated chitin which is at least 70% deacetylated and preferably at least 80% deacetylated. Chitosan (>75% deacetylated) is avai!abie from Sigma-Aidrich, The chitosan may be used in an amount effective to stimulate the immune response of the intranasally administered virus A antigen. Typical concentrations of the chitosan used in the vaccine composition of the invention are in the range of from 0.05 to 5%, preferably from 0.1 to 2.0%, more preferably 0.2 to 1.0%, by weight based on the volume of aqueous vaccine composition,

The vaccine compositions of the invention, including those formulated for intranasal administration, can be formulated as liquids or as dry powders according to procedures known in the art, Preferably, the vaccine compositions will be formulated as liquids, especially as aqueous dispersions, suspensions or solutions, for administration as drops or aerosols. The use of the vaccine composition of the invention in an aqueous system is especially preferred in the treatment of poultry since this makes if possible to carry out effective, mass vaccination programmes whereby large numbers of birds may be treated quickly and relatively inexpensively by droplet or aerosol administration, intranasal application, for example by the application of a single droplet of aqueous vaccine to one nostril of a bird being treated, is eas to carry out in the field by unskilled operators. Furthermore, because of the increased levels of mucosal IgA which result from intranasal vaccination, subsequent protection against the natural route of infection is enabled. When chitosan is used as an adjuvant in an aqueous vaccine composition of the Invention, it is beneficial, in order to maintain the solubility of the chitosan in the aqueous medium while ensuring that the antigen component of the vaccine is not adversel affected, that the aqueous vaccine composition has a pH in the range of from about 5.0 to 6.6, preferably about 5.0.

According to a especially preferred embodiment, the vaccine composition according to the invention contains inactivated split H5N1 virus antigen, siaiidase and hyaluronidase and preferabl a preservative, such as gentamycin suiphate, suspended in pH 5.0 acetic acid/acetate buffer, wherein the siaiidas and hyaluronidase are both obtained from frypsin-detoxified Clostridium petfringens type A strain 107 cell free centrifugate. Each 0.03 mi dose (for intranasal administration) contains 100 HA units of siaiidase and 48 units of hyaiuronidase. Preferably, the virus antigen is a mixture of two or more different isolates of the same virus, typically a 1 :1:1 mixture of three isolates. A preservative, if used, will typically be gentamycin sulphate in an amount of about 30 ug/ml.

The vaccine of the invention will be administered to a subject in an amount effective to elicit an immune response to the virus. It is within the skill and knowledge of the person skilled in the art to determine suitable and effective doses to be administered to a subject. The subject to whom the vaccine is administered may be human, According to a preferred embodiment of the invention, the vaccine is formulated for administration to birds, especially poultry.

The procedure used in a vaccination programme may be different for different subjects. For instance, in the vaccination of poultry, we have found that the vaccinatio procedure used to treat broilers successfully differs from that used in the treatment of laying hens. Typically, broiiers will be vaccinated intranasafly, using the vaccine composition of the invention, at 12 days of age and again at 23 days of age to give maximum protection to the birds in deep litter environment where the conditions of virus dissemination are high. However, laying hens housed in cages will typically receive one tntranasally- administered dose of the vaccine composition of the invention only when clinical signs of avian Influenza are observed, Normally, clinical signs of disease have disappeared within about 18 hours from administration of the vaccine and the birds recover and egg production returns to normal.

The treatment of or prevention of a viral infection in a human or animal subject, wherein the viral infection is an infection of a virus selected from Orthomyxovirus and Paramyxovirus, is achieved by a method which comprises administering to the subject a vaccine composition according to the invention, as described above. Preferably, the method comprises administering the vaccine mucosally, more preferably intranasally. it is believed that the siatidase-containing, proteolytic enzyme-detoxified bacterial exotoxin component of the vaccine composition has the action, following mucosal administration, of specificall binding to and degrading the sialated epithelial attachment sites in the subject, thus effectively denying access to these sites by the viruses which use this route for infection. According to a preferred embodiment, the method comprises administering the vaccine composition of the invention intranasa!iy to a bird, wherein the vaccine composition comprises an inactivated antigenic component of a virus selected from influenza type A subtype H5N1 virus and Newcastle disease virus.

