JP2011205968A - Method for producing influenza vaccine by cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a viral strain having high proliferation ability suitable for producing an influenza virus vaccine by a cell culture method, and to provide a method for efficiently producing the vaccine using the influenza viral strain.SOLUTION: A method for selecting an influenza viral strain having high proliferation ability is provided, comprising the following process: species of influenza virus, after diluted, is inoculated into TR7 cells to make a culture (first-generation subculture) of the resultant cells; and further, a virus-containing liquid obtained by the culture is diluted and then inoculated into TR7 cells (NITE AP-922) to make a culture of the resultant virus; and the latter procedure (subculture) is repeated several times.

Description

The present invention relates to a method for selecting an influenza virus strain having a high proliferation ability, a method for growing an influenza virus strain obtained by the method by cell culture, a method for producing an influenza virus vaccine obtained by the growth method, and the like.

  From the recent situation, it has become urgent to build a system that can rapidly produce vaccines against highly pathogenic avian influenza, swine influenza, and conventional seasonal influenza.

  In the past, the development of cell lines intended for use in the production of such influenza vaccines has been attempted. For example, in Japanese translation of PCT publication No. 2000-507448 (WO97 / 37001: Patent Document 1), MDCK-33016 strain capable of suspension (floating) culture in a serum-free medium by acclimation from an MDCK cell strain (ATCC CCL34 strain). And virus production methods using the same are described. In addition, US Pat. No. 6,852,036 (Patent Document 2) similarly uses the B-702 strain that is established from the MDCK cell line (ATCC CCL34 line) and can be suspended in a serum-free medium, and the same. Virus production methods are described.

  Furthermore, “Suspension MDCK Cell Culture on EX-CELL MDCK Serum-Free Medium”, SAFC Biosciences Technical Bulletin (2006) (Non-patent Document 1) includes an adherent MDCK cell line (ATCC CCL34) as a serum-free medium. It is described that the cells are adapted to suspension cells by subculture for 6-7 times. Also, Vaccine, Vol.26, pp6852-6858 (2008) (Non-patent Document 2) describes an example in which MDCK-33016 strain is inoculated with A / Kumamoto / 102/2002 (H3N2) to produce virus. ing.

The present inventors also produced a floating TR7 cell line from the MDCK cell line (ATCC CCL34 line) (Reiko Tsutsumi, Shigemi Fujisaki, Masanori Shozushima, Koichi Saito and Shigehiro Sato, Cytotechnology (2006), 52:71. -85 “Anoikis-resistant MDCK cells carrying susceptibilities to TNF-α and verotoxin that are suitable for influenza virus cultivation”: Non-patent document 3).

Currently, influenza virus vaccine strains are selected by public institutions according to the epidemic strains of the year. In recent years, vaccine strains used for the production of highly pathogenic avian influenza vaccines have been limited in number because of their strong pathogenicity. That is, NIBSC supplies three strains as H5N1 vaccine strains. These strains are reassortant viruses (combined binding viruses) with a total of 8 RNAs, 6 genes from the Puerto Rican strain with good egg growth and 2 genes from the H5N1 influenza virus isolated from humans. . Human-derived viral RNA is an NA gene and an HA gene that is directly involved in high pathogenicity. The latter modifies the sequence to reduce virulence. It has been pointed out all over the world that these vaccine strains are suitable for the production of vaccines using conventional embryonated chicken eggs and are not proliferative when infected with cultured mammalian cells.

In order to overcome this reality, Kawaoka et al. Have reported efforts to obtain highly proliferative viruses by modifying the base sequences of six genes derived from Puerto Rico strains (Non-patent Document 4).

