FR2506615A1 - Vaccine against brucellosis in humans - contg. phenol insoluble fraction of defatted Brucella abortus cells - Google Patents

Abstract

Vaccine against brucellosis comprises, in a suitable vehicle, the phenol-insoluble fraction (A) of Brucella abortus, esp. the strain B19. Pref. (A) is 1.7-2.3, esp. 2, mg (dry basis) per ml solvent and the vaccine is esp. formulated in unit doses of 0.5 ml. The pref. vehicle is a phenol-contg. physiological solvent. The vaccine is made from intact B.abortus cells by (1) removal of lipids; (2) drying; (3) extracting with phenol (esp. phenol satd. with water), at about 60 deg.C; (4) washing the filtered and purified prod. and (5) forming into a homogeneous, sterile suspension. The vaccine is suitable for admin. to humans, e.g. farm workers or veterinary surgeons, and is effective against B. melitensis and B. suis as well as B. abortus. It is nontoxic and induces only very mild hypersensitivity reactions.

Description

 The present invention relates to a new vaccine against human brucellosis as well as to a process for the preparation of this vaccine.

 We know that brucellosis, which is a major zoonosis, mainly affects cattle, sheep and goats, more rarely pigs. Among the Brucella species that cause human infections, Brucella melitensis is the most serious form of infection. Due to the numerous sources of contamination existing in the infected areas and the resistance of these bacteria, there are, particularly in the breeding areas, real endemic foci in which a high-risk population made up of professionals such as as farmers, veterinarians, butchers, dressers, laboratory technicians. Besides, apart from the medical problems posed by the disease itself, one must also take into account the fact that the man with brucellosis, cured of a clinical infection or inapparent then remains in a delayed type hyperactivity state. These subjects are allergic and can become hyperallergic so that, especially in professionals constantly exposed to contamination, there is in infected environment a real pathology of brucellian allergy.

 We have therefore already proposed a certain number of anti-mucosal vaccines. Some consisting of killed bacterial cells do not appear to have provided adequate immunity. Live vaccines have had better results but it is believed that they are not safe and, moreover, they create states of allergy with all the drawbacks that have been seen.

 In order to remedy these drawbacks, J. ROUX and A. SERRE proposed a vaccine prepared from a strain of Brucella melitensis in which the whole bacteria are first defatted by a mixture of equal parts of alcohol and ether and then treated with phenol, which finally makes it possible to obtain a fraction insoluble in phenol, called phenol-insoluble fraction or PI fraction, insoluble in water. This fraction is put into very homogeneous suspension and thus makes it possible to obtain an effective vaccine.

 However, this vaccine has some drawbacks. In particular, being propagated from a particularly pathogenic strain, its brbrication is not easy and relatively dangerous and therefore storytelling.

 The present invention therefore proposes to provide a new brucellosis vaccine which has the advantages of the above-mentioned vaccine1 and in particular very good vaccine immunity while being easy to manufacture and of reduced cost price.

 Yet another object of the invention is to provide such a vaccine which is effective not only against Brucella melitensis infections but also Brucella abortus and Brucella suis infections.

 The subject of the invention is a vaccine against brucellosis, characterized in that it comprises, in a suitable vehicle, the phenol-insoluble fraction of Brucella abortus.

 Among the Brucella abortus strains, the Brucella abortus B19 strain was preferred. This strain is well defined in the literature (see for example Bulletin of the Institut Pasteur, 1972, 70 p. 163-165).

Brucella abortus 319, which was mainly developed for bovine brucellosis vaccines, is available, among other things, from the United States administration Unités States Department cf Agriculture, Animal and Plant
Heaith Inspection Service Veterinary Service Post Office
Bos 70 Ames Iowa 500 lO USA

 The vaccine according to the invention, when it is ready for administration, comprises in a liquid vehicle a homogenized suspension of said phdnol-insoluble fraction of Brucella abortus. The vaccine advantageously comprises between 1.7 mg and 2.3 mg of dry extract per ml of solute and preferably, a suspension of approximately 2 mg of dry extract per ml of solute is adopted. The suitable physiological solution is preferably a phenolated physiological solution.

 Preferably, the vaccine is distributed in unit packages, ampoules or syringes, in a volume of 0.5 ml.

It has been found that the vaccine according to the invention provides protection not only against the homologous species
Brucella abortus but also against the heterologous species Brucella mélitensis and Brucella suis. This therefore provides a particularly effective broad spectrum vaccine.

 In addition, at the doses used, the vaccine is devoid of toxicity and causes only extremely discreet and acceptable hypersensitivity reactions.

