IL28696A - Attenuated live rubella virus vaccine and method of production - Google Patents

Description

28696/2 ins1* Attenuated live rubella virus vaccine and method of production RECHERCHE ET INDUSTRIE THERAPEUTIQUES "R.I.T." C. 27250 This invention relates to an attenuated live rubella virus vaccine capable of inducing active immunity against rubella and to a process for producing such vaccine.

The term 'attenuated live virus' employed in this specification refers to a rubella virus (RV) strain, the virulence of which has been attenuated by at least 15 serial passages on primary rabbit kidney tissue cultures.

According to the procedure described hereafter in this specification, there is obtained a modified virus and thereby a vaccine presenting high antigenicity while producing no appreciable undesirable reaction when inoculated to children.

The presence of undesirable reactions and, among them, more particularly the possible dissemination of the virus by the vaccinated people is one of the major problems in the develop-ment of a rubella live type vaccine.

It is known that besides complications such as encephalitis and thrombocytopenic purpura which are severe but quite rare and also arthritis which is not uncommon but generally self-limited and of short duration, the most dramatic complica-tion of this disease is of teratogenic nature. Rubella indeed is a serious problem for women in the first trimester of pregnancy because of its incidence on congenital malformations in the foetus, stillbirths or abortions.

In this prospect, it is essential that a child who receives a rubella live-type vaccine does not constitute a potential danger to any pregnant woman with whom he might come into contact .

Up to now, the only possible prevention means against such foetal rubella complications were either administration of gamma globulin in large doses to exposed pregnant women or exposure of young girls to rubella before they reach the child-bearing age .

The administration of gamma globulin is far from being considered by each specialist as an effective treatment and, for obvious reasons, the exposure to the disease at an earlier age is not an acceptable answer to the problem.

It has now surprisingly been found that serial passages of rubella virus in primary rabbit kidney (RK) tissue cultures not only involves attenuation of the virulence but also provides a method for preparing an attenuated live rubella virus vaccine presenting no appreciable undesirable reactions.

By the term 'no appreciable undesirable reactions' as cited above, it is understood not nnly that the inoculation of the vaccine according to this invention stimulates immunity without inducing the usual pathological symptoms of regular rubella but also that in the clinical trials conducted with a vaccine according to this invention, no serological or virolo-gical evidence of infection has been detected in susceptible intimate contacts.

Thus, it is an object of the present invention to provide a method for attenuating the virulence of rubella virus without loss of antigenicity, said method consisting in passagin serially a rubella virus strain at least 15 times on primary rabbit kidney tissue cultures.

It is a further object of the present invention to provide (1) attenuated live rubella virus obtained by said attenuation method and (2) a vaccine active against rubella, comprising as active ingredient attenuated rubella virus obtained by said attenuation method.

In other words, the present invention affords a method for preparing a vaccine active against rubella, said method consisting in passaging serially a rubella virus strain at least 15 times on primary rabbit kidney tissue cultures and using the harvested attenuated rubella- virus as active ingredient for a rubella vaccine, according to any technique known to the art for vaccine formulation.

According to this invention, rubella virus undergoes a sufficient number of passages in primary rabbit kidney tissue cultures until attenuation is obtained. It has been found that attenuation requires at least 15 passages and preferably from about 20 to about 60 passages, said passages being conducted at a temperature not exceeding 36°C.

According to a preferred embodiment of this invention, the duration of each passage is comprised between 3 and 6 days.

A vaccine is then prepared from an appropriate passage in primary R cells using any technique known to the art for such preparation.

The rubella virus employed for carrying out this invention is isolated from a typical clinical case, using for instance throat swabs or urine or gargle samples. The samples are either frozen immediately and maintained at - 60°C until used or immediately inoculated into an adequate tissue culture system, e.g. primary African green monkey kidney (GM ) monolayers or any other tissue culture system known to the art for such isolation.

The presence of rubella virus is checked by challenging cultures for instance with an enterovirus such as Echovirus 11 or Coxsackievirus A 9 on the 9th or 10th day after inoculation.

Using specific antiserum prepared in rabbits against a known RV strain, it is possible to identify rubella virus by specific neutralization test in susceptible cells such as GM cells. The absence of adventitious simian viruses is checked in G-MK cells after neutralization by specific RV antiserum prepared in an other cell system.

The primary rabbit kidney cell cultures for the serial passages are preferably prepared from kidneys of animals not older that 3 weeks old. A preferred growth medium for primary RK cultures is Hanks' balanced salt medium supplemented with inactivated calf serum, lactalbumin hydrolysate and tryptose phosphate broth but other media known by those skilled in the art can also be employed. The incubation temperature is not above 36°C. The serial passages are carried out by inoculating RK monolayers with aliquots of the supernatant fluid from the previous passage and preferably harvesting the virus between the 3rd and the 6th day after inoculation. Titration of the harvested virus can be carried out using the interference method in GMK culture tubes, using for instance Echovirus 11 or Coxsackievirus A 9 as indicated above for the isolation step.

