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• • 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
  • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
  • A61K39/25—Varicella-zoster 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
• • 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
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16761—Methods of inactivation or attenuation
  • C12N2710/16764—Methods of inactivation or attenuation by serial passage

Definitions

• Varicella commonly referred to as chickenpox
• This disease is marked by slight fever and an eruption of macular vesicles, which appear in crops, and are super- ficial and rarely umbilicated. They rarely become pustular, but dry up, and are only occasionally followed by scars. The duration of the disease is about a week.
• varicella is a relatively mild illness and rarely causes complications in normal children, when it occurs in a ward of a children's hospital, it may be serious and sometimes lethal in patients with leukemia or other malignancies, as well as in patients receiving immunosuppressive treatments and those with congenital immunodeficiencies. Because of such problem, numerous attempts have been made to prevent or modify the disease by passive immunization with convalescent serum, gamma- globulin, or zoster immune globulin. Adenenine arabinoside has also been used for treatment of disseminated zoster and varicella in compromised patients. However, effective- ness of these treatments varies, and in some instances clinical use of these agents may cause problems.
• OMPI fibroblasts ( I-38) , after passage in G.P.E. cells, reportedly was also effective in inducing immunological response without clinical reactions in normal susceptible children (see Takahashi et al. "Live Vaccine Used to Prevent the Spread of Varicella in Children in Hospital” Lancet (Nov. 30, 1974) 1238-90).
• the present invention provides a vaccine which i capable of inducing immunity against varicella, and thus i useful in protecting humans, especially children, against this source of human misery.
• the immunity the vaccine provides in hospital patients , such as those with leukemia or other malignancies, as well as in those patients receiving immunosuppressive treatmen and those with congenital immunodeficiencies, in which patients the disease may be serious, and, in some instance lethal.
• the vaccine of this invention advantageously does not evidence spread of the virus to contacts and does, not produce clinical reactions in normal susceptible children.
• the vaccine by reason of its mode of preparation, does not possess the capability of transmitting latent animal virus to vaccinees.
• the invention also consists in a novel process for preparing the vaccine.
• the vaccine of the present invention possesses the several above-described advantages in large measure by virtue of its mode of preparation.
• An essential featur of the process for preparation of the vaccine is the seria passaging of the varicella virus in human diploid lung fibroblasts, particularly WI-38 and MRC-5 fibroblasts .
• the WI-38 fibroblasts were originally derived from a single human lung; they are pedigreed in the sense that they have been extensively characterized biologically biochemically, virologically, and genetically.
• the MRC-5 fibroblasts were similarly derived from a single human lung, but of a different individual: and have been pedigreed in like manner. These two cell lines are standardized, in contrast to conventionally used primary animal cells.
• WI-38 has been described in Exper. Cell Res. 25, 585 (1961) and has been deposited with the American Type Culture Collection and assigned designation ATCC CCL- 75.
• MRC-5 has been described in Nature 227., 168 (July 11, 1970) .
• WI-38 has been made available to laboratories and may be obtained from the collection. The use of these cell lines for the propagation of the virus minimizes the likelihood of latent animal viruses being transmitted to a vaccinee by means of the vaccine.
• the Webster strain of varicella virus a preferred strain for use in preparation of the vaccine, was isolated in 1970 from a vesicle of a three year old female child at the onset of varicella (symptoms: fever and eruption of macular vesicles, in crops).
• This strain was isolated by Stanley A. Plotkin, M. D., and has been deposited with the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania, to afford permanence of the deposit and ready accessibility thereto by the public should a patent be granted on this application.
• other strains of varicella virus may be used.
• Propagation of the human diploid lung fibroblasts may be carried out by any of the standard methods described in the literature. Specific examples of such propagation techniques are disclosed in Exper. Cell Res. 25_, 585 (1961), and Virology 1(5, 147 (1962) .