Example 1

Experimental Methods

1 - Preparation of inactivated virus antigen

Highly pathogenic H5N1 homologous strains isolated and identified from infected chickens were propagated in eleven day old incubated specific pathogen-free embryonated chicken eggs. Using a working seed virus containing 128 HA units/25pl, the eggs were inoculated, by injection into the allantoic sac of each egg, with 0.2ml of a 1/1000 dilution in phosphate buffered saline pH 7.4 (PBS) + kanamycin 20mg/mi.

The eggs were, then, incubated at 37°C for 72 hours. Embryo death was noted within 25-27 hours.

The dead embryos were chilled at 4°C and the allantoic fluid harvested and clarified by centrifugation at 3000 rpm for 15 minutes to remove unwanted debris.

After centrifugation, the precipitated matter was discarded and the supernatant containing the virus was shown, on testing, to have haemagglutination activity (by causing the agglutination of chicken red blood ceils).

The supernatant was treated to inactivate the virus by the addition of 0.1 % formalin followed by incubation at 37°C for 18 hours. The virus was then split with 0.5% v/v chloroform for 18 hours with constant stirring at 4^C'C, Residual chloroform was removed under a vacuum of lOOmbar for 2 hours at 28 G and the sample was retested for haemagglutination activity. ίθ

Loss of virulence was demonstrated by inoculating eleven day old incubated ^■embryonated chicken eggs with 0.2ml of the inactivated virus suspension. AH embryos were afive after 5 days further incubation at 37°C.

The virus fluid was clarified by filtration and stored at -20°C.

2. Preparation of siattdase-containing exotoxin

(a) A culture medium was preparing by combining:

Oxoid L37 peptone 2%

Lactalbumin Hydrolysate 1%

Yeast extract 0.5%

NaCf 1.0%

Water to give a volume of 4,0 litre

The culture ingredients (pH 7.4) were placed in a 10L Pyrex bottle and autoclaved at 121 °C for 30 minutes. Aqueous glucose (50% w/v sterile aqueous glucose solution) was added to the autoclaved culture ingredients to give a glucose content of 1.0% in the final medium, The medium was then cooled rapidly to 37°C to maintain reducing conditions.

An inoculum of Clostridium petfringens A strain 107 was prepared by reconstituting freeze-dried seed in Robertson's cooked meat broth. The inoculum was added to 400m! of the culture medium and the mixture was then added back to the bulk culture medium to a total volume of 4.0L, The pH of the medium was maintained at 7.0 by addition of 5/N NaOH during a 3½ hour growth period and the temperature was maintained at 37°C. After the growth period, the culture was cooled and centrifuged to remove cells and other solid matter. The ceil-free supernatant was collected.

3. Preparation of^" slaiidase-.containi^'na enzyme-detoxified exotoxin

The cell-free supernatant obtained above was treated with 0,3% w/v trypsin 1 :250 USP for one hour at 37°C.

The activity of the trypsin-inactivated alpha toxin was determined by noting the haemolytic action of doubling dilutions against a fixed volume of chicken red blood cells in rnicrop!ates. The trypsin-treated supernatant was considered suitable for use when haemolysis ceased after a dilution of 1/250. The titer of the receptor destroying enzyme (ROE), i.e. sialidase, in the toxoid was determined by the following procedure.

Using a 98 well U-bottom microplate, doubling dilutions of the trypsin-treated toxin were made in 12 wells leaving 25μΙ of each dilution in each of the wells. To each of the dilutions in the wells were added 25μ| of 1% washed chicken red b!ood cells (RBCs) in pH 7,4 PBS followed by gentle mixing of the contents of each well. The sialidase was allowed to adsorb onto the RBCs for VA hours at 28°C. 25μί of 4HA units of formalin inactivated H5N1 haemagglutinin were added to each well and the contents of each well were left for 3 hours, maintaining the temperature at 28°C, The contents of each of the wells were then studied, noting the highest dilution of the trypsin-treated toxin which inhibited agglutination of the chicken RBCs, The degree of inhibition of agglutination (i.e. the titer of the RDE) was recorded as the reciprocal of the highest dilution of sialydase which inhibited agglutination. The sialidase RDE contained 512 units in 25pl, i.e. 20,480 RDE μ/ml.

4. Assay of the trypsin-inactivated ssaiidase-containing toxin for hyaluronidase

A culture of Paste rella multoclda type A, a strain sensitive to hyaluronidase decapsulation, was grown overnight in Oxoid brain heart infusion broth. The culture opacity was adjusted to a McFarland opacity of 0.5 and then diluted further ¼ in phosphate buffer saline. A fixed volume of Oxoid blood agar base was poured into a Petri dish arranged on a levelled metal plate to give a standard agar thickness in the dish. The ¼ diluted cuiture was spread evenly over the surface of the agar layer and then dried briefly at 37°C.

