GB2308367A - Non-haemolytic protein, ex. Aeromonas sp., having cellular immunosuppressive (CIF) activity but lacking glycerophospholipid cholesteryl acyltransferase (GCAF) - Google Patents

Abstract

A non-haemolytic protein, which may be obtained from Aeromonas sp., preferably A.salmonicida , having CIF activity, but lacking GCAF activity, is characterised by: (i) a molecular weight of ca . 30kDa.; (ii) a pI value of ca . 5.5; (iii) cytotoxic to ca . 100000 lymphocytes at levels of ca . 200ng.; (iv) cytotoxic to ca . 100000 AS2 tissue culture cells at levels of ca .200ng. The protein may be in vaccine formulation, especially for immunisation of fish against furunculosis.

Description

VACCINE COMPONENT The present invention relates to a vaccine component.

In particular, the present invention relates to an immunogenic component expressed by microorganisms and to vaccines containing such a component.

More in particular, the present invention relates to an immunogenic component obtainable from an Aeromonas organism and to a vaccine composition comprising the component and a vaccine, especially a vaccine against furunculosis.

The vaccine composition of the present invention is useful for administration to animals, especially fish.

Furunculosis is a significant disease that can infect many species of fish, examples of which include all species of salmon, rainbow trout, brook trout, brown trout, carp, goldfish and wrasse. In the case of salmon, flirunculosis is caused by the bacterium Aeromonas salmonicida.

As furunculosis can occur in many species of fish which are of commercial significance, so the intensive rearing of fish such as salmon, which is practised today on a wide scale, suffers from the disadvantage that the entire stock of fish in a facility can become infected with disease. Moreover, the commercial consequences of a serious disease outbreak can be enonnous, particularly in the farming of Atlantic salmon (Salmo salar).

Thus, the desirability of immunising farmed fish against infections and diseases, such as fiuunculosis, has long been recognised. However, despite the fact that the Aeromonas organism has been the subject of much scientific research, technical progress in preparing a suitable vaccine against furunculosis has been slow. In this regard, various workers have tried to identify the factors, such as extracellular proteins, which might cause the organism to be toxic to fish and which might provide the basis for vaccine compositions to generate immunity against the organism. But the position is complicated because the Aeromonas organism appears to comprise or to generate a wide range of factors which have differing toxic or immunosuppressive activities in fish.Thus, previous research has tended to concentrate on the factors which the organism produces in greatest abundance.

At present there are a few commercially-available vaccines which are recommended for use against furunculosis, but the effectiveness of these vaccines often requires the use of oil-based adjuvant.

The present invention seeks to provide a vaccine component that can enhance the effectiveness of a vaccine, especially a vaccine against furunculosis.

According to a first aspect of the present invention there is provided a non-haemolytic protein having cellular immunosuppressive activity but no glycerophospholipid cholesteryl acyl transferase activity, wherein the protein is obtainable from an Aeromonas organism and has the following characteristics: (i) a molecular weight of about 30,000 daltons; (ii) a p1 value of about 5.5; (iii) is cytotoxic to about 105 lymphocytes at levels of about 200 ng; and (iv) is cytotoxic to about 10S AS2 tissue culture cells at levels of about 200 ng.

According to a second aspect of the present invention there is provided a composition comprising a vaccine and the protein according to the present invention, and optionally a suitable carrier, excipient or diluent.

According to a third aspect of the present invention there is provided Aeromonas salmonicida strain S9 (NCIMB 40736).

According to a fourth aspect of the present invention there is provided Aeromonas salmonicida strain S21 (NCIMB 40737).

According to a fifth aspect of the present invention there is provided a protein according to the present invention or a composition according to the present invention for use in or as a vaccine composition for immunising an animal, preferably a vertebrate animal.

According to a sixth aspect of the present invention there is provided the use of a protein according to the present invention in the manufacture of a vaccine composition for immunising an animal, preferably a vertebrate animal.

According to a seventh aspect of the present invention there is provided the use of a protein according to the present invention to suppress the cellular immune response in an animal, preferably a vertebrate animal, preferably when the protein is present in a vaccine composition.

According to a eighth aspect of the present invention there is provided the use of the protein according to the present invention in a vaccine composition to enhance the resistance to fish to filrunculosis.

According to a ninth aspect of the present invention there is provided a method for enhancing the resistance of fish to furunculosis, wherein the protein according to the present invention is administered to fish.

Thus, in its broadest sense, the present invention relates to a hitherto unknown immunogenic protein produced by Aeromonas organisms. In the following commentary, the immunogenic protein according to the present invention is sometimes referred to as the Aeromonas Cellular Immunosuppressive Factor, or Aeromonas CIF for short.

Preferably, the protein is obtainable from Aeromonas salmonicida.

