GB2023420A - Preparations containing antigen from pasteurella haemolytica - Google Patents

Abstract

A vaccine against infections caused by Pasteurella haemolytica comprises at least one capsular associated antigen obtained from Pasteurella haemolytica. The vaccine generally contains capsular associated antigens obtained from more than one serotype of Pasteurella haemolytica, the serotype chosen depending on the species of animal for which the vaccine is intended and the geographical location of the animals. The vaccine may also comprise other microbial or viral antigens, the choice of other antigen(s) again being dependent on the intended use of the vaccine. The invention also provides an antiserum against a capsuler associated antigen of Pasteurella haemolytica, which antiserum may be used for passive immunization against Pasteurella haemolytica infections.

Description

SPECIFICATION Pasteurella haemolytica vaccines The present invention relates to vaccines against infections produced by Pasteurella haemolyticaand to such vaccines which also comprise other microbial antigens.

Pasteurellosis is currently the major infectious disease causing mortality in sheep in the U.K. The disease is produced by varous serotypes of Pasteurella haemolytica which may give rise to septicaemia or local pneumonic lesions, depending on the specific serotype implicated and the age of the affected animal.

Infections produced by gram negative bacteria, such as Pasteurella haemolyticaare more difficult to combat by chemotherapy than are gram positive bacteria, and attempts have been and are being made to produce vaccines against such microorganisms, for use in humans and in animals. The Pasteurella haemolytica vaccines currently available contain whole bacterial cells as the antigenic component, but there is little experimental evidence as to their efficacy, even though such vaccines are used extensively.

The present invention provides a vaccine which comprises a capsular associated antigen obtained from Pasteurella haemolytica. We have found that the use of this antgen instead of whole cells in a vaccine results in an improvement in the immune response and a decrease in toxicity.

The vaccine may comprise one or more capsular associated antigens and/or it may comprise one or more other microbial antigens, for example, bacterial cells or fragments thereof, viruses, toxins and toxoids. The vaccine preferably comprises in adjuvant, generally aluminium hydroxide, preferably in gel form. It may also comprise any one or more of the usual additives, for example, preservatives and buffers, and it may be lyophilized.

The invention also provides a process for the production of a vaccine, which comprises subjecting Pasteurella haemolytica bacteria to treatment to release capsular associated antigen, separating the cells and/or cell fragments and the liquid phase, which comprises the capsular associated antigen, and bringing the liquid phase into the form of a vaccine.

The bacteria may be produced by cuitivation in a suitable fluid culture medium, using shake flasks or fermenter techniques, or on the surface of solid media, and the resulting crude culture may be subjected to such techniques as, for example, differential temperature, for example, heat treatment or freeze thawing, or to alternative physical or chemical treatments, such as, for example, ultrasonication and solvent fractionation respectively.

The differential heat treatment is carried out, for example, at a temperature within the range, preferably from 40"C to 650C, and advantageously at 560C.

The duration of the heat treatment is generally from 1 to 1000 minutes, preferablyfrom 15to60 minutes and advantageously 30 minutes.

Freeze thawing may be carried out, for example, at temperatures within the range of -200 C to 125"C.

In order to bring the resulting capsular associated antigen-containing liquid phase into vaccine form after, for example, heat treatment, the culture is preferably centrifuged, the supernatant dialysed and concentrated if necessary by suitable chemical or physical means, sterile filtered and its biological activity assessed.

One or more further purification techniques, for example, selected from solvent fractionation, ion exchange and molecular exclusion chromatography, may be incorporated at this stage.

The biological activity of the supernatant is generally assessed by serological means, utilizing, for example, a haemagglugination inhibition test, and the activity is expressed in, for example, H.A.I. units per ml. The vaccine ultimately comprises, for example, from 1 to 10,000 H.A.I. units per ml, preferably from 10 to 200 H.A.I. units per ml, and advantageously 100 H.A.I. units per ml, of the capsular associated antigens of each serotype.

In the case of a multivalent vaccine comprising more than one capsular associated antigen, it is generally necessary to produce and standardise each antigen separately.

