AU511331B2 - Vaccines containing both ribosome and saccharide microorganism cell fractions - Google Patents

Description

-4r

(Note: This is a comprehensive form-'and parts inappropriate to a particular appli-ation should be deleted. Thieq form Australian Patent Attorney. No legalisation is necessary).

g~o orms 1,2, 4 and 5' iXY00LARS. Lq

/7 AUSTRALIA iq Patenits Act 1952 INSTRUCTIONS CONVENTION OR NON-CON VENTI APP ICATION FORA PATENT OR PATENT OF IN f6

Insertfull nameis, of applicantfs) l/We(a)...'PIERRE FABRE S.A.

*0 00

f Ift~ert (fo *addoenj(Ls. applicant(,)

o

hereby apply for the grant of a patentp for an invention entitled *0 005 (d Vaj(."Xjg_'5S. BASED ON RIBONUCLEIC ACIDS RNA COMBINED WITH POLYSACCH.ARIDES EXTRACTED FROM MICROBLiS",

00 which is described in the accompanying_(c) ~~~complete specification. I /We request that the patent may be granted as a patent of addition to ic) the patent applied. for on application No. in the name of patent No.J Ciclaffiee (Cf

fdf fnsertnumbe of first basi appliato o

apliato wasmad iW re at the term of the patent of addition be the same as that of the patent for the IserC ae ri n ivenionor so much of the term of the patent for the main invention as is unexpired, bai aplcto Fhis application is a Convention application and is based on the following application or Aspplications for a patent or patents or similar protection made in the following country or ountries on the following date or dates:-

N o. (g 76..2 197.6 N No. in on 19 No. in on 19 W/Our address for service is oare of CLEMENT HACK CO., Patent Attorneys, 140 William Street, Melbourne, Victoria, 3000, Australia. Inseif dict Dated this 1ST day of AUGUST, 1977.

-LAUSTRILIAi- RF .Z (ki) Signature(s)Of applicaisi(3) Ifopanty, to be exeeutedin a imanner binding ontheCO 2 AUG17 Company according tn itsArticfe.%of CEEJ'1UA1~'K Associato i thelaws i tf,\ I of the country (If Seafof the Companv fif any.. f.

To: The Commissioner of Patents

CLEMENTF HACK CO,, Patent Attorneys, 140.William Street, Melbourne, Victoria, 3000, Australia

Form COMMON~WEALTH OF AUSTRALIA

J~q Liu ;PjlIu4~

Forms 7 and 8

COMMONWEALTH OF AUSTRALIA Patents Icd 1952VW DE CLARtATION IN SUPPORT OF A CONVENTION OR NON-CONVENTION WNSIMUCTIONfI APPLICATION FOR, A PATENT OJR PATENT OF A1)DrP109' 'T Xnacrt Na. If In support of the application No. (a) eSA~., 7~ uivalable. Thetcrt full rtumo(s) made by 1n3 PIERRE FABRE S.A. of nvllcant(a). (b PIERRE FABEE c A,

Inzeart 0o of for a paetXW1m~t0 for an invention entitled(c- invention. "PREPA 4 ATIOil OF VACCINES BASED ON RI.BONUCLEIC ACIDS RNA COMBINED WITH- POLYSACCHARIDES EXTRACTEDFROM MICROBES". Insert full,;aa TF1 Wd' POn.T AM'c~ of declnrnilL~q) Wbo Inuct PEASON or PEIZONS, NOT ol uede jla2~i nd r a nLcarPoratabody. 4.,cc bad noz. 75016 PARIS, Fria.nce

(f D declrentey I do solexnviy and sincerely declare as follows:- ft ic n Iz

1. I am authorized by the abovernenitioned applicant for the patent~v;Mtxb*

Dlete entirey If F2. :0 M to make this declaration on its behalf. The basic application o as defined 3)y Section 141 of the Act was/Mleiiade in the following country or couintries on the followving date by the following applicant (a) (1 Inv r cou ntry I which fir~t bic namely:- Innrat d or rt. in France on 6 lIw f71%' PIERRE FABRE S.A. J PIERRE FABRE S.A. in Wi) N

()Ieetc entirely If by (Il- CInveno nlocty (i) in M Insert culr n~

of a)lnctdateo 3. x x e j M x ppnvnt(a).I 3. DUS SOTRD DIINTERLAND,. Gerard NOMH4ER, Anne Marie appllcant NOT PINEL and Jacques DURAND *cn)*,naert full na() of actual Inventor(s) I apzjllcanilt(el NOT Ia nventor(s). ruIe F rancis Poullenc 31 12ASTRES,_EFrace; 22, ue-es apuins- CASTP.ES, France and .arn 1 CATRES France, respect ively k;are the actual inventor(s) of the invention and the facts upon which the applicant(g) is/'Nexentitled to make the application are as follows:- Rtecite manner In -as regards entitlement tinder Section 34 of the Act:-(o)T2Lsaid PIERRE FABP.E which apvllcnnt(n) derive(s) titlo frou' S.A.-Is the assL~nee of the said INVENTORS. actual1 Inveritorfa) If appllcant(c) NO0T lnventor(a).

licietc entirely If Convention priority NOT claied.