Preferably, the protein is purified under conditions wherein the protein is stabilised.

Preferably, therefore, the protein is purified by use of a species that is capable of stabilising the protein. Preferably, the species is a detergent, preferably a detergent that forms two phases at an elevated temperature.

Preferably, the protein is stored in an environment that prevents or hinders proteolytic degradation thereof.

Preferably, the protein has been stored before use at low temperature, for example at about 4"C.

Preferably, the protein is obtainable from Aeromonas salmonicida strain NCIMB 40736 or NCIMB 40737.

Preferably, the vaccine is a vaccine for a fish.

Preferably, the animal is a fish.

Preferably, the fish is a Salmonid.

Preferably, the Salmonid is salmon, preferably Atlantic salmon.

Preferably, in the composition, the vaccine is a vaccine for immunising against furunculosis.

Preferably, the furunculosis is caused by Aeromonas salmonicida.

Preferably, the vaccine composition is suitable for oral administration to the animal.

Preferably, the protein of the present invention is present in an amount of from about 0.0008 %w/v to about 0.003 %w/v, preferably about 0.0015 %w/v, in the vaccine composition.

Preferably the protein of the invention is present in the composition in an amount of at least 0.001% by weight of the composition.

One of the key advantages of the present invention is that the Aeromonas CIF can enhance the immunising effect of vaccines for animals, in particular those vaccines used for fish, especially vaccines used for immunising against furunculosis.

It is known that the adaptive immune response of vertebrates can involve both hXumoral and cellular responses to the administration of foreign antigens. The humoral response involves the production of antibodies whereas the cellular response, or cell-mediated immunity, involves stimulation of T lymphocytes. The protein of the present invention is advantageous because it is non-haemolytic and is capable exhibiting a cellular immunosuppressive activity.

As indicated the present invention is particularly advantageous for enhancing the immunising effect of a vaccine for administration to salmon for immunising the salmon against furunculosis. However, a further advantage of the protein of the present invention is that it can enhance the effect of a vaccine for immunising other animals against furunculosis as well enhancing the effect of other vaccines for immunising salmon or other animals. With reference to fish (such as salmon, rainbow trout, brook trout, brown trout, carp, goldfish and wrasse), the protein of the present invention is capable of enhancing vaccines against vibriosis (caused by Vibrio anguillarum), Hitra disease or cold-water vibriosis (Vibrio salmonicida), enteric redmouth (Yersinia ruckeri), salmonid rickettsial septicaemia (Piscirickettsia salmonis which is a problem at present in Chile) and viral diseases such as infectious pancreatic necrosis (IPN), infectious haematopoietic necrosis (IHN), viral haemorrhagic septicaemia (VHS), pancreas disease (PD) and plasmacytoid leukaemia.

In this regard, there are some vaccines that are used against vibriosis, cold-water vibriosis and enteric redmouth. Even though these vaccines are effective, as they induce a humoral response to the bacterial lipopolysaccharide, they can be made more effective if admixed with the protein of the present invention.

The terms "Aeromonas Cellular Immunosuppressive Factor" or "Aeromonas CIF" as used herein do not necessarily mean that the protein must be obtained from Aeromonas as the protein may be obtained synthetically or by use of recombinant DNA techniques wherein the gene coding for the protein can be expressed in a suitable host organism, such as E. coli. Alternatively, the protein can be prepared synthetically.

The terms "non-haemolytic" and "not haemolytic" as used herein mean that at most the protein is capable of exhibiting a minimal haemolytic effect. Preferably, the terms mean that the protein is not capable of exhibiting any haemolytic effect.

The term "no glycerophospholipid cholesteryl acyl transferase activity" as used herein means that at most the protein is capable of exhibiting minimal glycerophospholipid cholesteryl acyl transferase activity. Preferably, the term means that the protein is incapable of exhibiting any glycerophospholipid cholesteryl acyl transferase activity in accordance with the protocol of Lee and Ellis (1990).

The term "cytotoxic to 10S AS2 tissue culture cells at levels of about 200 ng" is not necessarily indicative of an upper or lower level of activity. Instead, it indicates one level wherein cytotoxic activity can be easily observed.

Likewise, the term "cytotoxic to 10S lymphocytes at levels of about 200 ng" is not necessarily indicative of an upper or lower level of activity. Instead, it indicates one level wherein activity can be easily observed.

The term "animal" as used herein includes any animal, such as a mammal or a fish.

Preferably the term means a fish.

The term "bacterin" as used herein is used in its normal sense - i.e. killed bacteria, usually by treatment with formaldehyde or sometimes by heating, when used as a principal antigenic component of a vaccine.

The present invention therefore relates to Aeromonas CIF per se and its use in vaccine compositions.