A centrai 0.5cm diameter well was cut into the middle of the cuiture/aga layer and a further 10 wells were cut peripherally equidistant around the centrai well and numbered 1 to 10. Into the central well was placed 50μΙ of Sigma hyaluronidase (22 units). Into the peripheral well No.1 were placed 50μ! of sterile filtered 0.3% w/v trypsin solution in water. A ceil-free supernatant of the sialidase-containing exotoxin was obtained according to 2} above. A sampie of this was treated with 0.3% w/v trypsin for one hour at 37°G. Further samples were treated with 0.3% w/v trypsin for each of 2, 3, 4, 5, 6, 7, 8 and 9 hours.

50μΙ of the trypsinised toxins were added to the peripheral we!is as foilows:

Weil No. Trvpsinized toxin at 37°C

2 irypsinized for 1 hour

3 " 2 hours

4 ^B 3

5 '^■' 4

6 " 5

7 " 6

8 " 7

θ " 8

10 " 9

The substrate containing the wells was incubated at 37°C overnight and the resulting zones of inhibition were measured and recorded. The results were as follows.

Well Zone of inhibition (mm)

Centra! (positive control) 20

1 (negative control) Zero

2 16

3 18

4 18

5 18

6 17

7 17

8 18 9 16

According to these results, the C. perftingens toxin trypsinised for 3 hours at 37^BC produced a zone of inhibition of 18mm compared to a zone of 20mm obtained for the positive hya!urohidase control of 22 units hyaluronidase. This clearly shows that the C. perfnngens culture contains hyaluronidase.

5. Stock chitosan solution

85% ieacetylated chitosa sourced from marine crustaceans (Nova atrix) was prepared as a 0.5% w/v solution in 1% v/v pH 5,0 aqueous acetic acid, sodium acetate buffer and sterilised by autoclaving at 121°G for 20 minutes in a tightly sealed bottle.

6, Preparation of vaccine

To every 100,000 HA units of the inactivated virus prepared above were added 3.0 ml (20480 RDE units) of the sialidase-containing trypsin-detoxified Clostridium toxin preparation. The inactivated virus and sialidase-containing toxoid were mixed by stirring at 4°C for one hour. The mixture was then made up to a total volume of 30m! by the addition of 0.5% w/v acetic acid/acetate buffered sterile chitosan at pH 5.0 and was further stirred for one hour at 4°C. The final bulk vaccine was tested for sterility and placed in a sterile 30ml polypropylene bottle incorporating a dropper nozzle delivering 0.03 ml per drop, Each 0.03 ml dose, thus, consisted of 100 HA units of virus, approx. 20.5 RDE units of bacterial sialidase and approximately 12 units of hyaluronidase suspended in 0,5% w/v sterile chitosan, pH 5.0.

Example 2

Members of a flock of 100,000 poultry, on a commercial poultry farm, comprising 12-day old chicks, layers being between 24 and 90 weeks oid and pullets of about 20 weeks of age were observed to be suffering from an infection leading to mortality. The clinical sign of the infection that was observed was torticollis, which is a twisting of the head and neck of the birds. Avian influenza virus H6N1 was isolated from brains of dead birds showing torticollis.

All^■surviving birds in the flock were vaccinated rntranasall by the application of a 30μ1 drop of the vaccine prepared according to the procedure described in Example 1 above to one nostril. AH birds that had displayed the clinical symptoms of torticollis recovered within 24 hours. No further deaths or clinical signs of ill health were reported.

The dramatic overnight recovery of clinically sick birds following the intranasal vaccination described above demonstrates that the trypsin-treated, stabilised C, perfringens type A sialidase produced a profound actio in controlling the highly pathogenic avian influenza A subtype H5 1 disease in the flock. It is believed that the intensified action of the sialidase-containing, trypsin- detoxified O, perfringens toxin is able to occupy and degrade the sialidated a 2-3 sites in a bird's nasal mucosal epithelium preventing further virus attachment.

Example 3

The sialidase-containing trypsin-detoxified C. perfringens exotoxin, prepared in Example 1 , was tested against Newcastle disease virus according to the procedure described below.