• the tissue culture system usually comprises Eagle's basal medium (BME) or Eagle's minimal essential medium (MEM) in Eagle's balanced salt solution supplemented with prescreened calf serum containing a sterilizing amount of an antibiotic such as penicillin, streptomycin, chlortetracycline, or other antibiotic, or mixtures thereof, the system being buffered at a pH of about 6.8-8.5 with a conventional biological buffering agent such as an alkali metal bicarbonate, carbonate, or hydrogen phosphate.
• BME Eagle's basal medium
• MEM Eagle's minimal essential medium
• the virus is serially passaged in human diploid lung fibroblasts. In each passage the trypsinized infected whole cells from the
• OMPI previous passage are harvested and used to inoculate an uninfected culture. Each incubation proceeds for a perio of about 7 days and is conducted at a temperature of abou 35° C. After about 25 passages, the trypsinized culture of infected cells is subjected to sonication, and after centrifugation the cell-free supernatant containing infectious varicella virus is used to inoculate the next uninfected culture of human diploid lung .fibroblasts . Thi latter procedure is repeated for about 35 passages, each lasting about 4 to 7 days and conducted at a temperature o about 35° C.
• the total number of passages should be on t order of about 50, and preferably at least about 70, of which at least about 35 or more involve inoculation of eac uninfected culture with cell-free virus .
• the attenuated virus is then harvested and subjected to standard sterilit tests for the presence of bacteria, fungi, ycoplasma, an other contaminant agents .
• Attenuated virus refers to a virus of which the virulence has been altered by the method of culture so that it does not produce clinical symptoms when inoculated into humans although retaining its antigenicity, i.e. its ability to stimulate antibodies .
• the attenuated virus utilized as a vaccine is obtained by filtering the harvested material to remove cells or bacteria, and the filtrate is either used as is, frozen for later use, or lyophilized and subsequently reconstituted with a solvent such as water. Preferably a stabilizer such as sorbitol is used where the filtrate is lyophilized.
• the vaccine may be administered subcutaneous the minimum dosage of attenuated varicella virus being about 500 TCIDC Q . However, the dosage may be as high as 1000 TCID 50 .
• the varicella virus used is obtained from a normal three year old female child at the onset of clinical varicella. This strain is designated Webster and has been identified above.
• the vesicle fluid from the skin of the child is inoculated onto WI-38 human diploid fibroblasts.
• the nutrient medium used for tissue culture of the virus • is Eagle's minumum essential medium (MEM) with 2% of pre-screened calf serum added.
• the concentration of antibiotics in each ml. of medium is 60 eg. of penicillin and 10 eg. of gentamycin. Other antibiotics may be used in place of those specifically named.
• the harvest comprising supernatant fluid and virus infected cells after being trypsinized with 0,25% trypsin in a physiological saline solution is inoculated on stationary WI-38 human diploid lung fibroblasts.
• the nutrient medium used is the same as that employed for , tissue culture of the virus described above.
• the duration of each passage is 7 days, but passages on- the order of 4 to 7 days may be used.
• the temperature used in each passage is 35° C. However, temperatures in the range of 30° C to 37° C may be used.
• initial passages which preferably are 25 in number
• supernatant fluid containing virus infected cells is harvested from each preceding passage, after trypsinization is passaged on fresh uninfected culture comprising WI-38 human diploid lung cells .
• Approximately 507o of the cell-containing fluid from a preceding passage is used in each succeeding passage.
• the number of initial passages also referred to as the first series of passages, is such as to obtain a laboratory adapted virus.
• a somewhat smaller or greater number of passages than 25, e.g. 20 to 30 passages may be used.
• the supernatant fluid containing virus infected cells from the 25th passage is subjected to ultrasonic cell disruption, commonly referred to as sonication.
• a sonicator such as th made and sold by Branson Ultrasonics Corporation. Sonication generally takes place in about 30 seconds when such a sonicator is on setting No. 6.
