WO1996012734A1

WO1996012734A1 - Vaccine preparation recovered from bacteria grown in iron-rich media

Info

Publication number: WO1996012734A1
Application number: PCT/SE1995/001251
Authority: WO
Inventors: Andrew Cartner Barnes
Original assignee: Ewos Aktiebolag
Priority date: 1994-10-24
Filing date: 1995-10-23
Publication date: 1996-05-02
Also published as: AU3821095A; SE9403636D0

Abstract

The present invention relates to an improved vaccine and a process for its preparation, whereby the improved vaccine against bacterial diseases is recovered from culturing a bacterin-producing bacteria in a nutritive medium containing sources of iron at levels in excess of those normally and hitherto used for this purpose and subsequently preparing a vaccine from the cultivated bacteria and/or their component parts and/or components expressed by the bacteria and present in the growth medium.

Description

VACCINE PREPARATION RECOVERED FROM BACTERIA GROWN IN IRON-RICH MEDIA

Technical field

The present invention relates to improved vaccines and the preparation of vac- cines active against bacterial diseases, whereby a pathogenic bacteria is culti¬ vated, and a vaccine is prepared from the cultivated bacteria or its expression components present in the growth medium.

The object of the present invention is to obtain more efficient vaccines against bacterial diseases.

Background of the invention

Pathogenic bacteria possess on the outer surface different proteins or express different proteins and other components which proteins and components, when antigenic and/or immunogenic, can be used for vaccination of a host, either by inoculating the bacteria as such, and/or their expression components present in a growth medium in order to raise antibodies and thereby to increase immunity against said bacteria. The bacteria are said to produce a bacterin useful in creat¬ ing immunity. Some bacteria produce surface proteins which can be used in blocking receptors needed for the colonization and infestation of a host.

It is well recognised that animals have effective mechanisms for uptake of iron. This is important for their own metabolism and growth but also acts as a deterrent towards pathogenic microbes which also need iron to grow and infect the host.

The iron sequestering mechanisms need to be overcome by a pathogen which leads to the pathogen synthesing its own iron uptake mechanism which needs to be more efficient than that of the host. This is well recognised.

Typical responses of pathogens during infection are to synthesise siderophores (water soluble, low molecular weight iron chelating compounds which bind iron outside the pathogen cell) and one or several membrane receptor proteins which interact with the iron-loaded siderophores to take the iron into the pathogen cell. In culture in the laboratory it is possible to make the pathogen synthesise these components of its iron up-take mechanism by chelating the iron in the growth medium with an appropriate chelator. The cells then make the membrane recep¬ tors which are recognised as IROMPs (iron regulated outer membrane proteins). In some cases, at least, these IROMPs are antigenic and immunogenic and are therefore important components of vaccines.

There is an ever increasing demand of overcoming bacterial infections in ani¬ mals, as such infections, even if not fatal as they often are, cause heavy econo¬ mic losses in farming different animals for slaughtering purpose, whether this is a higher mammal or a fish. Thus there is a demand for efficient vaccines to treat animals in farming to eliminate or at least reduce the effects of bacterial infec¬ tions.

In the description and elsewhere the term "expression component" and "expres¬ sion components" means compound(-s) or substance(-s) which are bioactive and are expressed by the bacteria in the growth medium. In this context they are bioactive as vaccine(-s) when isolated or recovered from the medium or contain¬ ed in the medium, which is recovered and used.

Description of the present invention it has now surprisingly been shown possible to improve the efficiency of a vac¬ cine in accordance with the present invention which is characterised by culturing, for the purpose of producing a bacterin, bacteria in a nutritive medium containing sources of iron at levels in excess of those normally and hitherto used for this purpose and subsequently preparing a vaccine from the cultivated bacteria and/- or their component parts and/or components expressed by the bacteria and pre¬ sent in the growth medium.

Further characteristics are evident from the accompanying claims.

By preparing a vaccine in this way it has been demonstrated that the effective¬ ness of the vaccine is improved considerably, up to about eight fold.

Normal Fe content of TSB medium varies as there is a batch-to-batch variation but a "normal" Fe content in TSB dry concentrate is about 24 mg Fe/kg TSB, which corresponds to a con-centration of 12.9 microM Fe in TSB medium. In the present invention 100 microM FeC.3 is added, or from 10 microgram per ml to 100 mg/ml of medium, in excess of the normal content.

The invention will now be described in connection with the preparation of vac- cine from Paseurella piscicida, which is a fish pathogen.

Although described in connection with Paseurella piscicida other bacteria con¬ templated which are pathogenic in fish are:

Aeromonas salmonicida, Aeromonas hydrophila, Vibrio anguillarum, Vibrio ° salmonicida, Vibrio ordallai, Yersinia ruckeri, Edwardsiella tarda, Edwardsiella ictaluri, Flexibacter columnaris, Flexibacter psychrophilus, as well as other gram- negative and gram-positive pathogenic bacteria.