Using a 96 well U-bottom micropiate, doubling dilutions of the trypsin-treated toxin were made in 12 wells leaving 25μΙ of each dilution in each of the wells. To each of the dilutions in the wells were added 25ut of 1% washed chicken red blood cells (RBCs in pH 7,4 PBS followed by gentle mixing of the contents of each well. The sialidase was allowed to adsorb onto the RBCs fo 90 minutes at 28°C. 25μί of 4HA units of formalin-inactivated Newcastle disease haemaggiutin were added to each well and the contents of each well were left for 3 hours while maintaining the temperature at 28°C. The contents of each of the wells were then studied, noting the highest dilution of the trypsin-treated toxin which inhibited agglutination of the chicken RBCs. The inhibition of agglutination (i.e. the titer of the RDE) was recorded as the reciprocal of the highest dilution of sialidase which inhibited agglutination, it was, surprisingly, found that the agglutination inhibition titer obtained for the Newcastle disease virus was exactly the same as that obtained for the H5N1 influenza i section 3 of Example 1.

It can be concluded from these results that the trypsin-detoxified C. perfringens toxin is able to block ssalated sites on the chicken RBCs for both type A avian influenza virus and Newcastle disease virus, A vaccine, according to the present invention, containing the trypsin-detoxified toxin and inactivated antigenic component derived from Newcastle disease virus would be expected to be effective in treating or preventing Newcastle disease infection in birds. It is also expected that a similar therapeutic blocking action will be achieved for other paramyxoviruses, which use the same route of infection as Newcastle disease and which are the cause of a number of animal and human diseases.

Example 4

Comparison of different proteolytic enzymes

Using a cell-free supernatant containing C. perfringens exotoxin, produced as described in Section 2 of Example 1 , three different proteolytic enzyme- detoxified exotoxin compositions (toxoids 1 to 3 below) were prepared.

Toxoid 1 - prepared by adding papain (Promod™144P - Biocata!ysts Ltd., UK) to supernatant at a concentration of 0.3% w/v and at 37°C, mixing and maintaining the temperature at 37°G for one hour.

Toxoid 2 - prepared by adding trypsin (Trypsin T069P - Biccataiysts Ltd., UK) to supernatant at a concentration of 0.3% w/v and at 37^!iC, mixing and maintaining the temperature at 37°C for one hour.

Toxoid 3 - prepared by adding vegetable rennet ("VegeRen" - Just Wholefoods), obtained from Mucor miehei, to supernatant at a concentration of 0.01% v/v and at 37"C, mixing and maintaining the temperature at 37°C for one hour.

At the end of the one hour incubation time, each of the toxoids was tested for its activity to lyse chicken RBCs. Using a 96 wei! micropiate, 25 Ι of the toxoid was added to the first we!! and doubling dilutions, using norma! sa!ine, were made in successive welis leaving 25μ! of each dilution in each of the wells from ½ up to 1/2048. To each of the dilutions in the weils were added 25 Ι of 1 % washed chicken RBCs in pH 7.4 PBS followed by gentle mixing of the contents of each well. The procedure was carried out for each of the toxoids 1 to 3. Each micropiate was incubated at 37°C for one hour and the degree of haemolysis in the wells was recorded. The experiment was repeated using, as a control, untreated C. perfringens toxin.

The RDE activity of each of the toxoids was determined using the procedure corresponding to that described in Section 3 of Example 1 above. The experiment was repeated using, as a control, untreated C. perfringens toxin. The results are shown in the table below.

As shown in the table, all of the proteolytic enzymes tested detoxified the C. perfringens exotoxin, although the degree varies from ¼ for the papain - treated toxin to 1/64 for the vegetable rennet-treated toxin, in alt cases, the RDE sialidase titer is unaffected by the detoxification of the bacterial toxin.

