EP0000677A1 - Parainfluenza virus vaccine and its preparation - Google Patents

Parainfluenza virus vaccine and its preparation Download PDF

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Publication number
EP0000677A1
EP0000677A1 EP78400058A EP78400058A EP0000677A1 EP 0000677 A1 EP0000677 A1 EP 0000677A1 EP 78400058 A EP78400058 A EP 78400058A EP 78400058 A EP78400058 A EP 78400058A EP 0000677 A1 EP0000677 A1 EP 0000677A1
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EP
European Patent Office
Prior art keywords
parainfluenza virus
passage
virus
monkey kidney
type
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP78400058A
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German (de)
French (fr)
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EP0000677B1 (en
Inventor
Eugene Bernard Buynak
Maurice Ralph Hilleman
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Merck and Co Inc
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Merck and Co Inc
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Publication of EP0000677A1 publication Critical patent/EP0000677A1/en
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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
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18634—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
    • C12N2760/18734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention is concerned with the adaptation and propagation of parainfluenza types 1, 2 and 3 in tissue cultures prepared from embryonated hens' eggs. More particularly, this invention is directed to the development of live virus vaccines against the parainfluenza group of agents following serial passage in chick embryo tissue culture.
  • This procedure involves the steps of A) the isolation of the virulent viruses in any of a variety of cells in culture, and its adaptation to chick embryo tissue culture; B) the development of the attenuated viruses by a plurality of serial passages in chick embryo tissue culture; and C) the preparation of the vaccines from these attenuated live viruses. These steps will be separately explained.
  • Isolation and adaptation of parainfluenza virus can be accomplished in chick embryo tissue culture using virus previously propagated in another kind of cell culture, such as monkey kidney or a combination of monkey kidney and embryonated hens' eggs.
  • Isolation in the above-mentioned cell cultures can be from clinical material (e.g. throat swab).
  • Incubation of infected cultures can be carried out at any temperature between about 30° and about 38°C, preferably at 30-34°C (optimal 32°C) or at 35 to 38°C (optimal 36°C). From about 3 to about 20 serial passages may be employed to attenuate the virus.
  • Parainfluenza virus type 1 is isolated and adapted by at least 1 passage in monkey kidney cell culture and at least 1 passage in embryonated hens' eggs. Preferably at least 2 passages in monkey kidney cell culture and at least 2 passages in embryonated hens' eggs are employed.
  • Parainfluenza virus types 2 and 3 are isolated and adapted by at least 1 passage in monkey kidney cell culture.
  • parainfluenza virus type 2 is isolated and adapted by at least 2 passages in monkey kidney cell culture.
  • the virus which has been established in A. to be parainfluenza virus is added to glass bottles containing chick embryo tissue cultures prepared from minced and trypsinized approximately ten-day-old chick embryos.
  • the culture medium may be any of those which support cell growth and this may, for example, be the known medium 199 to which calf serum has been added.
  • the infected cell cultures are incubated in successive passages at 30-38°C and preferably at 30-34°C (optimal 32°C) and 35-38°C (optimal 36°C). During these passages the virus is replicated in large amount and becomes attenuated.
  • the parainfluenza virus harvested after this repeated serial passage is found to be nonpathogenic for monkeys and rodents, to cause little or no clinical reactions in human recipients, and to evoke a satisfactory level of neutralizing antibody.
  • the virus infectivity is stabilized by a suitable stabilizer such as sucrose, phosphate, glutamine, human albumin, or mixtures thereof.
  • a suitable stabilizer such as sucrose, phosphate, glutamine, human albumin, or mixtures thereof.
  • the virus pool is subdivided and filled into appropriate vials for use.
  • the product can be stored frozen or preferably dried from the frozen state and kept free of moisture.
  • the inoculum is parainfluenza virus type 1 which is obtained as described in A above after 2 passages in grivet monkey kidney cell culture, 8 passages in embryonated hens' eggs, and 9 passages in chick embryo tissue culture.
  • the washed tissue is trypsinized at 36°C using 0.25% trypsin (Difco 1:250) in tris saline buffer for approximately two hours.
  • the trypsin- cell suspension is harvested through two thicknesses of sterile cheese cloth and centrifuged at 1500 rpm for five minutes.
  • Growth medium consists of medium 199 (Morgan, J.F., Morton, H.J., and Parker, R.C., Proc. Soc. Exp. Biol. and Med., 73: 1-8, 1950) containing 2% unheated fetal calf serum and 50 mcg/ml neomycin. Bottle cultures are planted at a concentration of 700,000 viable cells per milliliter. Following incubation at 36°C for 48 to 72 hours, bottle cultures can be used for serial passage or vaccine preparation.
  • Chick embryo tissue cultures are prepared in glass bottles using medium 199 containing 2% unheated fetal calf serum as growth medium.
  • the growth medium is decanted and the cultures inoculated with 5.0-10.0 ml of undiluted or diluted seed virus per bottle.
  • 70 milliliters of medium 199 containing 2% agamma calf serum is added to each bottle, and re-incubated at 30-34°C.
  • the bottle cultures are washed four times with Hanks' BSS, 100 milliliters per wash. Following the washing procedure, l00 .
  • Appropriate harvest or harvests are selected following completion of infectivity titrations.
  • the selected material is removed from the freezer and thawed.
  • a sample is removed for control and safety testing.
  • the remaining fluid is clarified and a sample removed for monkey safety testing.
  • Appropriate additional stabilizer, as mentioned above, is added to the remaining fluid.
  • the fluids are distributed into individual vials and lyophilized. Following the lyophilization cycle the vials are capped, sealed and retained for reconstitution as a vaccine by the addition of sterile water (Water for Injection, U.S.P.).
  • the potency of the product is based on infectivity titrations in grivet monkey kidney cell cultures.
  • Example 1 The procedure of Example 1 is carried out but the incubation of the parainfluenza virus type 1 is in the 35-38°C range and close to 36°C.
  • Example 2 The procedure of Example 1 is repeated except substituting parainfluenza virus type 2.
  • the inoculum is parainfluenza virus type 2 which is obtained as described in A. above by employing 2 passages in grivet monkey kidney tissue, and 9 passages in chick embryo tissue culture. Similar clinical results are obtained.
  • Example 2 The procedure of Example 2 is repeated except employing parainfluenza virus type 2.
  • Example 3 The procedure of Example 3 is repeated except substituting parainfluenza virus type 3.
  • the inoculum is parainfluenza virus type 3 which is obtained as described in A. above by employing 1 passage in grivet monkey kidney tissue and 9 passages in chick embryo tissue culture. Similar clinical results are obtained.
  • Example 2 The procedure of Example 2 is repeated except employing parainfluenza virus type 3.

