USRE25721E - Swine erysipelas vaccine - Google Patents

Description

United States Patent 721 swmn nursuiunas VACCINE Dennis George Howell, Richmanswonth, Robert Parr, South 'Efliug, London, and Kathleen Rosemary'Heath,

Northolt, Greeu'r'ord, England, assignors to Glaxo" Group Limited, a British corporation No Drawing. Original No. 3,083,142, dated Mar. 26, 1963, Ser. No. 795,322, Feb. 25, 1959. Application for reissue Mar. 16, 1964, Ser. No. 362,544 Claims priority, application Great Britain, Feb. 27, 1958, 6,396/58; Aug. 14, 1958, 26,186/58 9 Claims. (Cl. 167-78) Matter enclosed in heavy brackets [1 appears in the original patent but forms'no part of this reissue specification; matter printed in italics indicates the additions made by reissue.

labour and hence expense involved in giving injections is considerable and to have to administer second or even third injections of vaccine may represent a substantial increase in the cost ofrearing the animals. This problem is particularly acute where range animals are concerned since the labour of rounding up the herds two or three times in the course of a few weeks is often prohibitive.

It has been suggested that adjuvants could be added to vaccine preparations for the purpose of delaying the release of antigen into the system and so thereby'reducing the number of doses necessary to set up a satisfactory antibody response. In particular, it has been proposed to present the antigenic material in a water-in-oil emulsion, the antigen being contained in the disperse aqueous phase and its absorption into the system so being delayed by the surrounding continuous oil phase. Such preparations have in many instances proved unsatisfactory in use as they frequently give rise to considerable undesired local reactions at the site of injection which may impede the desired release of antigen.

We have found that a vaccine dispersed in certain types of oil-in-watcr emulsion hereinafter referred to possess marked advantages over the hitherto proposed water-in-oil emulsion-based vaccines. The oil-in-water emulsions thus used for the purpose of the present invention are those which can be described as being stabilised and remaining substantially stable even following injection into muscle tissue. In experiments, we have carried out, we find that many emulsions, when injected into living muscular tissue, rapidly break down into separate oil and Water phases with the result that the oil phase tends to cause necrosis or other tissue damage, whilst the separated aqueous phase no longer has the protecting action of the oil intended to delay release of antigen; this phenomenon is noted even with emulsions which in vitro would normally be considered as quite stable. For the purpose of the present invention, therefore, we use emulsions which are stablilised, i.e. so formulated that theyv remain substantially stable for some time following injection into anirnal' rnuscle tissue.

The vaccines prepared according to the invention cause substantially less tissue damage than vaccines hitherto prepared with oily adjuvants and give rise to a more reliable and usually better delayed release of antigen.

It has also been observed that vaccine preparations Re. 25,721 Reissued Jan. 26, 1965 according to the invention are in general easier to inject than many hitherto proposcdvaccines based on water-inoil emulsions, being capable of production in a more readily flowing form.

According to the'present invention we provide an injectable vaccine preparation comprising antigenic material dispersed in a parentcrally acceptable stable emulsion of an oil dispersed in a continuous aqueous phase.

The term antigenic material is used in this specifica tion to mean material derived from the culture of a pathogenic organism, which material on introduction into animal blood gives rise to antibodies serving to protect against or combat infection.

The oil-in-water emulsions according to the invention will generally be prepared with the aid of parenterally acceptable wetting agents which serve to form and stabilise the desired emulsion.

As is well known, the formation of an oil-in-water emulsion as distinctfrorn a watcr-in-oil emulsion involves suitable choice of wetting agent or agents, having regard to the relative proportions of the oil and Water phases and their exact nature. The consistency of an emulsion and hence its suitability for injection is also dependent on its constituents. Thus for the purpose of the present invention it is necessary so to choose the constituents of the emulsion that an oil-in-watcr emulsion (as distinct from a water-in-oil emulsion) is formed and that its consistency is such that it can be injected. It is further necessary that the emulsion be stable and remain as an emulsion after injection into muscle tissue.'

The wetting agents used in the preparation of the stabilised emulsion forming the base of the vaccines according to the invention are preferably of the non-ionic type. With such wetting agents more reliable stability of the emulsion on injection is secured and there is less tendency for the Wetting agent to be precipitated from the aqueous phase by components of the antigen preparation than when a cationic or anionic wetting agent is used.