At a certain passage level, not before the 15th and preferably between about the 20th and about the 60th passage, the supernatant fluid of the inoculated cultures is discarded by the 6th day after inoculation and the monolayers are washed with a buffered saline solution, for instance Eagle's solution or Hanks' solution and further incubated with a maintenance medium, e.g. Hanks' medium supplemented with casein hydrolysate. After a further incubation for 3 days, the supernatant fluid is harvested and clarified by filtration or by centrifugation.

Alternatively, harvesting of the virus is carried out after freezing, thawing and shaking of the culture in the maintenance medium and subsequent centrifugation or filtration.

The harvested RV supplemented or not with a stabilizin additive may be stored either in the frozen state, for instance at about - 60°C or in the freeze - dried state. The obtained vaccine is administered by subcutaneous or intramuscular route, if necessary after reconstitution .

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

Example 1 A 3 weeks-old rabbit from a breeding colony is examined for absence of pathological lesions and sacrificed. Both kidneys are aseptically removed and cut into little pieces which are brought into contact with a buffered saline solution of trypsin (2.5 g/l.) and the mixture is continuously stirred for 10 minutes at a temperature of 37°C. The liquid is then poured off and replaced by an equal volume of fresh trypsin solution. Trypsinization is then continued while stirring until exhaustion of the tissue , the cells suspended in the liquid being removed from time to time and then centrifuged .

The cell sediment obtained is resuspended in Hanks1 solution and again centrifuged. This last step is repeated twice and the final cell sediment is suspended in growth medium consisting of Hanks balanced salt medium with 10 $ inactivated calf serum, 0.5 $ lactalbumin hydrolysate and 0.1 Aliquots (1 ml) of the obtained cell suspension containing each about 10 cells, are poured into 10 culture tubes (12 mm diameter) which are incubated in a slightly slant position at 37°C for 4 days. After that incubation period, a complete monolayer is developed. The nutrient medium is then replaced "by an equal volume of fresh one just before virus inoculation- ..

The same nutrient medium is used before and after virus inoculation and each primary RK cell culture indicated in this example is prepared according to the hereinabove described technique..

Aliquots (0.1 ml) of a rubella virus strain isolated in primary GMK cultures and passaged three times in this system for further characterization is inoculated into primary RK culture tubes which are incubated at 34°C on a slant position.

On the sixth day after inoculation, the supernatant fluid is harvested and the harvested virus is identified and titrated using the interference method described above.

The titer in GMK cells after this first passage on RK cells is 102*83 per ml.

Aliquots of the pooled supernatant fluid from the first passage are inoculated into other primary RK culture tubes which are incubated at 34°G.

On the 6th day after inoculation, the supernatant fluid is harvested. The virus is titrated as indicated at the end of the first passage. This procedure is repeated up to the 20th passage, the incubation period of each individual passage ranging between from 3 to 5 days.

Prom the 9th passage onwards, a cytopathic effect is observed in primary RK cells.

For the 21st passage in RK cells, primary RK cell cultures are prepared in 500 square centimeter Roux flasks, using the technique described above.

After an incubation period of 3 days at 36°C, a complete monolayer is obtained. The supernatant fluid is then discarded and replaced by an equal volume of the same growth medium and inoculated with aliquots of the viral material from the 20th passage .

After further incubation for 6 days at 34°C, the supernatant fluid is discarded and replaced by an equal volume of maintenance medium consisting of Hanks* solution supplemente with 0.3 Samples are taken for titration, identification and safety testing and the virus is stored at - 60°C.

The virus titer is 10 I D^Q per ml as tested in primary GM cells by the interference system.

This virus material is then subjected to safety testing including bacterial sterility and absence of adventitio agents by inoculation into rabbits, hamsters, guinea-pigs, mice and monkeys .

Additional safety testing is performed in different tissue culture systems.

Preliminary potency testing is performed by inoculati rabbits and monkeys with WJ 5 InD^Q. The serumneutralization (SN) tests are performed in GMK cells.

Results are given in the following Tables I and II.

Table I Antibody response of monkeys inoculated intramuscularly 103'5 InDcn ND = Not determined days after inoculation Table II Antibody response of rabbits inoculated subcutaneously with l03'5InDCA The virus preparation is divided into glass vials each of them containing one ml of fluid .

The vials are then freeze dried and sealed.

After reconstitution by adding one ml of distilled water, the vaccine is inoculated by subcutaneous route to susceptible subjects, the individual doses being about 125 InD5Q.

