Bivalent vaccine as well as preparation method and application thereof

Abstract

The invention provides a bivalent vaccine which comprises a rabbit hemorrhagic syndrome virus antigen, a rabbit hepatitis E virus antigen and a microbial freeze-dried slow-release agent, and provides a preparation method which comprises the following steps: culturing the rabbit hepatitis E virus by using passage cells, obtaining the rabbit hepatitis E virus antigen, inoculating the rabbit hemorrhagic syndrome virus, and carrying out freeze-drying on the rabbit hemorrhagic syndrome virus. A rabbit hemorrhagic syndrome virus antigen is obtained, the rabbit hemorrhagic syndrome virus antigen and a rabbit hepatitis E virus antigen are mixed in proportion, a freeze-drying protective agent is supplemented, and the bivalent vaccine is obtained. The invention further provides an application experiment result of the compound, and the compound is used for preventing or treating rabbit hemorrhagic syndrome and rabbit hepatitis E. According to the invention, the two antigens are proportioned properly, so that the prepared vaccine at least achieves the efficacy of preventing and controlling immune epidemic diseases. The bivalent vaccine provided by the invention can prevent two diseases by one injection, reduce immune stimulation, continuously enhance the immune effect of a body, promote the body to generate better immune response, reduce the immune cost, simplify the immune procedure, and is more economical and reliable.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal vaccines, and particularly relates to a bivalent vaccine and a preparation method and application thereof. BACKGROUND

[0002] Rabbit hemorrhagic disease (RHD) is an acute, highly infectious and highly lethal disease caused by rabbit hemorrhagic disease virus (RHDV), characterized by respiratory system hemorrhage, parenchymal organ edema, congestion and hemorrhage, which brings huge economic losses to the rabbit industry and was once a devastating infectious disease of rabbits and attracted much attention. The disease was first discovered in Jiangyin County, Jiangsu Province, China in 1984, and then spread to many provinces, cities and autonomous regions. So far, it has occurred in North Korea, India, Lebanon, Mexico, Cameroon, Austria, Belgium, Czech Republic, Denmark, France, Germany, Greece, Luxembourg, Netherlands, Poland, Spain, Sweden, Switzerland, etc. The disease often presents an outbreak, with a very high morbidity and mortality rate. The pathogenic rate of susceptible animals is as high as 90%, and the mortality rate is as high as 100%, which is one of the most serious diseases in the rabbit industry. There is no effective treatment for RHDV infection so far, and the only available treatment measures are vaccine vaccination and symptomatic treatment.

[0003] Hepatitis E (HE) is an acute infectious disease caused by HEV (Hepatitis virus, HEV) through fecal-oral route, and human infection with HEV presents acute jaundice hepatitis, liver decompensation, and serious harm to human health. HE has a high mortality rate, and shows an increasing trend compared with other types of viral hepatitis, and various animal infections have been reported. In 2009, rabbit HEV strains were first discovered and isolated in Gansu, China, and the rabbit HEV gene was sequenced, showing that the positive rate of rabbit infection with HEV was high; the United States, France and other countries also found that rabbits were infected with HEV, and the phenomenon showed an increasing trend year by year. In addition, rabbit HEV antibodies can be detected by human HEV antigen, suggesting that rabbit HEV may contain common antigen epitopes with known genotypes of HEV, and it is speculated that rabbit HEV may pose a serious threat to human health. Therefore, it is urgent to develop a rabbit HEV vaccine to prevent and control the occurrence and spread of the disease, reduce the possibility of animal disease and improve the quality of animal meat, which is more conducive to protecting human health and life safety.

[0004] Currently, the only commercially available vaccine for preventing and controlling rabbit diseases is the inactivated RHDV vaccine; there are no rabbit HEV vaccines or bivalent vaccines combining RHDV and rabbit HEV. Clinically, preventing these two animal diseases requires separate injections, which is not only labor-intensive and costly, but also causes greater stress to the animals with repeated immunizations, necessitating spaced immunizations. Therefore, developing a bivalent RHDV and rabbit HEV vaccine would be beneficial for preventing and controlling the occurrence and spread of animal diseases, ensuring the quality of animal products, and protecting human health.