We have found it preferable to employ a two-component wetting agent system, one of the wetting agents serving primarily to form the emulsion and the second primarily to stabilise the emulsion once formed. As is known, for example, the water-soluble or preferentially water soluble wetting agents generally act as emulsifying agents whilst wetting or surface active agents which are oil-soluble (or preferentially oil soluble) are known generally to exerta stabilising effect on emulsions and many different wetting agents are now offered on the market both for emulsifying and stabilising purposes.

The following types of wetting agents have been found to be particularly useful for the purpose of the present invention.

Type (a).P,olyhydric alcohols, or their anhydrides, esterified with fatty acids, for example, sorbitan monostearatc, propylene glycol monostearate, diglycol monostearate, mannitan mono-oleate, sorbitan sesqui'oleate, glyceryl' monostcarate, propylene glycol mono-olcatc, propylene glycol mono'laurate, diglycol monolaurate, diglycol mono-oleatc, diglycol monopalmitate, diglycol monoricinoleate, pentaerythritol monostearate, pentaerythritol distearate pentacrythritol dioleate, pentaerythritol glycerol oleate, pentaerythritol dioleostearate, low-molecular polyo-xyethylene oleate, glyceryl mono-groundnut acid ester, po-lyglyceryl monostearate, polyglyceryloleate, glycol mcnosteara-te, glyceryl mono-olcate, glyceryl monopalrnitate and low-molecular polyoxyethylene monostearate.

The wetting agents of the above type (a) are, in general, oil-soluble and hence suitable as stabilising agents The following type, type (b), however, are generally water-soluble andso suitable as emulsf'ying agents.

Type (b).Ployoxyalkylene derivatives of esters of olyhydric alcohols, for example polyoxyethylene sorbitan lonostearate, polyoxyethylene propylene glycol monoearate, polyoxyethylene glycol monolaurate, polyoxy- ;hylene glycol mono-oleate, polyoxyethylene glycol lonostearate, polyoxyethylene glyceryl stearate, polyxyethylene sorbitan monopalm-itate, polyoxyethylene soritan monolaurate, poly oxyethylene sorbitan trioleate 1d polyoxyethylene sorbitan mono-oleate.

Further types of Wetting agents which may be of use .clude the following:

Type (c).\Vater soluble fatty acid esters of polyhylene glycol, for example polyethylene glycol 300 diurate, polyethylene glycol 300 monostearate, polyethyll6 glycol distearate and nonaethylene glycol mono-oleate, wer molecular polyglycol ricinoleate, lower molecular )lyglycol monocaprate-caprylate, lower molecular polyycol monolaurate.

Type (d).Fatty alcohols condensed with ethylene :ide e.g. the material sold as Collone AC. or Abracol W. These wetting agents are water-soluble and hence itable as stabilising agents. The stabilising agents of type (a) are preferably used conjunction with the emulsifying agents of type (b). 1us, one especially suitable combination is sorbitan onostearate with polyoxyethylene sorbitan monostearate 1ile another is propylene glycol monostearate with poly- :yethylene monostearate, advantageously combined with glycol monostearate. The material Collone AC. is prefably used in conjunction with sorbitan monostearate d/or low-molecular polyoxyethylene monostearate. The exact proportion of Wetting agents to be used ll depend on their nature. However, using a combinarn of non-ionic emulsifying and stabilising agents, we .d in general that from 0.5 to 1.5% of emulsifying ent in combination with from 0.5 to 1.5% of stabilising ent gives satisfactory results. The ratio of emulsifying cut to stabilising agent can thus vary between 1:3 and 1. It should be noted that too great a proportion of :tting agent-either of the emulsifying or the stabilising peis undesirable and may cause instability or even eaking of the emulsion or phase reversal. The proportion of oil to water is important and preferly the proportion of oil to water in the emulsion should t be more than 65% but preferably not less than :sirably the proportion of oil to water should be within range of from 60%. The composition of the llllSlOIl base should, however, be correlated with the antity of water (if any) added with the antigen and final product, in order to be reliably stabilised, preftbly contains more than 10% of oil. In order to determine the suitability of a given set of .ulsion components for preparing a stable oil-in-water .ulsion of suitable thinness for injection one merely :nds the components in a homogeniser and leaves the .ulsion so formed to stand for a prolonged period. ,y tendency to separation or complete cracking can 11 be simply observed. Where there is doubt as to ether an oil-in-water or a water-in-oil emulsion has :n formed, one can add a dye which is soluble in only a phase and examine to see whether the disperse or continuous phase is coloured. Alternatively one can ermine the conductivity of the emulsion which would farlower for a water-in-oil emulsion than for an -in-water emulsion. The emulsion should be tested vivo; for example, it may be injected into the muscle ue of an experimental animal e.g. a mouse, and, after he days, the animal sacrificed and the tissue surrounding site of injection examined. If the emulsion is suffi- 1tly stabilised there should be no substantial necrosis I usually there will be some of the emulsion still sent. .h'e oils which may be employed to prepare the oilvater emulsions may comprise vegetable oils, for exple arachis, olive, sesame, soya bean, castor and cotton- :1 oils as well as oily esters e.g. ethyl oleate, dibutyl sebacate and isoamyl salicylate. However, we find that mineral oils of a pharmaceutical grade e.g. light liquid paraflin, are to be preferred as they in general, and surprisingly, cause even less tissue irritation than the vegetable oils. This we believe to be due to possible greater stability of mineral oil emulsions and hence less tendency for them to break after injection, the fine oil droplets being eventually dispersed by phagocytosis.