Aeromonas hydrophila is also a human pathogenic species. Other human patho- ⁵ genie bacteria are known, such as Streptococcus and Legionella, which are expected to respond in a similar way.

Vaccine preparations

Form iiniged cells 0 Paseurella piscicida MT1415 was removed from stock onto TSA and was incu^¬ bated at 22°C for 48 hrs.

1 litre of medium comprising 30 g of TSB (tryptophan soya broth) and 20 g of

NaCI (designated TSB2) was prepared and aliquoted 250 ml each into 500 ml ⁵ conical flasks, which were autoclaved at 121°C for 15 min. The flasks were allowed to cool whereupon the following filter sterilized additions were made aseptically into separate flasks:

100 mM 2,2'-dipyridyl dissolved in ethanol (250 microlitres);

200 mM EDDHA in 0.5 M NaOH (250 microlitres); ⁰ 100 mM FeCl3 (250 microlitres); and left as TSB2.

The flasks were incubated at 22°C while shaking for 1 hr to disperse the addi^¬ tives. The iron restricted media with 2,2'-dipyridyl turned reddish during this pe^¬ riod. In a flow cabinet 10 ml of liquid were removed from each broth into glass 5 universals.

10 ml universal starter cultures were inoculated with 1 microlitre of MT1415 taken from TSA plates. The incubates were shaken (140 rpm) at 22°C for 24 hrs. These starter cultures were inoculate broths which were incubated with shaking (140 rpm) at 22°C for 48 hrs. The microorganism cells were removed from the shaker and formalin was added to 0.2%. Then incubation continued at 4°C for 24 hrs. Cells were harvested at 3500 x g for 45 min. The pellets were washed once with PBS and resuspended to A540= 1.00 in sterile PBS.

Lipopolysaccharide

4 litres of medium comprising 120 g of TSB (tryptophan soya broth) and 80 g of NaCI was prepared and aliquoted 1.0 I each into 2.0 I conical flasks, which were autoclaved at 121°C for 15 min. The flasks were allowed to cool whereupon the following filter sterilized additions were made aseptically: 100 mM 2,2-dipyridyl dissolved in ethanol (1 ml); 200 mM EDDHA in 0.5 M NaOH (1 ml);

100 mM FeCl3 (1 ml); and left as TSB2

The flasks were incubated at 22°C while shaking for 1 hr to disperse the additi¬ ves. The iron restricted media turned reddish during this period. In a flow cabinet 10 ml of liquid were removed aseptically from each broth into glass universals.

10 ml universal starter cultures were inoculated with 1 microlitre of MT1415 taken from the TSA plates. The incubates were shaken (140 rpm) at 22°C for 24 hrs.

These starter cultures were inoculate broths which were incubated with shaking

(140 rpm) at 22°C for 48 hrs.

The EDDHA culture showed some, although an insufficient growth even after 5 days. Cells were harvested at 3500 x g for 45 min and resuspended in 25 mis of acetone. The solvent was allowed to evaporate over night in fume hood. The dry resultant cells were ground using a mortar and the residue was resupended in

20 mis Milli-Q water. The mixture was heated to 68°C whereupon 20 mis of 90% phenol, preheated to 68°C, were added. The solution was incubated for 1 hr with regular mixing and then transferred onto ice to allow phase separation to occur, (30 mins - 1 hr). The cells were separated by centrifugation at 3500 x g for 30 min in glass universals.

The upper aqueous phase was carefully removed and transferred for dialysis on membranes, The solution was dialysed for 24 hrs against running tap water to remove phenol. The solution was then spun at 15,000 x g for 30 min to remove larger par-tides. The solution was then transferred to ultracentrifuge tubes and spun at 125,000 x g for 3 hrs to pellet LPS. This pellet was clear. The pellets were resuspended in 3 ml sterile distilled water to prepare an inoculum for fish.

Outer membrane protein

4 litres of broth cultures of MT1415 were prepared as for aqueous phenol extrac- tion, above. The cells were harvested at 3500 x g for 45 min. and were washed in PBS. The pellet was resuspended in PBS + 20 micromolar PMSF, 1 unit DNase, and 1 unit RNase. Incubation was carried for 30 min on ice whereupon sonica- tion was carried out at 8 micrometer (6 x 30 s with 15 s of cooling intervals). Sar- kosyl was added to 0.7 % and the solution was incubated at ambient tempera- ture for 30 min. The lysate was cleared by centrifugation at 15,000 x g for 20 min. The supernatant was retained and the outer membrane proteins (OMPs) were harvested at 100,000 x g for 1 hr. The pellet obtained was resuspended in 0.7 % sarkosyl by reflux through a 26 gauge needle and harvested as above. The pel¬ let was washed in 1 ml of Milli-Q water. Then 2 ml of 1.5 x PBS were added to the OMP suspension.