Our initial studies relating to the present invention revealed that Aeromonas CIF is a very minor protein component of the total extracellular protein and so the presence of Aeromonas CIF in naturally-occurring strains of Aeromonas salmonicida is masked by other proteins. Examples of those other proteins include proteolytic enzymes and factors which are produced in abundance by Aeromonas strains. An example of one proteolytic factor is the 64kD serine protease (which used to be called the 70kD protease) as reported by Whitby, P.W., Landon, M. and Coleman, G. (1992). Sheeran, B. and Smith, P.R. (1981) have also reported on a second extracellular protease associated with the fish pathogen Aeromonas salmonicida.

Our further studies relating to the present invention revealed that Aeromonas CIF can be recovered from a culture of Aeromonas organisms and, in particular, via use of a transposon mutant wherein the proteolytic enzymes and factors, especially the 64 kd serine protease, have been eliminated. Alternatively, the proteolyic enzymes and factors can be inhibited. In this regard, an example of a inhibitor of protease activity has been reported by Sandvik and Hwaal (1992) who showed that broth cultures of A.

salmonicida contain inhibitors of the 64kD serine protease and other proteases such as trypsins from various animal species. Other examples include use of the inhibitors that can be found in two commercial vaccines, namely Aquavac Furovac 1M (which is a trade mark of and is supplied by Aquaculture Vaccines Ltd.) and Furogen b (which is a trade mark of and is supplied by Aqua Health Ltd.).

Our subsequent studies relating to the present invention revealed that the Aeromonas CIF has a molecular weight of about approximately 30,000 daltons as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (1970). The Aeromonas CIF has a pI value of about 5.5 as determined after preparative electrophoresis by the method of Radola (1974). In addition, we found that the Aeromonas CIF is cytotoxic to macrophages and lymphocytes of the Atlantic salmon and to AS2 tissue culture cells. In this regard, 200 ng of Aeromonas CIF causes detectable cytotoxicty with 105 AS2 cells or lymphocytes. The Aeromonas CIF is not haemolytic. The Aeromonas CIF has no glycerophospholipid cholesteryl acyl transferase activity.

Another aspect of the present invention is stabilised Aeromonas CIF.

As the Aeromonas CIF is capable of enhancing the protective properties of vaccines for animals - especially vaccines for fish, more particularly vaccines against furunculosis - the present invention also provides a composition useful as a vaccine against diseases or ailments, such as furunculosis, wherein the composition comprises a vaccine and the protein of the present invention.

Preferably, the composition comprises a bacterin derived from one or more strains of Aeromonas containing Aeromonas CIF, preferably in an amount exceeding 0.001% by weight of the total extracellular Aeromonas proteins of the composition. The composition may be administered to the animal, for example a fish, in any suitable form. For example, the composition can be an injectable composition comprising the Aeromonas CIF in a pharmaceutically acceptable liquid carrier, diluent or excipient, e.g. an aqueous composition. Alternatively, the composition can be an oral composition, comprising the Aeromonas CIF in or on or as an edible carrier, such as an animal feed, especially as a fish feed. The composition may also be an immersion vaccine.

A preferred embodiment of the present invention therefore relates to an immunogenic component and a vaccine containing such a component, wherein the vaccine is for administration to fish.

A highly preferred embodiment of the present invention therefore relates to an immunogenic component and a vaccine for a fish containing such a component, wherein the vaccine is against organisms implicated in causing furunculosis.

In a highly preferable embodiment of the present invention the protein is not in its natural environment (i.e. when it has been expressed by the gene coding for the protein when that gene is in its natural environment and is under the control of the promoter with which it is normally associated which is also in its natural environment). Thus, in this highly preferred embodiment, the protein has been isolated from Aeromonas or has been prepared by use of recombinant DNA techniques.

In one preferred embodiment the present invention therefore provides Aeromonas CIF substantially free from extracellular Aeromonas active protease proteins. With this aspect, there need not be totally pure Aeromonas CIF, although a pure form can be prepared if desired. Rather, it is generally sufficient if most of the proteases have been removed (i.e. eliminated) or have been inactivated.

In one aspect of the present invention there is provided a sample consisting substantially of Aeromonas CIF. In this sample, the protein content of the sample preferably comprises at least about 80%, and more preferably at least about 90% Aeromonas CIF. Thus, in this embodiment, the sample can be an Aeromonas culture extract from which at least the bulk of non-CIF Aeromonas extracellular protein has been removed.

A highly preferred embodiment of the present invention relates to the use of Aeromonas CIF to enhance the resistance of fish to furunculosis.

In addition, the present invention also provides a method for enhancing the resistance of fish to furunculosis wherein Aeromonas CIF is administered to fish in conjunction with a suitable vaccine.