In the preparation of the vaccines according to the invention it is convenient to prepare the oil-in-water emulsion by, for example, blending the oil, water and wetting agents in a homogeniser often at elevated temperature, for example at about C., and to mix this emulsion with the antigenic material in the desired proportions.

The final vaccine must not contain unwanted organisms and it is thus necessary to sterilise the various components of the vaccine. Where heat is used for sterilisation, the emulsion or other components must be sterilised in the absence of the antigenic material, which would be de stroyed by the high temperature. If the emulsion does not withstand heat sterilisation it is possible to sterilise the oily and aqueous components before emulsification. In general, the oil may be sterilised by heating to about C. for one hour while the aqueous medium may be sterilised in the autoclave at 15 lbs. pressure for 30 minutes. The emulsion should be cooled before mixing with the antigenic material since few antigens can with stand temperatures in excess of 40 C. and are best kept at room temperature or below. However, it is often preferable to emulsify the oil with an aqueous suspension of the antigenic material, particularly where a high concentration of antigenic material in the final vaccine is required. Thus if the antigen is obtainable only in the form of an aqueous suspension and the emulsion is prepared separately, it is necessary to add a substantial quantity of aqueous suspension to the emulsion to obtain the required high antigen concentration whichmay seriously alfect the stability of the emulsion. In such cases it is often possible to avoid the elevated temperatures generally used in preparing the emulsions since, when a water soluble wetting agent, such as polyoxyethylene monostearate, is added to the anitgen suspension and a more oil soluble wetting agent, such as propylene glycol monostearate, to the oil, it is generally possible to prepare the emulsion by blending at room temperature.

It is also possible to prepare the oil-in-water emulsions from a gel of the oil. The gelling agent is usually a metal soap of a long chain fatty acid eg aluminium stearate, aluminium oleate, aluminium palmi-tate, lithium stearate, calcium oleate etc. In general it is possible to use a somewhat lower proportion of oil in the emulsion when a gel is used. One suitable gel comprises arachis oil containing between 1.5 and 2.5% aluminium stearate.

While the antigenic material incorporated in the preparations according to the invention may be of substantially any kind or in any form, the following have been found particularly suitable for presentation with oil-inwater emulsions.

(1) Bacterial toxoids from which dead bacteria have been removed, for example, toxoids, from Staphylococcus aureus. Clostridial toxoids, in particular toxoids from C1. welchii, types B, C and D, which give protection in various combinations against lamb dysentery and pulpy kidney disease and enterotoxaemia in sheep and toxoids from C1. tetani which give protection against tetanus.

(2) Bacterial vaccines containing dead bacteria, for example preparations from Erysinelothrix rhusiopathiae which give protection against erysipelas in pigs and turkeys, also Brucella, Salmonella and Leptospiral vaccines. (3) Viral vaccines, for example, Swine fever virus, Foot and-Mouth virus, Loupin ill virus', Enzootic abortion virus, Infectious Larynys tracheitis virus, Newcastle disease virus, and Infectious Bronchitis virus.