JP 2000-507448 US Pat. No. 6,852,036

SAFC Biosciences Technical Bulletin (2006) Vaccine, Vol.26, pp6852-6858 (2008) Cytotechnology (2006), 52: 71-85 Murakami, S. et al., J Virol. 2008 Nov; 82 (21): 10502-9. Epub 2008

  The problem to be solved by the present invention is to select a virus strain having a high growth ability suitable for producing an influenza virus vaccine by a cell culture method, and proliferate the virus strain thus selected using MDCK-derived TR7 cells. For example, to produce vaccines efficiently.

According to the present inventors, the above-mentioned TR7 cell line is a cell that has extremely low tumorigenicity, has a higher influenza virus growth than the parental MDCK cell, and is delayed in cell death due to influenza virus infection. confirmed.

As a result, the present inventor can proliferate the TR7 cells in a large amount and rapidly using a serum-free medium, and more efficiently by adding an appropriate growth factor (such as epidermal growth factor (EGF)). It has been found that more advantages can be obtained by growing and adherent culture. The present inventor has further found out a method capable of selecting a virus strain having a high cell growth ability without performing genetic modification of the virus.

That is, the present invention relates to each aspect shown below.
[Aspect 1] TR7 cells (NITE AP-922) are prepared after diluting the seed influenza virus solution and inoculating and culturing the cells by inoculating TR7 cells (passage 1), and further diluting the virus-containing solution obtained after this culture. A method for selecting an influenza virus strain having a high growth ability by repeating the operation of inoculating and culturing (subculture).
[Aspect 2] The method according to Aspect 1, wherein the influenza virus is H5N1 influenza virus strain or H1N1 influenza virus strain.
[Aspect 3] The method according to Aspect 1 or 2, wherein the subculture is repeated until the second passage, the third passage, or the fourth passage.
[Aspect 4] The method according to any one of claims 1 to 3, wherein the dilution ratio of the virus-containing solution used for each subculture is 100 to 10,000 times.
[Aspect 5] The method according to any one of Aspects 1 to 4, wherein each subculture is performed for 4 to 6 days.
[Aspect 6] An influenza virus strain having a high proliferation ability selected by the method according to any one of Aspects 1 to 5.
[Aspect 7] A method for producing an influenza virus vaccine, comprising inoculating TR7 cells with the influenza virus strain having high proliferation ability according to claim 6 and culturing the cells in a serum-free medium to proliferate the virus.
[Aspect 8] The production method according to Aspect 7, wherein the serum-free medium contains a growth factor.
[Aspect 9] The production method according to Aspect 8, wherein the growth factor is epidermal growth factor (EGF).

  According to the method of the present invention, a virus strain having a high growth ability suitable for the production of an influenza virus vaccine by a cell culture method is selected, and the virus strain thus selected is propagated using MDCK-derived TR7 cells to produce a vaccine. Can be easily and efficiently mass-produced.

Dilute the virus solution in each well of the MDCK E1M1 10 <-4> plate to obtain E1M2 10 <-4> (10 ^-4 ), E1M2 10 <-3> (10 ^-3 ), E1M2 10 <-2 > Indicates the operation to pass to a total of 3 (10 ^-2 ). The graph of the passage result about NIBRG 121 in Example 1 is shown. The graph of the passage result about NIBRG 121 in Example 1 is shown. The graph of the passage result about NIBRG 12 in Example 1 is shown. The graph of the passage result about NIBRG 12 in Example 1 is shown. The graph of the passage result about NIBRG 23 in Example 1 is shown. The graph of the passage result about NIBRG14 in Example 1 is shown. The passage result about the plaque formation ability of NIBRG 14 having high proliferation ability is shown.

In the present invention, the seed influenza virus solution is diluted and then inoculated and cultured in the TR7 cells (passage 1st passage), and the virus-containing solution obtained after this culture is diluted and then inoculated and cultured in the TR7 cells. The present invention relates to a method for selecting an influenza virus strain having a high growth ability by repeating the operation (passage culture) several times.