Another highly preferred embodiment of the present invention relates to a composition comprising a vaccine composition comprising a bacterin derived from one or more strains of Aeromonas, containing Aeromonas CIF.

Another highly preferred embodiment of the present invention relates to the production of a composition for immunising an minimal, preferably a fish, comprising a vaccine and the protein of the present invention, in which process a culture of, or an extract derived from, one or more strains ofAeromonas is enriched by the addition thereto of Aeromonas CIF.

Another highly preferred embodiment of the present invention relates to the use of Aeromonas CIF in a vaccine to enhance the resistance of fish to furunculosis.

An improved vaccine, such as for immunising against furunculosis, can preferably therefore be prepared by screening existing Aeromonas salmonicida strains to identify one or more that are comparatively high producers of Aeromonas CIF. A bacterin based on such a selected strain will contain an elevated CIF level. Alternatively, an otherwise standard bacterin derived from an Aeromonas salmonicida strain can be improved by the addition of a pure, or concentrated, Aeromonas CIF extract.

To prepare a suitable Aeromonas-CIF-rich extract, a CIF-producing strain of Aeromonas, such as a strain ofAeromonas salmonicida, can be cultured by growth in a nutrient-rich medium, such as brain heart infusion broth or tryticase soy broth. An extract from the culture can then be prepared, e.g. by harvesting the culture supernatant and concentration by ultrafiltration.

If desired, pure Aeromonas CIF can be recovered from the extract by, for example, isoelectric focusing followed by hydrophobic interaction chromatography.

An alternative preferred method for preparing an improved vaccine, such as for immunising against furunculosis, is to induce a natural Aeromonas strain to produce enhanced levels of CIF by mutation, selective breeding or by genetic engineering.

Indeed, in a further alternative, Aeromonas CIF can be produced in a different organism, such as Escherichia coli, by transformation of that organism with a gene encoding the Aeromonas CIF protein in active form, or in a form that is non-toxic but nevertheless capable of inducing in fish an immune response.

At present it is believed that in the living Aeromonas organism the CIF is naturally associated with the lipopolysaccharide (LPS). If the Aeromonas organism is cultured and a typical bacterin formulation extracted from the culture, the CIF can become separated from the LPS, and it is believed that the CIF may not be as stable in this naked form. However, the extracted CIF can be rendered stable or more stable by, for example, adding a species that will mimick the natural LPS environment. This can be achieved by, for example, purification and/or storage in the presence of suitable detergents, e.g.Triton X-100, Triton X-114 or n-octylglucoside. [Triton X is a trade mark of Union Carbide.] Without wishing to be bound by theory it is believed that the effectiveness of the protein of the present invention is attributable to the interaction of the A eromonas CIF with the macrophages or lymphocytes of the animal, such as a fish, to stimulate them to an enhanced responsiveness. The cytotoxic effect of Aeromonas CIF against AS-2 cells, and probably also against lymphocytes, is labile at 40C and even at -200C in unstabilised preparations (see Table 3 below) whereas the potentiating activity in AVL vaccine is stable for at least a month at 4"C. Thus, the two activities are different although they could be properties of the same molecule.

The fact that the protein could have two activities could be plausible because other, but different, proteins are believed to exhibit two activities. One such other, different protein is Bordetella pertussis adenylate cyclase toxin. Bordetella pertussis adenylate cyclase toxin is an enzyme very different from the protein of the present invention, however workers have found that Bordetella pertussis adenylate cyclase toxin has both enzymic (adenylate cyclase) and cytolytic (haemolytic) activities. Those workers have also shown that these properties of Bordetella pertussis adenylate cyclase toxin are associated with different domains of the molecule, the enzymic activity being associated with (approximately) residues 1 to 400 and the cytolytic activity being associated with the remainder of the molecule, residues 401 to 1706.Those workers also produced truncated Bordetella pertussis adenylate cyclase toxin molecules which possessed either adenylate cyclase or cytolytic activities respectively, thus confirming that two quite different activities could be associated with a single toxin.

Therefore, in the highly preferred embodiment, it is believed that use of the protein of the present invention increases the effectiveness of furunculosis vaccines by inducing an effective response in fish against the other factors which the organism produces in relatively small quantities but which have significant physiological impact.

A strain of Aeromonas salmonicida, namely S9 (as defined below), was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 lRY, United Kingdom, on 8 June 1995 and has the allocated Deposit No. NCIMB 40736.

Transposon mutant S9 of A. salmonicida strain 80628, which is a virulent field isolate from a case of furunculosis in Atlantic salmon, was generated by conjugation with Escherichia coli strain Hub 101 containing the suicide vector pUW964 (pRK2013 Km::Tn7 xyz: Tn5,TpR, SpR, KmR) (Weiss et al. 1983).