 The invention also relates to a process for the preparation of this vaccine, characterized in that one carries out a delipidation of whole Brucella abortus bacteria, that the solid fraction is collected, that this fraction is dried beforehand with a extraction with phenol, preferably saturated with water and at a temperature of the order of 600C, then washing the final product, purified and filtered, preferably with alcohol and then with ether, and l A homogenized and sterilized suspension is prepared from this product.

 The invention will now be described in more detail with regard to a particular embodiment made by way of nonlimiting example.

Strain used
Brucella abortus St 19 culture ORIGINAL SEED (freeze-dried) serial 15202, produced in February 1972, by
Biological Reagents Section, Animal Realth Diagnostic,
APHIS MADL Ames Ioaza 50010 USA The strain is in the smooth phase.

 With this strain, a seed batch is first prepared by taking up the strain in a tube with soy trypticase medium, then after subculturing and new cultures, the bacterial suspension is collected and, after addition of a lyophilization substrate, it is distributed in ampoules. and we freeze it.

Culture of bacteria
An inoculum is then prepared containing the contents of one ampoule of the seed lot in a few milliliters of the medium and it is transplanted into a tube. After culturing in the tube, a 100 ml Erlenmeyer flask is still inoculated, still containing the soy trypticase medium, and culture is carried out for 24 hours. hours of 370C with stirring.

 From the culture of the bottle, a 40 liter pre-fermenter of useful volume is seeded by transfer.

The culture is continued for 24 hours at 370C with stirring, then the entire suspension of the prefermentor is introduced into a fermenter of 400 liters in volume.

The medium used, the composition of which is within the reach of those skilled in the art, comprises the following products in demineralized water
Peptone, trypticase, yeast extract, sodium chloride, sodium glutamate, sodium thiosulfate, magnesium sulfate, ferrous sulfate, manganese sulfate, vitamins B1, 35, PP, B12, anhydrous glucose, disodium phosphate and monopotassium phosphate.

 The culture in the fermenter is carried out for 24 to 48 hours, then when the concentration of germs is satisfactory, the contents of the tank are centrifuged at low temperature and the bacterial pellet is washed. It is then resuspended in physiological saline buffered so as to obtain a bacterial concentrate.

PrEDaration of the WhEnolfinsoluble fraction
The bacterial concentrate in physiological solution is taken up in an alcohol ether mixture (in equal volumes) at the rate of one volume of concentrate for one volume of mixture and kept under stirring at + 40C for 48 hours.

 The mixture is filtered and then taken up again in an alcohol ether mixture at the rate of 125 g of solid fraction per liter of mixture and left at room temperature. It is then filtered and dried.

 This phase constitutes the delipidation phase of bacteria which remain whole, that is to say non-lysed. They are thus in particular detoxified.

 The dry extract is taken up at the rate of 60 g per 300 ml of distilled water. The suspension obtained reheats to 600C is added an equal volume of saturated phenol at the same temperature and the mixture obtained is stirred for 15 minutes and then centrifuged. The centrifugation pellets are subjected to a second phenolic extraction then the centrifugation pellet reextracted and dialyzed 96 hours against distilled water.

 This extraction, which causes bacterial lysis, makes it possible to obtain a pellet composed of the desired phdnol-insoluble fraction.

 This purified and dialyzed fraction is then filtered then dried with alcohol then with ether and finally dehydrated under vacuum. It then appears as a dry powder that can be stored at + 40C.

 Each dry extract is a powder essentially consisting of peptidoglycan, rid. toxic lipids and containing a large part of the immunogenic elements of the bacteria.

Vaccine preparation
Each dose of 1 mg of extract corresponds to approximately total bacteria and represents the human dose to be used for use, suspended in 0.5 ml of phenol physiological solution.

 For this, a suspension is first prepared from the dry fraction which is taken up in buffered physiological solute. This suspension is homogenized and then sterilized by heating 1200C for 10 minutes. After cooling, a saturated solution of phenol in water is added at the rate of 100 g of phenol per 10 g of water and the whole / in ampoules or syringes whose extractable volume is not less than 0.5 ml. The dry matter concentration is 2 mg per milliliter of solute.

Pharmacological toxicity tests
With regard to primary toxicity, the following tests have been performed
- Abnormal toxicity. It has been researched on mice and guinea pigs and attempts to culture Brucella after sacrifice of the animals. The treated animals received 0.5 ml of vaccine by intraperitoneal injection. No symptoms appeared as much in the mice as in the treated guinea pigs, the control animals receiving the same volume of solute alone. The weights recorded showed a normal increase.

 After sacrifice of the animals, no organic or peritoneal lesion was recorded and no culture of Brucella could be made.