(q)3lcltta manner In 4. The basic application x) referred to in paragraph 2 of this Declaration Ias/xNiM der] 70(s) MOOI. from the first applicat~ion(,Kr made in a Convention country in1 respect of the invention the ,le rpzllerznL(h) If rznprlicant NOT subject of thie application. 1:141c nrypllcazt(s).

Signara)o Declared at P ai S, 14th day of July, 1977. ducaan() GHD.lo al or ,tznn imprerslon 1.3 bt, aprliL-d).

tet hiI-m To, The Conmmissioner of Patents, Commonwealih of Australia.

CtA~ET HAK O.,~Patent Attorneys, i,

(11) AU-BI-27,538/77

(12) PATENT SPECIFICATION ABRIDGEMENT (19) AU

(21) 27,538/77 511,331 (22) 2.8.77 (23) 2.8.77 (24) 6.8.76 (31) 7624124 (32) 6.8.76 (33) FR (43) 8.2.79 (44) 14.8.80 2 (51) A61K 39/02 A61K 31/73 A61K 35/74 C12D 7/00 C12D 13/04 C12D 13/06 (54' Vaccines containing both ribosome and saccharide microorganism cell fractions (71) Pierre Fabre S.A. (72) D'Hinterland, Normier, Pinel, A.M, and Durand, J. (74) HA (56) 27,862/71 (57) Claim 1. An acellular vaccine for prevention and treatment of bacterial diseases in humans and animals, which comprises a combination of: ribosomes isolated from the bacteria corresponding to the disease against which prevention and/or treatment is desired, and saccharides, consisting of polysaccharide and lipo- polusacch&ride, extracted from the membrane of one or more bacterial strain selected from the genera Klebsiella, Serratja and Corynebacterium. Claim 10. A method of making an acellular vaccine for prevention and treatment of bacterial disease in humans and animals comprising the steps of: isolating ribosomes of the strain of bacteria corresponding to the disease against which a prevention and/or treatment is desired by at least one centrifugation at 30,000 g of a suspension of crushed bacteria, coll.ection of supernatant layer and precipitation of ribosomes contained in this layer by addition of polyethyl glycol and ethyl alconhnr ,J2

30:5

(11) AU-BI-27,538/77 2-

(ii) recovering the ribosomes; (iii) isolating the membrane of at least one bacterial strain selected from the group consisting of the genera Klebsiella, Serratia,and Corynebacterium by crushing a culture of the strain, and centrifugating the membrane at 30,000 g after the removal of the cellular debris; (iv) extracting saccharides, consisting of poly- saccharides and iipopolusaccharides from the isolated membrane; and combining the ribosomes with the saccharides.

CLEMENT HACK CO., Patent Attorneys, 140 William Street, Melbourne, Victoria, 3000, Australia

Form 4, lf COMMOPIWEALTH OF AUSTRALIA PATENTS ACT 1952-W

If COMPLETE SPECIFICATION (OR IGINAL)

FOR OFFICE USE: Application Number Class Int. Class Lodged:

Complete Specification Lodged Accepted Published Priority Crc, ts x uWA

.ton49. Related Art

-4 S Crc for priiti

Nam ofApl0nts

PIERRE FABRE S.A.

Address of Applicant(s) 125, rue de la Faisanderie, ~0 75016 PARIS FRANCE 000

0* 000

o 09

0 ual Inventor(s) LUCIEN DUSSOURD D' H1NTERLAND GERARD NORMIER ANNE MARIE PINEL JACQUES DURAND ~0 0 09

Address for Service: CLEMENT HACK CO. .4*1WMXSDPeW 140 William Street, Melbourne, Victoria, 3000 Australia.

Complete Specification for the invention entitled: "PREPARATION OY VACCINES BASED ON RIBONUCLEIC ACIDS RNA COMBINED WITH POLISACCHARIDES EXTRACTED FROM MICROBES".

The following statement is a full description of this invention, including the best method of performing it knowvn to me/us: LtlwocF~wr MJ.TD.

