A foot-and-mouth disease virus and its application

Abstract

The invention discloses a foot-and-mouth disease virus and its application. It relates to the technical field of vaccines. The deposit number is CCTCCNO: V202293. It also provides an immune preparation based on the foot-and-mouth disease virus and a preparation method. The invention provides a relatively stable foot-and-mouth disease virus strain that is both heat-resistant and acid-resistant, thereby improving the antigen stability and thus the stability and effectiveness of the vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, and more particularly to a foot-and-mouth disease virus and application thereof. Background Art

[0002] At present, foot-and-mouth disease is a highly contagious disease that is highly contagious. Once it breaks out, it will bring devastating losses to the breeding industry, so the disease is listed as a Class I infectious disease in the world. The best prevention and control measure for this disease is immunization, so this disease is listed as one of the compulsory immunization diseases in my country. At present, the vaccines on the market are mainly inactivated foot-and-mouth disease vaccines, and safety, efficiency, uniformity and stability are the characteristics that vaccines must have. Stability is crucial for vaccines, and the stability of vaccines mainly depends on the integrity and stability of the antigens.

[0003] Foot-and-mouth disease virus is a non-enveloped virus that is not sensitive to organic solvents, but is sensitive to temperature and acidity. When the temperature rises or the pH changes, the virus is prone to lysis, which leads to a decrease in the content of the active ingredient-antigen in the vaccine, and ultimately leads to a decrease in the effectiveness of the vaccine, and fails to achieve the expected immune effect. Therefore, the key to improving the stability of the vaccine is to improve the stability of the vaccine strain, thereby improving the quality of the vaccine.

[0004] Therefore, whether a suitable foot-and-mouth disease virus can be provided is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a foot-and-mouth disease virus and its application. Through a heat-resistant and acid-resistant domestication and breeding method, a foot-and-mouth disease virus strain that is relatively stable to temperature and acidity is bred, and is also suitable for the domestication and breeding of foot-and-mouth disease viruses of various serotypes.

[0006] Preservation information: Foot and mouth disease virus OJMS-S (Foot and mouth disease virus OJMS-S) is deposited in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: V202293, and the preservation date is December 22, 2022.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The invention discloses a foot and mouth disease virus OJMS-S (Foot and mouth disease virus OJMS-S), with the deposit number being CCTCC NO: V202293.

[0009] The present invention also provides an immune preparation of the foot-and-mouth disease virus OJMS-S.

[0010] Preferably, the immune preparation is a vaccine.

[0011] Preferably, an adjuvant is also included.

[0012] Preferably, the adjuvant is 206 adjuvant.

[0013] The present invention also provides a preparation method based on the above vaccine, wherein the mass ratio of the purified antigen of the foot-and-mouth disease virus to the 206 adjuvant is 1:1, and the vaccine is prepared by emulsification at 30° C. for 30 minutes.

[0014] Through the above technical solutions, it can be known that compared with the prior art, the present invention discloses a foot-and-mouth disease virus and its application, and the technical effect achieved is to provide a relatively stable foot-and-mouth disease virus strain that is both heat-resistant and acid-resistant, improve the antigen stability, and thus improve the stability and effectiveness of the vaccine. According to the comparative experimental data of the antigen storage period, the selected strain was stored at 37°C for 36 hours, and 3% of the antigen had not yet been degraded; while the original strain was stored for 24 hours, and the antigen was completely degraded; the selected strain was stored at room temperature for 60 days, and it was not completely degraded, while the unselected strain was 100% degraded for 30 days; under 4°C storage, the selected strain degraded slower than the unselected strain. In the immunogenicity comparison experiment, compared with the O-type strain OJMS in the currently common foot-and-mouth disease O-type inactivated vaccine (OJMS strain), the r value was 0.9, and the selected strain also had good immunogenicity. The vaccine was stored for 15 months, and the degradation rate of the effective antigen content of the selected strain was slower than that of the current vaccine strain. The vaccine virus and the selected strain were placed at 37°C and pH 5.0 overnight. The vaccine strain was almost completely inactivated, but after the selected strain was diluted 10,000 times, surviving viruses could still be detected. The normal culture temperature of foot-and-mouth disease virus is 37°C and the pH is 7.6-7.8. The selected strain and the vaccine strain are now continuously passaged at 38.5°C and pH 6.5. The selected strain can be stably passaged, and the pathological time is gradually shortened and the virus value is increased; while the vaccine strain cannot be continuously passaged. The above comparative experimental data prove that the strains bred using this technology have obvious improvements and advantages in heat and acid stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1 The accompanying drawing is a schematic diagram comparing the storage effects of antigens provided by the present invention at 37°C.