Putative porin preparation

4 litres of TBS2 containing 100 micromolar 2,2-dipyridyl were prepared and ino¬ culated with MT1415. After growth as reported above, the cells were harvested, washed and sonicated and the lysate was cleared as in accordance with the OMP preparation above. The OMPs were harvested and the pellet was resus¬ pended in SDS buffer to solubilize proteins not associated with peptidoglycan. The solution was spun at 100,000 x g for 1 hr to precipitate PG and associated proteins. The pellet was resuspended in SDS buffer + 2 M NaCI to solubilize the proteins non-covalently bound with the peptidoglycan. The PG was spun out as above and the supernatant containing porins was retained. The solution was concentrated and desalted in Microcon 30. It was then diluted threefold with 3xPBS.

5 100 microlitres of each of the preparations above were injected intraperitoneally in fish.

Ten groups of fish at 28 fishes per group were injected, and each group was di¬ vided between four tanks and incoming fresh water temperature was maintained 0 between 14 and 18°C. All fish were vaccinated intraperitoneally with OJ ml vac¬ cine or control (sterile PBS). Two control groups were maintained comprising 28 and 30 fishes, respectively.

The fishes were starved 24 hrs before vaccination. 5 The different groups were:

1. Control, PBS (30 fishes)

2. l-bacterin (DP) (the bacterin injected had been grown with the Fe-chelator 2,2'-dipyridyl)

3. l-bacterin (EDDA) (the bacterin injected had been grown with the Fe chelator 0 EDDA (ethylenediamino diacetic acid))

4. l+bacterin (FeC-3) (the bacterin injected had been grown with added FeCtø)

5. l-norm bacterin (the bacterin injected had been grown with the normal medium without added Fe or chelator)

6. I+LPS (FeC.3) (the fish were injected with a preparation of LPS 5

(lipopolysaccharide) from the membranes of cells grown with added FeCl3 (cf 4. above)).

7. I Norm LPS (the fish were injected with a preparation of LPS from the membranes of cells grown with normal medium without added Fe or chelator (cf 0⁵-»-

8. I-OMP (DP) (the fish were injected with a preparation of OMP (outer membrane proteins) from the membranes of cells grown with the Fe-chelator 2,2"-dipyridyl (cf 2.)).

9. I+OMP (FeCl3) (the fish were injected with a preparation of OMPs from the 5 membranes grown with added FeC.3 (cf 4.)). 10. 1 norm OMP (the fish were injected with a preparation of OMP from the mem¬ branes of cells grown with normal medium without added Fe or chelator (cf 5.)). 11. l-porin (the fish were injected with a specific OMP called porin isolated from a culture grown with the Fe-chelator 2,2'-dipyridyl. 12. Porin buffer control (control for 11. using the same buffer but no porin)

The fish groups were injected on day 1 , and were then challenged on day 69 af¬ ter having raised the water temperature in the tanks to 25°C during the last four days before challenge, by injecting intraperitoneally a preparation of Pasteurella 0 piscicida as follows. Pasteurella piscicida MT1415 taken from a stock at -80°C was grown on TSA + 2% NaCI (TSA2) at 22°C for 48 hrs. From this a cell sus¬ pension of 1θ5 cfu/ml was prepared in sterile saline and aliquots (0,1 ml) were spread onto forty fresh TSA2 plates which were then incubated at 22°C for 48 _ hrs. Cells were harvested from the plates and a suspension of 10⁹ cfu/ml was prepared in sterile PBS (phosphate buffered saline). This was then diluted ten¬ fold and OJ ml aliquots (10⁷ cells) per fish were injected intraperitoneally. Morta¬ lities were removed daily and frozen before identification and bacteriological sampling.

Results

Mortalities commenced 1 day post challenge. The experiment was concluded 14 days post challenge.

Jablfi

.GiΩi-iβ % survival after 14 days

1. 0.0

2. 22.078 3. 2.507

4. 61.04

5. 7.468

6. 0.0

7. 22.078 8. 22.078

9. 2.597

10. 0.0

11. 7.468

12. 0.0

Pasteurella piscicida was recovered from all mortalities sampled during the challenge period.

As evident from the above table preparations of bacterin obtained from cultures grown on Fe-supplemented media show a considerably higher rate of survival than did normal preparations and those prepared after growth on iron-chelated media. 3. is in this respect odd, but the result is probably due to bad growth of the cells in the preparation.