This invention relates to acne

type described in French Patent No. 75 10252 entitled:

"Preparation of vaccines based on ribonucleic a. s PRNA

combined with polysaccharides extracted from microbes".

The invention relates more particularly to new

vaccines based on ribosome fractions combined with the

saccharide fractions of cell membranes, consisting of membrane

lipopolysaccharides and polysaccharides.

S.The old vaccines, consisting of attenuated dead

i bacteria or of microbial lysates, are inferior in their

vaccinating power to vaccines consisting of living bacteria.

Attempts have therefore been made to find new

vaccines with a more powerful antigenic activity than that of

the old vaccines.

The present invention is the outcome of research

showing that the ribosomes of bacterial cells carry the

vaccinating activity and that this activity nmanifests itself

only in the presence of an immunity adjuvant. In the present

case, this soluble adjuvant is extracted from the cell membranes

of certain genera of bacteria in the form of a saccharide C.

fraction.

The present invention thus provides an accellular

vaccine for prevention and treatment of bacterial diseases in

humans and animals, which comprises a combination of:

ribosomes isolated from the bacteria

2

No. 27538/77 Proposed: 23 JUN 1980 I0l1 corresponding to the disease against which prevention and/or treatment is desired, and saccharides, consisting of polysaccharide and lipopolysa--haride, extracted from the membrane of one or more bacterial strain selected from the genera KlebsielLa, Serratia and Corynebacterium. e.

The present invention relates in particular to a

vaccine in which the saccharide fraction is extracted from the

S bacterial membranes of at least one bacterial in selected

from the following species: Klebsiella pneilmoniae

Klebsiella rhinoscleromatis

Serratia corallina

Serratia indica

Serratia keilensis

Serratia kiliensi)s

Serratia marcescens

Serratia plymutnica

o** Corynebacterium avidum

Corynebacterium bovis

Corynebacterium enzymicum

Corynebacterium equi

Corynebacterium fascians

Corynebacterium flaccumfaciens

3

No. 27538/77 Proposed: 23 JUN 1980

"h'

Corynebacterium flavidum

Corynebacterium fusiforme

Corynebacterium granvlosum

Corynebacterium helvolum

Corynebacterium hypertrophicans

Corynebacterium insidiosum

Corynebacterium liquefaciens

Corvnebacterium parvum

Corynebacterium parvum infectiosum

"L Corynebacterium paurometabolum

Corynebacterium pyogenes

Corynebacterium tumescens

Corynebacterium xerosis

In one particular embodiment of the invention, the *oo

saccharides are extracted from the membranes of the bacteria

S" from which the ribosomes are extracted in addition to saccharides *0

extracted from one or more of the above-mentioned bacteria. The present invention also provides a method of making an acellular vaccine for prevention and treatment of *00*

bacterial disease in humans and animals comprising the steps of:

isolating ribosomes of the strain of bacteria

corresponding to the disease against which a prevention and/or

treatment is desired by at least one centrifugation at 30,000 g

of a suspension of crushed bacteria, collection of supernatant

layer and precipitation of ribosomes contained in this layer

4

/No. 27538/77 Proposed: 23 JU 1980

C L 1.

by addition of polyethyl glycol and ethyl alcohol;

(ii) recovering the ribosomes;

(iii) isolating the membrane of at least one

bacterial strain selected from the group consisting of the

genera Klebsiella, Serratia and Corynebacterium by crushing

a cult're of the strain, and centrifugating the membrane at

30,000 g after removal of the cellular debris; (iv) extracting sacc' irides, consisting of

Spolysaccharides and lipopolysaccharides from the isolated

-l membrane; and

combining the ribosomes with the saccharides.

In the vaccines according to the present invention,

the proportion by weight of ribosome fraction to saccharide

fraction is preferably between 0.5 and 1. It should be understood, however, that the unit

dose of vaccine may be varied according to the requirements o

4A

No. 27538/77 Proposed: 23 JUN 1980 of the treatment or the method of administration.