For TR7 cells, MDCK cells (CCL 34) purchased from ATCC are cultured for 6 months in Eagle's MEM (Gibco) medium containing 50 μg / ml MEP (metalloendopeptidase: derived from Streptomyces griseus) (twice a week). The floating cells that survived the medium change were named 6M-4. This is a cell line prepared by culturing 6M-4 cells in 0.3% soft agar (Difco Agar Noble) medium (MEM) and selecting colonies that had grown after 10 days.

  The above TR7 cells are dated March 30, 2010, 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818, Japan. And has been given the receipt number NITE AP-922.

Influenza virus is not particularly limited, but in addition to seasonal influenza, H5N1 influenza virus strain (bird type) such as NIBRG12, NIBRG14 and NIBRG 23 strain or H1N1 influenza virus strain (swine type) such as NIBRG 121 strain I can list them.

Seed influenza virus solution can be obtained by any method known to those skilled in the art, such as inoculating the chorioallantoic cavity of an embryonated chicken with an influenza virus vaccine strain available from a public organization such as NIBSC (National Institute for Biological Standards and Control). Can be prepared (choremic fluid).

  The influenza virus strain having a high growth ability refers to a cell strain having, for example, 128 or more, preferably 256 or more, more preferably 512 or more as a value indicated by the HA titer described below.

The dilution ratio of the virus-containing solution (culture solution) used for each subculture can be appropriately determined by those skilled in the art according to the type of virus, but is usually about 100 times to about 10,000 times, and is a high dilution. Rate is preferred. In addition, it is not necessary that the dilution ratios of the virus-containing liquid used for the subculture of each generation are the same, and can be adjusted as appropriate.

Conditions for each subculture and each operation can be carried out according to any method well known to those skilled in the art (for example, the method described in Non-Patent Document 3). Reach. It is preferable to repeat such subculture until the second passage, the third passage, or the fourth passage.

By carrying out the above method, an influenza virus strain having a high growth ability as described in the examples of the present specification can be selected. Therefore, the present invention also relates to a method for producing an influenza virus vaccine, which comprises inoculating such an influenza virus strain and TR7 cells with the influenza virus strain and culturing the cell in a serum-free medium to proliferate the virus. It is concerned.

Virus inactivation (β-propiolactone, UVC irradiation, etc.) itself in cell culture methods and vaccine production can be carried out by any method known to those skilled in the art. For example, a growth factor such as epidermal growth factor (EGF) can be included in the serum-free medium. Furthermore, any culture method known to those skilled in the art such as suspension culture method, microcarrier culture method, and adhesion (attachment) culture method can be used.

  The present invention will be described in detail with reference to the following examples, which are merely provided for explaining the present invention. Accordingly, these examples do not limit or limit the scope of the invention disclosed herein. It is easily understood by those skilled in the art that various embodiments based on the technical idea described in the claims can be made in the present invention.

experimental method
Virus
Three H5N1 (bird type) influenza virus vaccine strains NIBRG12, NIBRG14 and NIBRG 23 strains purchased from NIBSC (National Institute for Biological Standards and Control) and one type of H1N1 (swine type) influenza virus vaccine strain NIBRG121 were used.

Primary growth of virus in embryonated eggs <br/> Incubated eggs (10-day eggs) were purchased from Koiwai Farm (Iwate Prefecture), and the virus stock solution (E0) was added to PBS (-) (phosphate buffer (Mg, Ca-free). 200 μl was inoculated into the chorioallantoic cavity. NIBRG12, NIBRG14 and NIBRG 23 were inoculated with 5 of each virus. For NIBRG121 strain, 100 μl of double dilution was inoculated into one egg. Forty-eight hours after inoculation, the eggs were transferred to a refrigerator and left overnight for more than one hour to stop tissue hardening and infection. The chorioallantoic fluid was collected and those inoculated with the same virus were collected and used as a virus pool solution (E1).