The bacterin recovered, either as a whole cell product, component part or ex¬ pression component, is normally prepared in solution form for administration by injection, whereby adjuvants, carriers and vehicles used in connection with vac¬ cine preparations and well known to the one skilled in the art are added. The vaccine is then packed in ampoules, vials, or glass bottles for storage and distri- bution either as a single dose units, or multiple dose units.

The iron added in the cultivation of bacteria may be present as a ferrous salt, fer¬ ric salt, an iron complex or even as iron as such. Ferrous salts are ferrous sul- phate, ferrous chloride, and ferrous nitrate, and others. Ferric salts are ferric chlo¬ ride, ferric sulphate, ferric nitrate and others, such as mixed salts.

The present invention is not restricted to TSB medium but can be excersied with any commercial, or individually, laboratory composed growth medium used or contemplated within the art of growing bacteria.

The present invention has been demonstrated using an injectable form of a vaccine. However, the invention is not restricted to injectable vaccines only, but can be applied for oral, nasal, and immersion vaccines. Thus for humans and land animals injection and oral administrations are preferred, while when fish is concerned oral, injection, and immersion administrations are contemplated.

It is possible that an improvement is achieved in connection with plant pathogens as well, although different mechanisms for immunization are met.

Claims

CLAI MS

1. An improved vaccine against bacterial diseases, characterised in that the vaccine is recovered from culturing a bacterin producing bacteria in a nutritive medium containing sources of iron at levels in excess of those normally and hitherto used for this purpose and subsequently preparing a vaccine from the cultivated bacteria and/or their component parts and/or components expressed by the bacteria and present in the growth medium.

2. A vaccine according to claim 1 , wherein the bacterium is selected from a group known to be animal pathogens.

3. A vaccine according to claim 1 , wherein the bacterium is selected from a group known to be human pathogens.

4. A vaccine according to claim 1 , wherein the bacteriaum is selected from a group known to be fish pathogens.

5. A vaccine according to claims 1 and 4, wherein the vaccine is a fish vaccine.

6. A vaccine according to claim 5, wherein the vaccine is effective against furυnculosis (Aeromonas salmonicida).

7.A vaccine according to claim 5, wherein the vaccine is effective against vibriosis (Vibrio angυillarum).

8. A vaccine according to claim 5, wherein the vaccine is effective against cold water vibriosis (Vibrio salmonicida).

9. A vaccine according to claim 5, wherein the vaccine is effective against pasteurellosis (Pasteurella piscicida).

10. A vaccine according to claim 5, wherein the vaccine is effective against infection caused by Aeromonas hydrophila.

11. A vaccine according to claim 5, wherein the vaccine is effective against infection caused by Vibrio ordallai.

12. A vaccine according to claim 5, wherein the vaccine is effective against infection caused by Yersinia rupkeri.

5

13. A vaccine according to claim 5, wherein the vaccine is effective against infection caused by Edwardsiella tarda.

14. A vaccine according to claim 5, wherein the vaccine is effective against 10 infection caused by Edwardsiella ictaluri.

15. A vaccine according to claim 5, wherein the vaccine is effective against infection caused by Flexibacter columnaris.

¹5 16. A vaccine according to claim 5, wherein the vaccine is effective against infection caused by Flexibacter psycrophilυs

17. An improved process for preparing vaccines against bacterial diseases, characterized in that the improvement comprises the cultivation of bacteria in a 0 nutritive medium containing sources of iron at levels in excess of those normally present in such growth media, and subsequently preparing a vaccine from the cultivated bacteria or their component parts or components synthesised by the bacteria and accumulated in the growth medium, and optionally adding effective adjuvants, carriers, and vehicles for administration. 5

18. A process according to claim 18, wherein the bacteria are selected from a group known to be animal pathogens.

19. A vaccine according to claim 18, wherein the bacteria are selected from a 0 group known to be human pathogens.

20. A vaccine according to claim 18, wherein the bacteria are selected from a group known to be fish pathogens.

5

21. A process according to claims 18, 19, or 20, wherein the nutritive medium consists of one selected from those commercially available.

22. A process according to claims 18, 19, or 20, wherein the nutritive medium consists of individually selected components or combinations thereof including sources of nitrogen and carbon suitable for the cultivation of bacteria.

23. A process according to claims 21 or 22, wherein an additional amount of iron, iron containing salt, and/or iron containing complex is added.

24. A process according to claim 23, wherein the additional iron source is a ferrous salt.

25. A process according to claim 23, wherein the additional iron source is a ferric salt.

26. A process according to claims 23 to 25, wherein the additional iron is added to a level of 10 microgram/ml to 100 mg/ml above that present in the nutritive medium.

27. A process according to claim 23, wherein the additional iron is added to a level of 100 microM FΘCI3.