Below are given specific examples of doses which

may be used according to the invention but which should be

regarded as non-limiting: Example 1 A broncho-otolaryngeal vaccine having the following

composition:

Ribosome fractions

Ribosomes of Klebsiella pneumoniae 3.5 pg

Ribosomes of Diplococcus pneumoniae 3.0 pg

.*10 Ribosomes of group A1 2 Streptococcus pyogenes 3.0 pg 12

Ribosomes of Haemophilus influenzae 0.5 Pg 0* 10.0 pg

Soluble adjuvant

Membrane saccharide fraction of Klebsiella

pneumoniae 15.0 ug

1 dose 25.0 pg

of pure active constituent. *O A dental vaccine having the following composition:

Ribosome fractions

Ribosomes of Actinomyces viscosus 1.2 pg

Ribosomes of Rothia dentocariosus 1.2 pg

Ribosomes of Streptococcus mutants 0.8 pg

Ribosomes of Streptoccocus salivarius 1.0 pg

Ribosomes of Lactobacillus casei 1.2 pg

5.4 pg

mg

-F-

Soluble adjuvant

Membrane saccharide fraction of

Klebsiella pneumoniae 9.6 pg

1 dose 15.0 pg

of pure. active constituent.

A veterinary vaccine having the following composition:

Ribosome fractions

Ribosomes of Pasteurella haemolytica H 1 3.0 pg

Ribosomes of Pasteurella multocida A 1 1048 5.0 pg 0 Ribosomes of Pasteurella multouida A A13 3 0g o 9.0 pg a 9 Soluble adjuvant

Membrane saccharide fraction of S e

Klebsiella pneumoniae 13.5 pg

Membrane saccharide fraction of

Pasteurella haemolytica H 1 1.0 pg 0*

9* Membrane saccharide fraction of

*0 Pasteurella multocida A I 1048 1.0 pg

Membrane saccharide fraction of

Pasteurella multocida A A13 1.0 pg

16.5 pg

1 dose 25.5 pg

of pure active constituent.

In the vaccines according to the present inventi'on,

the saccharide fraction may be obtained, for example, by extracting an aqueous suspension of membranes with phenol, the saccharides being isolated from the aqueous phase after removal of the phenolic phase. This phenolic extraction is preferably carried out at a temperature of between 60 and

70 more preferably at 650C, with a 90% phenol solution.

The aqueous suspension of membranes used for

carrying out the extraction described above may be obtained,

as has been described in the aforementioned French Patent

No. 75 10252 from a bacterial culture which is crushed and

e10 then centrifuged to bring about selective sedimentation of

*the membranes which are then again suspended in an aqueous

solution. Sedimentation of the membranes may be carried out,

for example, by centrifuging at an acceleration of

substantially 30,000 g after removal of the cellular debris.

The ribosome fraction may be prepared by extracting

crushed bacterial cells with an acid saline buffer containing

hydrochloric acid, MgCl 2 NaC1 and sodium dodecyl sulphate.

An aqueous solution is thereby obtained which is

precipitated with polyethylene glycol and ethyl alcohol, the

ribosome fraction being contained in the precipitate. In

order to obtLin a more highly purified product, the precipitate

is preferably taken up in a buffer containing hydrochloric

acid, MgCl 2 NaCI and sodium dodecyl sulphate, and this

aqueous phase is reprecipitated with ethyl alcohol,

preferably 95% ethyl alcohol.

1- i

The precipitate obtained in this way may bc used

as the basis for the vaccines according to the invention by

mixing it with the above-mentioned saccharide fraction in

the proportions which have already been defined above and

which will become clearer in the course of the following

description.

It is to be understood that the vaccines according

to the present invention may be used according to their

constituents for the treatment or for the prevention of

"10 various diseases, as is well known in the field of vaccines.

.9 Thus, the bronchootolaryngeal vaccine defined earlier may

be used for the treatment or the prevention of the various

forms of chronic rhinotiacheobronchitis, of asthma 9.

complicated with bronchitis, of bronchiectasis, and of viral

pneumopathy with secondary infections as well as for the

treatment of the various forms of sinusitis and of rhinopharyngo-

laryngitis.

The dental vaccine can be used for preventing the

major common iinfections of the mouth.

20 The vaccines according to the present invention are

preferably presented in the form of aerosols but they may

also be provided in other medicinal forms. Some of the

vaccines according to the invention may be prepared for

parenteral administration, for example, by injection.

;t-~ur _P _y

I

It is not necessary at this point to provide a list

of components which may enter into the composition of the

excipient for the vaccine according to the invention since

they are well known in the art of preparing vaccines or

pharmaceutical preparations

The two components of the vaccine are generally

kept in the form of a solution but they may be freeze dried

when preparing the vaccine, and all that is then necessary

is to mix the various components of the vaccine in the *a required proportions. C C~ The analytical standards of the product obtained Q C *C may be determined, as follows: The concentration of ribosomes *0

of the ribosome fraction is calculated from the RNA concentration C as follows: S concentration of RNA x ±00 Ribosome concentration

C. O0C

0* The RNA concentration is in turn measured by spectro- CO

*CC photometric determination of the phosphomolybdic complex

compared with an RNA sample of Klebsiella of known concen-

tration measured by determination of the phosphorus. S The concentration of proteins in the ribosome

fraction is determined colorimetrically with biuret by

comparison with a range of standard samples of albumen.