• the resultant sonicate is centrifuged at 2000 rpm for about 20 minutes t separate cellular debris, and the cell-free supernatant
• a somewhat lesser or greater number of passages employing the sonication st may be from about 30 to 50.
• the temperature may vary from 30° to 37° C; preferably a temperature of 35° C being used.
• the duration of each passage will be on the order of 4 to 7 days, a passage duration of 7 days being preferred.
• the cell-free supernatant containing attenuated varicella virus from the final passage preferably at the time of maximum titer, and, as previously noted, after separation of cell debris, is used as such as a vaccine, is frozen and kept at a temperature of -70° C until time of use, or lyophilized for subsequent reconstitution with a solvent, e.g. water.
• a solvent e.g. water
• the varicella vaccine grows on human fibroblasts such as WI-38 and MRC-5 fibroblasts.
• Its cytopathic effec (CPE) is characterized by the lysis of cells into formatio of elliptical plaques of 1-2 mm. in diameter. The nuclei sometimes contain inclusions.
• the vaccine should be stored at -70° C in a solution of sorbitol (one volume of virus to one volume of stabilizer) .
• the vaccine is tested according to the procedures described by the Bureau of Biologies for testing live virus vaccine in tissue culture and in animals .
• the vaccine (Webster strain) produced as a result -of a total of 70 passages (25 first series, 45 second series as described above) was administered subcutaneously (1000 plaque-forming units) , and each patient was observed daily for one month. Serum specimens were taken 42 days after inoculation and subjected to varicella-zoster antibody to membrane antigen (VZ-FAMA) testing.
• VZ-FAMA varicella-zoster antibody to membrane antigen
• MRC-5's human embryonic lung fibroblasts
• growth medium consisting of minimum Eagle's Medium, supplemented with 7.5% fetal calf serum, 1.25% glutamine and penicillin-gentamycin- amphotericin-B
• Monolayers are routinely split from 1 ⁇ 2 to 1:5 once a week.
• Confluent monolayers are refed with maintenance medium as above with 2% fetal calf serum. The cells are incubated at 37° C.
• the infected culture is harvested by washing for 10 seconds with 5 mis, of 0,25% trypsin - 0.1% versene at 37° C and then incubating the cells at 37° C until the cells detach ( ⁇ 5 minutes), The infected cells are collected in 3.0 ml, of growth medium, of which 0,3 ml. is used to infect a new monolayer and the rest for FAMA as described below.
• the cells may also be stored. frozen in a suspension of growth medium, with 10% DMSO at -70° C and quick-thawed immediately before use. Frozen cells can be stored for FAMA testing for up to 2 months. They are more convenient to use than fresh cells, but preparations made with fresh cells are easier to read.
• FAMA testing is done in microtiter U-plates (Linbro, polystyrene) - A microtiter dropper is used to add 0.025 ml. PBS to each well. Serum is transferred by microtiter loop (0.025 ml.) which is then used to make two-fold dilutions of the serum from 1:2 to 1:64, or further, as necessary. A positive and negative control serum are included per plate.
• the cells are prepared for microscopic examinati by placing one drop of the cell suspension on a glass slid and covering it with a cover slip.
• the slides are examine with a Leitz fluorescent microscope. Positive cells appe rounded with a bright apple-green halo of fluorescence on the perimeter. A field is considered positive if the cell fluoresce with an intensity of at least +2 on a scale of 0 to +4. The fluorescence should diminish with dilutions of serum and a distinct end-point dilution should be observed.

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• 1981
  • 1981-01-21 JP JP56500729A patent/JPS57500149A/ja active Pending
  • 1981-01-21 EP EP19810900474 patent/EP0043847A4/de not_active Withdrawn
  • 1981-01-21 WO PCT/US1981/000075 patent/WO1981002107A1/en not_active Application Discontinuation
  • 1981-01-21 GB GB8123471A patent/GB2077590A/en not_active Withdrawn
  • 1981-01-26 IT IT8147637A patent/IT8147637A0/it unknown

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