FR2564320A1 - METHOD FOR PRODUCING ANTIRABIC VACCINE ON NON-TUMOROUS RABBIT CELL LINE CULTURES, ANTIRABIC VACCINE OBTAINED BY THIS PROCESS AND NOVEL CELL LINES FOR USE IN THE PRODUCTION OF SAID VACCINE - Google Patents

Abstract

THE PRESENT INVENTION RELATES TO A PROCESS FOR THE PRODUCTION OF ANTIRABIC VACCINE. THE ANTI-RABIES VACCINE IN ACCORDANCE WITH THE PRESENT INVENTION IS CHARACTERIZED IN THAT IT IS PRODUCED BY CULTURE OF A FIXED STRAIN OF RABIES VIRUS ON A CULTURE OF A DIPLOID CELL LINE OF RABBIT SKIN EMBRYONAL FIBROBLASTS (CALLED FELT CELLS). THE PRESENT INVENTION ALSO RELATES TO A CELL LINE SUITABLE TO SERVE AS A SUBSTRATE IN THE PRODUCTION OF ANTIRABIC VACCINE AND TO THE PROCESS FOR OBTAINING IT. APPLICATION TO ANTIRABIC VACCINATION.

Description

 The present invention relates to a new method for producing rabies vaccine on cultures of rabbit non-tumorigenic cell lines, to the new cell lines used for the production of said vaccine and to the rabies vaccine obtained by the above method.

 Rabies remains a zoonosis of global significance today.

 The fight against the natural reservoir of the disease, constituted by wild animals, is difficult and doubtful in the long term. On the other hand, preventive vaccination of domestic animals is possible, effective, and of indisputable interest both economically and in terms of human protection.

 In particular, vaccination of dogs must allow perfect control if not complete elimination of canine rabies and, consequently, the disappearance of the vast majority of cases of human contamination. Until this result, the objective of a global campaign by the O.M.S., is reached, many human anti-rabies treatments (vaccination after biting) must be implemented.

 Large quantities of vaccine are therefore necessary, both for veterinary use and for human use.

 The only technology acceptable for the present or for the near and far future (depending on the country and their state of scientific and technological development) is the production of vaccines on cell cultures.

Many types of cell cultures are currently proposed and used for the preparation of rabies vaccines. In particular, it has been proposed to grow strains of rabies virus on cells of first explantation obtained from 1. Primary cells of mammals, and in particular hams
ters, kidney of bovine embryos, kidney of monkey 2. Primary cells of avian origin and in particular of eggs
embryonated chicken or quail 3. Human diploid cells in lines; 4. Simian diploid cells (from Rhesus monkeys); 5. Rabbit diploid cells (FEL) 6. Heteroploid cells (BHK or VERO).

 However, the various cell culture systems proposed do not, as a general rule, simultaneously meet all of the main criteria for choosing an appropriate cell culture, that is to say, in particular, the criteria for ease of culture. , adaptability to mass production, virus productivity (antigen), safety (absence of oncogenic power), relatively low cost price, etc.

 The object of the present invention is to provide a method for producing a rabies vaccine which simultaneously satisfies all the criteria for choosing an appropriate cell culture, in that it uses a culture of embryonic cells of a mammal. very widespread, in particular in the main industrialized countries, in that the cell cultures used as substrates for the production of rabies vaccine in accordance with the invention are very stable since they seemingly replicate easily and unlimitedly over time, with regular and uniform growth, without undergoing morphological transformation, in that their safety is total and in that the ease of culture of these cell lines and their suitability for mass production in no way harm their immunogenicity.

 The subject of the present invention is a rabies vaccine for human or veterinary use, characterized in that it is produced by culture of a fixed strain of rabies virus on a culture of a line of diploid cells of embryonic rabbit skin fibroblasts ( say FEL cells).

 According to a preferred embodiment of the rabies vaccine in accordance with the present invention, said vaccine is an inactivated vaccine, the inactivation of which is preferably carried out using ss-propiolactone.

 In accordance with the present invention, said rabies vaccine results from the culture of a fixed strain of rabies virus on a line of diploid cells of rabbit skin embryonic fibroblasts, obtained by trypsinization of a rabbit embryo, then culture of the collected fibroblasts , in an appropriate Dulbecco medium and at least eleven passages of the primary culture in the same medium.

According to another preferred embodiment of the rabies vaccine in accordance with the present invention, the fixed strain of rabies virus used to be cultured on a line of diploid cells of rabbit embryonic fibroblast skin, with a view to obtaining said rabies vaccine , is taken from the group which includes the fixed virus strain PV, which is the rabies virus strain isolated by PAS
TEUR, and strains derived from the latter, such as the strain PV-BHK, the strain PV / RV / 2P VERO, the strain PV / RV / 2P
VERO / 2P FEL, in particular.