Measurement of virus concentration The chicken preserved blood (Nippon Biotest Laboratories) is washed three times with PBS (-) (1,600 rpm for 10 minutes). The leukocyte layer on the erythrocyte surface is removed at each centrifugation. Take 1 ml of blood cell pellet and add to 200 ml of PBS (-) to make 0.5% (V / V) erythrocyte suspension. Then, the blood cell count is measured with a blood cell counter (NIHON KOHDEN), adjusted to 3 × 10 ⁷ RBC / ml, and used for the test. Add 50 μl of PBS (-) to each well of the 96-well U-shaped plate, and add 50 μl of the test virus solution to A1, B1, C1, D1, E1, F1, G1, H1. Make a 2-fold dilution series while mixing from each row 1 to 11, and discard 50 μl after stirring 11 holes. 12 holes are for control. Add 50 μl of erythrocyte suspension to each well, and then stir for 30 seconds on a horizontal shaker. Visually observe after 1 to 2 hours. Record the position of the positive agglutination hole, and use the specimen dilution factor in that hole as the HA value and virus concentration. That is, if the positive reaction is up to A2, the HA value is 4, and if up to A5, it is 32.

Plaque formation test
Culture 6-well microplate cells and conduct infection experiments on the day of confluence. Agarose (DIfco Agarose Noble, BD) 2% (W / V) Aqueous solution is autoclaved in advance and placed in a 45 ° C water bath. Place 2 × MEM containing trypsin (SIGMA 93630, 2 μg / ml) in a 37 ° C. water bath. Make a 10-fold dilution series of virus in trypsin-containing MEM. Rinse the cell culture with PBS (-) once. Add 250 μl of each diluted virus solution to the cells and place in a 37 ° C. CO ₂ thermostat. Stir the microplate every 15 minutes. After 90 minutes, mix an equal amount of the agarose solution and 2xMEM that have been kept warm above, add some 3 ml to each well after cooling down and place at 25 degrees. When the agarose has hardened, place it in a 37 ° C CO ₂ constant temperature bath and leave it for 7 days. After 7 days, add 10% formalin solution containing 1% gentian purple and leave for 2 days. Rinse the formalin solution and gel with water.

Preparation The NIBRG12, NIBRG14, NIBRG23 and NIBRG121 virus solutions (E0) purchased from NIBSC to make seed virus stock solutions were diluted and inoculated into hatched chicken eggs, respectively, as described above. The collected virus solution was designated as a seed influenza virus solution (E1). As a result of measuring HA titer of E1 using chicken erythrocytes, it was 512 (NIBRG12), 512 (NIBRG14), 256 (NIBRG23), and 1024 (NIBRG121) (positive control: HA titer of diagnostic antigen 512). These were values that could be judged to be sufficiently high titer virus solutions.

Procedure (1) After seeding MDCK and TR7 cells in a 12-well microplate (about 5 × 10 ⁴ / ml) and making them confluent according to a standard method known to those skilled in the art (for example, the method described in Non-Patent Document 3), After removing the medium and washing with physiological saline, it proceeded to the next operation.
(2) A 10-fold virus dilution series solution (MEM-trypsin) of E1 was prepared, and 1 ml of 10 ⁻² , 10 ⁻³ , and 10 ⁻⁴ dilutions were added to each plate as illustrated. From the first passage, the ones with the least amount of inoculated virus are E1M1 (-4), E1M1 (-3), E1M1 (-2), and E1T1 (-4), E1T1 (-3), E1T1 (- 2).
(3) On the 4th or 6th day, the infection progressed, and the virus production did not increase even if the retention days were increased. At that time, a part of the culture solution was collected for measurement of HA value.
(4) simultaneously diluting the virus solution of each well in the MDCK and TR7 cells prepared above for passaging second generation, ^10-2 (10 <-2>), ^10-3 (10 <-3>), 10 ^-4 (10 <-4>) was inoculated (Fig. 1). Specifically, 96-well plates were used to prepare 100-fold diluted solutions of 24 wells of E1M1 ^10-4 . E1M2 (-4 to -4) and E1M2 (-4 to -3) were respectively added to the 24-well cells to which 1 ml of MEM-trypsin solution had been added in advance. To E1M2 (-4 to -2), 10 μl was transferred from each hole of the E1M110 ^-4 plate.
(5) The same operation was performed on MDCK and TR7 cells prepared for the third passage.
Table 1 shows an image of each operation described above. Moreover, the result obtained by the above operation is shown in FIGS.