It will be appreciated that it is often advantageous to administer to an animal two vaccines giving protection against ditferent diseases and that it is convenient to form.

combine. these into a single vaccine for simultaneous administration. Thus the toxoid from C1. welchii type D toxin, which protects against pulp-y kidney diseasein lambs, can be combined with toxoid from Cl. .tetani, which protects against tetanus, so as to protect young lambs against both these diseases.

The antigenic material may be incorporated into the oil-in-water emulsion in substantially any convenient It may thus be a dried solid, if desired purified, an adsorbate on a parenterally acceptable adsorbant, for example aluminium phosphate, or aluminium hydroxide, pumice or kieselguhr or a suspension in a parenterally acceptable liquid; The final vaccine should, however, be pyrogen free and should contain as little material as possible which gives rise to unwanted reaction in the body. The aqueous phase of the final vaccine is also preferably substantially isotonic, that is the sodium chloride conconcentration in the aqueous phase is preferably about 0.85%, and it may contain preservatives such as phenol or thiomersalate.

The invention will now be described with reference to two particular embodiments thereof. It will be understood that these embodiments whilst representing vaccines which are of considerable use are only examples of many which can be prepared according to the invention.

VACCINE FOR THE CONTROL OF ERYSEPELAS IN- SWINE Erysipelas in swine is caused by the organism Erysipelothrix rhusiopathae and hitherto vaccines for the immunisation of swine against the disease have been prepared by culturing the organism on suitable nutrient media, killing the organism and adsorbing the immunising antigens so obtained on a suitable adsorbent, e.g. alumina, which is then suitably formulated for injection. The adsorbate of antigens has previously been proposed to be administered in various media, the principal use in Great Britain having been of simple aqueous suspensions; such preparations have usually been of unsatisfactory potency, and have given only relatively short immunity. To overcome these difiiculties a vaccine has been proposed which is in the form of a water-in-oil emulsion but it was not acceptable due to the severe local reactions which occurred after injection and was, furthermore, difficult to inject.

We have found that a particularly effective vaccine preparation for the control'of erysipelas in swine can be formulated comprising a stabilised emulsion of a parenterally. acceptable oil in water, having dispersed therein an adso-rbate of immunising antigenic material derived from the culture of Ery-sipelothrix rhusiopathae upon an injectable adsorbent. Such vaccines in accordance with the present invention can readily be prepared and have good antigenicity and are stable, readily injectable, do not, in general, give rise to-marked'reactions and give improved prolongation of action as compared with simple aqueous suspensions of an adsorbate of the antigens. In particular they are surprisingly better from various points of view, especially ease of injection, reduced tendency to cause reactions, etc., than erysipelas vaccines based on water-in-oil emulsions previously proposed.

In the preparation of the vaccines according to the invention, it is preferred to prepare separately an adsorbate of the antigenic material and blend this with an emulsion of the chosen oil in water; preferably the adsorbate of the antigens is first suspended in water containing a bactericidal agent, e.g. formalin or thiomersalate, and made isotonic with blood e.-g. withthe addition of sodium chloride. This suspensionis then-blended with the emulsion;

Preferably the proportion of oil to water in the emulsion before addition ofthe antigenic material is not more than 65% and is advantageously within the range of from -60%.

The proportion. of adsorbate' isalso preferably withii the range of from525%, conveniently 10%.

The adsorbate of the antigens. may be formed from an suitable adsorbent, examples of which are aluminum hy droxide, aluminum phosphate, pumice and kieselguhr The adsorbate may be prepared in any desired way, fo: example by culturing the. organism on a suitable nutrien medium, inactivating the culture containing antigens witl for example formalin or thiomersalate, adding adsorben' to adsorb'the antigens, the resultant adsorbate being so arated, for example by centrifugation.

A virulent strain of organism should naturally be user for the production of this vaccine and, in general, organisms showing no substantial filamentous growth are suit able. We have found it preferable to culture the organism at or about 30 C., instead of the more usuai 37 C., as in this way a more virulent culture is obtained. The medium used'for the culture organism may be of standard type.