[0017] Figure 2The accompanying drawing is a schematic diagram comparing the room temperature storage effects of antigens provided by the present invention.

[0018] Figure 3 The accompanying drawing is a schematic diagram comparing the effects of 4°C storage of antigens provided by the present invention.

[0019] Figure 4 The accompanying drawing is a comparative schematic diagram of the neutralizing antibodies provided by the present invention.

[0020] Figure 5 The accompanying figure is a schematic diagram of the comparison of 146S antigen mass concentration during the shelf life of the vaccine provided by the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The embodiment of the invention discloses a foot-and-mouth disease virus and application thereof.

[0023] Example 1

[0024] (1) Venom heat treatment See Table 1: Take the venom of foot-and-mouth disease type OJMS strain and dispense it into 15 ml centrifuge tubes, 3 ml of venom in each tube, for a total of 6 tubes, and place them in a 56°C or 60°C water bath for 30 min, 45 min, and 60 min, respectively.

[0025] Table 1 Virus value after strain treatment (10 n TCID 50 / 0.1ml)

[0026]

[0027] (2) Virus strain passaging See Table 2: Take the T75 BHK21 cells that have grown into a monolayer after treatment in (1) and pass them at a ratio of 1% to 3%. When the cell lesions reach 80% to 90%, harvest them; if the cells do not show typical lesions, harvest them blindly after 72 hours. After freezing and thawing the harvested venom twice, continue to pass them until the lesions, harvest time and venom titer return to the original level.

[0028] Table 2 Subculture after the first heat treatment

[0029]

[0030] (3) Heat-treat and subculture the venom F3 obtained in (2) for the second time according to the methods in (1) and (2); perform this treatment at least three times. The data are shown in Tables 3 and 4.

[0031] Table 3 Subculture after the second heat treatment

[0032]

[0033] Table 4 Subculture after the third heat treatment

[0034]

[0035] (4) Shake flask breeding (see Table 5): The strains that were subcultured after the last heat treatment until they recovered to their original levels were subjected to shake flask experiments. A 250 ml shake flask was inoculated and the cell density was greater than or equal to 6.0 × 10 6 The viability was above 90%, the inoculation ratio was 0.5%, and the lesions reached more than 80%. The samples were taken and the toxin titer was determined. The remaining venom was inactivated with BEI at 30℃ for 28h, and the samples were taken to determine 146S. The strains with higher toxin titer and 146S were selected, that is, treated at 56℃ for 30min.

[0036] Table 5. Shake flask test results after heat treatment

[0037]

[0038] (5) First acid treatment: Take the strain selected in (4) and perform acid treatment. Take an appropriate amount of maintenance solution to adjust the pH to 5.0 and dispense into 15 ml centrifuge tubes, 9 ml per tube, and dispense into 7 tubes; take 7 ml of the venom to be treated and add it to 7 tubes containing 9 ml of pH = 5.0 maintenance solution, 1 ml per tube, 3 of which are placed in a 37°C incubator for treatment for 30 min, 60 min and 120 min respectively; the remaining 4 tubes are placed at 4°C for treatment for 2 h, 8 h, 17 h and 24 h respectively. After treatment, adjust the pH of the liquid in the tube to 7.6 and set aside.

[0039] (6) Virus strain passaging: The venom treated in (5) was inoculated into a confluent monolayer of BHK21 cells for passaging at a ratio of 1% to 3%. When the cell lesions reached more than 80%, the cells were harvested; if the cells did not show typical lesions, they were harvested blindly after 72 hours. The harvested venom was frozen and thawed twice, and then continued to be passaged until the lesions, harvest time and venom titer returned to the original levels.

[0040] Table 6. Subculture after the first acid (pH = 5.0) treatment

[0041]

[0042] (7) Second acid treatment (see Tables 7 and 8): Take the strains and F3 generations that have recovered to the original level after the acid treatment in (6), and perform a second treatment and passaging according to the methods in (5) and (6); this treatment should be repeated at least three times.

[0043] Table 7 Subculture after the second acid (pH = 5.0) treatment

[0044]

[0045] Table 8 Passaging after the third acid (pH = 5.0) treatment

[0046]

[0047] (8) Shake flask breeding: The strains that were subcultured after the third acid treatment until they recovered to their original levels were subjected to shake flask experiments. A 250 ml shake flask was inoculated and the cell density was greater than or equal to 6.0 × 10 6 The viability was above 90%, the inoculation ratio was 0.5%, and the lesions were harvested when they reached more than 80%, and samples were taken to determine the venom titer; the remaining venom was inactivated with BEI at 30℃ for 28h, and samples were taken to determine 146S. The strains with higher venom titer and 146S were selected.

