Virus strain and application thereof in preparation of rabies vaccine

Abstract

The invention discloses a virus strain and application thereof in preparation of rabies vaccines, and belongs to the technical field of biomedicine. The virus strain disclosed by the invention is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: V202406, the preservation time is January 24, 2024, and the preservation address is No. 299 on eight road in Wuchang District, Wuhan City, Hubei Province. The invention also discloses application of the virus strain in preparation of rabies vaccines.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, specifically relates to the field of vaccine technology, and in particular to a virus strain and its application in preparing rabies vaccine. Background Art

[0002] Rabies is an acute infectious disease caused by the lyssavirus, which is usually transmitted to humans through bites or scratches from infected animals, especially dogs and cats. The rabies virus belongs to the genus Lyssavirus of the family Rhabdoviridae. It is a ribonucleic acid-type rhabdovirus, shaped like a bullet, with a diameter of 65-80nm and a length of about 130 to 240nm. At present, there is no effective treatment for rabies. Once humans become ill, the mortality rate is almost 100%. Therefore, vaccination is still the most effective means of preventing rabies.

[0003] Vaccine strains in the prior art are mainly divided into Pasteur strains (PAS, PM, PV), Flury strains, SAD strains, aG strains, CTN strains and their derivatives. These strains have been adapted to in vivo or in vitro culture. The PM strain was formed by the Pasteur strain of rabies virus in the rabbit brain, which was then transferred to the National Institutes of Health and continued to be passaged in the rabbit brain. Finally, Dr. WIK-TOR of the Wistar Institute in the United States successfully obtained the PM strain through cell passage adaptation.

[0004] Human diploid cells are cells derived from normal human tissues cultured in vitro. They maintain a diploid karyotype during the passage process, contain no exogenous proteins and exogenous DNA components, and do not have the potential tumorigenicity and carcinogenicity risks of animal-derived passaged cells. Human diploid cells have good application prospects in virus culture and viral vaccine preparation, and are considered to be ideal cells for producing human vaccines. The World Health Organization also recommends that human diploid cells be considered first in vaccine production. However, the development of human diploid cell rabies vaccines is limited by their low production, high cost, and lack of price advantages.

[0005] Therefore, it has become an urgent problem to be solved by technicians in this field to provide a rabies virus human diploid cell-adapted strain with good immunogenicity and high titer, which is safe, effective, stable and suitable for large-scale production. Summary of the invention

[0006] The purpose of the present invention is to provide a virus strain which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: V202406 and is used for preparing rabies vaccine, which can provide good immune protection and has good safety.

[0007] Another object of the present invention is to provide the use of the virus strain in the preparation of rabies vaccine.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: On the one hand, the present invention discloses a virus strain, which is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: V202406, a preservation time of January 24, 2024, and a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] On the other hand, the present invention discloses the use of the above virus strain in the preparation of rabies vaccine.

[0010] In some embodiments of the present invention, the rabies vaccine is an inactivated vaccine.

[0011] In a third aspect, the present invention discloses a rabies vaccine comprising the above-mentioned virus strain.

[0012] In some embodiments of the present invention, the virus strain is an inactivated virus strain.

[0013] In some embodiments of the present invention, the rabies vaccine further comprises an adjuvant.

[0014] The adjuvants described in the present invention are prior art.

[0015] In some embodiments of the present invention, the administration of the vaccine includes intramuscular injection or intradermal injection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the virus strain of the present invention is used to prepare the virus seed, the virus titer is high, and it is used for the production of rabies vaccine, which is conducive to the later production and amplification. The virus strain of the present invention has good passage stability and immunogenicity.

[0017] The virus strain library prepared using the virus strain of the present invention meets the virus strain requirements of the "Human Rabies Vaccine" in Part III of the 2020 edition of the "Pharmacopoeia of the People's Republic of China". BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attached Figure 1 This is a graph showing the results of investigating the virus titer of different generations of the virus strain of the present invention.

[0019] Attached Figure 2 The present invention is a process flow chart for constructing a master virus seed library using the virus strain of the present invention.

[0020] Attached Figure 3 The present invention is a flowchart of the process for constructing a working virus seed library using the virus strains of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] The working bank cells described in the embodiment of the present invention are KH-02 cells Homo sapiens., which are derived from the China Center for Type Culture Collection, with a preservation number of CCTCC NO: C2023325, and were deposited in the China Center for Type Culture Collection on November 1, 2023. The preservation address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0023] Example 1 This embodiment discloses a method for obtaining the virus strain of the present invention, which is as follows: Step 1. Cell recovery: Take the working bank cells, recover them into a T75 culture flask, add culture medium to the culture volume, and culture them in a 37°C, 5% carbon dioxide incubator.

[0024] Step 2. Cell digestion: After the cells grow densely, digest them with 0.02% EDTA-0.25% trypsin solution to make a cell suspension.