Another strain of Aeromonas salmonicida, namely S21 (as defined below), was deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 lRY, United Kingdom, on 8 June 1995 and then redeposited on 15 December 1995 and has the allocated Deposit No. NCIMB 40737.

Transposon mutant S21 was generated using, as the host, the field isolate A.

salmonicida strain 0508 and E. coli BRD 327 containing the suicide vector pRT733 (pJM103.1 Knit:: TnphoA, oriR6k, mob for RP4) (Miller and Mekalanos, 1988). S21 does not produce the 64kD serine protease mentioned above (Whitby, P.W., Landon, M. and Coleman, G. (1992)).

The present invention will now be described only by way of example.

A. CULTURING OF AEROMONAS SALMONICIDA The protease-deficient strain of Aeromonas salmonicida S21 was cultured in brain heart infusion broth (Oxoid) for 24h at 20"C in 1 litre volumes contained in 2 litre baffled conical flasks.

B. PREPARATION OF AEROMONAS CIF An inoculum of 10 mls of an overnight culture from the above-mentioned culture was used. The culture supernatant was harvested by centrifugation and the supernatant was concentrated 80-fold using a Millipore "Minitan" ultrafiltration system with a membrane having a cut-off size of 30 kilodaltons. The concentrated culture supernatant was dialysed overnight at 4"C against 4 litres 1% glycine solution.

Preparative electrofocusing was carried out in a layer of Sephadex G-200 (Pharmacia) gel in 1% Ampholines (pH range 3.5 - 10; Pharmacia). The Sephadex gel was prepared by slowly adding 2.5 g dry, prewashed fine grade Sephadex G-200 to the concentrated culture supernatant (95 ml supernatant and 5 ml 20% Ampholine solution). After rehydration of the gel for 1 hour at 40C the gel slurry was poured into the mould of an LKB Multiphor II preparative electrofocusing apparatus. After addition of filter paper wicks, electrofocusing was carried out for 18 hours at 1 0C (final voltage 1000V). Thirty fractions were obtained and samples were eluted from the Sephadex with 0.9% saline and assayed for cytotoxicity, A280 and pH.Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was also done and cytotoxic fractions (pH range 5.5 - 5.9) were bulked, concentrated to < 1 ml by dialysis against a 7.5% w/v solution of polyethylene glycol (PEG 20000, BDH Ltd.) and loaded onto a column of phenyl-Sepharose CL-4B (30 ml bed volume) equilibrated in 1 M (NH4)2SO4 solution in 0.02M phosphate buffer, pH 7.0. Adsorbed proteins were eluted with a linear gradient of (NH4)2SO4 decreasing from 1 M to zero molar in 0.02M phosphate buffer, pH 7.0 and a gradient of Triton X-100 increasing from 0 to 0.5% in 0.02M phosphate buffer, pH 7.0, total gradient volume, 400 ml).

Fractions of 5 ml were collected and fractions 76 and 77 contained the protein of the present invention.

C. MOLECULAR WEIGHT DETERKNATION OF AEROMONAS CIF Molecular weight estimation was by SDS-polyacrylamide gel electrophoresis using the method of Laemmli (1970) with Sigma low molecular weight standards (bovine serum albumin, 66kD; egg albumin, 45kD; glyceraldehyde-3-phosphate dehydrogenase, 36kD; carbonic anhydrase, 29kD; trypsinogen, 24kD; trypsin inhibitor, 20.1kD and alphalactalbumin, 14.2kD). Resolving gels contained 12% (w/v) acrylamide and stacking gels contained 4.5% (w/v) acrylamide.

Protein bands were visualised by silver staining (Oakley et al., 1980). The measured Mr was about 30,000 Daltons as determined by SDS-PAGE.

D. ISOELECTRIC POINT DETERMINATION OF AEROMONAS CIF The isoelectric point of the protein of the present invention was determined after preparative isoelectric focusing of the proteins when present in concentrated crude culture supernatants in a Sephadex granulated gel following the method of Radola (1974, supra). After elution of proteins from the Sephadex support gel with 0.9% saline, the pH of each fraction was measured at the same temperature as for separation (i.e. 1"C). The protein of the present invention had a measured pI value of about 5.5.

E. EXTRACTION OF AEROMONAS CIF USING DETERGENT The following commentary describes the extraction of the protein of the present from culture supernatant using Triton X-114.

Since it is thought that the protein is amphipathic and readily associates with detergent micelles, phase separation was used to simplify the partial purification of the protein.

These studies showed that Triton X-114 (and similar detergents) is an effective stabiliser of the protein of the present invention (see Table 1 below), has a low CMC (0.02%), is inexpensive and is not toxic to fish (at 0.2% w/v). Furthermore, this detergent forms a single phase with water at temperatures below 30"C but separates into detergent-rich and aqueous phases at temperatures above 30"C, thereby allowing easy recovery of the amphipathic protein of the present invention in the detergent-rich phase.

