CH639420A5 - Process for the production of an RS virus - Google Patents

Description

The present invention relates to a method for producing an attenuated RS virus (Respira tory syncytial virus) in human diploid lung fibroblasts. The aim of the invention is to develop a live, attenuated vaccine containing RS viruses.

The WI-38 fibroblasts, which were originally derived from a single human lung, have been extensively characterized biologically, biochemically, virologically and genetically. The MRC-5 fibroblasts, likewise derived from a single human lung but from a different individual, were also standardized. WI-38 fibroblasts are described in «Exper. Cell Res. », 25, p. 585 (1961) and are deposited with the American Type Culture Collection (ATCC CCL-75). MRC-5 fibroblasts are described in "Nature", 227, p. 168 (July 11, 1970) and are deposited with the American Type Culture Collection (ATCC CCL-171). Reproduction of human diploid lung fibroblasts can be carried out by any of the standard methods described in the literature. For example, human diploid lung fibroblasts are made in glass containers using BME (GIB diploid) supplemented with 10% unheated calf serum as the growth medium. After incubation at 36 ° C for 48-72 h, the cultures can be used for serial passage or for the preparation of the vaccine.

The method according to the invention is defined in claim 1 and comprises, for example, the following stages:

A. isolation of the virulent virus in any type of cell culture and its adaptation to human diploid lung fibroblasts,

B. Development of the attenuated living virus through a variety of passages through human diploid pulmonary fibroblasts and

C. Preparation of the vaccine from these attenuated live viruses.

These process steps are described in detail below.

A. Isolation and adaptation of the virulent virus

Isolation and adaptation of the RS virus can be carried out in human diploid lung fibroblasts using a virus that has previously been conventionally grown in another cell culture such as the monkey kidney. The isolation in cell culture, such as in the monkey kidney, can come from clinical material (e.g. neck smear). The isolation can be carried out through one or more row passages in such a cell culture. After isolation, the virus can be subjected to 3-30 series passages, preferably 4-15 series passages, in human diploid lung fibroblasts. These passages are used to adapt and weaken the virus. The incubation of the infected cultures in human diploid lung fibroblasts can be carried out at any temperature between 30-38 ° C, preferably 30-34 ° C (optimally about 32 ° C), or from 35-38 ° C (optimally about 36 ° C) will.

B. Development of the attenuated live RS virus

The virus, which was isolated and adapted as described under A, is introduced into glass containers containing human diploid lung fibroblasts. The culture medium can be any that supports cell growth, such as a culture medium known and known as "Eagle's basal" medium (BME) or "Eagle's minimal essential" medium (MEM) in Eagle's balanced saline solution ( BSS) supplemented with previously screened calf serum. After the virus has been added, the infected cell cultures are incubated in successive passages at 30-38 ° C. in a number which is sufficient to weaken the virus but to retain its antigen and immunogen activity. Generally, 3-30 consecutive passages at 30-38 ° C, preferably 30-34 ° C (optimally around 32 ° C), or from 35-38 ° C (optimally around 36 ° C) are required. The virus is preferably incubated in 4-15 successive passages. The virus is replicated and weakened in large quantities during these passages.

The aforementioned series passages can be carried out using an undiluted or diluted inoculum and a variety of yields are obtained at different intervals. Titrations can be performed in HEP-2 cell cultures either in tubes or Falcon microtiter plates.

The virus obtained can be stored in frozen form or at low temperature to maintain its potency. Before freezing, a stabilizer or a combination of stabilizers, such as sorbitol or gelatin, can be added in such an amount as determined by the stability test of the frozen or lyophilized viral product.

C. Production of attenuated virus vaccine

The RS virus, which was obtained after repeated series passages (in particular about 3-30), was found not to be pathogenic to monkeys and rodents, vers

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causes little or no clinical response in humans and develops a sufficient level of neutralizing antibody. After titration to determine its potency, the total mass of the virus is divided and filled into containers for its use. The product can be stored in the frozen or preferably freeze-dried state and in the absence of moisture.

