DK141955B - Process for the preparation of a piglet colibacillosis vaccine. - Google Patents

Description

(11) PUBLICATION REFERENCE 1 ^ 1955 DENMARK (51) lnt Cf3 A 61 K 39/108 '(21) Application No 6524/75 (22) Filed on 5 Dec. 1975 (23) Live day Dec 5 1975 (44) Do not submit any submitted and submitted publication published on 28 Jul. 1980

DIRECTORATE OF

PATENT AND TRADEMARK (3 °) Priority baked from it

4. dec. 1972, 511998, US

<71> INQUIRY AND INDUSTRY THEREPEUTIftUES R.I.T., RUe du Tilleul, 15, H-152O Genval, BE.

(72) Inventor: Lucia Dobrescu, Avenue des Eperviers, 90, B-H50 Brussels, BE: Constant Huygelen, Vossekoten, 21, B-5055 Huldenberg, BE.

(74) Plenipotentiary in the proceedings:

Pinnaet Chas. Hude.

(64) Process for the preparation of a piglet colibacillosis vaccine.

The invention relates to a method of preparing a mammalian colibacillosis vaccine for intramuscular or subcutaneous administration to pregnant sows.

Neonatal colibacillosis in pigs is a predominant cause of mortality in piglets, and numerous attempts have been made to control this disease by active immunization of the animal mother over and above Escherichia coli (E.coll), either with killed or live vaccines or with to give the pigs E. ooli antiserum.

2 141955

Active immunization of the sows has been made by parenteral administration of E. coli vaccines (see, e.g., MRWilson and J.Svendsen, Amer.J.Vet.Res. 32, 1971 »891-898), but then E. The coli strain or strains used for immunization can only produce specific antibodies, while there may be several and different strains associated with a given outbreak, these antibacterial immunity-based prophylaxis attempts have provided inconsistent and generally unsatisfactory results. Although good protection was sometimes obtained, it was limited to the E. coli serotypes present in the vaccine.

Administration of antiserum (see, e.g., O.P.Miniats, Can.Vet.J. 11, 1970, 52-56) cannot constitute a practical prophylactic agent for mammalian colibacillosis. The protection afforded by the administration of antiserum is, in fact, of short duration and still requires administration to the piglets.

The process of the invention is characterized by isolating heat-labile (LT) E.coll. enterotoxin in the form of a cell-free and substantially lipopolysaccharide-free extract from cells of a heat-labile, enterotaxin-producing known method with an adjuvant to vaccine.

Surprisingly, it has been found that when administering the vaccine thus prepared to pregnant sows from three to six weeks before mating instead of administering whole cells of E. coli, the vaccinated sows not only develop antibodies in their colostrum and milk, but also thereby protect the piglets from the action of enterotoxins produced by E. coli serotypes, either homologous or heterologous to the E. coli strain from which the heat-labile (LT) enterotoxin was isolated.

In other words, the administration of the heat-labile (LT) E. coli enterotoxin to the mammal protects piglets, not only against colibacillosis due to the heat-labile (LT) E. coli enterotoxin, but also against colibacillosis due to the heat-stable (ST) enterotoxin.

141955 3

It is therefore an object of the invention to protect piglets against co-libacillosis, which consists of administering to the sows, together with an adjuvant, a heat-labile (LT) E. coli enterotoxin preparation isolated from a culture of an enteropathogenic E. co-li serotype. Such an enteropathogenic E. coli serotype is deposited with The American Type Culture Collection, Rockville, Maryland, U.S.A., where it was given accession number 21,972.

With the vaccine prepared by the method of the invention, an effective amount of the heat-labile (LT) E. coli enterotoxin preparation is administered with adjuvant to pregnant lungs by intramuscular or subcutaneous route. The dosage unit is at least 5 mg and preferably from 10 to 150 mg of lyophilized extract.