[0048] Table 9 Shake flask verification results after acid (pH = 5.0) treatment

[0049]

[0050] (9) Directed cloning screening: The strains with high yield verified by shake flask in (8) (4℃2h and 4℃24h) were subjected to directed cloning screening; the original strain was used as a control and cloned under the same conditions. The venom was diluted 10 times and 10 -2 , 10 -3 , 10 -4 , 10 -5 1 mL of 4 titer dilutions were added to a 6-well plate of BHK21 cells that had grown a monolayer, and adsorbed for 1 hour. After adsorption, the adsorption solution was discarded, and 2.5 mL of agar medium with a pH of 6.5 was preferably added to each well. After the medium solidified, it was inverted in CO2 culture, and the temperature was preferably set to 38.5°C. After 48 to 72 hours of culture, the virus strain selected under the conditions of 48 hours of culture, 4°C for 2 hours, and 4°C for 24 hours showed plaques, and the original virus strain had no plaques after 72 hours of culture.

[0051] (10) Picking plaques: The plaques picked in (9) are propagated by adherent cytotoxic subculturing. The pH of the maintenance medium is preferably 6.5 and the culture temperature is preferably 38.5°C during subculturing; subculturing is continued until the sixth generation of cloning, i.e., K1F6.

[0052] Table 10 Continuous passage of cloned viruses at 38.5°C pH = 6.5

[0053]

[0054] (11) Reactor scale-up (see Table 10): Take the K1F7 venom from the directed cloned virus in (10) and inoculate it into a 14 L reactor with a cell density of 6.0×10 or more. 6 , the viability is above 90%, the inoculation ratio is 0.5% to 1%, the inoculation volume is 5L, the dissolved oxygen is controlled at about 40%, the temperature is controlled at 37°C, and the pH is maintained at about 7.6. When the cell pathological rate reaches 80-90%, harvest and take samples to test the virus titer and 146S. If the test results are not lower than the current level, continue to conduct immunogenicity and vaccine shelf life experiments; otherwise, continue to select and breed according to the above method until a qualified strain is selected, and the selected qualified strain is named OJMS-S.

[0055] Deposit of patent procedures.

[0056] Table 11. 14L reactor validation results after cloning

[0057]

[0058] (12) Preparation of purified antigen prepared from OJMS-S: The venom of OJMS-S (4°C for 24 h) selected in (11) was inactivated by inactivating with BEI at 30°C for 28 h; the inactivated solution was centrifuged, the precipitate was discarded, and PEG-6000 (purchased from Sinopharm) with a final concentration of 7% was added, and the solution was placed on a magnetic stirrer and stirred at 4°C for 4 h, and allowed to stand overnight; the solution was centrifuged, the precipitate was taken, and resuspended with a phosphate buffer of pH 7.6 to 7.8, and concentrated 10 times; 3% chloroform (purchased from Sinopharm) was added to the 10-fold concentrated solution, and after thorough mixing, the solution was centrifuged, the precipitate was removed, and the supernatant was taken. This supernatant was the purified and concentrated OJMS-S antigen.

[0059] Comparative experiment 1

[0060] Antigen preservation period experiment: The (10-fold purified and concentrated) antigen of OJMS-S was placed at 37°C, room temperature, and 4°C, and samples were taken regularly to detect 146S; at the same time, an untreated strain (original strain OJMS, the same below) was set as a control, and stored and sampled in the same way as the preserved OJMS-S, and 146S was measured.

[0061] The stored test data in Tables 12 to 14 and Figures 1 to 3 It can be seen that the strain OJMS-S selected after heat and acid resistance treatment degrades more slowly than the original strain OJMS under the storage conditions of 37°C, room temperature (20-25°C), and 4°C. At 37°C, 16.8% of the antigens were still not degraded after 24 hours, while the original strain was completely degraded; after 30 days of storage at room temperature, the original strain was completely degraded, but the selected strain was not completely degraded; at 4°C, the degradation rate of the original strain was higher than that of the selected strain.

