CA1153310A

CA1153310A - Antigenic composition derived from bacteriodes nodosus

Info

Publication number: CA1153310A
Application number: CA000357009A
Authority: CA
Inventors: Charles M. Thorley
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1979-07-27
Filing date: 1980-07-25
Publication date: 1983-09-06
Also published as: NL8004297A; ZA804525B; IT8049346A0; FR2462166A1; AU6074980A; NZ194448A; IT1128549B; AU529322B2; FR2462166B1; DE3028248A1; BE884487A

Abstract

ABSTRACT
Antigenic Compositions A two-phase water-in-oil emulsion containing antigenic material derived from or consisting of Bacteroides nodosus organisms and an aluminium adjuvant in the aqueous phase of the emulsion may be prepared by standard methods known in the art for the preparation of emulsions of biological materials. The composition or a vaccine comprising a sterile preparation thereof may be used for the treatment or prophylaxis of foot-rot infections of sheep.

Description

~53310 _1_ A~03 ANTIGENIC COMPOSITIONS

This invention relates to antigenic compositions, vaccines thereo*, their preparation and use for the prevention and treatment of foot-rot, especially in sheep.
5. Ovine foot-rot is a widely-occurring, contagious disease, affecting the epidermal tissues of the foot and caused by the synergic action of two Gram-negative anaerobic bacteria, Bacteroides nodosus (Fusiformis nodosus) and 10. Fusobacterium necrophorum (Fusiformis necrophorum). The disease occurs only when both these organisms are present.
F. necroPhorum is fol~nd normally in the alimentary tract, and is excreted in the 15. faece~, so the organism i9 usually available in the immediate environment of the sheep's feet to participate in the infection. B. nodosus, on the other hand, is unable to survive under natural conditions for more than a few days 20. outside the lesions of foot-rot. The infected foot is its only natural habitat and accordingly B. nodosus is described frequently as the specific causal agent of the disease. The JDM/LM/2 July 1979 -- ~153310

-2- A603 elimination of B. nodosus from a flock of sheep, which could be accomplished by curing all cases of foot-rot present or by the specific destruction of the organism, would eradicate the disease since F. necrophorum cannot cause foot-rot in the absence of B. nodosus.
In past years, foot-rot has been controlled by isolation of the affected animals, followed by treatment comprising extensive paring of the affected areas of the feet and external application of disinfectants or external or parenteral admini-stration of suitable antibiotics. More recently, the successful large-scale culture of B. nodosus has permitted the vaccination of sheep using emulsion or aluminium adjuvenated vaccines, see for example UK
Patent Specification No 1 375 544. In many instances of the use of such vaccines, incomplete control of the disease has been achieved, and intense efforts have been made to discover the cause of failure and to improve the efficacy of existing vaccines.
It has now been found that an antigenic preparation of the present invention produces a greater antibody titre and a better control of the disease than B. nodosus vaccines used hitherto. Accordingly, the present invention provides an antigenic composition comprising a two-phase water-in-oil emulsion containing antigenic JDM/LM/23 June 1980 ~3310 _3_ A603 material derived from or consisting of Bacteroides nodosus organisms and an aluminium adjuvant in the aqueous phase of the emulsion.
The antigenic material of the composition may comprise an immunogenic extract of the organisms but preferably the material comprises the whole killed organisms in the form of an anaculture or harvested cells. Any one immunogenic strain or variant OT any combination of any number of strain(s) and/or variant(s), representative of any one or more B. nodosus serotypets), may be used. Satisfactory protection has especially been obtained by using organisms grown from fimbriate colonies in which the organisms are highly piliated (see for example J A Short et al, J. appl. Bact.
1976, 40, 301-315).~_. nodosus organisms grown on solid media are highly piliate'd, a~d in the performance of the present invention, growth conditions in liquid culture should be chosen so as to ensure the maximal degree of piliation in order to enhance the efficacy of the resultant vaccines. By highly piliated B. nodosus organisms, as described herein, are meant organisms having at least 20 pili per organism, and preferably more than 100 per organism. For maximal efficacy, the antigenic compositions and vaccines derived therefrom should consist wholly of highly piliated variants of B. nodosus, but as a practical matter adequate protection is obtained where 90~ of the organisms are JDM/~M/23 June 1980 .