The adjuvant, whose role it is to ensure a high immune response, may be any product or composition known to those skilled in the art as having an adjuvant effect on vaccines. Examples of adequate adjuvants are aluminum compound gels such as aluminum hydroxide, aluminum phosphate and "ALHYEGROGEL" ® (the trade name for an aluminum hydroxide gel manufactured and sold by Superfos Export Co., Copenhagen, Denmark), and water-in-oil emulsions, such as 65 (a water-in-oil emulsion of peanut oil antigen, e -> - mutilated by the addition of mannid monooleate and stabilized by the addition of aluminum luminescent stearate), and the complete Freund's adjuvant (a light mineral oil water-in-emulsion , emulsified by mannid monooleate and supplemented with killed Mycobacterium tuberculo = sis).

Said heat-labile (LT) E. coli enterotoxin preparation can be isolated from any E. coli serotype. which can induce mammalian coli bacillosis and which necessarily contains the heat-labile (LT) E. coli enterotoxin. An example of such an E. coli serotype is the E. coli strain ATCC 21,972, but many other examples of E. coli strains containing the LT enterotoxin have been cited in the literature review, e.g. by W.J. Soybean in Annales de Médecine Vétérinaire 116 * 377-446, 1972, which lists E. coli strains G 7, G 205, G 4/66, G 491, E 68 type I, G 1253 and Abbots-townJ by E.M. Kohier in Ann.

NEW. Acad. Sci. 1976: 212-19, 1971, which indicates E. coli strains Arnold, Emery, 263, 407 and P 307; by H.W. Smith and C.L. Gyles in J.

With. Microbiol. 3: 387-401, 1970 referring to 25 E. coli strains, 4 HI 955 showing various serotypes of L, Dobrescu & al. i Zentralbl. Vet. With. (B) 20: 222-29, 1973, which lists 4 E. coli strains freshly obtained without restriction from the Institut National de Recherches Vété-rinaires, Brussels (Belgium), Faculty of Veterinary Medicine, University and Ghent (Belgium), and Veterinaruntersuchungs-und. Tiergesund Heitsamt, Dresden (East Germany).

The method of isolating the heat-labile (LT) E. coli entotoxin is well known. It is described by Gyles and Bamum (J. Infect. Dis. 120, 1969, 419-426), and variations of this method are described e.g. by HWSmith and CLGyles (J.Hed.Microbiol. 3, 1970, 387-401), HWSmith and S.Halls (J.Path.Bact. 93, 1967, 531-543) and MRWilson, C.Gyles and J.Svendsen (Can.J.Comp. Med. 35, 1971, 294-297).

Each of these methods, as well as any other equivalent known to those skilled in the art, may be used for the purpose of preparing the heat-labile (LT) E. coli enterotoxin isolate used in the process of the invention.

This invention does not require obtaining pure heat-labile (LT) E. coli enterotoxin that could possibly be obtained, e.g. either by electrophoresis or density gradient ultracentrifugation or adsorptiojaVelution of hydrophilic, cross-linked dextrans such as "Sephadex" S (a product manufactured and sold by Pharmacia Pine Chemicals AB, Uppsala 1, Sweden).

The invention essentially requires only that the enterotoxin is liberated and separated from the cells of E. coli and that it is essentially free of lipopolysaccharides.

The following examples illustrate the invention. However, they should not be construed as limiting its scope.

1/141955 5

Example 1

Preparation medium (solid)

Water (10 liters) is added to 130 g, raftCTO-LEKXPTOSE, - sup.pe and 150 g of "BACDO-AGAR" (BACT0-TRYPT0SE and BACTO-AGAR are trade names of products manufactured and sold by DIFCO Laboratories, Detroit 1, Michigan, USA). The mixture is heated to 121 ° C with stirring for 60 minutes. 20 g of "BfiCTO-nEXTROSB" (trade name of a product manufactured and sold by DIFCO Laboratories) is dissolved in 20 ml of distilled water and the solution is passed through a 0.45 micron Millipore Sterilization Filter (Millipore Corporation, Bedford, Massachusetts, USA ). Both preparations are brought together and 110 ml aliquots are partitioned into 500 cm 2 Roux flasks.