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

Abstract

By serially passaging a previously isolated and adapted virulent parainfluenza virus in tissue culture prepared from embryonated hens' eggs, a live, non-pathogenic but antigenic parainfluenza virus is produced. This virus is useful in preparing a live virus vaccine.

Description

  • In general terms, the invention is concerned with the adaptation and propagation of parainfluenza types 1, 2 and 3 in tissue cultures prepared from embryonated hens' eggs. More particularly, this invention is directed to the development of live virus vaccines against the parainfluenza group of agents following serial passage in chick embryo tissue culture. This procedure involves the steps of A) the isolation of the virulent viruses in any of a variety of cells in culture, and its adaptation to chick embryo tissue culture; B) the development of the attenuated viruses by a plurality of serial passages in chick embryo tissue culture; and C) the preparation of the vaccines from these attenuated live viruses. These steps will be separately explained.
    • A. Isolation and adaption of virulent virus
  • Isolation and adaptation of parainfluenza virus can be accomplished in chick embryo tissue culture using virus previously propagated in another kind of cell culture, such as monkey kidney or a combination of monkey kidney and embryonated hens' eggs. Isolation in the above-mentioned cell cultures can be from clinical material (e.g. throat swab). Incubation of infected cultures can be carried out at any temperature between about 30° and about 38°C, preferably at 30-34°C (optimal 32°C) or at 35 to 38°C (optimal 36°C). From about 3 to about 20 serial passages may be employed to attenuate the virus. Parainfluenza virus type 1 is isolated and adapted by at least 1 passage in monkey kidney cell culture and at least 1 passage in embryonated hens' eggs. Preferably at least 2 passages in monkey kidney cell culture and at least 2 passages in embryonated hens' eggs are employed. Parainfluenza virus types 2 and 3 are isolated and adapted by at least 1 passage in monkey kidney cell culture. Preferably parainfluenza virus type 2 is isolated and adapted by at least 2 passages in monkey kidney cell culture.
    • B. Development of attenuated live parainfluenza virus vaccine
  • The virus which has been established in A. to be parainfluenza virus is added to glass bottles containing chick embryo tissue cultures prepared from minced and trypsinized approximately ten-day-old chick embryos. The culture medium may be any of those which support cell growth and this may, for example, be the known medium 199 to which calf serum has been added. After the addition of the virus, the infected cell cultures are incubated in successive passages at 30-38°C and preferably at 30-34°C (optimal 32°C) and 35-38°C (optimal 36°C). During these passages the virus is replicated in large amount and becomes attenuated.
  • The above serial passages are performed using undiluted or diluted inoculum and multiple harvests are collected at various intervals. Infectivity titrations are performed in grivet monkey kidney tissue cultures.
    • C. Preparation of vaccine from attenuated virus
  • The parainfluenza virus harvested after this repeated serial passage is found to be nonpathogenic for monkeys and rodents, to cause little or no clinical reactions in human recipients, and to evoke a satisfactory level of neutralizing antibody. The virus infectivity is stabilized by a suitable stabilizer such as sucrose, phosphate, glutamine, human albumin, or mixtures thereof. After titration to establish its potency, the virus pool is subdivided and filled into appropriate vials for use. The product can be stored frozen or preferably dried from the frozen state and kept free of moisture.
  • The following examples illustrate the present invention without, however, limiting the same thereto.
    • EXAMPLE 1