~ ;33~0 highly piliated variants. The highly piliated variant of strain CSIR0 No 198 (Wellcome Laboratories Culture No 6476) ~deposited with the American Type Culture Co-llection on 19 July 1979 under No 31545) is especially valuable. Preferably9 protection may be afforded against homologous challenge (i.e.
challenge with one or more serotype ~s~ homologous with the B. nodosus serotype (s) of the composition) since protection against heterologous challenge is weaker.
Thus, challenge with organisms representative of m~re than one B. nodosus serotype will preferably be met with a composition comprising more than one strain,each strain being representative of each serotype comprising the challenge organisms.
The B. nodosus organisms for use in compositions -of the present invention may be grown or cultured by any method known in the art, for example by the method described in UK Patent Specification No 1 375 544; or by the methods described in the paper of Short et al (ibid) which produces highly piliated organisms.
The aluminium adjuvant is one which in its own right enhances the antibody response to the antigenic material, and may be chosen from those well Xnown in the art such as potash alum, aluminium hydroxide and aluminium phosphate. Particularly preferred are thixotropic ~-type aluminium hydroxide gels ~' ~;3310 which carry a positive charge in the absence of electrolytes. An example of such a gel is 'Alhydrogel' (Trade Name3. Preferably the compositions should contain between 0.25 and 5 . "4, o % vv/v of the aluminium adjuvant."
The emulsion has a single oil continuous phase and a single aqueous dispersed phase (that is to say, it is a ~w'o-'ph'a'~e wa`të.r-i''n-oi'l emulsion), and may be prepared by the use of 10. one or more emulsifiers and an oil. Naturally, the emulsifier or emulsifiers for inclusion in the compositions must be non-toxic and compatible with the antigenic components of thè
vaccine. They are preferably of the non-ionic 15. type so that there is less tendency for them to be precipitated by components of the antigen preparation than when a cationic or anionic emulsifier is used. There is also less danger of denaturation of the protein of the 20. antigen if a non-ionic emulsifier is used~ The combination, quantities and proportions of emulsifier chosen should also be those which, when included in the vaccine,produce the maximal degree of enhancement of antigenicity and 25. stability of the emulsion, and preferably should con~ist of a lipophilic and a hydrophilic JDM/LM/2 July 1979 ~1533~0 emulsifier.
The lipophilic emulsifiers are preferably non-ionic surface active agents having a low hydrophile-lipophile balance of between 8 and 20.
5. ~William C Griffin, 'Calculation of HLB values of non-ionic surfactants', Journal of the Society of Cosmetic Chemists (1954) volume 5, pages 249-256; Osipow, 'Surface Chemistry ACS
Monograph 153', pages 295-314, Reinhold, 1962.
10. This is the outer limit of the range of HLB
values of emulsifiers most suitable for incorporating in the oil phase and an optimal limit of HLB values is 2 to 6. Among suitable lipophilic emulsifiers are di- and tri- esters 15. of polyhydric alcohols and fatty acids, oxidised ~ fatty oils, and partial esters of common fatty acids, e.~g. palmitic, lauric, stearic and oleic acids with hexitol anhydrides derived from sorbitol or mannitol. Examples of specific 20. emulsifiers are mannitan and sorbitan fatty acid esters such as mannide monooleate (Arlacel A, HLB 4.3), sorbitan monooleate (Arlacel 80, HLB 4.3), sorbitan monopalmitate (Arlacel 40, HLB 6.7), sorbitan monostearate (Arlacel 60, 25. HLB 4.7), and sorbitan sesquioleate (Arlacel C, HLB 3.7)-JDM/LM/2 July 1979 ~ fra~ ~a~