The protein of the present invention after purification by isoelectric focusing was then concentrated by dialysis against polyethylene glycol 20000 to 5.2 mg/ml protein and 100p1 of this solution was added to 900pL1 1% Triton X-114 in phosphate buffered saline at 0 C. After periodic mixing for 20 min the solution was incubated at 37"C for 10 min and the phases separated by centrifugation at 12000 x g for 10 min. By SDS-PAGE it was shown that the protein was recovered in high yield in the detergent phase with only traces partitioning in the aqueous phase.The protein could also be extracted from culture supernatants (or whole cultures) by addition of Triton X-114 to a concentration of 1%, incubation of the culture at 0 - 40C for 20 min, incubation at 37 C for 10 min and recovery of the separated phases by centrifugation. From crude cultures or culture supernatants the protein of the present invention was recovered as the dominant protein.

F. CYTOTOXICITY OF AEROMONAS CIF TO AS2 CELLS Cells from the established tissue culture cell line of Atlantic salmon (AS-2) were removed from monolayer culture by trypsinisation and resuspended to a concentration of 106 cells ml-t in Eagle's Minimal Essential Medium (MEM; Eagle, 1959), pH 7.3, containing 10% foetal calf serum, 2 rnM L-glutamine, 0.075% sodium bicarbonate, 20 mM Tris /HC1 buffer, Non-Essential Amino Acids, 100 intemational units/ml penicillin, 100 llg/ml streptomycin and 2.5 llg/ml amphotericin B.Volumes of 100curl of the cell suspension were dispensed into the wells of a 96-well microtitre plate (flatbottomed wells) and after culture for 24 h at 200C the medium was removed and 100111 of serial doubling dilutions ofAeromonas CIF in serum-free MEM were added.

After incubation for 24 h at 200C the cell monolayers were inspected with an inverted microscope. The end-point in the titration was the dilution of a sample which destroyed 50% of the monolayer in comparison to MEM-treated control wells.

The results showed that the protein of the present invention when purified in the absence of a stabiliser routinely had a titre of 1/128 in the assay, equivalent to 200 ng causing detectable cytotoxicity with 105 cells. However, surprisingly, when the protein of the present invention had been stabilised by purification in the presence of 0.1 % Triton X-100 the protein was active in the above assay at dilutions of > 1/2048, such that lOng Aeromonas CIF in a volume of 100p1 serum-free MEM caused a cytotoxic effect with 105 cells.

G. CYTOTOXICITY OF AEROMONAS CIF TO LYMPHOCYTES AND MACROPHAGES Fractions of cells enriched for lymphocytes and macrophages were obtained by gradient centrifugation of the peripheral blood of Atlantic salmon. In assays such as that described above for Atlantic salmon cells the protein of the present invention had a similar cytotoxic titre towards lymphocytes as AS-2 cells (titre of native protein = 1/128 in both cases) but a much lower cytotoxic activity against macrophages (titre of protein = 1/4 to 1/8). Thus lymphocytes appear to be 16-32 times more sensitive to the protein of the present invention than macrophages.

H. GCAT ACTIVITY STUDIES OF AEROMONAS CIF Following the protocol of Lee, K.-K. and Ellis, A.E. (1990) the protein of the present invention was investigated to determine if it had glycerophospholipid cholesteryl acyl transferase (GCAT) activity. Glycerophospholipid cholesterol acyl transferase is a major lethal exotoxin and cytolysin of Aeromonas salmonicida. The experiments showed that the protein of the present invention does not have glycerophospholipid cholesterol acyl transferase activity according to this protocol.

I. STUDIES OF STABILISATION OF AEROMONAS CIF BY DETERGENTS In these studies, detergents were added to the protein of the present invention when it had been purified by isoelectricfocusing. The results of these studies were that the activity of protein was enhanced up to 8-fold. The activity of 5 detergents tested is shown in Table 1. From the results, it would appear that the degree of stimulation is related to the critical micelle concentration (CMC) of the detergents used. Greater recoveries of the protein of the present invention, measured by cytotoxicity titre against AS-2 cells, were obtained by incorporating detergent during isoelectricfocusing.

The requirement for detergents to be present at at least (preferably greater than) the CMC is confirmed for N-octylglucoside, which has a CMC of 0.7% (see Table 2).