The percentage of RNA in the sum of RNA proteins

varies approximately from 55 to

The concentration of saccharides in the adjuvant

is determined from the glucose concentration measured

colorimetrically with anthrone, as follows:

concentration of saccharides concentration of glucose x 100

A standard preparation of highly purified

saccharide was used to determine the glucose content of the

saccharides, which is 50% by this method of determination.

:The preferred method of carrying out the process of

the present invention to prepare vaccines according to the

*10 present invention is described below by way of example.

Preparation of an aqueous solution of ribosomes

e. The various strains from which it is desired to

S" extract the ribosomes are cultivated in known manner on

suitable growth media in fermentation apparatus.

'o*a* 15 After cultivaticn, the bacterial cells are

collected on a Westfalia or Sharples centrifuge and washed

by centrifuging with cold physiological serum at a

temperature of substantially

The bacterial concentrate obtained in this way may be stored frozen at -20°C if it is not required for

immediate use.

As in the case of the extraction of saccharides,

extraction of the ribosomes may be carriedout on a mixture

of various strains but it is preferable to treat each strain

separately so that the product obtained can be more easily controlled; in that case, the ribosomes and/or saccharides are mixed at the end of extraction to constitute the vaccine.

A sample is removed from the bacterial concentrate

Aed to determine the dry weight of the cells after stove

drying (the ratio of Ary cells to wet cells various between

and

Another sample is removed to test the purity of

the culture and to identify the strain.

10 The bacterial concentrate obtained as described

S: above is then disintegrated as follows:

If only a small quantity of wet cells is present

(less than 50 the cells are dispersed in a proportion

of 1 g (moist weight) in 4i ml of the following buffer:

tris-HCl M/100, pH 7.2, containing: MgCl ;6 H 0 0.01 M

NaCl 9 g/ a S

S. sodium dodecyl sulphate 5 g/l

When the suspension has been thoroughly homogenised

0 it is disintegrated by ultrasonic treatment for three periods

of 10 minutes each while it is kept in an ice bath. An ultra-

sonic apparatus Sonnimasse 500 T, for example may be used.

If larger quantities of cells are to be treated

(more than 50 the defrosted cells are dispersed in the

same b'iffer as used above and in the same proportions, and are thenr hoinoen b vigorouis stirring for 10 minutes in a i Iffji-sp-lod reactor.

Taey are then crushedl in the cold in a Manton-Gaulin m!1 (5cycles Tor each strain). The coarsest cellular debris

is el imiinatecl by passing uhe sample o-er a Sharples apparatus, aiid th'- suipern- tant layer is then centrifuged at 4I0 C for miiot'ts and 730,000 g (Beckman 521-T3 cenitrifuge with rot'or 0 x 25-0 nil JA Ili).-

Theo sodliment formed by cen-brifuging is discarded. *10 and theC SUIpernlatant layer rckntai ninjg the ri bosomes is stored 1b 0

9 Ext. ru tion of teribosomes

100~t g/I of polyethylene glycol. 'j000 (PEG 4i000 'a Merck are acdt-d to the aforementioned supernatant layer

and, after complete soliut:Lon, I volume of 9%ethyl alcohol is added at -2000.

The mixture is left to stand in -the cold for

a mintetF. a 'd the precipitate is then collected on a Sharples apparatuis ait 4iC (the sul-ernwttant layer is discarded).

The p. .ecip-itate is then redissolved in the same voltume of buffer as that used for crushing (tris-HC1, MgCl 2 NaCl SI)S 501) by agJ I ation in -the reactor at room temp- eraturo for 30 to 60 minutes (the purpose of waslng at room temperatu-ce is to remove non-ribosomie proteins).

0.8 Vo:Lume, of 95% ethyl al.cohol at -20 0 C is then mIIP added and the substance is left to precipitate at 100 C for minutes.

The ribosome precipitate is collected on the

Sharples apparatus and the supernatant layer is discarded

(without refrigeration in order not to precipitate the SDS).

The sediment of ribosomes is taken up in buffer (MgC1 2 0.02 M;

KC1 0.02 M; pH 7) at Ji°C in a proportion of 2 ml for 1 gram of

moist cells originally used (stirring on average for 2 to 4

hours). The sediment taken up in the buffer is then

centrifuged at 30,000 g for 45 minutes at O°C (removal of

residual SDS).