 The PV-BHK, PV / RV / 2P VERO and PV / RV / 2P VERO / 2P FEL strains are strains derived from the fixed PV strain, adapted to different cellular substrates: the PV-BHK strain is adapted to the BHK cell- 21 t the PV / RV / 2P VERO strain is the PV strain adapted to the bovine embryo kidney cell and having undergone two successive passages on the VERO cell and the PV / RV / 2P VERO / 2P FEL strain results from the adaptation of the previous strain to the FEL cell, by making it undergo two additional passages on the FEL cell.

 According to yet another preferred embodiment of the rabies vaccine according to the present invention, it results from the culture of a fixed strain of rabies virus on an FEL cell line which has the characteristics of non-tumorigenicity, of stability in the time in that the cells of said line show no sign of morphological transformation and replicate easily, with regular and uniform growth, even after more than 100 successive passages.

 The present invention further relates to a line of diploid cells of embryonic rabbit skin fibroblasts, called FEL cells.

 According to an advantageous characteristic of the FEL cell line, said line is a line established from fibroblasts of embryonic rabbit skin cultured in DULBECCO medium supplemented with approximately 10 8 calf serum, the primary culture being subjected to a plurality of passages in the same medium supplemented with approximately 5 to 10% of newborn calf serum, for the establishment of a cell population exhibiting rapid and regular growth, which is collected, and possibly frozen, to constitute the primary seed.

 According to an advantageous arrangement of the invention, the primary semen is collected after eleven passages.

 According to another advantageous characteristic of the FEL cell line in accordance with the present invention, said line is distinguished by: - its non-tumorigenicity after 21 days, after inoculation in athymic mice with 106 FEL cells / mouse, collected after 11 at 50 passages; - its stability, represented by the ability of cells to replicate without any sign of morphological transformation and following a regular and uniform growth, even after 100 successive passages.

 The FEL cell line in accordance with the present invention was deposited on May 16, 1984 under the number I-304 with the National Collection of Cultures of Microorganisms held by the Pasteur Institute.

According to yet another advantageous characteristic of the FEL cell line in accordance with the present invention, said line is capable of being used as a substrate in the production of rabies vaccine, to allow the obtaining of usable rabies virus, after inactivation, as a rabies vaccine with a high infectious titre without requiring concentration, devoid of latent contaminating viruses, having an infectious power (before inactivation) of 5.106 to 5.107 PFU / ml (PFU = virus-forming units) and immunogenicity - or protective power - comparable to that of a rabies vaccine obtained by culture on heteroploid cells such as cells
VERO, for example, without the inconvenience resulting from the use of these latter cultures.

 The present invention also relates to a method for producing rabies vaccine using the line of diploid FEL cells in accordance with the invention, by culture of a fixed strain of rabies virus on said line, characterized in that one infects a line of diploid cells of embryonic rabbit skin fibroblasts (FEL) maintained in an appropriate medium, with a strain of rabies virus, by allowing them to incubate for one hour + 15 minutes approximately, at + 37 "C i 2 "C, in that the medium is eliminated in the presence of which the infection was carried out and replaced by fresh medium supplemented with newborn calf serum, in which the culture is maintained for 18 to 30 hours at 34 "C i 2" C, in that this medium is then replaced by medium containing bovine albumin, in which the culture is maintained at 34 "C + 2" C, in that the cell supernatants are harvested at appropriate time intervals and by inactivating the suspension of v rabid irus thus harvested.

 According to an advantageous mode of implementation of this process, the medium for maintaining the cell lines, into which the strain of rabies virus to be cultivated is introduced, is complete DULBECCO medium supplemented with approximately 5 to 10% calf serum new born.

 According to another embodiment of the method for producing rabies vaccine according to the invention, the fixed strain of rabies virus is introduced in an amount of 0.1 to 0.3 LD50 / cell.

 By LD50 is meant the 50% controlled intracerebral lethal dose in mice.

According to another method of implementing the rabies vaccine production method in accordance with the invention, the medium for replacing the infection medium, for maintaining the virus culture, is medium
DULBECCO complete with approximately 5% newborn calf serum.

 According to another embodiment of the method for producing rabies vaccine in accordance with the invention, the medium for replacing the medium for maintaining the culture of the rabies virus is a complete DULBECCO medium optionally containing approximately 0.3% of albumin. bovine.