[0025] Step 3. Virus inoculation and adsorption: Take the PM strain virus, melt it and inoculate it into the cell suspension. Adsorb the virus-cell mixed suspension at 37℃ for 60 minutes.

[0026] Step 4. Virus culture and harvest: Transfer the virus-cell suspension after adsorption to a culture bottle, add culture medium to the culture volume, and culture in a 37°C, 5% carbon dioxide incubator. After the cells grow densely, transfer to a 33°C, 3% carbon dioxide incubator for further culture. After 7 days of culture, harvest the supernatant, which is the P1 generation.

[0027] Step 5. Virus adaptation by passage: Continuously passage the virus for 28 to 32 generations in the same manner as above to obtain the PM strain of rabies virus KH-02 cell adapted strain.

[0028] Test Example 1 The virus strains were passaged according to the method of Example 1 to obtain virus strains of generations P1 to P32. The virus titers of virus strains of generations P1 to P32 were measured by plaque assay. The results are as follows: Figure 1 shown.

[0029] The results showed that the virus titer was low when the virus was just adapted to passage, and the virus titer increased with the increase of passage number, and tended to be stable at P10. The virus strain of the present invention has a high virus titer and good passage stability and immunogenicity.

[0030] Example 2 This embodiment discloses a method for constructing a main virus seed library using the virus strain of the present invention, and the process flow is shown in the attached figure. Figure 2 As shown, the specific method is as follows: Step (1) Cell recovery: Take the working bank cells, recover them in a T75 culture flask, and culture them in a 37°C, 5% carbon dioxide incubator.

[0031] Step (2) Cell passaging: After the cells have grown densely, pass the cells to 2T75 at a ratio of 1:2.

[0032] Step (3) Virus inoculation and culture: After the cells grow densely, take 1T75 and digest it with 0.02% EDTA-0.25% trypsin solution to make a cell suspension. Take the PM strain P10 virus, melt it and inoculate it into the cell suspension. Adsorb the virus-cell mixed suspension at 37°C for 30 minutes. Transfer the virus-cell mixed suspension after adsorption to a culture bottle and culture it in a 37°C, 5% carbon dioxide incubator. After the cells grow densely, transfer it to a 33°C, 3% carbon dioxide incubator for further culture. Culture for 7 days.

[0033] Step (4) Cell passaging: Another densely grown T75 cell was passaged to 2T75 at a passaging ratio of 1:8.

[0034] Step (5) Cell passaging: After the cells in 2T75 grow densely, take 1T75 cells and pass them to 6T75 at a passaging ratio of 1:6.

[0035] Step (6) Virus seed passaging: harvest the virus supernatant after 7 days of culture, digest the infected cells, and digest two bottles of densely grown T75 cells. Mix the cell suspension, infected cells and supernatant, adsorb at 37°C for 30 minutes, and pass to 2T300. First, culture in a 37°C, 5% carbon dioxide incubator, and transfer to a 33°C, 3% carbon dioxide incubator for further culture after the cells grow densely. Culture for 7 days.

[0036] Step (7) Cell passaging: Take the 2T75 cells from step 5 and pass them to 2T300 cells at a ratio of 1:8.

[0037] Step (8) After the cells grow densely, take 1T300 and subculture it to 6T300 at a subculture ratio of 1:6.

[0038] Step (9) Virus seed passaging: harvest the virus supernatant after 7 days of culture, digest the virus-carrying cells, and digest four bottles of densely grown T300 cells. Mix the cell suspension, virus-carrying cells and supernatant, adsorb at 37°C for 30 minutes, and pass to 2T300. First, culture in a 37°C, 5% carbon dioxide incubator, and transfer to a 33°C, 3% carbon dioxide incubator for further culture after the cells grow densely. Culture for 7 days.

[0039] Step (10) Virus seed harvesting: After 7 days of virus culture, the supernatant is harvested, and the virus-carrying cells are digested and mixed with the supernatant to obtain the main virus seed. The main virus seed is then packaged into cryopreservation tubes and stored in a refrigerator at ≤-70°C to obtain the main virus seed library.

[0040] The main virus strain library was sent to the Quality Inspection Department of Chengdu Kanghua Biological Products Co., Ltd. for testing. The results showed that the sterility test, mycoplasma test, and exogenous viral factor test were in compliance with the regulations. The neutralization index was 794, the protection index was 794, and the virus titer was 6.01lgPFU / ml, which met the virus strain requirements of the "Freeze-dried Human Rabies Vaccine (Human Diploid Cells)" in the "Pharmacopoeia of the People's Republic of China" (2020 edition, Volume III).

[0041] Example 3 This embodiment discloses a method for building a working virus seed library using the main virus seed library viruses of Example 2. The process flow is shown in the attached figure. Figure 3 As shown, the specific method is as follows: S1. Cell recovery: Take the working bank cells, recover them in a T75 culture flask, and culture them in a 37°C, 5% carbon dioxide incubator.

[0042] S2. Cell passaging: After the cells grow densely, pass the cells to 4T75 at a ratio of 1:4.