In all cases it is preferably to incorporate an adjuvant in the vaccine. Preferably the antigen is adsorbed onto aluminium hydroxide, advantageously 3%(w/v) aluminium hydroxide gel suitably diluted to give 20% by volume of the gel in the final vaccine. The final vaccine generally has a pH between 5.5 to 8.0, preferably from 6.0 to 7.0, and advantageously from 6.1 to 6.5. A preservative is generally incorporated, for example, thiomersal in a final concentration of from 0.010 to 0.015%, especially 0.013%(2/v).

The vaccine may be lyophilized.

If desired, suitably treated whole bacterial cells may be incorporated in the vaccine, but no improvement in efficacy appears to result from this.

The vaccine is generally filled, adhering to accepted sterile techniques, into unit or multidose containers, the size of the individual dose ranging according to the animal to which it is to be administered, for example, a suitable unit dose for sheep, goats, calves and young pigs may be 2 ml, whereas 5 ml is a more suitable dose for adult pigs and cattle. (The preferred antigen content per ml is as given above.) A primary course of vaccination may be, for example, 1-10 doses of vaccine adminstered 1-90 days apart, preferably 1-3 doses administered 10-60 days apart and advantageously, two doses of vaccine administered 14 to 45 days apart.

As indicated above, the vaccine of the invention may be univalent or multivalent. The choice and combination of serotypes depends on the intended use of the vaccine, for example, P. haemolytica vaccines may be formulated for use primarily in sheep, or in cattle: for use in sheep, the vaccine preferably contains each of the serotypes Al, A2, T3, T4, A6, A7, A9, and T10, whereas for use in cattle, the preferred combination of serotypes is Al and A2.

These serotypes may be varied in accordance with prevailing epidemiological trends.

However, respiratory disease in cattle and sheep in some parts of the world is often caused by a com bination of P. haemolytica and Pasteurella multocida, so for such an indication, a combination vaccine preferably comprises the capsular associ ated antigens of appropriate serotypes of P.

haemolytica and also antigens, for example, whole bacterial cells or fragments thereof, of P. multocida.

The strains of P muttocida incorporated will depend on the species and geographical location for which the vaccine is intended.

Similarly, a univalent or multivalent P.

haemolytica vaccine may also be combined with live, attenuated or inactivated viral antigen(s): viral infections may predispose to secondary bacterial respiratory disease in most species of animal, including sheep and cattle, Viruses implicated in respiratory disease in sheep include Parainfluenza Type 3 and in cattle, Pi3, Rhinovirus and Respiratory Syncitial Virus, In addition, Adenovirus, Reovirus, Bovine Virus Diarrohea virus and Infectious Bovine Rhinotracheitisvirus have been implicated in respiratory disease in cattle. The serotypes of P haemolytica included in such a vaccine would be as noted above, the species of viruses included depending on the species and geographical location for which the vaccine is intended.In such a vaccine combination, other suitable adjuvants such as double stranded RNA may be incorporated in addition to/instead of aluminium hydroxide gel.

In many instances, it is of considerable practical importance to be able to protect sheep, goats, cattle and pigs against Clostridial disease and Pasteurellosis simultaneously by using a single vaccine comprising antigens from both classes of microorganisms.

One of the main indications for such a vaccine combination is in fattening lambs, when the appropriate P. haemolytica serotypes are combined with purified formol toxoids of Cl. tetani, Cl- septicum and Cl. welchii Type D together with the purified formalin killed cultures of Cl. chauveoi.

Another is in adult breeding sheep (pregnant or otherwise) when the appropriate multivalent P.

heemolytica component is combined with purified formol toxoids of Cl. welchii Types B, C, and D, Cl.

septicum, Cl- tetani and CL oedematiens Type B together with the purified formalin killed cultures of CL chauveoi.

Antigens of other appropriate Clostridial species/types, for example. Cl. oedematiens Type D and antigens of appropriate strains of P. multocida, may be added to such vaccine combinations depending on the species of animal and geographical location for which the vaccine is intended.

