Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/20—Rubella virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5254—Virus avirulent or attenuated
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/36011—Togaviridae
  • C12N2770/36211—Rubivirus, e.g. rubella virus
  • C12N2770/36234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• ABSTRACT Rubella virus is passaged in primary rabbit kidney tissue at least 15 times, preferably 51-61 times, at temperatures of 28-36 C, to obtain an effective vaccine.
• 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.
• Attenuated live virus employed in this specification refers to a rubella virus (RV) strain, the virulence of which has been attenuated by at least serial passages on primary rabbit kidney tissue cultures.
• RV rubella virus
• gamma globulin The administration of gamma globulin is far from being considered by each specialist as an efiective treatment and, for obvious reasons, the exposure to the disease atan earlier age is not an acceptable answer to the problem.
• 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.
• 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 20 to 60 passages, said passages being conducted at a temperature not exceeding 36C.
• the duration of each passage is comprised between 3 and 6 days.
• a vaccine is then prepared from an appropriate passage in primary RK 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 -60C until used or immediately inoculated into an adequate tissue culture system, e.g., primary African green monkey kidney (GMK) monolayers or any other tissue culture system known to the art for such isolation.
• GMK primary African green monkey kidney
• rubella virus is checked by challenging cultures for instance with an enterovirus such'as Echovirus l l or Coxsackievirus A 9 on the 9th or 10th day after inoculation.
• enterovirus such'as Echovirus l l or Coxsackievirus A 9 on the 9th or 10th day after inoculation.
• 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 withinactivated 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 36C.
• 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.
• the supernatant fluid of the inoculated cultures is discarded by the 6th day after inoculation and the monolayers are washed with a bufiered saline solution, for instance Eagles 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.
• a bufiered saline solution for instance Eagles solution or Hanks solution
• a maintenance medium e.g., Hanks medium supplemented with casein hydrolysate.
• 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 stabilizing additive may be stored either in the frozen state, for instance at about 60C or in the freeze-dried state.
• the obtained vaccine is administered by subcutaneous or intramuscular route, if necessary after reconstitution.
• EXAMPLE 1 A 3-week-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 37C. 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.
• trypsin 2.5 g/l.
• the cell sediment obtained is resuspended in Hanks 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 percent inactivated calf serum, 0.5 percent lactalbumin hydrolysate and 0.1 percent tryptose phosphate broth so as to secure a concentration of about 10 cells per ml.
• each primary RK cell culture indicated in this example is prepared according to the hereinabove described technique.
• 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 10 InDm per ml.
• 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 th passage, the incubation period of each individual passage ranging between from 3 to 5 days.
• RK cell cultures are prepared in 500 square centimeter Roux flasks, using the technique described above.
• the supernatant fluid is discarded and replaced by an equal volume of maintenance medium consisting of Hanks solution supplemented with 0.3 percent casein hydrolysate.
• the supernatant .fluid' is harvested and clarified by centrifugation.
• Samples are taken for titration, identification and safety testing and the virus is stored at 60C.
• the virus titer is 10 InD per ml as tested in primary GMK cells by the interference system.
• This virus material is then subjected to safety testing including bacterial sterility and absence of adventitious agents by inoculation into rabbits, hamsters, guinea pigs, mice and monkeys.
• Preliminary potency testing is performed by inoculating rabbits and monkeys with 10 InD
• the serumneutralization (SN) tests are performed in GMK cells.
• the vials are then freeze dried and sealed.
• the vaccine After reconstitution by adding 1 ml of distilled water, the vaccine is inoculated by subcutaneous route to susceptible subjects, the individual doses being about 125 InD Results of a serological response testing conducted in a group of 25 seronegative children (15 receiving vaccine and controls living in close contact) are given in Table III.
• a vaccine is prepared therefrom and safety testing is performed as described in Example 1.
• the potency of the preparation is tested in animals (monkeys).
• the obtained vaccine is safety tested and inoculated into monkeys and rabbits for potency testing.
• the supernatant fluid is harvested, clarified by centrifugation and mixed with an equal volume of stabilizing solution consisting of 30 g of potassium glutamate, 200 g of sucrose and 50 mg of chloramphenicol per liter of distilled water and the mixture is distributed into glass vials containing 1 ml of water.
• the vials are then freeze-dried and sealed.
• the vaccine After reconstitution by adding 1 ml of distilled water, the vaccine is inoculated by subcutaneous route to 65 seronegative subjects, the titration of the individual 'doses reaching about l0"-InD in GMK cells or 10" PFU (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 means antibody titer of 1/ 128 as assayed in HA! (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 percent casein hydrolysat e and containing 50 mcg of chloramphenicol per ml of Hanks solution.
• the supernatant fluid is harvested, clarified by centrifugation and mixed with an equal volume of stabilizing solution consisting of 30 g of potassium glutamate, 200 g of sucrose and 50 mg of chlora'mphenicol per liter of distilled water and the mixture is distributed into glass vials containing 1 ml of water.
• the vials are then freeze-dried and sealed.
• the vaccine After reconstitution by adding 1 ml of distilled water, the vaccine is inoculated by subcutaneous route to seronegative subjects, the titration of the individual doses reaching about l0 -"InD in GMK cells or 10" PFU in RK 13 cells.
• a serological response testing conducted in a group of seven 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.
• a method for attenuating the virulence of rubella virus without loss of antigenicity comprising passaging serially a rubella virus strain for 3 to 6 days 51 to 61 times in primary rabbit kidney tissue cultures at 28-36 C 2.
• a process for attenuating a live rubella virus comprising serially passaging a rubella virus strain, after isolation and characterization, solely in primary rabbit kidney tissue culture from 51-61 times inclusive for 3 to 6 days at about 34 to produce an antigenically active, non-communicable, minimal side effect-producing, live rubella virus.
• a process for producing an attenuated live rubella virus which comprises the steps of introducing a virulent, live rubella virus into a rabbit kidney tissue cell culture, incubating said tissue culture at a temperature compatible with growth of said tissue and said virus, harvesting at least a portion of the virus so produced, reintroducing said harvested virus into fresh culture of the tissue, and repeating such tissue culture passage for at least 50 passages to produce an antigenically active, non-communicable live rubella virus.
• a method for preparing a rubella vaccine comprising passaging serially a rubella virus strain for 3 to 6 days 51 to 61 times in primary rabbit kidney tissue cultures at 2836C, and then combining the resulting attenuated virus with a vehicle.