The sediment is discarded and the supernatant layer

is kept.

A sample (I to 2 ml) is removed to determine the

RNA concentration, the concentration of proteins and the

number of doses per ml.

The ribosomes are stored in sterile.plasma bottles

at 20 0 C until required for use.

SDS sodium dodecyl sulphate e

Preparation of an aqueous suspension of membranes

After culture, the cel'. are collected by centrifuging

in a Westfalia or Sharples apparatus and then washed by centri-

fuging with cold physiological serum. The bacterial concentrate

obtained may be stored at -200C.

;-I

The dry residue is determined on a sample by

drying it in a stove and the identity and purity of the

sample are controlled bacteriologically.

The defrosted cells are suspended in co d buffer

in a proportion of 1 g of cells to 4 ml of buffer. The buffer has the following composition:

tris-HCl 0.01 M, pH 7

NaCl 0 g/l

MgCl2,. 6 H20 0.01 M

The cells are crushed in a Manton-Gaul.in apparatus 9

(3 cycles at maximum pressure). Both the intact cells and the cellular debri.s are removed in a Sharples apparatus and the @0

supernatant layer is stored at 4°C. 9.

The membranes are then collected by centrifuging

for. 15 minutes at 30,000 g and 4'C (Beckman J 21 B, rotor

JA 14).

The supernatant layer is discarded (a portion may be kept back for the preparation of a ribosome RNA standard for

analysis). The sediment is kept to extract the soluble

20 adj'a-n'. from it. The various sediments are resuspended in distil.t-i' .ater used in an amount of 2.5 ml for each gram of

wet cells used as starting material (homogenisation in a Turrax

apparatus for 1 to 2 minutes).

Extraction of the saccharides

One volume of aqueous phenol (90o phenol) il.ich has

mg 1i ji

T T~ i p

previously been adjusted to a temperature of approximately

800C is added to the aqueous suspension obtained as

described above, and the mixture is heated to 65°C for

minut-es with vigorous stirring.

This is immediately followed by rapid cooling to

0°C.

In the case of small volun-es, the aqueous phase is

separated by centrifuging at 5000 revs/min at a temperature

4;,00 of 0 C for 5 minutes in stainless steel containers. The 61 69

upper, aqueous phase is then collected. 6 *6

For larger volumes, the aqueous phase is separated a00

by passing the sample over a liquid-liquid Westfalia or S

Sharples separator.

The phenolic phase is discarded.

The residual phenol in the aqueous phase is removed

by dialysis against running water for 24 to 48 hours, either

in a dialysis tube if the volumes are small, or on a plate o0 dialyser (Sartorius type) in the case of larger volumes.

The slight precipitates which form during dialysis

are then removed by centrifuging at 10,000 g and 0°C for

minutes.

The supernatant layer is kept in sterile plasma

bottles at -20 0 C.

A sample of 1 or 2 ml is removed for analytical control.

The vaccines according to the present invention

may be used both in human and in veterinary medicine.

1) lnrestigation of the immunogenic power of the veterinary

vaccine mentioned above

This investigation is carried out for the purpose

of checking the appearance and persistence of specific anti-

bodies in the serum of vaccinated mice.

Female mice of Swiss breed weighing 20 2 g are used

for the investigation. They are kept in cages of 10 in a

*10 room kept at a substantially constant temperature of 22± 1 0C

VI 0 and a relative humidity of o10to 60/o and given food in the form of pellets (UAR mouse diet) and drinking water. 0

UAR Usine d'Alimentation Rationelle, France. I

Immunisation of the animals

Each animal is injected subcutaneously with the

dose of vaccine made up to a volume of 0.2 ml.

The injections are carried out at the following O.

intervals:

5 inoculations in 15 days one injection a ee

every 3 days),

1: week's rest, 2 inoculations in 8 days (booster dose),

puncture through the tetro-orbital sinuses, at

least 10 days and not more than 15 days after

the last booster injection.

i

-C 91 I

Method of detecting the antibodies

The serum of each vaccinated mouse is studied by

Laurell's technique of immunoelectrodiffusion.

Each animal serum is studied against the Pasteurella

antigen from which the membrane saccharides were obtained:

Pasteurella haemolytica I- and Pasteurella multocida A3

Fach serum is then studied in the same manner against

all the Pasteurellae together from which the ribosomes of

the preparation were extracted.

The chosen antigen is incoriprated in a 1o agar gel buffered with veronal at pll 8.6. This gel is poured over

a glass plate measuring 1.5 x 90 x 110 mm.

7 pJ of the serum from each mouse are deposited in

o pits previously formed in the gel.