Seed Preparation E. coli strain ATCC 21.972 is hydrated with sterile saline and incubated for 18 hours at 37 ° C in Petri dishes, each containing 20 ml of "TKXETOSE-A3KR", w as a solid, prepared by mixing 26 g. miK ¥ PTaSE "Soup, 30 g * AG8R DIKD" (TRYPTOSE Soup and AGAR DIFCO are products manufactured and sold by DIFCO Laboratories) up to 1 liter with water and the mixture is heated to 115 ° C for 45 minutes.

A liquid culture medium (designated PP3) is then prepared as follows: Proteosepteptone # 3 (a product manufactured and sold by DIFCO Laboratories) (30 g), yeast extract (4 g) and dextrose (5 g) is dissolved in 1 liter. water at 60 ° C. After cooling NaCl (5 g), NaHPOPO (5.05 g) and KH₂PO0 (1.2 g) are added. The medium, whose pH is 6.9-7.0, is filtered on Seitz EKS filter and distributed in 100 ml culture flasks.

These culture flasks are seeded with the smooth colonies that have emerged on the Petri dishes, using one colony per ml. 20 ml of liquid medium PP 1 and incubated for 6 hours at 37 ° C with shaking on cradle shelves (22-24 w / min).

E. coli preparation

4 ml of aliquot portions of the resulting E. coli culture are seeded in liquid medium PP 2 (i.e., 4 x 1Cr bacteria) in each 500 cm Roux flask containing the production medium which is then incubated for 24 hours at 37 ° C. shaking on cradle shelves (22-24 cradles per minute). Each cell culture is harvested in 25 ml of distilled water and the yields of 15 Roux flasks (one series) are combined in one liter bottle. One sample (1 ml) is taken from each series for purity test.

For this purpose, 0.5 ml of "TRXPTOSE-AfiftR" soup is poured into Petri dishes and incubated for 7 days at 34 ° C, and the other 0.5 ml fraction is poured into Petri dishes containing 20 ml of Sabouraud dextrose agar, prepared by dissolving 65 g of Sabouraud dextrose agar DIFCO (a product manufactured and sold by DIFCO Laboratories) in 1 liter of hot, distilled and further distilled water, and the culture is incubated for 7 days at 22 ° C.

The yields are supplemented with a sterile 1 µl neomycin sulfate aqueous solution (1.2 ml neomycin sulfate solution per ml). 100 ml of the harvested product) and the cells are ruptured by sonication of the medium for 30 minutes in a Branson Europa sonicator model J 22 (Branson Europa N.V., Soest, The Netherlands), holding the medium in a molten ice bath.

After rupturing the cells, the suspension is centrifuged for 2 hours at 2,550 rpm. at the temperature of 5 ° C to remove the cell debris.

The above liquid is passed successively through a clearing filter and through a 0.45 micron Millipore sterilization filter (Millipore is a trade name of Millipore Corporation, Bedford, Massachusetts, U.S.A.).

Pre-sterilized ammonium sulphate with ethylene oxide is added to the filtrate to obtain 50 ° / one saturation (380 g of ammonium nitrate per liter of filtrate) and allowed to stand for 3 hours at room temperature . The resulting precipitate is centrifuged for 2 hours at 2,550 rpm. at the temperature of 5 ° C.

The sediment is poured into a cellophane bag and dialyzed with running water until the concentration of ammonium sulfate is lowered to between 0.1 and 0.01 °, testing the ammonium sulfate contents with Nessler's reagent.

The enterotoxin preparation is sterilized by passing through a 0.45 micron Millipore sterilization filter (Millipore is the trade name of Millipore Corporation) and freeze-dried.

Example 2

One 1 g aliquot of freeze-dried enterotoxin preparation prepared as the final product of Example 1 is rehydrated by the addition of 20 ml of a sterile phosphate buffer saline solution (pH 7.4) containing NaCl (8 g), KCl (2 g) , Na 2 HPO 2 (11.5 g), KH 2 PO 4 (2 g), CaCl2.2H2 O (1.325 g), MgCl2.6H2 O (lg) and distilled water (10 liters).