 - Local tolerance and dermonecrotic power.

Tests carried out on guinea pigs, a very sensitive species, did not reveal any point of necrosis. Only small, rapidly disappearing congestive lesions were observed.

 - Acute short-term toxicity in a single administration. The tests on mice and guinea pigs as well as on Macacus cynomolgus monkeys were carried out using on mice and guinea pigs a dose double the human dose and eight human doses for the monkey. All animals survived and none showed general or local disturbance or organic damage.

 - Long-term chronic toxicity in repeated long-term administration. The tests were carried out on the dog and on the monkey and all the animals survived, none having shown any significant general or local disorder or organic lesions.

Secondary toxicity was sought as follows
- Direct anaphylactic toxicity (hypersensitivity type 1): guinea pigs received 0.5 ml subcutaneously then fourteen days after the same dose by cardiac route. The guinea pigs survived after showing symptoms of anaphylaxis.

 - Direct allergic toxicity: The tests were carried out on guinea pigs. There are individual differences in reactivity but the average of the results is low and is not significantly different from that of the controls. The short-term reactogenic power (Ten weeks) after brucellosis infection on the guinea pig has shown a real but weak reactogenic power vis-à-vis a sensitizing brucellosis infection with virulent Brucella abortus. In the long term (one year of vaccination in the guinea pig) we see that the vaccine has a power of sensitization compared to itself real but discreet.

Protective Power: The vaccination-test activity test was razBté applied in mice on the one hand with homologous test, that is to say infection from the virulent Brucella abortus 544 strain immunologically corresponding to the strain original from which the vaccine is derived, and by heterologous test against the strains
Brucella melitensis (M15) and Brucella suis (1330) virulent, all strains pathogenic for humans. The mice were immunized subcutaneously with 0.5 ml of vaccine diluted 1/10 in physiological buffered phenolic water Twenty days after vaccination the vaccinated mice were inoculated by subcutaneous injection of a milliliter of bacterial suspension containing 106 CFU (colony forming units). The vaccine shows specific homologous and heterologous protective activity.

 Clinical expertise was conducted on a group of 262 people without delayed hypersensitivity according to the biological balance sheet (tintradermordaction to melitin and to the phenol-insoluble fraction and serological study). The vaccine dose was 0.5 ml containing one mg of dry extract.

Each subject received a first injection followed by a second injection fourteen days later.

 80% of the subjects presented a local reaction (erythema, induration or pain) t the frequency is about 20% if one considers the association of two types of reaction in the same person and lower than 10% if we consider the subjects having presented the three components of the local reaction.

 About 70% of the subjects presented at least one of the three general reactions (temperature, discomfort or chills) t less than 4% of the subjects presented all the general reactions.

 The epidemiological study in the vaccinated subjects which is in progress, shows that the vaccination is effective if one considers the statistics of morbidity.

Although the inversion has been described in connection with a particular embodiment, it is understood that it is in no way limited thereto and that various modifications can be made to it. Thus, vaccines according to the invention can be prepared using strains of Brucella abortus different from the strain 319, such as for example
Brucella abortus 544 and Brucella abortus 99.

Claims (10)

RESELLXCATZONS  1. Vaccine against brucellosis, characterized in that it comprises, in a suitable vehicle, the phenol-insoluble fraction of Brucella abortus.  2. Vaccine according to claim 1, characterized in that it comprises the phenol-insoluble fraction of Brucella abortus B 19.  3. Vaccine according to any one of claims 1 and 2, characterized in that it comprises between 1.7 mg and 2.3 mg of dry fraction per ml of solute and in particular 2 mg per ml.  4. Vaccine according to any one of claims 1 to 3, characterized in that it is distributed in unit packaging in a volume of about 0.5 ml.  5. Vaccine according to claim 4, characterized in that said dose comprises approximately 1 mg of dry fraction.  6. Vaccine according to any one of claims 1 to 5, characterized in that the vehicle comprises a phenol physiological solution.  7. Method for preparing the vaccine according to any one of claims 1 to 6, characterized in that one carries out a delipidation of whole Brucella abortus bacteria, that the solid fraction obtained is collected, that this fraction is subjected previously dried with phenol extraction, in particular with phenol saturated with water and at a temperature of the order of 6O0C, then washing the purified and filtered final product and preparing from this product a homogenized and sterilized suspension.  8. Method according to claim 7, characterized in that the delipidation is carried out in a mixture of ether alcohol.  9. Method according to any one of claims 7 and 8, characterized in that the washing is carried out with alcohol and then with ether.  10. Method according to any one of claims 8 and 9, characterized in that the vaccine is sterilized by heating at 1200C for about 10 minutes.