VACCINE FOR THE CONTROL OF LEPTOSPIROSIE IN CATTLE AND OTHER ANIMALS Leptospirosis can be caused by the organism Leptospira pornona, the disease occurring in two phases. In the first phase, which is the acute phase, the infected animal shows clinical manifestations of the disease with accompanying fever, haemoglobinuria, and loss of condition which sometimes results in death. In the second phase the leptospira organisms colonize the kidney tubules and the animals, although apparently free from infection, become carriers, shedding viable leptospira organisms in the urine. They are thus a source of infection to any susceptible animal in the vicinity.

We have found it possible to formulate a particularly useful vaccine active against leptospirosis comprising a stabilised parenterally acceptable oil-in-water emulsion containing antigenic material derived from the culture 0 Leptospira pornona.

We find that by formulating the new vaccine in a stabilised oil-in-Water emulsion a product is obtained which gives good protection against both phases of'leptospirosis and has reasonably prolonged action. Thus our new vaccine is more reliable and'effective than leptospirosis vaccines formulated in various other vehicles. In particular the new vaccine is more acceptable than a similar-product in the form of a water-in-oil emulsion.

The vaccine according to the invention is prepared by culturing Leptospira pomona, preferably a virulent strain thereof, in a culture media therefor, killing the organism and incorporating the antigenic material thereby obtained into a s tabilised oil-in-water emulsion according to this invention. Preferably the Whole culture broth is used in the formulation of the vaccine.

We have found furtherthat'thechoice of means used to kill the organism is'of importance'if best results are to be 0 tained. Thus, whilst for example thiomersalate, formalin or heat may be used for this purpose, much-improvide results are obtained by the use of a phenol. Such phenols include phenol itself, as well as the cresols resorcinol and the xyleno-ls, of which phenol is preferred.

Various media may be used to support the growth of Leptospira pomona as-Willbe well known to those skilled in the art. As with allmicroorganisms the cultural requirements of'Leptospirapomona essentially comprise a source of nitrogen, a source of carbon an'd'energy and nutrient salts. Very many materials are now known for this purpose, so thatthe choice of nutrients is very Wide and it is a simple matter to choose nutrients suitable forv [he culture is preferably carried out in at least two and ferably three stages. For example in the first or priry stage a culture is made from a suitable source of organism e.g. an infected animal and grown up to m' a suitable inoculum for a secondary or development ge. The culture from the secondary stage is then used inoculum for a production stage. The media used are iveniently of the same constitution for each stage.

a suitable temperature for the culture is 27 C. Following the culture the organisms are killed and the )th is then suitable for formulation.

in the formation of the vaccine we prefer first to form oil-in-water emulsion base and then incorporate the :igenic material therein care being taken to keep the .ulsion as an oil-in-water emulsion.

?referably the proportion of oil to water in the emuln and before addition of the antigenic material is not re than 65% and is advantageously within the range from 40-60%.

After formation of the emulsion base, the antigenic .t'erial preferably as the whole culture broth is incorrted therein by any convenient method, preferably by z of a high speed blender or homogeniser. The prortion of culture broth to be incorporated in the emul- 11 base is preferably from 40 to 60% of the base.

if desired instead of first forming an emulsion base and :n incorporating the anhydrous material therein, the .ture broth may for example be simply diluted as necary and the oil emulsified therein. In this case it is :ferable to add a water-soluble wetting agent to the ueous suspension and an oil-soluble agent to the oil. The use of vaccine adjuvants, such as aluminium hy- )xide gel or aluminium phosphate is not necessary acrding to the invention, but such adjuvants may if deed be used. Thus for example the antigenic material Ly be adsorbed on to some suitable adsorbent and the sorbates incorporated into the emulsion base. The dit incorporation of the whole culture broth into the se is however preferred.

For the better understanding of the invention we now 'e the following examples by way of illustration only.

Zxample 1.Preparation of Swine Erysipelas Vaccine (a) PREPARATION F THE CULTURE BROTH ver extract (as above) 8%.

Z. amine (type E) 4% in distilled water. dium chloride 0.5%.

ucose 0.05%.

Heat to dissolve adjust the pH to 7.5. Filter while 11 hot through Whatrnans No. 54 filter paper. Sterie by autoclaving at lbs. pressure for minutes.

id sterile inactivated horse serum, rnls. per litre. cubate at 37 C. to test for sterility.

) THE PREPARATION OF ALUMINIUM HYDROXIDE This is prepared by the method described in the World :alth Organisation report No. 61. The gel is precipited from aluminium sulphate by ammonia in the presce of ammonium sulphate, washed free from soluble lts and sterilised by autoclaving.