 According to yet another embodiment of the method for the production of rabies vaccine according to the invention, the cell supernatants containing the rabies virus are harvested every 2 to 4 days approximately.

 According to another embodiment of the method for producing a rabies vaccine in accordance with the invention, the rabies virus present in the harvested cell supernatants is inactivated by an appropriate inactivation agent such as, in particular, ss-propiolactone at the final dilution of about 1/7000 to 1/10000 and preferably about 1/8000.

 The present invention further relates to a method for producing a line of diploid cells of embryonic rabbit skin fibroblasts, which is characterized in that a rabbit embryo of at least 15 days old and no more than 20 days, to a trypsinization treatment to collect rabbit skin fibroblasts, which are cultured in complete DULBECCO medium supplemented with approximately 10% of newborn calf serum, the initial primary culture being subjected to a plurality of passages, preferably 10 to 12 passages, in complete DULBECCO medium supplemented with approximately 5 to 10% of newborn serum.

 According to a preferred embodiment of this method, the cell line thus established is frozen to be stored for later use.

 According to another preferred embodiment of this process, the cell line thus established is maintained in DULBECCO medium supplemented with approximately 5-10% of newborn calf serum, until inoculation with rabies virus.

 In addition to the foregoing arrangements, the invention also comprises other arrangements which will become apparent from the description which follows.

 The invention will be better understood using the additional description which follows, which refers to an example of implementation of the method which is the subject of the present invention.

 It should be clearly understood, however, that this example of implementation is given solely by way of illustration of the subject of the invention, of which it in no way constitutes a limitation.

EXAMPLE OF ANTIRABIC VACCINE PREPARATION
A. ESTABLISHMENT OF FEL CELLS
The FEL cells (rabbit embryonic fibroblasts) were established by putting rabbit skin fibroblasts obtained by trypsinization of a rabbit embryo (Fauve de Bourgogne) for 17 days, in culture in complete DULBECCO medium supplemented with 10% of calf serum, then by subjecting the primary culture obtained to 11 successive passages in complete DULBECCO medium supplemented with 10% newborn calf serum.

 In practice, the line established after 11 successive passages is frozen to serve as primary seed; it is thawed in a complete DULBECCO medium supplemented with 5-10% newborn calf serum, each time it is desired to use it as a substrate in the production of rabies vaccine.

B. PREPARATION OF THE VACCINE
The FEL cell line obtained, established in the middle DULBECCO complete supplemented with 5-10% of newborn calf serum, is infected with a strain of rabies virus at a rate of 0.1 to 0.3 LD50 / cell (doses lethals 50% controlled in mice).

 After one hour of incubation at + 37 "C, the infection medium is eliminated and replaced with complete DULBECCO medium supplemented with 5% newborn calf serum, in which the culture of the virus is continued for 24 hours at temperature of 34 "C.

 At the end of this time, the medium containing 5% of serum is eliminated and replaced by a complete DULBECCO medium possibly containing 0.3% of bovine albumin, in which the culture of the rabies virus continues at 34 C.

 Cell supernatants containing the rabies virus in suspension are harvested approximately every three days.

 The virus thus harvested is inactivated by B-propiolactone (Kp10: 47-510C; d = 1.14) at the final dilution of 1/8000.

C. REPLICATION OF THE RABIC VIRUS ON FEL CELLS
The sensitivity of FEL cells and their ability to produce virus and rabies antigens were tested under the following three conditions of immobilization of FEL cells
1. In stationary glass and plas bottles
tick.

 2. In glass and plastic roller bottles.

3. In a semi-industrial "Gyrogen" type device
(Chemap Company) consisting of a glass tank and
of uniformly rotating glass tubes
on which the cells are immobilized.

 a) Production of infectious particles.

FEL cells have been shown to be sensitive to several fixed strains of rabies virus adapted to different cell substrates. These strains are derived from that isolated by PASTEUR (PV strain deposited at ATCC under the number "VR 320) and are
1. The PV strain adapted to the BHK-21 cell (C13):
called "PV-BHK".

2. The PV strain adapted to the embryonic kidney cell
bovine called "PV / RV" (used for the produc
the Pasteur Production Institute vaccine).

She underwent two successive passes on the cell
VERO (PV / RV / 2P VERO).

3. The same strain as that used in 2. above,
but with two additional passages on the cel
lule FEL (PV / RV / 2P VEROj2P FEL).

 The maximum of infectious particles released into the culture medium (5.106 to 5.107 PFU / ml, units of virus forming plaques) is obtained between the sixth and the tenth day following infection.

 b) Production of rabies antigens.