Respiratory and enteric disease complexes often affect animals at similar times, thus it is useful to protect animals simultaneously against, for exam ples, disease produced by P. haemolytica, P.

multocida and Salmonella spp. Other microbial infections occurring at similar times to/associated with P. haemolytica infections, include those caused by E. coli, Bordetella bronchiseptica and various enteric viruses.

Capsular associated antigens of appropriate serotypes of P. haemolytica may then be combined with other microbial antigens, examples of which are noted above, in order to produce multivalent vaccines against various disease complexes, the formulation depending on the species of animal, its management and geographical location.

In all cases, the amount of capsular antigen of each of the serotypes of P. haemolytica present in the vaccine is as indicated above, and the other antigen(s) should be present in sufficient amount and should be of sufficient quality to pass any statutory potency test required bythe country or state for which the vaccine is intended.

All of the combination vaccines indicated above are part of this invention.

The invention also provides an antiserum comprising antibodies directed against the capsular associated antigen obtained from Pasteurella haemolytica.

The antiserum may be obtained in the usual manner, for example, by administering the capsular associated antigen, preferably in the form of a vaccine of the invention, to an animal, bleeding the animal after a suitable time has elapsed, and preparing the antiserum from the blood.

The antiserum may be used for the passive immunisation of animals againstPasteurella haemolytica infections, in which case it is brought into a form suitable for parenteral administration.

A further use for the anti-capsular associated antigen-serum, and for the capsular antigen itself, is as a diagnostic agent for the detection of pasteurellosis. Any suitable immunological diagnostic method may be used.

The following Examples illustrate the invention.

Example 1: DivalentPasteurella haemolytica vaccine containing Capsular associated antigens of serotypes Al and T10.

Antigens of each serotype were grown individually using the following procedure: A lyophilized culture of each serotype was reconstituted in 10 ml brain heart infusion broth, and incubated at 370C for 6 hours.

2.5 ml of this seed culture was inoculated into each of 4 x 150 ml brain heart infusion broths, which were then incubated at 37"C for 18 hours on a rotary shaker revolving at 40 r.p.m.

Each culture flask was then subjected to heattreatment at 560C for 30 minutes, the cultures then being centrifuged at 12,500 r.p.m. for 60 minutes, at4 C.

The culture supernatants were then decanted, dialyzed against distilled waterfor 24 hours at 4"C, and freeze dried in 100 ml amounts in 500 ml flasks.

The cell deposits remaining after centrifugation were resuspended in 0.7% formol tris maleic buffered saline, incubated at 370C for 6 hours, then washed twice in 0.7% formol maleic buffered saline, by centrifugation. Sterility of the cell deposits was checked using standard techniques.

3.9 gm, of the freeze dried supernatant of each serotype were reconstituted individually in 26 ml tris maleic buffered saline (pH 7.4) and sterile filtered through 0.22,am Millipore filters.

The biological activity of each reconstituted serotype supernatantwas assessed by titrating the capsular associated antigens contained therein, utilising a haemagglutination inhibition test, against previously standardised antiserum.

On this basis, the supernatant of the Al serotype, contained 1280 H.A.I. units per ml and the T10 supernatant 320 H.A.I. units per ml.

The final vaccine was formulated as follows: 300 ml tris maleic buffered saline (pH 7.2) 25 mlP. haemolytica Al supernatant 25 ml P. haemolytica T10 supernatant 100 ml Alhydrogel (containing 3% aluminium hydroxide) - pH 5.6 25 mlP. haemolytica Al washed cells (1010 cells/ml) 25 mlP. haemolytica T10 washed cells (10'0 cells/ml).

Final vaccine thus contained 64 H.A.I. units per ml of Al capsular associated antigen and 16 H.A.I. units per ml of T10 capsular associated antigen.

Final vaccine pH was 6.4 The sterility of the vaccine was confirmed using standard techniques.

Toxicity was checked by administering 1 ml of the vaccine subcutaneously to each of four mice, and 2ml subcutaneously to each of two guinea pigs.

The vaccine was filled into sterile 50 ml collapsible polyethylene containers, and sealed with a combination disc seal.

This vaccine was used in an experimental study to assess its efficacy in protecting lambs against an intratracheal challenge with P. haemolytica serotype Al.