[0043] S3. Virus inoculation and culture: After the cells grow densely, take 1T75 and digest it with 0.02% EDTA-0.25% trypsin solution to make a cell suspension. Take the main virus seed virus of Example 2, melt it and inoculate it into the cell suspension. Adsorb the virus-cell mixed suspension at 37°C for 30 minutes. Transfer the virus-cell mixed suspension after adsorption to a culture bottle, culture it in a 37°C, 5% carbon dioxide incubator, and transfer it to a 33°C, 3% carbon dioxide incubator for further culture after the cells grow densely. Culture for 7 days.

[0044] S4. Cell passaging: 3T75 cells that have grown densely are passaged to 3T300 cells at a ratio of 1:4.

[0045] S5. Cell passaging: When the cells grow densely, pass 3T300 to 18T300 at a ratio of 1:6.

[0046] S6. Virus seed passaging: harvest the virus supernatant after 7 days of culture, digest the virus-carrying cells, and digest a bottle of densely grown T300 cells. Mix the cell suspension, virus-carrying cells and supernatant, adsorb at 37°C for 30 minutes, and pass to 8T300. First culture in a 37°C, 5% carbon dioxide incubator, and transfer to a 33°C, 3% carbon dioxide incubator for further culture after the cells grow densely. Culture for 7 days.

[0047] S7. Cell passaging: The remaining 17T300 cells from S5 were passaged into 3CF10 cell factories at a ratio of 1:4.

[0048] S8. Cell passaging: After the 3CF10 cell factory cells grow densely, pass them to two cell tanks (15g mc / tank) at a passaging ratio of 1:4 for microcarrier culture. Set the control parameters: temperature 36.0-37.0℃, pH 7.2-7.6, speed (Motor) 40-60rpm, dissolved oxygen (DO) 50%, continue to culture for 4 days, and replace half of the culture volume of MEM growth medium every day.

[0049] S9. Virus Passaging: S9.1 Harvest the virus supernatant after 7 days of culture, digest the virus-carrying cells, and mix the cell suspension and supernatant to prepare a virus seed-cell suspension.

[0050] S9.2 Digest the cells on the two cell tanks, use a cartridge filter (150μm filter element) to filter the microcarriers and collect the cell suspension, mix the cell suspension with the virus seed-cell suspension, adsorb at 37℃ for 30min, and shake every 10min. After the adsorption, pass the cells evenly into two BC-15L cell tanks (60g mc / tank) for culture. Set the control parameters: temperature 36.0~37.0℃, pH7.2~7.6, speed (Motor) 40~60rpm, dissolved oxygen (DO) 50%, change MEM growth medium 4L every day, change the medium continuously for four days until the cells grow densely, and set the control parameters: temperature 33.0~34.0℃, pH7.2~7.6, speed (Motor) 40~60rpm, dissolved oxygen (DO) 50%.

[0051] S10. Virus harvest: Starting from the fourth day, two 15L culture tanks will each collect 8L of liquid every two days, and add 2% serum growth liquid to the original culture volume. Transfer the collected virus harvest liquid to a 2-8℃ cold storage for temporary storage. Harvest 5 times in total. Harvest all the culture liquid for the fifth time, and no longer add 2% serum growth liquid for cultivation. Mix all the virus harvest liquids to make several working virus strains. Divide into thread-mouth bottles and store in a refrigerator ≤-70℃ to make the working virus strain library.

[0052] The working virus strain library was tested, and the results showed that the sterility test, mycoplasma test, and exogenous viral factor test were in compliance with the regulations, the neutralization index was 1000, the protection index was 1259, and the virus titer was 7.0lgPFU / ml, which met the virus strain requirements of the "Freeze-dried Human Rabies Vaccine (Human Diploid Cells)" in the "Pharmacopoeia of the People's Republic of China" (2020 edition, Volume III).

[0053] The detection of the main virus seed library and the working virus seed library in the embodiments of the present invention are carried out according to the "Virus Seed" detection method under the "Lyophilized Human Rabies Vaccine (Human Diploid Cells)" of the "Pharmacopoeia of the People's Republic of China" (2020 edition, Part III).

[0054] The above description is only a preferred embodiment of the present invention, which is merely illustrative of the present invention and does not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A virus strain, characterized in that: It is deposited in China Center for Type Culture Collection with the accession number CCTCC NO: V202406.

2. Use of the virus strain described in claim 1 in the preparation of rabies vaccine.

3. The use according to claim 2, characterized in that: The rabies vaccine is an inactivated vaccine.

4. A rabies vaccine, characterized in that: The invention comprises the virus strain described in claim 1.

5. The rabies vaccine according to claim 4, characterized in that The virus strain is an inactivated virus strain.

6. The rabies vaccine according to claim 4 or 5, characterized in that Adjuvants are also included.

7. The rabies vaccine according to claim 4 or 5, characterized in that The administration of the vaccine includes intramuscular injection or intradermal injection.

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