The aluminium hydroxide content is calculated to eight in grams per ml. of gel.

((3) OIL-IN-WATER EMULSION This is a sterile liquid paraffin emulsion syringeable rough a No. 26 (British standard wire gauge) needle epared from:

the

Light liquid paraflin (medicinal grade) a 44.5% w./v. Grill 8 (emulsifying agent) 1.1% w./v. Crill 3 (stabilising agent) 0.9% w./v. Sodium chloride 0.85% Vehicle Thiomersalate 0.015% to Distilled water to 100% 100% Sterilise the parafiin by heating at 160 C. for one hour. Disperse the crills and dissolve the sodium chloride and thiomersalate in the water, then sterilise at 15 lbs. pressure for 30 minutes.

Add the water to the oil when both phases are at a temperature of 60 C. Homogenise. The final product contains of this emulsion.

(d) VACCINE PREPARATION Culture and incubation.-The strain employed for vaccine production is maintained in a freeze dried state (lyophilised) and when required is reconstituted in liver serum broth medium, incubated overnight at 30 C. and tested for purity. This culture is then used to inoculate the amount of broth required as an inoculum for the vaccine.

An 18 hour liver serum broth culture is used, adding 10 mls. of inoculum to 1 litre of medium. This is incubated at 30 C. for 48 hours.

Killing of the bacteria.Add thiomersalate to the culture to give a final dilution of 0.02% or formalin 0.25%. Leave the culture in the incubator at 30 C. for 3 days and then test for any live bacteria by adding a quantity of the culture to a digest meat broth containing 0.1% cysteine hydrochloride to counteract the presence of any thiomersalate.

Adsorption.When the culture is proved to be completely dead, enough aluminium hydroxide gel is added to give a final concentration of 1 mgrn. per ml. The vaccine is shaken well and left overnight at +4 C. to adsorb.

Concentration.-The vaccine is now put through a centrifuge and the supernatant fluid removed. The deposit is scraped from the bowl of the centrifuge and thiomersalate saline is added. (The deposit from litres of culture is made up to 2 litres with thiomersalate saline. This constituted 10% of the final vaccine.) A mechanical stirrer is used to break up the deposit, which is then added to the emulsion and homogenised. The deposit, from 100 litres of culture being made up to 20 litres with theemulsion so concentrating five times.

Example 2.Preparation of Leptospirosis Vaccine Culture medium: Grams Bacto-peptone 0.8 Sodium chloride 1.4 Sodium bicarbonate 0.02 Potassium chloride 0.04 Calcium chloride 0.04 Potassium dihydrogen phosphate 0.24 Disodium hydrogen phosphate 0.69

Distilled Water to 1 litre.

The medium is boiled to dissolve the constituents, autoclaved to allow any phosphate to precipitate and filtered. The pH is adjusted to 7.2 and the medium is bottled and autoclaved.

56 for two hours is added to the medium.

CULTIVATION OF VACCINE Before inoculation 10% sheep serum deactivated at These flasks are incubated to maximum growth which is generally achieved in from seven to fourteen days.

Final vaccine cultures.T he leptospira are grown in 1 litre flasks containing 500 ml. of medium and 50' ml. of

serum. One secondary culture is added to each vaccine flask and the whole incubated for seven days. Tests for sterility are carried out on the fifth day. The cultures in all the flasks are pooled after seven days growth. An aliquot is removed and counted. Phenol to 0.5% is then added to kill the leptospira and the pooled vaccine is refrigerated at 4" C.

After seven days the vaccine is then mixed in 50:50 proportions with an oil-in-water emulsion prepared as follows:

OIL AND WATER EMULSION This is a sterile liquid paraffin emulsion syringeable through a No. 26 (British standard wire gauge) needle prepared from:

Light liquid paraflin (medicinal grade) 44.5% w./v. Grill 8 (emulsifying agent) 1.1% w./v. Grill 3 (stabilising agent) 0.9% w./v. Sodium chloride 0.85 Thiomersalate 0.015% g Distilled water to 100% o 7 Example 3 Parts Sorbit'an monostearate (stabilising agent) 1.5 Polyoxyethylene sorbitan monostearate (emulsifying agent) 0.5 Light liquid paralfin 45.0 Saline 53.0

Example 4 Polyoxyethylene monostearate (emulsifying agent) 0.5 Propylene glycol monostearate (stabilising agent) 2.0 Diglycol monostearate 0.75 Light liquid parafiin 43.75 Saline 53.0

In this example the light liquid parafiin may be replaced with an equal amount of arachis oil.