From the results shown in these figures, the virus production of all virus strains was low in MDCK and TR7 cells at the first passage, and did not reach the HA value of E1 solution at all. In comparison, the amount of virus production in TR7 cells was high, and it was found that there was a difference in mean values (horizontal line: significant in statistical processing in the figure) (FIG. 2, FIG. 4, FIG. 6, FIG. 7). ). In addition, it was found that with E1M2, a virus solution with a medium HA value becomes low when subcultured to E1M3 (FIGS. 3 and 5).

Selection of highly proliferative cell lines of influenza virus A / H5N1 vaccine strain NIBRG14: Evaluation results by HA titer (Fig. 7) are shown in Table 2 (comparison between passage 1 and passage 2) and Table 3 (passage). Comparison between the 1st generation and the 3rd generation). Each value is the mean (Mean), standard deviation (SD), minimum value (Min), maximum value (Max), t-test significance test (Unpaired t-test), and F Distribution test (F) is shown. Here, it seems that the fact that the average value of yield is higher in TR7 than MDCK and that there is a significant difference between the two groups reflects the high productivity of the TR7 strain. In addition, it should be noted that there is a significant difference between MDCK and TR7 in the F distribution, which is a numerical value indicating the degree of variance (the distance from the average value). In statistical processing, there is a qualitative difference between the two groups (the cells themselves), which means that the degree of dispersion of the measurement is not considered a coincidence. Therefore, it is considered that a highly proliferative virus could be efficiently selected only by using TR7 cells.

  The hyperproliferative virus strains selected by the method of the present invention showed cell growth as compared with the purchased NIBRG14 (E0) grown in embryonated chicken eggs by the present inventors (E1), as shown by the plaque forming ability (FIG. 8). The sex is much greater. Since the present inventor has determined the gene sequence of the high-growth strain and confirmed that there is no mutation in the sequence corresponding to the HA and NA proteins involved in antigenicity as a vaccine strain, Sex virus strains are considered very useful as vaccine strains.

Influenza vaccine can be easily and efficiently produced in large quantities by mass-producing and rapidly growing influenza virus strains selected by the method of the present invention with MD7-derived TR7 cells. It becomes.

Claims (9)

Inoculate TR7 cells after diluting the seed influenza virus solution (passage 1st generation), and further inoculate TR7 cells (NITE AP-922) after diluting the virus-containing solution obtained after this culture. A method of selecting an influenza virus strain having a high growth ability by repeating the operation of culturing (subculture). The method according to claim 1, wherein the influenza virus is an H5N1 influenza virus strain or an H1N1 influenza virus strain. The method according to claim 1 or 2, wherein the subculture is repeated until the second passage, the third passage, or the fourth passage. The method as described in any one of Claims 1-3 whose dilution rate of the virus containing liquid used for each subculture is 100 times-10,000 times. The method according to any one of claims 1 to 4, wherein each subculture is performed for 4 to 6 days. An influenza virus strain having a high proliferation ability selected by the method according to any one of claims 1 to 5. A method for producing an influenza virus vaccine, comprising inoculating TR7 cells with the influenza virus strain having a high proliferation ability according to claim 6 and culturing the cells in a serum-free medium to proliferate the virus. The production method according to claim 7, wherein the serum-free medium contains a growth factor. The production method according to claim 8, wherein the growth factor is epidermal growth factor (EGF).