The resulting vaccine is thoroughly mixed under sterile conditions with 20 ml of sterile complete Freund's adjuvant (ie, a water-in-oil emulsion of light mineral oil emulsified with mannid monooleate and added killed Mycobacterium tuberculosis), and the resulting adjuvanted vaccine is distributed. 4 ml ampoules, which are either sealed or closed tightly to give an (LT) enterotoxin amount equal to 100 mg of freeze-dried preparation per day. ampoule.

The contents of each ampoule correspond to one vaccine dose unit. The vaccine is administered to pregnant sows by intramuscular or subcutaneous route from 3 to 6 tigers before farrowing.

Alternatively, divide the adjuvanted vaccine into larger ampoules to obtain whole numbers for the amount of dosage units so that similar multidose vaccine preparations can be obtained.

Example 3

The technique is as described in Example 1 and comprises the incubation of E. coli ATCC 21.972 for 6 hours at 37 ° C in PP 1 liquid medium.

The preparation of E. coli is then carried out in a liquid production medium as follows.

6 mm aliquot portions of the E. coli thus obtained (i.e., ca.

6 x 10 5 bacteria) are inoculated into production flasks containing 300 ml of PP · liquid medium and the cultures are incubated for one day while shaking on cradle shelves (22-24 wicks per minute).

Combine the yield of 5 (1 series) flasks and centrifuge each series for 2 hours at 2,550 rpm, suspending the sediment in 150 ml of distilled water and examining a sample of the purity suspension as set forth in Example 1. .

The cell suspension is sonicated for 30 minutes in a Branson Europa applicator, model J 22, keeping the medium in a melting ice bath. After rupturing the cells, the suspension is centrifuged for 2 hours at 2,550 rpm. at 5 ° C to remove the cell debris. The above liquid is passed through a 0.45 micron Millipore sterilization filter (Millipore is a trade name of Millipore Corporation) to give 100 ml of filtrate whose pH is adjusted to 6.4 with normal hydrochloric acid. Thiomersal is added at a final concentration of 0, Q2? 6.

Example 4

Mix 40 ml of a 2% aqueous solution of "ALHYDROGEL" ® (a product manufactured and sold by Superfos Export Company) thoroughly with 80 ml of the filtrate obtained as the final product of Example 3 and stop the adsorption of the enterotoxin on ^ by precipitation experiments with trichloroacetic acid.

20 ml of the above liquid is poured off and the remaining mixture is partitioned into 10 (10 ml) vials which are sealed.

The content of each ampoule (9 ml) corresponds to a vaccine dose unit of 100 mg (dry weight basis) of heat-labile (LT) E. coli enterotoxin.

141955 9

The vaccine is administered on pregnant lungs by intramuscular or sub-cutaneous route from 3 to 6 weeks before delivery.

Alternatively, the adjuvanted vaccine is distributed in larger ampoules to obtain whole numbers for the dose unit amount so that corresponding multi-doses of the vaccine preparations can be determined.

Example 3

The technique is as in Example 3, but the mixture is distributed in 100 vials which are sealed.

The content of each ampoule (0.9 ml) corresponds to one vaccine dose unit of 10 mg (dry weight basis) of heat-labile (LT) E. ooli enterotoxin extract.

The vaccine is administered on pregnant lungs by intramuscular or sub-cutaneous route from 3 to 6 weeks before delivery.

Example 6

Prevention of mammalian colibacillosis has been demonstrated by subcutaneous administration of heat-labile (LT) E.coll entero = toxin preparations prepared by the method of the invention in the following experiments in, II and III.

In each of these experiments, the sows were vaccinated from 3 to 6 weeks before farrowing and a sow was used as a control. Twenty-four hours after birth, the piglets were provoked, i.e. is exposed to infection with 400 mg Ag weight of LT E. coli entemboxin prepared according to Example 1, using a stomach tube for administration.