Sixteen, hysterectomy derived, colostrum deprived lambs were maintained in isolation accomodation. All were fed from birth to slaughter on a purely milk diet (Carnation Milk diluted 1:3 with sterile tap water) fed twice daily such that the mean intake per lamb per feed was two pints.

The lambs were divided into two groups of eight on the basis of age - each group being housed in slatted floored adjacent pens in the same building.

All lambs in one of the groups were vaccinated with 2 ml of the above vaccine subcutaneously on each of two occasions - at four and at six weeks of age.

All lambs were challenged intratracheally at eight weeks of age with 10 ml of a washed suspension of P. haemolytica serotype A1, containing 5.6 x 107 cells per ml.

The humoral and cell mediated immune responses to vaccination were assessed using standard serological and lympocyte transformation assays.

Lambs which survived challenge were euthanised at 72-96 hours post challenge and post mortem examination carried out, including viable bacterial counts from the lungs.

No signs of toxicity were apparent in any of the vaccinated lambs.

Results: a) A significant serological response to P.

haemolytica Al was evident in all vaccinated lambs, a response to P. haemolytica T10 occur ring in 5/8 of the vaccinates - no response to either serotype occurred in the controls.

b) A cell mediated immune response to the capsular associated antigens, as assessed by lymphocytic transformation techniques, was demonstrable in the vaccinated lambs.

c) One control lamb succumbed to challenge - all vaccinates survived.

d) Respiratory symptoms were more severe/longer lasting in the control group.

e) In the unvaccinated animals, post mortem examination revealed evidence of septicaemia (1), pleurisy (1), polyarthritis (2), and laryngeal lesions (4) in addition to gross pulmonary pathology.

The area and extent of the pneumonic lesions in the vaccinated animals was significantly less than that in the controls- no other significant lesions being observed in the vaccinates.

f) The mean count of P. haemolytica serotype Al from the lungs of vaccinated lambs at post mortem was 3.5 log,O per gram less than that of the controls.

Example 2: Bival ent Pasteurella haemolytica vaccines contain ing capsular associated antigens of serotypes Al and T10.

Three variants of the vaccine produced as per Example 1 were prepared with the following mod ifications: a) Identical to Example 1.

b) Identical to Example 1, but bacterial cells were not included-the volume of such cells being replaced by additional tris maleic buffered saline.

c) As Example 1, but using a 3% Arlacel A in mine ral oil adjuvant instead of Alhydrogel.

d) As (c) but with the exclusion of bacterial cells the volume of such cells being replaced byaddi- tional tris maleic buffered saline.

Each of these four vaccine variants contained 64 H.A.I. units Al capsular associated antigen and 16 H.A.I. units T10 capsular associated antigen per ml.

Each was subjected to sterility and toxicity tests as described above.

The potency of each vaccine was tested utilising a challenge procedure in mice, known to correlate with the results of challenge of specific pathogen I free lambs-the results being compared with those obtained using two commercially available vaccines, XandY.

0.1 ml of each vaccine was administered sub cutaneously to each of ten mice at 4 weeks and 2 weeks prior to challenge.

The vaccinated mice and ten untreated control mice were challenged intra-peritoneally with 0.5 ml of a ten hour broth culture of P. haemolytica Al in gastric mucin (1:4)- 1.2 x 107 colony forming units I per mouse.

Five mice in each group was killed at T = 0 and T = 5 hours. Livers were removed aseptically, macerated and diluted for P. haemolytica Al viable counting.

The results are expressed as log,0 counts of P.

haemolytica Al per liver, each value being the mean of 5 mice.

Mean log,O count Vaccine T=0 T=5 Controls 4.88 7.87 Commercial'X' 5.63 7.64 Commercial'Y' 6.16 7.78 Experimental a) 5.74 5.95 Experimental b) 5.23 4.94 Experimental c) 5.58 5.08 Experimental d) 5.78 5.73 The response occurring in mice treated with commercially available vaccines did not differ significantly from that occurring in control mice, whereas, in all groups vaccinated with experimental vaccines a, b, c and d, the growth of P. haemolytica serotype Al was significantly retarded.