Table 1 The effect of various detergents on the cvtotoxic activitv of Aeromonas CIF on AS-2 cells Deterqent Cytotoxicity titre detergent* + cytotoxin detergent alone none 64 0 Triton X-100 256 4 Triton X-114 512 32 N-octylglucoside 64 0 N-dodecylglucoside 64 32 N-dodecylmaltoside 128 32 * all detergents were incorporated to 0.1% (w/v) Table 2 The effect of N-octvlelucoside concentration on the cytotoxic activity of Aeromonas CIF on AS-2 cells Concentration of detergent Reciprocal of cytotoxicity titre (% w/v) of neak fraction none 128 0.1 256 0.2 512 > 2048 [0.1 Triton X-100] > 2048 The cytotoxicity of Aeromonas CIF was retained when stored in the presence of 0.1% Triton X-100 (Table 3). Loss of activity of the native Aeromonas CIF on storage at 20"C was correlated with progressive conversion of the single 30kD band observed on SDS-polyacrylamide gel electrophoresis into a doublet.

Table 3 The stabilitv of the cytotoxic activitv of Aeromonas CIF prepared in the presence or absence of Triton X-100 Sample - Cytotoxicity titre of CIF stored at -200C for: 0 days 7 days 28 days CIP purified without Triton X-100 128 32 0 CIF purified with 0.1t Triton X-100 > 2048 * > 2048 > 2048 * greatest dilution tested J. PREPARATION OF AEROMONAS CIF BY RECOMBINANT DNA TECHNIOUES The gene encoding A eromonas CIF is identified by, for example, preparation of a gene bank from the DNA of A. salmonicida. Then clones containing the gene for Aeromonas CIF are isolated. The gene is then subcloned and expressed in E. coli following, for example, the teachings of Sambrook et al (1989).In this regard, DNA from the bacteria is partially digested with suitable endonucleases, separated on sucrose gradients and cloned into an appropriate cloning vector such as phage Xgtl 1.

Phages carrying and/or expressing the gene for Aeromonas CIF are then easily detected using antiserum to Aeromonas CIF. Suitable clones are subcloned and the DNA is transferred to an expression vector for production of sufficient Aeromonas CIF for incorporation into an appropriate vaccine.

K. VACCINE COMPOSITION PRODUCTION A quantity (2.5 mg) of Aeromonas CIF was recovered by electrofocusing of a 20 fold concentrate of 4 litres of 24 h culture supernatant of Aeromonas salmonicida S9 as described above. The Aeromonas CIF solution was adjusted to protein concentrations of 6.2, 3.1 and 1.6 pg/ml with sterile 0.9% saline and 8 ml of saline.

These preparations therefore contained the protein of the present invention in an amount of 0.003 %w/v, 0.0015 %w/v and 0.0008 %w/v, respectively, in the vaccine composition.

The Aeromonas CIF solutions were then mixed with an equal volume of the furunculosis vaccine AVL 013 (supplied by Aquaculture Vaccines Ltd., Saffron Walden, U.K.). Vaccine AVL 013 contains formolised (i.e. formaldehyde-treated) cells of A. salmonicida mixed with an adjuvant which is probably aluminium phosphate.

L. TESTING OF VACCINE COMPOSITION IN FISH A trial was conducted in salmon using the vaccine composition prepared above.

Groups of 60 Atlantic salmon, mean weight 30 g were anaesthetised by addition of benzocaine, final concentration 15-20 parts per million, to the tank water and injected with 0.2 ml vaccine. Fish were distributed into four 560 litre tanks with equal numbers from each group in each tank. Fish were maintained at a temperature of 11 13"C for 6 weeks and a furunculosis cohabitation challenge was initiated by addition of Atlantic salmon injected with 10S colony forming units of Aeromonas salmonicida strain FCC (1 infected fish per 10 vaccine trial fish). Mortalities were recorded daily for 43 days and bacteriological culture was carried out on the kidney of all dead fish to confirm the presence of Aeromonas salmonicida.

In the control, unimmunised group of 60 fish, 85% died as a result of the challenge and the Relative Percentage Survival (RPS = 100 (1 - (% mortality in vaccinated fish/ % mortality in unvaccinated control fish)) was calculated for each vaccine group (Table 4).

A similar vaccine trial was carried out with fish immunised with vaccines containing Aeromonas salmonicida CIF which had been stored at 40C for 4 weeks. In this trial mortalities were monitored for 20 days after challenge by cohabitation with A eromonas salmonicida-infected fish (Table 4).

Table 4 Relative Percentage Survival of fish immunised with vaccines containing Aeromonas salmonicida CIF Vaccine Relative percentage survival with vaccine containing: freshly prepared CIF CIF stored 4 weeks at 4"C AVL 013 21.6 23.5 AVL 013 + 6.2g CIF 25.5 39.2 AVL 013 + 3.lug CIF 29.4 51.0 AVL 013 + 1.6pg CIF 27.5 45.1 (CIF = Aeromonas CIF) The results of Table 4 indicate that a vaccine based on the Aeromonas CIF enhanced the protection obtained using a conventional furunculosis vaccine. The results also indicate that storage of Aeromonas CIF at 40C for 4 weeks before incorporation into AVL vaccine did not diminish its ability to potentiate the activity of AVL013. In contrast to this, the RPS values were greater for all doses of Aeromonas CIF tested.