~.~L5i3310 The hydrophilic emulsifier for use in the compositions should preferably have an HLB value of between about 9 and20 and optimally between 11 and 18. Suitable classes are polyglycol fatty 5. acid esters~ polyoxyethylene modified fatty acid esters, polyoxyethylene-polyol fatty acid esters, polyoxypropylene fatty alcohol ethers, and --- polypropylene glycol fatty acid esters. A
particularly useful class are polyoxyalkylene 10. derivatives of polyhydric alcohol fatty acid esters such as polyoxyethylene derivatives of partial esters of lauric, palmitic, stearic and oleic acids and hexitol anhydrides. Examples of emulsifiers are polyoxyethylene (20) sorbitan 15. ~ monolaurate (Tween 20, HLB 16.7), polyoxyethylene ,. , ~
(20) sorbitan monopalmitate (Tween 40, HLB 15.6), ' polyoxyethylene ~20) sorbitan monostearate (Tween - 60, HLB 14.9), polyoxyethylene (20) sorbitan monooleate (Tween 80, HLB 15)S polyoxyethylene 20. (20) sorbitan trioleate (Tween 85, HLB 11.0) and polyoxyethylene (8) laurate (G 2127, HLB 12.8).
Any non-toxic oil of vegetable or mineral origin compatible with the antigenic material may be used in the compositions of this invention.
25. Mineral oils of a pharmaceutical grade are especially preferred as they in general produce JDM/LM/2 July 1979 ff~Je ~ k ~ 1 ~3310 -~ -8- A603 a substantially more stable emulsion and less tissue irritation. Light liquid paraffin oils ~` (Bayol F and Drakeol No 6R) have produced satisfactory emulsions and compositions.
The liquid of the aqueous phase may consist of the culture medium or, where the cells are extracted or harvested, aqueous saline or water.
The compositions of the invention may be made by any of the techniques known in the art for the preparation of emulsions of biological materials and may in fact be prepared by VigoTous agitation of an aqueous antigen/aluminium adjuvant mixture with the oil and emulsifier~s). The aqueous antigen/
aluminium adjuvant mixture is prepared by inactivating the B. nodosus organisms, using for example formalin , concentrating the culture or anaculture to about 30~
pre~erably of the original culture volume, but~no less than 10~, by any of the techniques known in the art and adding the aluminium adjuvant to the aqueous antigen. The aluminium adjuvant may be added after concentration or the concentration process may itself use an aluminium adjuvant. An example of the former technique is isoelectric point precipitation and an example of the latter technique is co-precipitation with a flocculating agent.DIsoelectric point precipitation is especially applicable to antigenic material comprising highly piliated B. nodosus organisms. In this technique, the pH of the culture or anaculture JDM/LM/23 June 1980 ~ ~r~ ~v~

~9~ A603 is lowered, by the addition of acid, to approximately the isoelectric point of the cellsj although a lower pH may be used. Suitable acids may be chosen from mineral acids such as hydrochloric, phosphoric and especially sulphuric acids and strong organic acids such as acetic and lactic acids. The pH is suitably less than 6.0, preferably less than 5.6 and more preferably in the range of from 4.8 to 5.2. Below pH 4.8, precipitation should be complete but, depending upon the particular antigenic material used, damage to the antigen may occur. After lowering of the pH, the temperature of the culture or anaculture should be lowered to between 4 and 12C.k o-precipitation using a flocculating agent may be a,chieved by adding a 5 sterile solution of the flocculating agent to the for example anaculture and the pH adjusted~with alkali to give optimal precipitation. After settling in the cold ~say 2 days or more) the clear supernatant liquor is removed to waste, thereby effecting a concentration of the cells and proteinaceous matter (including pili) in the anaculture. Although some of the flocculating agent may be discarded with the supernatant liquor, it is preferable to restrict the quantity used for precipitation since the larger the amount used the more appears in the precipitate (cell concentrate) and the more irritant the ensuing vaccine. Suitable flocculating agents include the sulphates, chlorides and insoluble hydroxides of polyvalent cations, and polyanionic JDM/LM/23 June 1980 331~