[0062] Table 12. Antigen shelf life test results (37°C)

[0063]

[0064] Table 13. Antigen storage period test results (room temperature: 20-25°C)

[0065]

[0066] Table 14. Antigen shelf life test results (4°C)

[0067]

[0068] Comparative test 2

[0069] Immunogenicity experiment: The purified antigen prepared from OJMS-S in (12) was emulsified at 30°C for 30 min in a mass ratio of 1:1 between the antigen and 206 adjuvant (purchased from Sepico, France) to prepare a vaccine; at the same time, the antigen prepared from the O-type strain OJMS in the currently commonly used foot-and-mouth disease O-type inactivated vaccine (OJMS strain) was prepared into a vaccine in the same way. The antigen content of the vaccine was 5 μg / 2 ml. Five rabbits were immunized with the two vaccines, each vaccine was injected into the leg muscle, and 1 ml was injected each time. The antibody changes in the rabbits were monitored 14 days, 21 days, and 28 days after immunization, and the cross-neutralization results of the strains before and after treatment were determined with the sera of 28 days, and the parentage relationship between the strains after treatment and the strains before treatment, i.e., the r value, was calculated. The r value neutralization experiment was repeated at least three times.

[0070] Note: The r value is the serological correlation coefficient between the strain to be tested and the reference strain. It is a one-way relationship value that reflects the degree of cross-reaction of the antiserum of one strain to the antigen of another strain). Its purpose is to compare the antigenic relationship between the strain to be tested and the reference strain. The closer the r value is to 1, the closer the antigenic relationship between the strains is; conversely, the more distant the antigenic relationship is, the greater the difference between the strains. Calculation of r value: The relationship between the strain to be tested and the reference strain, that is, the formula for the r value is:

[0071] r = the reciprocal of the titer of the reference serum against the strain to be tested / the reciprocal of the titer of the reference serum against the reference strain

[0072] From the experimental data in Tables 15 and 16, Figure 4 It shows that the immunogenicity of the selected strains is comparable to that of the original strains, and the antigenic relationship between the strains is very close, indicating that although the stability of the strains has been improved, the immune response effect of the antigen has not been changed.

[0073] Table 15 Antibody monitoring in immunogenicity experiments

[0074]

[0075] Table 16 r values ​​of selected strains and unselected strains

[0076]

[0077]

[0078] Comparative experiment 3

[0079] Vaccine shelf life verification: The vaccine obtained in comparative experiment 2 was stored at 2-8°C for 1, 3, 5, 7, 9, 11, 12, and 15 months, and the antigen content 146S test was performed to verify the stability of the vaccine. The results are shown in Table 17. Figure 5 , indicating that the effective content of antigen in the vaccine made from the selected strains and stored in the refrigerator for 15 months is higher than that of the vaccine strains.

[0080] Table 17 Results of antigen content during vaccine shelf life

[0081]

[0082] Comparative test 4

[0083] The strain (OJMS-S) obtained by breeding was placed at 37℃ and pH 5.0 overnight, and the vaccine strain (OJMS) was used as a control and treated in the same way. After treatment, the virus titer was measured, and the results showed that the virus titer of the selected strain (OJMS-S) was significantly higher than that of the vaccine strain (OJMS). Thus, a foot-and-mouth disease strain that is both heat-resistant and acid-resistant was obtained.

[0084] Table 18 Virus testing results when the strains were placed at 37°C and pH 5.0

[0085]

[0086] Comparative experiment 5

[0087] The currently commonly used foot-and-mouth disease O-type inactivated vaccine (OJMS strain) OJMS was continuously passaged at a temperature of 38.5 and a pH of 6.5. The passage results in Table 19 show that obvious lesions were produced in the first passage, the lesion time was prolonged in the second passage, and there was no obvious lesion in the third passage, and the virus titer gradually decreased, indicating that this strain cannot replicate and survive in a hot and acidic environment. Compared with the passage results in Table 10, OJMS-S is more heat-resistant and acid-resistant than OJMS.

[0088] Table 19 Vaccine strains continuously passaged at 38.5°C pH = 6.5

[0089]

[0090]

[0091] The invention obtains a high-quality strain OJMS-S, and through comparison and verification, the heat and acid resistance of the selected strain is higher than that of the vaccine strain OJMS, thereby obtaining a heat-resistant and acid-resistant foot-and-mouth disease strain. The strain can be further prepared into related preparations and vaccines.

[0092] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0093] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A foot and mouth disease virus OJMS-S (Foot and mouth disease virus OJMS-S), characterized in that: The deposit number is: CCTCCNO: V202293.

2. Use of the foot-and-mouth disease virus OJMS-S according to claim 1 in preparing vaccines.

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