Example 5 Parts Sorbitan monostearate (stabilising agent) 2.0 Polyoxyethylene sorbitan monostearate (emulsifying agent) 1.0 Aluminium stearate/arachis oil gel (2.4%) 35.0 Saline n 62.0

We claim:

7 1. An injectible vaccine preparation in the form of an oil-in-water emulsion comprising a preformed emulsion base which is stable in the presence of body fluids upon intramuscular injection and is an oil-in-water emulsion containing an aqueous phase substantially isotonic to the blood and an oil phase consisting essentially of from 30% to 65% of mineral oil, from 0.5% to 1.5% of a watersoluble nonionic emulsifying agent and from 0.5% to 1.5% of an oil-soluble nonionic stabilising agent; and an antigenic material consisting of dead Erysipelothrix [rhu siopathol] rhusiopathae, the entire preparation containing more than 10% of mineral oil.

2. A preparation as claimed in claim 1 in which salt antigenic material is an adsorbate on a parenterally ac ceptable adsorbent.

3. A preparation as claimed in claim 1 in which sair emulsifying agent is a polyoxyalkylene derivative of hexitan ester and said stabilising agent is a fatty acid este: of a polyhydric alcohol.

4. A preparation as claimed in claim 1 in which sair emulsifying agent is polyoxyethylene sorbitan mono stearate and said stabilising agent is sorbitan mono stearate.

5. A preparation as claimed in claim 1 in which thr proportion of oil to water in the emulsion base is betweei 40 to 60% p 6. A preparation as claimed in claim 1 in which sair oil includes a gelling agent.

7. A preparation as claimed in claim 6 in which salt 1 gelling agent is a metal soap of a long chain fatty acid 8. An injectible vaccine preparation in the form of a1 oil-in-water emulsion comprising a preformed emulsiot base which is stable in the presence of body fluids MP0) intramuscular injection and is an oil-in-water emulsiol containing an aqueous phase substantially isotonic to tlu blood and an oil phase consisting essentially of from 30% to 65% of mineral oil, from 0.5% to 1.5% of water-soluble nonionic emulsifying agent and from 0.5% to 2.0% of on oil-soluble nonionic stabilising agent; am an antigenic material consisting of dead Erysipelothri; rhusiopathae.

9. An injectible vaccine preparation in the form of at oil-in-water emulsion comprising a preformed emulsiol base which isstuble in the presence of body fluids upo-t intramuscular injection and is an oil-in-water emulsiol containing an aqueous phase substantially isotonic to tht blood and an oil phase consisting essentially of iron 30% to 65% of mineral oil, from 0.5% to 1.5% of t water-soluble nonionic emulsifying agent and from 0.9% to 2.0% of an oil-soluble nonionic stabilising agent; am an antigenic material consisting of dead Erysipelothri: rhusiopathae.

References Cited by the Examiner The following references, cited by the Examiner, arc of record in the patented file of this patent or the origina patent.

UNITED STATES PATENTS 2,057,623 10/36 Beard 167-7 2,529,461 11/50 Schneiderwirth 1677E 2,675,343 4/54 Clymer 16782 2,756,176 7/56 Maurer et a1. 1677E FOREIGN PATENTS 425,406 3/35 Great Britain.

OTHER REFERENCES Freund: Annual Review of Microbiology, vol. 1 1947 (pp. 291-308, page 304 is particularly pertinent) Annual Reviews, Inc., Stanford, Calif.

Hoag et al.: Am. Jour. Vet. Res., 16 (60), pp. 381-385 (1955).

Trim et al.: Surface Activity and Permeability at Factors in Drug Action, pp. 111-142, in Symposia of the Society For Experimental Biology, No. III, Selective Toxicity and Antibiotics, 1949, Academic Press, Inc. New York, N.Y.

Woodhour et al.: Development and Application 01 New Parenteral Adjuvants v. Comparative Potencies ol Influenza Vaccines Emulsified in Various Oils, 1. Immunology, vol. 86, No. 6, pp. 681-689 (1961).

LEWIS GOTTS, Primary Examiner.