polymers such as sodium carboxymethyl cellulose and hydroxypropylmethyl cellulose. Especially suitable for the present invention is potash alum since this may also function as the aluminium adjuvant. It has been found that 1 volume of 10% w/v potash alum added to 7 volumes of anaculture ~1.25% w/v alum overall) and with the pH adjusted to 5.4, a precipitate of suitable properties and clear supernatant liquor develops. The higher the pH te.g. 5.8, 6.4) the longer the time required for complete clearing of the supernatant. The lower the concentration of alum the longer the clearing time required. The bulk of precipitate also is dependent on the amount of alum added to the system; thus the addition of 1 volume of 10%iw/v alum to 11 volumes of anaculture tO.83~ alum w/;v overall) results in a barely acceptable precipitate with an inconveniently long settling time, whereas the addition of 1 volume of alum added to 4 volumes of anaculture t2.0% alum w/v) results in a very bulky precipitate which does not pack sufficiently well without centrifugation to be of use in the harvesting of cells. In similar manner the purified ~a~
aluminium hydroxide substance 'Alhydrogel' tTrade N~ffle) when added to anacultures of B. nodosus, remains in the dispersed phase for a long while, so that harvesting of absorbed cells/protein needs to be accomplished by centrifugation ~Another technique for concentrating the culture or anaculture besides centrifugation is ultra-filtration, and either of these latter two JDM/LM/23 June 1980

3~0 -ll- A603 techniques may be used in conjunction with isoelectric point precipitation or co-precipitation.
~In` the case where more than one serotype of B. nodosus is comprised in the antigenic material, an aqueous antigen/aluminium adjuvant mixture is prepared as described above for each strain or variant,then about equal amounts of each mixture are blended prior to emulsification. Preferably, the emulsion is prepared by mixing the oil with the lipophilic emulsifier, the aqueous antigen/aluminium component with the hydrophilic emulsifier, and combining the oil and aqueous phases. It is preferable to add the aqueous phase slowly to the oil phase with agitation in order to minimise any tendency to foTm an oil-in-water emulsion. Finer dispersions of the aqueous phase can be obtained by homogenising or milling in a colloidal mill, and this may be continued until no further decrease in particle size occurs.

JDM/LM/23 June l980 , .

~L53310 The optimal proportion of oil to water phase in a vaccine will depend on a variety of factors such as the concentration of antigenic material 5. in the aqueous phase, the desired viscositv of the vaccine., and the antigenic~ty of the antigenic material.
The aqueous phase preferably does not exceed 600/o and does not fall below 10% of the total volume of the emulsion. A greater proportion 10. of water is undesirable because of the increase in viscosity and instability of the emulsion, and a proportion of less than 10% makes it difficult to incorporate sufficient antigen without having an unacceptably large dose 15. volume. It has been found that satisfactory ~ , .
vaccines are obtained when the aqu~ous phase comprises, from 20 to 50/0 by volume of the whole - - - vaccine. If a large proportion of water is required, for example above 40%~ then precautions 20. should be taken to avoid formation of an oil-in-water emulsion and instability of the emulsion.
It has been found that such emulsions containing polyoxyethylene (20) sorbitan trioleate may be stabilised by the addition of Falba - a stabiliser 25. containing beeswax paraffin oils of various viscositi~s and oxycholesterins extracted from lanolin.
JDM/LM/2 July 1979 ~;33~0 -13- ~603 The amount of emulsifier needed in the respective phases will depend on a variety of factors and particularly on the HLB values of the emulsifiers and the choice of oil, and it 5. is estimated th~t from 2.5 to 15% by volume of a lipophilic emulsifier and from 0.5 to 10.0%
by volume of a hydrophilic emulsifier in the respective phases will provide vaccines of the properties described herein. A considerable 10. enhancement of the antigenicity of the antigens has been obtained using from 5 to 12% by volume of the oil phase and from 0. 2 to 7~ by volume of theaqueous phase, as the respective emulsi~iers.
The compositions of the present invention 15~ may be formulated into a vaccine suitable for administration by incorporating the composition in an appropriate concentration into a container as a sterile preparation.
It is necessary to sterilise the composition 20. after emul ification or to sterilise the individual components beforehand and emulsify under sterile conditiong, ~ompositions of the present invention may be sterilised by the use of heat and filtration to sterilise the components, or by the use of 25. gamma-radiation without any loss of potency or stability.