15 Deposition takes place at the cathode. Migration

takes place for 9 hours under a continuous current with a

potential difference of from 2 to 3 volts/cm.

oo5 At the end of this migration, the plates are washed

and dried and the immunoprecipitates are shown up by

colouration with qoomassie Brilliant Blue.

Results

The presence of speciflic immunoprecipitates between

the antibodies of the serum of treated mice and the detector

antigen is seen in the form of a "rocket", the height of

which represents the quantity of antibodies contained in the

serum of the vaccinated animals.

Highly significant results are seen on all the

plates with the serum of vaccinated mice compared with the

sera of control inice, thus confirming the high vaccinating

power of the compositions according to this invention.

2) Investigation of the immunogenic power of the dental

vaccine mentioned above

Female mice of Swiss breed weighing 2012 g, kept

in cages of 5 ind given food in the form of pellets (UAR

mouse diet) and drinking water in an animal room kept at

a constant temperature of 2211 C and a humidity of /i0 to

60%o are used in the same way as under above.

Immunisation of the animals Each animal is injected subcutaneously with the

dose of vaccine in a volume of 0.2 ml (it should be noted

"15 that tests carried out with 1:1, -1:1.5 and 1:2 ratios of

ribosome fraction to saccharide fraction showed that the

best results are obtained with the 1:1.5 composition S* mentioned above).

The injections were carried out at the following 00** intervals:

5 inoculations in 15 days (one injection every 3

days,

1 week's rest,

2 inoculations in 8 days (booster injection),

puncture through the retroorbitol sinuses at

least 10 days and not more than 15 days after

the last booster injection.

Metehod of detecting -the anrtibodies

TPhe sertim of each v~accinaed inouse is studied by

Ull Ire I Is jimilimoe I ec t roddif fusion too hn i que.

Thle antigen corresponsding 'to each type of bacteria

fCrom whrlich thle rib~osoes are obtained is embodied in a. 1%6

agar go] burireii with. -eronal at pliI 8.6; This gel is

potred over- a glass I e measuring x 90 x 110 111111

7 pI of the seruin from each mnouse are deposited

s a in the pits proviousIV hollowedd out of the gel. rr a a

a aa 10 Deposition taes place at the catthode. Migration a a

a u lasts hoitrS Linder a continuoiis current wsith a potential o a e a r a

9 difforonce of 2 to 3 vo"A.ts/cfm. r a o r A't tihe end oXf thle mir~ation, tie plates are w~ashed a a a a a and Oriecl e and thle :iimimnoproc-i p i. tat es are shoin Lip, by

coloration wdith Coomanssie Brilliant lue.

,o; Results1; a

a, Thle presence of specific immunorecipitate betw~een a

thle antibo~ies of thei sertim of the treated mrice and thee a a detector antigen -is seen in th.e formn of a "roc~et" the

height of whrlich represents the quantity of antibodies o a contained in the seruim of thee vaccinaed animals.

Highly~ significant results are observed on all the

pl~ates w~ith *thle Seruim of the vaccinated mice, comnpard with

sera from~ control ic, -thus dellionstrating the high

vaccinaing po~er bf -the raccines according to the invention.

Claims (1)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. An acellular vaccine for prevention and treatment

   of bacterial diseases in humans and animals, which comprises

   a combination of:

   ribosomes isolated from the bacteria

   corresponding to the disease against which

   prevention and/or treatment is desired, and

   0 saccharides, consisting of polysaccharide 000 and lipopolysaccharide, extracted from the @0 0

   membrane of one or more bacterial strain 00

   selected from the genera Klebsiella, Serratia

   *o 0 and Corynebacterium.

   2. An acellular vaccine according to claim 1, in f 0 which the saccharides are extracted from the membranes of at o

   least one bacterial strain selected from the group consisting

   of:

   Klebsiella pneumoniae

   Klebsiella rhinoscleromatis

   oo Serratia corallina

   Serratia indica

   Serratia keilensis

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   No. 27538/77 Proposed: 23 JUN 1980

   Serratia kiliensis

   Serratia rarcescens

   Serratia plyrnuthica

   Corynebacteriun aviclum

   Corynebacteriun bovis

   Corynebac t eriii ei'i7ynhicum~

   Corynebacteriuml egui.

   Corynebacterium fascians

   Corynebacteriuni flaccuinfaciens

   Corynebacterium flaviduni

   Corynebacterium fusiforme

   Corynebacterium granulosum

   Corynebacteriun heivolum Coryncbacteriun hypertrophicans

   Corynebacterium insidiosum Corynebacterium liguefaciens

   ~Corynebacterim parvum

   Corynebacteriui parvum infectiosui

   Corynebacterium paurometabolum

   Corynebac terium -pyogenes Corynebacterium turnescens

   Corynebacterium xerosis.