For 3 pig litters, as indicated in the following tables, some piglets were also provoked with heat stavit (ST) E. coli entero = toxin prepared by strain ATCC 21,972 using 660 mg of ST enterotoxin.

The clinical development of piglets was followed regularly, observing the appearance of typical symptoms of colibacilli.

In the following tables - Vaccine A represents the vaccine of Example 2 at a dosage unit of 100 mg (dry weight basis), - the vaccine B vaccine of Example 4 at a dosage unit of 100 mg (dry weight basis), and - Vaccine C represents the vaccine of Example 5 at a unit dose of 10 mg (dry weight basis).

Experiment # 1 __So___ Pig Pig__:] Diarrhea. Weight 24

Vaccine- Provoke- Number of hours Lasting- Dead- hours after-

No. right with No. dish with ml. provocation is similar to procrastination and other- (timed vocation __fall__mer) __ 2-10 LT - - before change.

2-11 LT - + 3.5% 2-12 LT - -, + 9? 5% 2 A 2-13 LT - - no change.

2-14 ST - - + 3.6% 2- 15 ST - - no change

2-16 ST - - - + 2.8% 3- 17 LT - + 10.6% 3-18 LT - + 11.8% 3-19 LT - - - + 6.3% 3 A 3-20 LT - + 5.8% 3-21 ST - + 10% 3-22 ST - + 11.9% 3-23 ST - - - + 10.4% _ 3-24 ST - + 7.5% 10- 01 LT 6 24 10% 10-02 LT 6-12 24 - - 10.9% 10-03 LT 6 12 - 5.3% 10-04 LT 6 96 + 14.7% 10 - 10-05 LT 6- 12 10 7.6% trol) 10-06 ST - - no change.

10-07 ST 1 3 no change.

10-08 ST - - no change.

10-09 ST 6 12 - no changes.

11 141956

Experiment # 2 _________So i___r __ Piglet ____ ^ ......

1 1 'Diarrhoea "P It / oct 24

Vaccine- Provoke- Number of hours' Durable- Dead- hours after-

No. right with No. dish with ml. provocation is similar to procrastination and other (tenure vocation ___________fall more) __ 47-12 LT - - no change.

47-13 LT - + 9.2% 47 A 47-14 LT - - - no change.

47-15 LT - - + 6.4% 47 - 16 LT - - - + 6.6% 48 - 08 LT - - + 5.2% 48-09 LT - - + 6.7% hQ B 48-10 LT - + 3.7% 48-11 LT - + 10.5% 50-01 LT 5 21 + - 19.8% 50-02 LT - - no change.

50-03 LT - - - + 2.6% 50 - 50-04 LT 6 14-21 + - 22.1% (con- 50-05 LT 14 48 + - 18.8% troi) 50_06 lt 21 10 _ - 3.2% 50-07 LT 6 21 + - 28.5% 12 141955

Experiment # 3

So___ Pattegris___ 'Diarrhea

Vaccine- Provo- Number of Hours Durable- Dead- Weight 24

No. right with No. candle ml. provocation equals hours with ring and other (times pro- - - - + 2% 13-122 LT - no change.

l3 B 13-123 LT - - - + 7.1% 13-124 LT 4 2 - + 8.1% 13-125 LT - - - + 8% 13-126 LT - + 5.8% 13-127 LT 8 40 + - 21% 13-128 LT - + 8.4% 21-110 LT 3 48 + - 15% 21-111 LT 3 6-11 - 6.9% 21-112 LT 4 48 + - 14, 3% 21 c 21-114 LT - - - - 5.9% (*) 21-115 LT 6 6 - - 6.3% 21-116 LT - - - - 3.6% 21-117 LT - - - - 2% 21-118 'LT 6 5 - 12.5% 15-129 LT - - - + 7.4% 15-130 LT - - - + 4.9% 15 C 15-131 LT - - - + 13 , 2% 15-132 LT - - - + 16% 15-133 LT - - - no change.