JDM/LM/2 July 1979 ~53310 The ~inal vaccine is introduced into sterile containers and sealed.
The vaccine desirably contains a bacteriostat of the kind generally employed in killed bacterial vaccines, and 0. 01% (w/v) thiomersal(sodium ethyLmercurithiosalicylate) may be incorporated for this purpose, in the aqu~ous phase of the composit~on.
Conveniently the final vaccine preparation contains, in total, from-2.5 x 107 to l x 1011, and desirably between i x 10 and 1.25 x 109, killed organisms per millilitre of the preparation.
The vaccines of the present invention are desirably administered to animals by subcutaneous, intramuscular or intraperitoneal injection. It is advantageous to administer the vaccine at a site where any ~ocal reaction upon administration is of little concern to the farmer, for example, ~0 on the anterior dorsal aspect of the neck or on the lower mandible. The most preferred dosage range is between 1 x 108 and 1 x 10 killed organisms per dose, in particular between 1 and 10 and 1 x 10 per dose, wherein the dose is the total quantity of antigenic material administered at one time to an animal such as a sheep in a suitable volume JDM/YA/2g July 1979 ~, ~53310 ~603 of liquid, for example 1 to 2 ml. The dose of vaccine may be administered to an animal as one unit or as a multiplicity of sub-doses, and the latter may be administered over a period of time, 5. or by simultaneous injections of the sub-doses at different sites. A suitable immuni-sation schedule may comprise a single inj ection of the ~ose, or two injections, each comprising a sub-dose, given simultaneously at. different sites 10. on the ~nimal. The optimal dosage schedule will of course vary according to the strains of organism chosen for the basis of the vaccine, : the total number of organisms employed, the nature of the adjuvant and t:he route of 15. administration. It has been found especially suitable to administer a vaccine comprising , 3 x 10~ organisms/ml in a single 1 ml or 2 ml do~e by the subcutaneous route followed by a second injection of the same volume and 20. strength and by the same route after a~ interval of at least 6 weeks, preferably of 12 weeks.
Boo~ter doses should be given just before the a~ticipated seasonal spread of foot-rot~
In addition to containing antigenic 25. material derived from B. nodosus, a vaccine of the present invention may also contain JDM/LM/2 July 1979 - "
.

331~

antigenic material derived from other bacteria that are causative organisms of diseases in, for example, sheep, in particular F. necorphorum, or clostridia. A particularly preferred combination is a multiple component vaccine comprising a preparation of one or more clostridial antigens described in UK patent specification No 1 143 545, in association with a B. nodosus antigenic composition produced as hereinbefore described.
According to the present invention there is therefore . , _ .. . . .. . .. . . .
provided: ~
(a) A~ antigenic composition comprising a two-phase water-in-oil emulsion containing antigenic material derived from or consisting of Bacteroides nodosus organisms and an aluminium adjuvant in the aqueous phase of the emulsion.
(b) A method of preparing an antigenic composition as defined in paragraph (a) above comprising~the emulsification of an aqueous medium containing antigenic . . . . . , . . . . . . . , . . . , . . ~ . _ . . . . . .
material derived from or consisting of B nodosus organisms and an aluminium adjuvant with an oil.
(c) A vaccine comprising a sterile preparation of a composition as defined in paragraph (a) above.
(d) A method for the prophylaxis or treatment of a foot-rot infection of sheep comprising the administration to the sheep of an effective, non-toxic dose of a vaccine as defined in paragraph (c) above.