In similar studies, the RPS values for vaccines with stored octylglucoside-stabilised Aeromonas CIF were shown to be greater than for AVL013 alone with 5 of the 6 doses of Aeromonas CIF employed.

Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

REFERENCES Eagle, H. (1959) Science, 130, 432.

Laemmli, U.K. (1970) Nature, 227, 680-685.

Lee, K.-K. and Ellis, A.E. (1990) Journal of Bacteriology, 172, 5382-5393.

Miller, V.L. and Mekalanos, J.J. (1988) Journal of Bacteriology, 170, 2575-2583.

Oakley, B.R., Kirsch, D.R. and Morris, R. (1980) Analytical Biochemistry, 105, 361363.

Radola, B.J. (1974) Biochim. Biophys. Acta, 386, 181-195.

Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press Sandvik, 0. and Hwaal, A.-B. (1992) Journal of Fish Diseases, 15, 131-141.

Sheeran, B. and Smith, P.R. 1981, FEMS Microbiology Letters, 11, 73-76.

Weiss, A.A., Hewlett, E.L., Myers, G.A. and Falkow, S. (1983) Infection and Immunity, 42, 33-41.

Whitby, P.W., Landon, M. and Coleman, G. 1992, FEMS Microbiology Letters 99, 65-72.

Claims (24)

1. A non-haemolytic protein having cellular immunosuppressive activity but no glycerophospholipid cholesteryl acyl transferase activity, wherein the protein is obtainable from an Aeromonas organism and has the following characteristics: (i) a molecular weight of about 30,000 daltons; (ii) a pI value of about 5.5; (iii) is cytotoxic to about 10S lymphocytes at levels of about 200 ng; and (iv) is cytotoxic to about 105 AS2 tissue culture cells at levels of about 200 ng.

2. A protein according to claim 2, wherein the protein is obtainable from Aeromonas salmonicida.

3. A protein according to claim 1 or claim 2, wherein the protein is purified by use of a species that is capable of stabilising the protein.

4. A protein according to claim 3, wherein the species is a detergent, preferably a detergent that forms two phases at an elevated temperature.

5. A protein according to any one of claims 1 to 4, wherein the protein is stored before use in an environment that hinders or prevents proteolytic degradation thereof.

6. A protein according to claim 5, wherein the protein is stored before use at low temperature, for example at about 4"C.

7. A protein according to any one of claims 1 to 6 wherein the protein is obtainable from Aeromonas salmonicida strain NCIMB 40736 or NCIMB 40737.

8. A composition comprising a vaccine and the protein according to any one of claims 1 to 7, and optionally a suitable carrier, excipient or diluent.

9. A composition comprising a vaccine and the protein according to any one of claims 1 to 8, wherein the protein is present in an amount of from about 0.0008 %w/v to about 0.003 %w/v, preferably about 0.0015 %w/v, in the composition.

10. A composition according to claim 8 or claim 9, wherein the vaccine is a vaccine for a fish, preferably wherein the fish is a Salmonid.

11. A composition according to claim 10, wherein the Salmonid is salmon, preferably Atlantic salmon.

12. A composition according to any one of claims 8 to 11, wherein the vaccine is for immunising against furunculosis.

13. A composition according to claims 8 to 12, wherein the furunculosis is caused by Aeromonas salmonicida.

14. Aeromonas salmonicida strain S9 (NCIMB 40736)

15. Aeromonas salmonicida strain S21 (NCIMB 40737)

16. A protein according to any one of claims 1 to 7 or a composition according to any one of claims 8 to 13 for use in or as a vaccine composition for immunising an animal.

17. Use of a protein according to any one of claims 1 to 7 in the manufacture of a vaccine composition for immunising an animal.

18. Use of a protein according to any one of claims 1 to 7 to suppress the cellular immune response in an animal, preferably when the protein is present in a vaccine composition.

19. The invention according to any one of claims 8 to 18 wherein the vaccine composition is suitable for oral administration to the animal.

20. The invention according to any one of claims 8 to 18, wherein the animal is an animal as defined in claim 10 or claim 11.

21. Use of the protein according to any one of claims 1 to 7 in a vaccine composition to enhance the resistance to fish to furunculosis.

22. A method for enhancing the resistance of fish to furunculosis, wherein the protein according to any one of claims 1 to 7 is administered to fish.

23. A protein substantially as hereinbefore described.

24. A vaccine composition substantially as hereinbefore described.