Results of a serological response testing conducted in a group of 25 seronegative children (15 receiving vaccine and 10 controls living in close contact) are given in Table III.

From the 15 vaccinated children, 13 had a rise of antibodies up to 1/32 or more (2 samples - indicated ND -were not available at the time of testing) . None showed clinical signs of infection. All 10 contacts remained serologically negative.

Table III Serological response expressed in seroneutralizing antibod titer .

V = vaccinated Example 2 The technique is that described in example 1 hut the passages are continued up to the 30th passage in primary RK cell cultures. Δ vaccine is prepared therefrom and safety testing is performed as described in example 1.

The potency of the preparation is tested in animals (monkeys) .

Results are given in Table IV.

Table IV Antibody response of monkeys inoculated intramuscularly with 102'5InDCA. days after inoculation Example 3 Starting from the viral material from the 21st passage at 34°C obtained in example 1 , 9 further serial passages are carried out in primary RK cell cultures but at 28°C, including the last step for vaccine preparation.

The obtained vaccine is safety tested and inoculated into monkeys and rabbits for potency testing.

Results are given in the following Tables V and VI.

Table V Antibody response of monkeys inoculated intramuscularly with 102·°ΙηΒΚΛ. ■ ■ days after inoculation Table VI Antibody response of rabbits inoculated intramuscularly with 102'°InDcn. days after inoculation Example 4 The technique is that described in example 1 but the passages are continued up to the 51st passage in primary RK cell cultures, the final maintenance medium consisting of Hanks' solution supplemented with 0.3 # casein hydrolysate and containing 50 meg of chloramphenicol per ml of Hanks' solution.

After incubation for 9 days at 34°C, the supernatant fluid is harvested, clarified by centrifugation and mixed with an equal volume of stabilizing solution consisting of 30g of potassium glutamate, 200g of sucrose and 50 mg of chloramphenico per liter of distilled water and the mixture is distributed into glass vials containing one ml of water.

The vials are then freeze-dried and sealed.

After reconstitution by adding one ml of distilled water, the vaccine is inoculated by subcutaneous route to 65 seronegative subjects, the titration of the individual doses reaching about 102'6InD5Q in G-MK cells or 10^* 7 PPU (Plaque forming units) in RK 13 cells. A group of 30 seronegative children was kept in intimate contact with the vaccinees for a period of 6 weeks.

A serological response testing conducted in the group of 65 vaccinated subjects showed that all but one responded to the vaccine with a mean antibody titer of 1/128 as assayed in HAI (Hemagglutination Inhibition) test. None showed clinical signs of infection.. All 30 contacts remained serologically negative .

Example 5 The technique is that described in example 1 but the passages are continued up to the 61st passage in primary RK cell cultures, the final maintenance medium consisting of Hanks' solution supplemented with 0 . 3 casein hydrolysate and containing 50 meg of chloramphenicol per ml of Hanks' solution.

After incubation for 9 days at 34°C, the supernatant fluid is harvested, clarified by centrifugation and mixed with an equal volume of stabilizing solution consisting of 30 g of potassium -glutamate, 200g of sucrose and 50 mg of chloramphenico per liter of distilled water and the mixture is distributed into glass vials containing one ml of water.

The vials are then freeze-dried and sealed.

After reconstitution by adding one ml of distilled water, the vaccine is inoculated by subcutaneous route to seronegative subjects, the titration of the individual doses reaching about 1O2'83InD50 in GM cells or 103 ' 9 PFU in R 13 cells.

A serological response testing conducted in a group of 7 seronegative subjects showed that all responded to the vaccine with a mean titer of 1/64 as assayed in HAI test.

None showed clinical signs of infection.

Claims (1)

1. HAVING NOW particularly described and ascertained the nature of our said invention and in what manner the same to,b performed, we declare that what we claim is A method for attenuating the virulence of rubella virus without loss of antigenicity, consisting in passaging serially a rubella virus strain at least 15 times on primary rabbit kidney tissue cultures. A method according to claim 1 wherein the number of serial passages on primary rabbit kidney tissue cultures is comprised between about 20 and about 60. / method according to any of claims 1 and 2 wherein the passages are conducted at a temperature not exceeding 36°C A method according to any of claims 1 to 3 wherein the duration of each serial passage does not exceed 5 days. A method for preparing a vaccine against rubella, said method consisting in passaging serially a rubella virus strain according to the method described in any of '¾£baims 1 to 4 and using the harvested attenuated live rubella virus as active ingredient for a rubella vaccine. A method for attenuating the virulence of rubella virus substantially as hereinbefore described, more particularly in the examples. A method for preparing an attenuated live rubella vaccine substantially as hereinbefore described, more particularly in the examples. Dated this 28th day of September, 1967