There thus appears to be no significant benefit obtainable from the addition of bacterial cells, confirming that the protective antigens of P.

haemolytica are associated with the capsule of the organisms and can be isolated from appropriately treated culture supernatants.

Example 3: Bivalent Pasteurella haemolytica vaccines containing capsular associated antigens of serotypes Al and T10.

Antigens of each serotype were grown individually using the following procedures: A lyophilized culture of each serotype was reconstituted in 10 ml brain heart infusion broth, and incubated at 37"C for 6 hours.

2.5 ml of this seed culture was inoculated into each of 4 x 150 ml brain heart infusion broths, which were then incubated at 37"C for 18 hours on a rotary shaker revolving at 40 r.p.m.

Each culture flask was then subjected to heattrea- tment at 56"C for 40 minutes, the cultures then being centrifuged at 12,500 r.p.m. for 60 minutes at 4 C.

The culture supernatant was decanted, dialyzed against distilled water at 4"C for 24 hours and the biological activity of each serotype supernatant, assessed by titrating the capsular associated antigens contained therein against standard antiserum, utilising a haemagglutination inhibition test.

The Al supernatant contained 160 H.A.I. units per ml and the T10 supernatant 40 H.A.I. per ml.

Concentration of capsular associated antigens was carried out-the Al supernatant being ultrafiltered through semi permeable membranes under vacuum for 8 hours, reducing the volume by a factor of 2.1 and increasing the biological activity by a similar amount-from 160 to 320 H.Al. units per ml.

- The T10 supernatant was concentrated utilising polyethylene glycol-4 hours at4 C- reducing the volume by a factor of 7.5 and increasing the biological activity from 40 to 320 H.A.I. units per ml.

Each concentrated supernatant was sterile filtered through 0.22,am Millipore membranes, and their sterility checked using standard techniques.

The cell deposits remaining after centrifugation were treated in a similar manner to that utilised in Example 1.

Final vaccine was formulated to contain 100 H.A.I.

units of both capsular associated antigens of serotypes Al and T10 per ml: 15.6 ml A1 concentrated supernatant 15.6 ml T10 concentrated supernatant 2.5 ml A1 cells (1010 per ml) 2.5 ml T10 cells (1010 per ml) 10 ml Alhydrogel (3% aluminium hydrnxide) - pi4 5.6 3.8 ml tris maleic buffered saline (pH 6.5) 1.0 ml 0.658% thiomersal.

The final vaccine pH was 6.4, Sterility was checked using standard techniques and toxicity performed by administering 1 ml subcutaneously to each of four mice- no signs oftoxicitywere observed.

2 ml of this vaccine was administered subcutane- ouslyto each of five lambs at six and again at eight weeks of age.

No signs of toxicity observed.

Example 4: Preparation of multivalentPasteurella haemolytica vaccines.

A lyophilized culture of each serotype of P.

haemolytica to be included in the vaccine, was transferret to blood agar and incubated at 37 C for 18 hours.

The growth on blood agar was used to seed 100 ml of brain heart infusion broth which was then incubated at 37"C for 18 hours.

A 20 litre fermenter medium was then seeded with the latter culture - the fermenter medium including 3% peptone 1% yeast extract 1% glucose Distilled water to 20 litres (pH 7.0) The culture was incubated at 37"C for 24 hours, being continually sparged with air or oxygen and agitated throughout. pH was automatically controlled, involving dilute sodium hydroxide addition to maintain pH at7.0.

The whole culture was then subjected to heattrea- tment at 1 000C for 10 minutes, centrifuged at 3000 r.p.m. for 20 minutes at room temperature, and the supernatant decanted.

The biological activity of each culture supernatant was assessed by titrating its contained capsular associated antigen against standard antiscrum, utilising a haemagglutination inhibition test.

Final vaccine was formulated so as to produce a sterile preparation at pH 6.2-6.8, and to contain a minimum of 100 H.A.I. units of the capsular associated antigen of each included serotype per ml, its sterility and toxicity being checked by standard techniques.