 Numerous studies have shown that the immunogenicity of an inactivated rabies virus suspension is linked more to the quantity of glycoproteins (proteins constituting the spicules) than to the infectious power of the preparation before inactivation.

The kinetic study of the production of glycoproteins and the examination of the physical state of the viral particles were carried out using an immunoenzymatic method. As a result, the maximum of glycoproteins is released at variable times after infection, depending on the strain: between the 4th and 7th day for PV / BHK and between the 14th - 19th day for
PV / RV / 2P VERO and PV / RV / 2P VERO / 2P FEL. During the period of high production of glycoproteins, harvests every two or three days can be carried out.

 The level of glycoprotein production with the FEL cell lines is substantially identical to that obtained with the VERO cell, regardless of the rabies strain used.

Under these conditions and in parallel with the cell
VERO, the following results were obtained with the cell
FEL
Immunogenicity: Protective Power
(International Units x)
CELL STRAIN FEL CELLS VERO
PV / RV / 2P VERO 0.87 0.53
PV / RV / 2P VERO / 2P FEL 2.6 to 5.0 7.7 to 8.5
PV / BHK 3.6Xx
x Reference vaccine (concentrated and purified): 12 Units
xx xx Production carried out with the "Gyrogen" device: Harvest
on the 7th day.

Given the variability of the immunogenicity evaluation test for a rabies vaccine, it can be concluded that FEL cells allow the production of a rabies vaccine of immunogenicity comparable to that obtained with cells
VERO.

D. The vaccine thus obtained can be conveniently lyophilized in the presence of an appropriate stabilizer known in itself.

 It is put in sealed ampoules containing the lyophilized powder which is put into injectable form extemporaneously by dissolution in a suitable vehicle.

E. CHARACTERIZATION OF FEL CELLS
To be used as a substrate in the production of inactivated viral vaccines, a cell line must meet the requirements of the WHO (WHO - Report
Technical 673. Revision 1981, p. 76-82).

Characterization studies carried out by
Inventors have demonstrated that FEL cells are suitable for vaccine production for the following reasons
1 ") Tumorigenesis. It was studied at the level of the 11th and 50th passages in nu / nu mice, in parallel with HeLa cells (positive control). The results show that the inoculation of 106 FEL cells per mouse does not did not produce tumors or metastases (macro- and microscopic examinations) in athymic mice after three weeks of observation. Under the same conditions of inoculation, the HeLa cells induced tumors in 100 percent of the animals. reach a thickness of 1/1 cm after three weeks.

 2-) Stability of FEL cells during passages.

To determine the lifespan of FEL cells (cell line with a limited lifespan or a line with an unlimited lifespan: stable line), the inventors carried out successive passages and showed that during 100 passages the cells replicate easily. During these passages, no sign of morphological transformation was observed.

The growth is always regular and uniform.

3 ') Sensitivity of FEL cells to interferon. The
The inventors have also demonstrated that only rabbit interferon acts on the viruses cultivated in FEL cells. This test shows the species identity of this cell.

 The rabies vaccine obtained in accordance with the present invention has the advantage of being able to be produced in large quantities, from an inexpensive and readily available raw material. The virus productivity is at least equal to that which the previously proposed methods make it possible to obtain, while ensuring that a vaccine having total safety is obtained, due to the origin and characteristics of the FEL line.

 The BHK-21 cell has been deposited under the number CCL 10 at ATCC.

Claims (10)