  • The inoculum is parainfluenza virus type 1 which is obtained as described in A above after 2 passages in grivet monkey kidney cell culture, 8 passages in embryonated hens' eggs, and 9 passages in chick embryo tissue culture. Nine to eleven- day-old chick embryos, after removal of the head and extremities, are finely minced under aseptic conditions and the minced tissued washed in several changes of Hank's Balanced Salt Solution (BSS). The washed tissue is trypsinized at 36°C using 0.25% trypsin (Difco 1:250) in tris saline buffer for approximately two hours. The trypsin- cell suspension is harvested through two thicknesses of sterile cheese cloth and centrifuged at 1500 rpm for five minutes. Packed cells are resuspended in growth medium for counting. Growth medium consists of medium 199 (Morgan, J.F., Morton, H.J., and Parker, R.C., Proc. Soc. Exp. Biol. and Med., 73: 1-8, 1950) containing 2% unheated fetal calf serum and 50 mcg/ml neomycin. Bottle cultures are planted at a concentration of 700,000 viable cells per milliliter. Following incubation at 36°C for 48 to 72 hours, bottle cultures can be used for serial passage or vaccine preparation.
  • Chick embryo tissue cultures are prepared in glass bottles using medium 199 containing 2% unheated fetal calf serum as growth medium. Three to four days post-planting, the growth medium is decanted and the cultures inoculated with 5.0-10.0 ml of undiluted or diluted seed virus per bottle. Following an adsorption period of one hour at 30-34°C, 70 milliliters of medium 199 containing 2% agamma calf serum is added to each bottle, and re-incubated at 30-34°C. Three to four days post-seeding, the bottle cultures are washed four times with Hanks' BSS, 100 milliliters per wash. Following the washing procedure, l00 . milliliters of medium 199 containing a suitable viral stabilizer is added to each bottle and the cultures in cubated at 30-34°C. Neomycin at a concentration of 50 mcg/ml is incorporated in the growth and maintenance medium. Multiple harvests are collected at 2-4 day intervals and the bottle cultures are refed with fresh maintenance medium containing stabilizer. Infectivity titrations of each harvest are performed in grivet monkey kidney tissue cultures. Each harvest is collected aseptically into a sterile container, samples removed for microbial sterility testing and the remainder is shell-frozen in a dry ice-alcohol bath. The virus-containing fluids are stored at 70°C in an electrically operated freezing unit prior to selection of a harvest or harvests for preparation of the vaccine.
  • Appropriate harvest or harvests are selected following completion of infectivity titrations. The selected material is removed from the freezer and thawed. A sample is removed for control and safety testing. The remaining fluid is clarified and a sample removed for monkey safety testing. Appropriate additional stabilizer, as mentioned above, is added to the remaining fluid. The fluids are distributed into individual vials and lyophilized. Following the lyophilization cycle the vials are capped, sealed and retained for reconstitution as a vaccine by the addition of sterile water (Water for Injection, U.S.P.).
  • The potency of the product is based on infectivity titrations in grivet monkey kidney cell cultures.
  • Tests in man. Clinical studies of parenterally administered parainfluenza virus type 1 (monovalent vaccine) was conducted in children. Good antibody responses were observed with little, if any, untoward clinical-reactions.
    • EXAMPLE 2
  • The procedure of Example 1 is carried out but the incubation of the parainfluenza virus type 1 is in the 35-38°C range and close to 36°C.
    • EXAMPLE 3
  • The procedure of Example 1 is repeated except substituting parainfluenza virus type 2. The inoculum is parainfluenza virus type 2 which is obtained as described in A. above by employing 2 passages in grivet monkey kidney tissue, and 9 passages in chick embryo tissue culture. Similar clinical results are obtained.
    • EXAMPLE 4
  • The procedure of Example 2 is repeated except employing parainfluenza virus type 2.
    • EXAMPLE 5
  • The procedure of Example 3 is repeated except substituting parainfluenza virus type 3. The inoculum is parainfluenza virus type 3 which is obtained as described in A. above by employing 1 passage in grivet monkey kidney tissue and 9 passages in chick embryo tissue culture. Similar clinical results are obtained.
    • EXAMPLE 6
  • The procedure of Example 2 is repeated except employing parainfluenza virus type 3.