20-101 LT 5 6 - 3.6% 20-102 LT 6 6 - 16.5% 20-103 LT 25 4 - 4.5% (**) 20-104 LT 19 6 - 5, 3% 20 - 20-105 LT 6 6 - 4.7% (con- 20-106 LT 3 '4-9 - no change.

tro1- 20-107 LT - - 3.2% 20-108 LT 6 6 - - 5.7% 20-109 LT 6 6 4.2% 141955 13 (*) Mastitis occurred 48 hours after farrowing.

(**) Weight loss 4 hours after the onset of diarrhea.

The results of the experiments show that 6 of the 7 pig litters from vaccinated sows are protected against oral provocation with E. coli entero = toxins. The protection provided was 90% for one litter and 100% for the other 5 litters. No litter of the control lakes showed resistance to the same provocation with enterotoxins, while the mortality and morbidity of the control mammals ranged from 71.4 to 88.8%.

In 5 of 7 litters from vaccinated sows, all the piglets were completely protected. In one litter, 8 out of 10 suckling pigs were completely protected. For the piglets provoked with LT-entero = toxin originating from control sows, the numbers were as follows: In one litter, 5 of 5 / day, r showed typical symptoms, in another litter showed 5 of 7 and in a third litter 8 of 9 typical symptoms.

Example 7

A 12 g aliquot of freeze-dried enterotoxin preparation, prepared according to the procedure described in Example 1, is suspended in 1 liter of pyrogen-free distilled water and the suspension is mixed with a solution of 20 g of "ALKSDEDGEL" (a product prepared and sold by Superfos Export Company) in 1 liter of pyro = non-distilled distilled water. The pH is adjusted to 6 with 1N hydrochloric acid and the mixture is allowed to stand in the dark at room temperature for 40 hours with intermittent stirring to adsorb the enterotoxin "AIJKDBOGEEEN" ®. The final pH is 6.3 ·

After centrifugation, the supernatant is poured off and the sediment rehydrated by adding 2.2 liters of a 50/50 mixture of physiological serum and buffer (22 g Na 2 HPO 2 (M / 10) and 78 g KHgPO 2 (M / 10) with 10). mg of thiomersal to 100 ml, pH 6.3).

The mixture is stirred at 4 ° C for 2 hours and divided into two 1100 ml batches, designated A and B. The batch A (containing 5.4 mg enterotoxin preparation and 9 mg "MjKXDEQGEL" ® per ml, respectively) is divided. in 5 ml aliquot portions (thus containing 27 mg of enterotoxin preparation) which are dispensed into sealed vials. Charge B is diluted by adding 303 ml of a 50/50 mixture of physiological serum and buffer (22 g Na 2 HPO 2 (M / 10) and 78 g KH 2 PO 2 (M / 10) with 10 mg thiomersal to 100 ml). pH 6.3) "to which 2.7 g" of "JCffiROGEL" has been added. Charge B (containing 4.3 mg of enterotoxin preparation and 9 mg of "µµεεοοε "ι" ® per ml) is divided into 2.5 ml aliquots parts (thus containing 10.75 mg of enterotoxin preparation) which are distributed in vials which are then sealed.

The vaccine preparation of Chargers A and B has been tested as follows on sows in breeding centers infected with colibacillosis diarrhea.

The sows were inoculated subcutaneously between 3 and 6 weeks prior to the single dose vaccine operation: 59 sows received a single dose of Charge A vaccine and 28 sows received a single dose of Charge B vaccine. 50 sows were used as control, receiving the same volumes of the same "ALHEDKOGEL" buffer buffer mixture without enterotoxin extract.

All piglets were observed for diarrhea.

From the second day of clinical symptom on disease, the piglets were treated with daily administration of chloramphenicol, ampicillin or sulfadoxine / trimethoprim.

The results are summarized in the following Table I, indicating stool consistency, duration of diarrhea, number of antibiotic administrations, and mortality from dehydration.

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