The following examples illustrate the invention in detail.

example 1

A respiratory syncytial virus was obtained from a cervical smear. This first inoculum is subjected to two passages in monkey kidney cell culture and four passages in human diploid lung fibroblasts WI-38. The human diploid lung fibroblasts WI-38 are produced in glass bottles using BME supplemented with a 10%, unheated, fetal calf serum as growth medium. 2 days after planting, the growth medium is decanted and the cultures are inoculated with 5.0 ml of an undiluted four passage seed virus per container (diluted seed virus can be used if desired). After an adsorption period of 1 h at 30-34 ° C, 100 ml MEM containing 2% unheated fetal calf serum are added to each bottle and incubated again at 30-34 ° C. 3 or 4 days after planting, the bottle cultures are washed four times with Hank's BSS, 100 ml per wash. After washing, 100 ml of MEM containing a viral stabilizer, e.g. human albumin, added to each bottle and the cultures incubated at 30-34cC. Neomycin is added to the growth and nutrient medium in a concentration of 50 µg / ml. A large number of yields are obtained at intervals of 2-4 days, and the bottle cultures are treated with new nutrient medium containing a stabilizer.

loaded.

A viral stabilizer, consisting of a mixture of equal parts of sorbitol and gelatin, is added before freezing and before storage at -70 ° C. After completing the infectivity titrations, one or more returns are received. The selected material is removed from the freezer and thawed. A sample is taken for control and for a safety test. The remaining liquid is emptied and a sample is taken for a safety test in the monkey. The liquid is distributed to individual vessels and lyophilized. To

639420

During the lyophilization cycle, the containers are sealed, sealed and stored for reuse as a vaccine by adding sterile water (water for injections U.S.P.).

The potency of the product is determined based on infectivity titration in a HEP-2 cell culture.

Example 2

The procedure of Example 1 is repeated, except that 9 series passages instead of 4 are carried out in human diploid fibroblasts WI-38.

Example 3

The procedure of Example 1 is repeated with the exception that the incubation temperature of the RS virus is in the range of 35-38 ° C instead of 30-34 ° C.

Test 1

A dose of the attenuated RS virus vaccine according to Example 1 is administered parenterally to 8 children who have no previous infection with the RS virus. Essentially all children developed a significant level of neutralizing antibody within 6 weeks of vaccination. No adverse clinical reactions have been observed.

Test 2

A dose of the attenuated RS virus vaccine according to Example 2 was administered parenterally to 11 children without previous infection with the RS virus. Essentially all children developed a significant level of neutralizing antibody 6 weeks after vaccination. No adverse clinical reactions have been observed.

Test 3

Samples of the virus, prepared as described in Examples 1 and 2, are frozen and stored at -70 ° C for more than 18 months. Upon thawing, it was found that the potency of the samples was essentially unchanged.

Test 4

Samples of the virus, prepared according to Examples 1 and 2, are lyophilized and stored at -20 ° C for more than 18 months. Examination of the potency of the samples showed that it was essentially unchanged.

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Claims (12)

639420 1. A method for producing an attenuated RS virus, characterized by serial passage of the RS virus through human diploid lung fibroblasts at an incubation temperature of 30-38 ° C in such a number of passages that causes an effective attenuation of the virus, its antigen and However, immunogen activity is not impaired, and recovery of the virus obtained. 2. The method according to claim 1, characterized by 3-30 row passages. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized by 4-15 row passages. 4. The method according to any one of claims 1 to 3, characterized in that the human diploid lung fibroblasts WI-38 are fibroblasts. 5. The method according to any one of claims 1 to 4, characterized by an incubation temperature of 30-34 ° C. 6. The method according to any one of claims 1 to 4, characterized by an incubation temperature of 35-38 ° C. 7. Attenuated RS virus produced by the method of claim 1. 8. Virus according to claim 7 in frozen or lyophilized form, together with a stabilizer. 9. vaccine, characterized by a content of RS virus according to claim 7. 10. Vaccine according to claim 9, characterized in that it also contains a viral stabilizer. 11. Vaccine according to claim 10, characterized in that the stabilizer is sorbitol, gelatin or a combination of sorbitol and gelatin. 12. Vaccine according to one of claims 9 to 11 in frozen or lyophilized form.