JDM/Y~ 25 July 1979 `~ ~1533~0 The following are Examples of this invention but should not be construed as comprising any limitation thereof.
5. Reference Example 1 - Growth of organisms B. nodosus (Strain CSIR0 198) organisms obtained from seed culture, were inoculated in the medium (described by Thorley,C.M.(1976) ' J. Appl, Bact. 40, 301-309 at page 302) 10. contained in screw-capped bottles, and the air displaced by a mixture of 10% carbon dioxide and 90% nitrogen. The cultures wer-~ maintained at 37 C for between 20 and 40 hours, and the organisms killed by addition of formalin (0. 5%
- 15- ' w/w). Examination of the organisms under the electron microscope showed that at least 90/0 were highly piliated (>100 pili per organism).
, ' Example 2,- AntiFenic com~osition A formolised anaculture (3,200 ml) of 20. highl~piliated B. nodosus organism~ obtained in the manner described in Example 1 was treated with an aqueous solution '( 10% w/v) of potash alum (320 ml) and the pH of the mixture adjusted to 6.2 by the addition of aqueous 25. sodium hydroxide (400/o w/v). After maintaining the mixture at 4C for 24 hours, supernatant JDM/LM/2 July 1979 liquid above the precipitated organisms was discarded until the volume was reduced to 1,200 ml.
Tween 80 (30 ml; 400/o v/v aqueous solution) 5. and thiomersal (1.2 ml; 10% aqueous solution) were added, and the mixture emulsified with a mixture (2.8 1) of Arlacel A (10% v/v) and Marcol (90% v/v).
Examples 3-6 - Anti~enic Compositions 10. Highly piliated B. nodosus organisms were collected by isoelectric point precipitation by treating the anacultures with sufficient dilute aqueous sulphuric acid to lower the pH to 5Ø
After standing for three days at 16-20C, the 15. organisms were concentrated 9-10 times by discarding 133.7 litres of supernatant. The or~anisms were used in the preparation of vaccine formulations of Examples 3-6 as shown in Table 1.
In each instance, the concentrated . 20. anaculture was diluted (Co.lumn 3) with aqueôus sodium chloride solution, and thiomersal added to the aqueous mixture (Column 7) to produce a concentration of 0. 01% w/v prior to emulsification with the oil (Column 8). In Exampl~ 4 to 6, the 25. pH was adjusted after the addition of the hydrophilic emulsifier.
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~ ~,5331() Example 7 - Antigenic Composition Following the procedure described in Example 2, a further composition was prepared using highly piliated B. nodosus organi~ms 5. (obtained in the manner described in Example 1), as shown in Table 1.
Exa le 8 - Vaccines mp .
The compositions described in Examples 3-7 may be filled into l, 2 and 10 ml sterile 10. vials, sealed and sterilised by gamma irradiation.
Example 9 - EfficacY
In this experiment, four groups of five sheep were vaccinated subcutaneously on the 15. lower mandible with 2 ml~vaccine prepared as described in Example 2 (Vaccine A) and with 3 other vaccine preparations ( Vaccines B, C
and D), Six weeks later, a second dose of vaccine similar in all aspects to the first 20. was given to all sheep, and five sheep from each of the vaccine groups were challenged by having a live culture of the vaccine strain applied directly to each (predisposed) foot.
Thereafter, each group of five sheep was 25. challenged at monthly intervals, 50 that the last challenge oc~urred twelve weeks post JDM/LM/2 July 1979 -2l- A603 second vaccination. Feet were assessed for clinical foot-rot three weeks after each challenge. A score of one or more feet affected with u~der-r~mning foot-rot or severe inter-5. digital dermatitis in 2 or more feet on two or more of the five animals was deemed to fail the vacci~e on grounds of insufficient protection, and further testing of that vaccine was omitted.
The four experimental vaccines (vaccines 10, A, B, C, and D} were tested simultaneously in comparison with a non-vaccinate control group of sheep to check the virulence of the challenge , culture., Five lots of 4 x 5 sheep were used. Blood , samp}es were collected at 0 and six weeks and at 15. the time of foot assessment. Agglutination titres were determined and geometric mean titres found.
- The results of the trial are given in Table 2.
It can be seen that piliate alum/oil vaccine 20. A of the invention was the only one to confer protection on sheep at the fourth challenge. Two weeks subsequent to foot assessment the foot-lesions in the one sheep to become infected in this vaccine group had resolved whereas those in 25. the five control sheep were as severe as ever.
Furthermore, a distinct difference was found JDM/L~/2 Jul~ 1979 ~L53310 between serum agglutination titres o~ sheep immunised with Vaccine A and sheep receiving the other vaccines.
~hallenge 2 was invalid on account of 5. three control sheep failing to become infected.
Challenge 4 was very severe, the control sheep being badly infected at a preliminary ten day-post-challenge examination.
lt is believed that this experiment has 10. demonstrated for the first time the possibility of protecting sheep by vaccination against foot-rot for a period of twelve weeks following the last dose of vaccine.
Vaccines B, C and D were identical to 15. vaccine A except in that:-Vaccine B: the organisms were non-virulent and were genetically non-piliated, that is to say~ none were found to have more than 20 pili per bacterium.
20. Vaccine C: the aluminium adjuvant was omitted.
Vaccine D: the aluminium adjuvant was omitted and the organisms were non-piliate as in Vaccine B.