   3.A vaccine according to claim 1 or 2,ivh-ioh-14 contains, are in addition, the saccharidesfrae44-eftextracted from the membranes of the bacteria from which the ribosomes are extracted.

   4. A vaccine according to any one of claims 1 to 3,

   iniwhich the proportion by weight of ribosomes relative to

   the total amount of saccharides is within the range of from

   0.5:1 to 1:1.

   A vaccine according to any of claims 1 to 4, in

   which both the ribosomes and saccharides are extracted from the membranes of Klebsiella pneumoniae.

   S; 6. A vaccine according to claim 5, which is a broncho-

   otolaryngeal vaccine containing ribosomes extracted from any

   one or more of the following:

   Klebsiella pneumoniae

   Diplococcus pneumoniae

   Group A 1 2 Streptococcus pyogenes and

   Haemophilus influenzae.

   7. A vaccine according to claim 5, which is a dental

   vaccine in which the ribosomes are extracted from any one or

   more of the following:

   Actinomyces viscosus

   Rothia dentocariosus

   Streptococcus mutans

   ,Streptococcus salivarius and

   Lactobacillus casei.

   8. A vaccine according to claim 4 or 5, which is a

   22

   No. 27538/77 Proposed: 23 JUN 190 veterinary vaccine containing as ribosomes the ribosomes extracted from any one or more of the following:

   Pasteurella haemolytica H 1

   Pasteurella multocida A, 1048 and

   Pasteurella multocida A 3 A13.

   9. A vaccine according to claim 1 substantially as

   hereinbefore described with reference to any one of the

   foregoing Examples 1 to 3. S A method of making an acellular vaccine for

   prevention and treatment of bacterial disease in humans and

   animals comprising the steps of:

   isolating ribosomes ci the strain of bacteria

   corresponding to the disease against which a prevention and/or

   treatment is desired by at least one centrifugation at 30,000 g of a suspension of crushed bacteria, collection of supernatant o** layer and precipitation of rib somes contained in this layer by

   addition of polyethyl glycol and ethyl alcohol;

   (ii) recovering the ribosomes;

   (iii) isolating the membrane of at least one

   bacterial strain selected from the group consisting of the

   genera Klebsiella, Serratia and Corynebacterium by crushing a

   culture of the strain, and centrifugating the membrane at

   30,000 g after removal of the cellular debris;

   (iv) extracting saccharides, consisting of

   polysaccharides and lipopolysaccharides from the isolated

   23

   No. 27538/77 Proposed: 23 JUN 1980 membrane; and combining the ribosomes with the saccharides. 11. A method according to claim 10, in which the saccharides are extracted from an aqueous suspension of membranes by means of phenol, the saccharides being removed from the aqueous phase.

   12. A method according to claims 10 or 11, in which 0 extracting is carried out at a temperature within the range of 9e ee from 60 to 700C.

   13. A method according to any one of claims 10 to 12, S" in wh5 tnit- bosomes are isolated by extraction of crushed

   bacte. x l with a buffer containing HC1, MgC1 2 NaC1 and

   sodium Jodecyl sulphate, and precipitation of the resulting

   homogeneous aqueous phase with polyethylene glycol and ethyl

   alcohol, the ribosomes being contained in the precipitate. 9 9 14. A method according to claim 13, in which the buffer

   comprises 0.01 M hydrochloric acid, 0.01 M MgCl 2 9 g/l of NaCl

   and 5 g/l of sodium dodecyl sulphate. 0

   15. A methor according to claims 13 or 14, in which the S precipitate obtained is taken up in a buffer containing HC1,

   MgC1 2 NaCI and sodium dodecyl sulphate, and is then reprecipi-

   tated with ethyl alcohol.

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   No. 27538/77 Proposed: 23 JUN 190

   16. A method according to claim 10 substantially as

   herein')efore described with reference to the illustrative

   Example.

   DATED THYS 23RD DAY OF JUNE, 1980.

   PIERRE. FABRE S.A. By Its Patent Attorneys:

   CLEMENT HACK CO.

   Fellows Institute of Patent 2attorneys of Au, Ltralia. 9 9 9 S.

   9 9 9 9

   9~ 9 999

   09 9 0 S

   99..

   9999

   *V 9 J *9

   9

   o 99

   25

   No. 27538/77 Proposed: 23 JUN 190