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• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Virology (AREA)
• Pharmacology & Pharmacy (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Chemical & Material Sciences (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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Citations (2)

• 1966
  • 1966-10-21 GB GB47399/66A patent/GB1135987A/en not_active Expired
• 1967
  • 1967-09-29 IL IL28696A patent/IL28696A/en unknown
  • 1967-10-05 CA CA001,858A patent/CA956232A/en not_active Expired
  • 1967-10-11 ES ES345972A patent/ES345972A1/es not_active Expired
  • 1967-10-11 US US674650A patent/US3686394A/en not_active Expired - Lifetime
  • 1967-10-12 LU LU54664D patent/LU54664A1/xx unknown
  • 1967-10-13 CH CH1432067A patent/CH477553A/fr not_active IP Right Cessation
  • 1967-10-17 FI FI2788/67A patent/FI43625B/fi active
  • 1967-10-18 FR FR1548489D patent/FR1548489A/fr not_active Expired
  • 1967-10-18 BR BR194003/67A patent/BR6794003D0/pt unknown
  • 1967-10-18 FR FR124840A patent/FR7321M/fr not_active Expired
  • 1967-10-18 YU YU2034/67A patent/YU31804B/xx unknown
  • 1967-10-19 DE DE1967R0047176 patent/DE1617775B1/de active Pending
  • 1967-10-19 AT AT946167A patent/AT279038B/de not_active IP Right Cessation
  • 1967-10-19 BE BE705342D patent/BE705342A/xx not_active IP Right Cessation
  • 1967-10-19 NL NL676714223A patent/NL148799B/xx not_active IP Right Cessation
  • 1967-10-19 NO NO170199A patent/NO125776B/no unknown
  • 1967-10-20 DK DK525067AA patent/DK121619B/da not_active IP Right Cessation
  • 1967-10-20 SE SE14398/67A patent/SE341451B/xx unknown
• 1972
  • 1972-03-07 CY CY63572A patent/CY635A/xx unknown

Patent Citations (2)

Non-Patent Citations (2)

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