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~ ~53310 Example 10 - Efficac~ of vaccines In this experiment, vaccines prepared from the compositions described in Examples 3 to 7 were compared with vaccines adjuvenated by alum 5. alone (Vaccines F and G). Sheep free of foot-rot were treated either with a single dose of a Vaccine of Examples 3 to 7 or with two doses of Vaccines F or G spread 6 weeks apart. At intervals post-vaccination, groups of the 10. vaccinated sheep were compared with non-vaccinate controls for resistance to infection against artificial challenge with a living culture of B. nodosus as described in Example 9, The ! . . results are given in Table 3.

JDM/LM/2 July 1979 ~11533J,0 TABLE

. ~
Vaccine Dose of Challenge post-vaccinationtsheep (ml) .affected) .
. 8 weeks12 weeks 15 weeks _ _ . _ Ex 3 1 1/5 o/4 _ .- . _ Ex 4 1 0/5 ¦ /5 _ . ,, . I . .
Ex 5 2 _ _ /5 _ . . , Ex 6 1 /5 _ /5 , _ . Ex 7 1 /5 1/5 _ .
Ex 7 2 . 0/4 /5 _ _ , F 2x2 4/5 G 2x2 4/5 _ . _ _ . .
Controls 4/5 5/5 4/5 JDM/LM/2 July 1979

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An antigenic composition comprising an emulsion containing antigenic material derived from or consisting of Bacteroides nodosus organisms, wherein the emulsion is a two-phase water-in-oil emulsion containing the antigenic material and an aluminium adjuvant in the aqueous phase of the emulsion.

2. A composition according to claim 1, wherein the Bacteroides nodosus organisms are highly piliated, having at least 20 pili per organism.

3. A composition according to claim 1, wherein the Bacteroides nodosus organisms are highly piliated, having at least 100 pili per organism.

4. A composition according to claim 1, wherein the Bacteroides nodosus organisms comprise a variant of the strain deposited with the ATCC under No. 31545.

5. A composition according to claim 3, wherein the Bacteroides nodosus organisms comprise a variant of the strain deposited with the ATCC under No. 31545.

6. A composition according to claim 1, 2 or 5, wherein the aluminium adjuvant is selected from potash alum, aluminium hydroxide and aluminium phosphate.

7. A composition according to claim 1, 2 or 5, wherein the aluminium adjuvant is selected from potash alum and thixotropic .beta.-type aluminium hydroxide gels.

8. A composition according to claim 1, 2 or 5, wherein the emulsion contains a non-ionic emulsifier.

9. A composition according to claim 1, 2 or 5, wherein the emulsion contains a lipophilic emulsifier.

10. A composition according to claim 1, 2 or 5, wherein the emulsion contains a lipophilic emulsifier selected from mannitan and sorbitan fatty acid ester.

11. A composition according to claim 1, 2 or 5, wherein the emulsion contains mannide mono-oleate as a lipophilic emulsifier.

12. A composition according to claim 1, 2 or 5, wherein the emulsion contains a lipophilic emulsifier in an amount in the range of from 5% to 12% v/v of the oil phase of the emulsion.

13. A composition according to claim 1, 2 or 5, wherein the emulsion contains a hydrophilic emulsifier.

14. A composition according to claim 1, 2 or 5, wherein the emulsion contains a polyoxyalkylene derivative of a polyhydric alcohol fatty acid ester as a hydrophilic emulsifier.