Influence of enzymes on HaemagqSutination inhibition

This test was carried out to determine whether the proteolytic enzyme, itself, has any action on the cell membrane of the chicken RBCs that would interfere with the agglutination of chicken RBCs by haemagglutinin from H5N1 virus. Each of the three proteolytic enzymes was diluted in distilled water to the same concentrations that were used in preparing the toxoids above (i.e. 0.3% w/v papain; .0.3% w/v trypsin and 0.01% v/v vegetable rennet). Using a 98 well micro plate, 25μΙ of phosphate buffered saline was placed in each well 25μί of proteolytic enzyme solution were added to the first well and doubling dilutions (from ½ to 1/2048) were added to the second and subsequent wells leaving 25μ! of each dilution in each of the wells, 25pi of 4 virus HA units were added to each well and 25μΙ of 5% chicken RBCs were added to each well and the microplate was then incubated for 1 hour at 37°C, This procedure was carried out for each of the three proteolytic enzymes, After the completion of the incubation, all wells displayed haemaggiutination indicating thai the receptors on the chicken RBCs were unaffected fay the proteolytic enzymes. None of papain, trypsin or vegetable rennet showed any interference with viral hae aggSutinin attachment. It can be concluded from these results that any residual proteolytic enzyme present in an intranasal dose of the vaccine of the present invention will have no effect on viral attachment to cell surface receptors on the nasal associated lymphoid tissue (NALT),

Example 5

Members of a flock of 80,000 layers (egg producing chickens) were observed to be suffering from upper respiratory tract infections leading to general ill- health and reduced egg production. Each member of the flock was vaccinated intranasally using a 0,03ml dose of a vaccine composition according to the present invention. Each dose consisted of 100HA units of the inactivated H5N1 virus antigen prepared as described in section 1 of Example 1 , approximately 41 RDE units of bacterial siaiidase and approximately 24 units of hyaluronidase, as provided by the detoxified Clostridium perfringen alpha toxin prepared according to section 3 of Example 1 above, in sterile pH 5.0 acetic acid/acetate buffer.

After 24 hours, the birds showed a dramatic improvement, respiratory symptoms disappeared and egg production rapidly returned to normal.

Claims

1 , A vaccine composition comprising

(b) a sia!idase-containing, proteolytic enzyme-detoxified bacterial exotoxin.

2, A vaccine composition according to claim 1 , further comprising hyaluronidase.

3, A vaccine composition according to claim 1 or claim 2, wherein the bacterial exotoxin is derived from Pseudo onas aeruginosa or Clostridium perfringens.

4, A vaccine composition according to claim 3, wherein the bacterial exotoxin is derived from Clostridium perfringens type A.

5, A vaccine composition according to claim 4, wherein the bacterial exotoxin is derived from Clostridium perfringens type A. strain 107.

8. A vaccine composition according to any one of claims 1 to 5, wherei the virus is an influenza virus.

7. A vaccine composition according to claim 6, wherein the virus is an influenza type A virus.

8. A vaccine composition according to claim 7, wherein the virus Is influenza type A subtype H5N1.

9. A vaccine composition according to any one of claims 1 to 5, wherein the virus is a virus of the Paramyxoviridae family.

10. A vaccine composition according to claim 9, wherein the virus is selected from

Genus Avulaviras type species Newcastle disease virus]

Genus Henipavirus type species Hendravirus and Nipavirus;

Genus Morbiltivirus type species Measles virus, Rinderpest virus, Canine distemper virus, phocene distemper virus and Peste des Pettis Ruminants virus;

Genus Ruhatavirus type species Mumps vims; and

Human Parainfluenza virus type 1 and 3.

1 . A vaccine composition according to any one of claims 1 to 10, wherein the antigenic component is selected from inactivated whole virus, disrupted virus and purified viraf membrane glycoprotein.

12. A vaccine composition according to any one of claims 1 to 11 , wherein the sialidase-containing, proteolytic enzyme-detoxified bacterial exotoxin is obtained by detoxifying the bacterial exotoxin which contains sia!idase by treatment with a proteolytic enzyme selected from trypsin, papain, bromelain, ficin, microbial rennet from Rhizomucor miehei_i Bacillus subtilis protease and Bacillus licheniformis protease.

13. A vaccine composition according to claim 12, wherein the proteolytic enzyme is trypsin or papain.

14. A vaccine composition according to any one of claims 1 to 13, further comprising chitcsan.

15. A vaccine composition according to any one of claims 1 to 14, which is in the form of an aqueous dispersion or solution.

18. A vaccine composition according to any one of claims 1 to 15, which is adapted for mucosa! administration.

17. A vaccine composition according to claim 16, wherein mucosa! administration is intranasal administration.

18. A method of treating or preventing a vira! Infection in a subject selected from a human or animal subject, said viral infection being an infection of a virus selected from Orthomyxovirus and Paramyxovirus, which method comprises administering to the subject a vaccine composition of any one of claims 1 to 17.

19, A method according to claim 18 wherein the subject is a bird and wherein the virus Is selected from influenza type A subtype H5N1 and Newcastle Disease virus.