Claims (11)

1. A process of preparing a live, attenuated parainfluenza virus comprising serially passaging an isolated and adapted parainfluenza virus in tissue'culture prepared from embryonated hens' eggs for from about 3 to about 20 passages.
2. A process according to claim 1 wherein the parainfluenza virus has been isolated and adapted by at least one passage in monkey kidney cell culture or by at least one passage in monkey kidney cell culture and at least one passage in embryonated hens' eggs.
3. A process according to claim 2 wherein the parainfluenza virus is type 1 and is isolated and adapted by at least one passage in monkey kidney cell culture and at least one passage in embryonated hens' eggs.
4. A process according to claim 2 wherein the parainfluenza virus is type 2 or type 3 and is isolated and adapted by at least one passage in monkey kidney cell culture.
5. A process according to claim 2 wherein the isolation and adaption is carried out at from about 30° to about 38°C.
6. A composition comprising the attenuated virus of claim 1 together with a viral stabilizer.
7. A composition according to claim 6 in frozen state or lyophilized state.
8. A parainfluenza vaccine comprising an attenuated yet antigenic and immunogenic parainfluenza virus.
9. A vaccine according to claim 8 wherein the parainfluenza virus is type 1, or type 2, or type 3, or a mixture of at least 2 types.
10. A vaccine according to claim 8 containing a viral stabilizer.
11. A vaccine according to claim 8 in frozen state.or lyophilized state.
EP78400058A 1977-07-22 1978-07-12 Parainfluenza virus vaccine and its preparation Expired EP0000677B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81795577A 1977-07-22 1977-07-22
US817955 1977-07-22

Publications (2)

Publication Number Publication Date
EP0000677A1 true EP0000677A1 (en) 1979-02-07
EP0000677B1 EP0000677B1 (en) 1981-08-05

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EP78400058A Expired EP0000677B1 (en) 1977-07-22 1978-07-12 Parainfluenza virus vaccine and its preparation

Country Status (9)

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EP (1) EP0000677B1 (en)
JP (1) JPS5444015A (en)
DE (1) DE2860895D1 (en)
DK (1) DK326878A (en)
EG (1) EG14362A (en)
ES (1) ES471766A1 (en)
IT (1) IT1105533B (en)
PT (1) PT68284A (en)
ZA (1) ZA784161B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0013188A2 (en) * 1978-12-29 1980-07-09 Merck & Co. Inc. Parainfluenza virus vaccine and its preparation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3852918T2 (en) * 1987-09-28 1995-07-13 Beecham Inc Pig TGEV virus as a vaccine for dogs.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4940926A (en) * 1972-08-25 1974-04-17

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4940926A (en) * 1972-08-25 1974-04-17

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0013188A2 (en) * 1978-12-29 1980-07-09 Merck & Co. Inc. Parainfluenza virus vaccine and its preparation
EP0013188A3 (en) * 1978-12-29 1981-01-07 Merck & Co. Inc. Parainfluenza virus vaccine and its preparation

Also Published As

Publication number Publication date
EP0000677B1 (en) 1981-08-05
ES471766A1 (en) 1979-10-01
JPS5444015A (en) 1979-04-07
IT1105533B (en) 1985-11-04
ZA784161B (en) 1980-02-27
DE2860895D1 (en) 1981-11-05
PT68284A (en) 1978-08-01
DK326878A (en) 1979-01-23
IT7850293A0 (en) 1978-07-13
EG14362A (en) 1984-03-31

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