FEL CLAIMS).  1 ") Rabies vaccine capable of ensuring immunity against rabies in humans and animals, characterized in that it is produced by culture of a fixed strain of rabies virus on a culture of a diploid fibroblast cell line rabbit skin embryos (called cells  2 ") Rabies vaccine according to claim 1, characterized in that it is an inactivated vaccine, the inactivation of which is preferably carried out using 8-propiolactone.  3) Rabies vaccine according to claim 1 or claim 2, characterized in that it results from the culture of a fixed strain of rabies virus on a line of diploid cells of embryonic rabbit skin fibroblasts, obtained by trypsinization of a rabbit embryo, then culture of the fibroblasts collected, in an appropriate Dulbecco medium and at least eleven passages of the primary culture in the same medium. BHK, the strain PV / RV / 2P VERO, the strain PV / RV / 2P VERO / 2P FEL, in particular.  4 ') Rabies vaccine according to any one of claims 1 to 3, characterized in that the fixed strain of rabies virus used to be cultivated on a line of diploid cells of embryonic rabbit skin fibroblasts, for the purpose of obtaining said rabies vaccine, is taken from the group which comprises the strain of fixed virus PV, which is the strain of rabies virus isolated by PASTEUR, and strains derived from the latter, such as strain PV  5) Rabies vaccine according to any one of claims 1 to 4, characterized in that it results from the culture of a fixed strain of rabies virus on an FEL cell line which has the characteristics of non-tumorigenicity, of stability over time in that the cells of said line show no sign of morphological transformation and replicate easily, with regular and uniform growth, even after more than 100 successive passages.  6) Cell line capable of serving as a substrate in the production of the rabies vaccine according to any one of claims 1 to 5, characterized in that it is constituted by a line of diploid cells of embryonic rabbit skin fibroblasts, called cells FEL.  7) Cell line according to claim 6, characterized in that said line is a line established from fibroblasts of embryonic rabbit skin cultured in DULBECCO medium supplemented with approximately 10% calf serum, the primary culture being subjected to a plurality of passages in the same medium, added with approximately 5 to 10% of newborn calf serum, for the establishment of a cell population with rapid and regular growth, which is collected, and possibly frozen, to constitute the primary seed.  8) Cell line according to claim 7, characterized in that the primary semen is collected after eleven passages. FEL / mouse, collected after 11 to 50 passages; - its stability, represented by the ability of cells to replicate without any sign of morphological transformation and following a regular and uniform growth, even after 100 successive passages.  9) FEL cell line according to any one of claims 6 to 8, characterized in that it is distinguished by: - its non-tumorigenicity after 21 days, after inoculation in athymic mice with 106 cells  11 ") Method for producing the rabies vaccine according to any one of claims 1 to 5, by culture of a fixed strain of rabies virus on a line of FEL diploid cells according to any one of claims 6 to 10, characterized in infecting a line of diploid cells of rabbit skin embryonic fibroblasts (FEL) according to any one of claims 6 to 10, maintained in an appropriate medium, by a strain of rabies virus, in that one leaves incubate for one hour to about 15 minutes, at + 37 "C * 2" C, in that the medium is eliminated in the presence of which the infection was carried out and replaced by fresh medium supplemented with new calf serum -born, in which the culture is maintained for 18 to 30 hours at 34 "C + 2" C, in that this medium is then replaced by medium containing bovine albumin, in which the culture is maintained at 34 "C t 2" C, in that the cell supernatants are harvested at appropriate time intervals opiate and inactivating the rabies virus suspension thus harvested.  10) FEL cell line according to any one of claims 6 to 9, characterized in that it was deposited on May 16, 1984 under the number "I-304 from the National Collection of Cultures of Microorganisms held by the Pastor Institute.  12) Method according to claim 11, characterized in that the maintenance medium for cell lines, in which the strain of rabies virus to be cultivated is introduced, is complete DULBECCO medium supplemented with approximately 5 to 10% calf serum new born.  13) Method according to any one of claims 17 and 12, characterized in that the fixed strain of rabies virus is introduced in an amount of 0.1 to 0.3 DLs0 / cell.  14 ") A method according to any one of claims 11 to 13, characterized in that the medium for replacing the infection medium, for maintaining the virus culture, is complete DULBECCO medium supplemented with approximately 5% of newborn calf serum.  15) Method according to any one of claims 11 to 14, characterized in that the medium for replacing the culture culture rabies virus maintenance medium is a complete DULBECCO medium.  16) Method according to any one of claims 11 to 15, characterized in that the cellular supernatants containing the rabies virus are harvested every 2 to 4 days approximately.  17) Method according to any one of claims 11 to 15, characterized in that the rabies virus present in the harvested cellular supernatants is inactivated by an appropriate inactivation agent such as, in particular, ss-pro-piolactone at the dilution final of about 1/7000 to 1/10000 and preferably about 1/8000.  18) Method for producing a line of diploid cells of embryonic rabbit skin fibroblasts according to any one of claims 6 to 10, characterized in that a rabbit embryo is subjected for at least 15 days and d '' at most 20 days, to a trypsinization treatment to collect rabbit skin fibroblasts, which are cultured in complete DULBECCO medium supplemented with approximately 10% calf serum, the initial primary culture being subjected to a plurality of passages, preferably 10 to 12 passages, in complete DULBECCO medium supplemented with approximately 5 to 10% of newborn calf serum.  19) The method of claim 18, characterized in that the cell line thus established is frozen to be stored for later use.  20) Method according to claim 18, characterized in that the cell line thus established is maintained in DULBECCO medium supplemented with approximately 5-10% of newborn calf serum until the inoculation of rabies virus.