Claims (30)

1. A vaccine which comprises at least one capsular associated antigen obtained from Pasteurella haemolytica.

2. A vaccine as claimed in claim 1, wherein the capsular associated antigens are obtained from more than one serotype of Pasteurella haemolytica.

3. Avaccine as claimed in claim 1 or claim 2, wherein the capsular associated antigen(s) is or are obtained from one or more of serotypes Al, A2, T3, T4, A6, A7, A9 and T10 of Pasteurella haemolytica.

4. A vaccine as claimed in claim 3, wherein the antigens are obtained from each of serotypes Al, A2, T3, T4, A6, A7, A9 and T10.

5. Avaccine as claimed in claim 3, wherein the antigens are obtained from serotypes Al and A2.

6. A vaccine as claimed in any one of claims 1 to 5, which also comprises one or more further microbial antigen(s).

7. A vaccine as claimed in claim 6, which comprises a Pasteurella multocida antigen.

8. A vaccine as claimed in claim 6 or claim 7, which comprises one or more viral antigen(s).

9. A vaccine as claimed in claim 8, wherein the viral antigen or antigens is or are selected from parainfluenza Type 3, Pal3, rhinovirus, respiratory syncitial virus, adenovirus, reovirus, bovine virus diarrhoea virus, and infectious bovine rhinotracheitis virus.

10. A vaccine as claimed in any one of claims 6 to 9, which comprises one or more clostridial antigen(s).

11. A vaccine as claimed in claim 10, wherein the clostridial antigen(s) is or are selected from purified formol toxoids of Cl. tetani, Cl. septicum, Cl. we/chil, Cl. sedematiens and purified formalin killed cultures of Cl. chauveoi.

12. A vaccine as claimed in claim 6 or claim 7, wherein the antigen(s) is or are selected from antigens of micro-organisms causing enteric and or respiratory disease.

13. A vaccine as claimed in claim 12, wherein the microorganism(s) is or are selected from Salmonella spp., E. coli, Bordetella bronchiseptica and enteric viruses.

14. A vaccine as claimed in any one of claims 1 to 13, which comprises from 1 to 10,000 H.A.I. units of the or each capsular associated antigen per ml.

15. A vaccine as claimed in claim 14, which comprises from 10 to 200 H.A.I. units of the or each capsular associated antigen per ml.

16. A vaccine as claimed in claim 15, which comprises 100 H.A.I. units of the or each capsular associated antigen per ml.

17. A vaccine as claimed in any one of claims 1 to 16, in unit dose or multidose form.

18. A vaccine as claimed in claim 17, wherein a unit dose comprises from 2 to 5 ml.

19. A vaccine as claimed in any one of claims 1 to 18, in lyophilized form.

20. A vaccine as claimed in claim 1, substantially as described in any one of the Examples herein.

21. Aprocessforthe manufacture ofavaccine as claimed in claim 1, which comprises subjecting Pasteurella haemolytica bacteria to treatment to release capsular associated antigen, separating the cells and/or cell fragments and the liquid phase, and bringing the liquid phase into the form of a vaccine.

22. A process as claimed in claim 21, wherein the treatment is differential temperature treatment, ultrasonication or solvent fractionation.

23. A process as claimed in claim 22, wherein the differential temperature treatment is heat treatment carried out at a temperature within the range of from 40 to 650C.

24. A process as claimed in claim 22, wherein the differential treatment is freeze thawing.

25. A process as claimed in claim 24, carried out at temperatures within the range of from -200 C to 1250C.

26. A process as claimed in any one of claims 21 to 25, wherein two or more serotypes of 1 - methyl N5 - [3 - [3 - [ are treated separately to release the capsular associated antigens, and the resulting liquid phases are brought into the form of a multivalent vaccine.

27. A process as claimed in claim 21, carried out substantially as described in any one of the Examples herein.

28. A vaccine as claimed in claim 1, whenever prepared by a process as claimed in any one of claims 21 to 27.

29. An antiserum which comprises antibodies directed against a capsular associated antigen obtained from 1 - methyl - N5 -[3 -[3 [.

30. A veterinary preparation which comprises an antiserum as claimed in claim 29 in a form suitable for parenteral administration.