15. A composition according to claim 1, 2 or 5, wherein the emulsion contains polyoxyethylene (20) sorbitan mono-oleate as a hydrophilic emulsifier.

16. A composition according to claim 1, 2 or 5, wherein the emulsion contains a hydrophilic emulsifier in an amount in the range of from 0.2% to 7% v/v of the aqueous phase of the emulsion.

17. A composition according to claim 1, 2 or 5, wherein the oil phase of the emulsion comprises a mineral oil.

18. A composition according to claim 1, 2 or 5, wherein the oil phase of the emulsion is a liquid paraffin oil.

19. A composition according to claim 1, 2 or 5, wherein the aqueous phase is in the range of from 10%
to 60% v/v of the emulsion.

20. A composition according to claim 1, 2 or 5, wherein the aqueous phase is in the range of from 20%
to 50% v/v of the composition.

21. A composition according to claim 1, 2 or 5, wherein the antigenic material is present in the range of from 2.5 x 107 to 1 x 1011 Bacteroides nodosus organisms per millilitre of the composition.

22. A composition according to claim 1, 2 or 5, wherein said aluminium adjuvant is selected from potash alum, aluminium hydroxide and aluminium phosphate and said emulsion contains a lipophilic emulsifier in an amount in the range of from 5% to 12% v/v of the oil phase of the emulsion.

23. A composition according to claim 1, 2 or 5, wherein said aluminium adjuvant is selected from potash alum and thixotropic .beta.-type aluminium hydroxide gels and said emulsion contains mannitan or sorbitan fatty acid ester as a lipophilic emulsifier in an amount in the range of from 5% to 12% v/v of the oil phase of the emulsion.

24. A composition according to claim 1, 2 or 5, wherein said aluminium adjuvant is selected from potash alum, aluminium hydroxide and aluminium phosphate and said emulsion contains a hydrophilic emulsifier in an amount in the range of from 0.2% to 7% v/v of the aqueous phase of the emulsion.

25. A composition according to claim 1, 2 or 5, wherein said aluminium adjuvant is selected from potash alum and thixotropic .beta.-type aluminium hydroxide gels and said emulsion contains a polyoxyalkylene derivative of a polyhydric alcohol fatty acid ester as a hydrophilic emulsifier.

26. A composition according to claim 1, 2 or 5, wherein the antigenic material is present in the range of from 2.5 x 107 to 1 x 1011 Bacteroides nodosus organisms per millilitre of the composition, said aluminium adjuvant is selected from potash alum, aluminium hydroxide and aluminium phosphate, said emulsion contains a lipo-philic emulsifier in an amount in the range of from 5% to 12% v/v of the oil phase of the emulsion, and the aqueous phase is in the range of from 20% to 50% v/v of the composition.

27. A composition according to claim 1, 2 or 5, wherein the antigenic material is present in the range of from 2.5 x 107 to 1 x 1011 Bacteroides nodosus organisms per millilitre of the composition, said aluminium adjuvant is selected from potash alum, aluminium hydroxide and aluminium phosphate, said emulsion contains a hydrophilic emulsifier in an amount in the range of from 0.2% to 7% v/v of the aqueous phase of the emulsion, and the aqueous phase is in the range of from 20% to 50% v/v of the composition.

28. A vaccine comprising a sterile preparation of an antigenic composition comprising an emulsion containing antigenic material derived from or con-sisting of Bacteroides nodosus organisms, characterised in that the emulsion is a two-phase water-in-oil emulsion containing the antigenic material and an aluminium adjuvant in the aqueous phase of the emulsion.

29. A vaccine according to claim 28, characterised in that the aqueous phase of the emulsion includes a bacteriostat.

30. A method of preparing an antigenic composition according to claims 1, 2 or 5, comprising dispersing the antigenic material derived from or consisting of Bacteroides nososus organisms in an aqueous medium and emulsifying the aqueous medium with an oil, characterised in that an aluminium adjuvant is present in the aqueous medium.