[0005] The materials used to prepare sustained-release vaccines exhibit good biocompatibility and are non-toxic. Upon release into the body along with the vaccine, they stimulate the body's immune response without producing harmful metabolites, demonstrating excellent safety. They combine adjuvant and antigen delivery functions, enhancing the body's immune response through dual regulation. Furthermore, they avoid the side effects commonly associated with other adjuvants. With numerous advantages such as good biocompatibility, flexible antigen delivery and targeted release, promotion of antigen uptake and presentation, and enhanced cellular immune responses, they have attracted considerable attention. Summary of the Invention

[0006] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a method for preparing a bivalent vaccine. This invention prepares a vaccine capable of preventing and controlling two diseases by achieving a suitable ratio of two antigens. Furthermore, the bivalent vaccine preparation method of this invention is simple, the vaccine has high immunogenicity, and vaccination is convenient and quick. Compared with existing technologies that require at least two injections to prevent or treat the two diseases, this invention achieves dual protection with a single injection, reduces immune stimulation, promotes a better immune response, lowers vaccination costs, saves immunization time, and is more economical, convenient, and reliable.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a bivalent vaccine, comprising the following components: rabbit hemorrhagic syndrome virus antigen, rabbit hepatitis E virus antigen, and vaccine freeze-drying protectant; The rabbit hemorrhagic syndrome virus antigen contains the rabbit hemorrhagic syndrome standard virus strain NJ-2009, GenBank sequence number HM623309.1; The rabbit hepatitis E virus antigen contains rabbit hepatitis E virus JSBL220, with GenBank sequence number JQ953664.

[0008] Preferably, the content of the rabbit hemorrhagic syndrome virus antigen is ≥10. 8 HA / Head portion.

[0009] Preferably, the content of the rabbit hepatitis E virus antigen is ≥10. 7 TCID 50 / Toufen.

[0010] Preferably, the content of the rabbit hemorrhagic syndrome virus antigen and the rabbit hepatitis E virus antigen are both ≥50 rabbit infection doses / dosage, and each dosage contains 0.01 mL of viral fluid.

[0011] The present invention also provides a method for preparing the above-mentioned bivalent vaccine, the method being: Rabbit hepatitis E virus was cultured using passaged cells to obtain rabbit hepatitis E virus antigen. Rabbit hemorrhagic syndrome virus was then inoculated to obtain rabbit hemorrhagic syndrome virus antigen. The rabbit hemorrhagic syndrome virus antigen and the rabbit hepatitis E virus antigen were mixed in a certain proportion and supplemented with a freeze-drying protectant to obtain a bivalent vaccine.

[0012] Preferably, the volume ratio of the rabbit hemorrhagic syndrome virus antigen to the rabbit hepatitis E virus antigen is 1:10.

[0013] The present invention also provides the application of the above-mentioned bivalent vaccine for the prevention or treatment of rabbit hemorrhagic syndrome and rabbit hepatitis E.

[0014] Compared with the prior art, the present invention has the following advantages: In the bivalent vaccine of this invention, by achieving a suitable ratio of the two antigens, the vaccine efficacy is at least equal to that of the original single vaccine. Furthermore, the preparation method of the bivalent vaccine of this invention is simple, the vaccine has a high potency, and immunization is convenient and quick. Compared with existing technologies that require multiple immunizations, at least two injections to prevent the above two diseases, this invention achieves dual protection with a single injection, reduces immune stimulation, promotes a better immune response, lowers immunization costs, saves immunization time and manpower, and is more economical, convenient, and reliable.

[0015] The present invention will be further described in detail below with reference to the embodiments. Detailed Implementation

[0016] Example 1 The bivalent vaccine of this embodiment includes the following components: rabbit hemorrhagic syndrome virus antigen, rabbit hepatitis E virus antigen, and vaccine freeze-dried protectant; The rabbit hemorrhagic syndrome virus antigen contains the rabbit hemorrhagic syndrome standard virus strain NJ-2009, GenBank sequence number HM623309.1; deposited at the China Institute of Veterinary Drug Control. The rabbit hepatitis E virus antigen contains rabbit hepatitis E virus JSBL220, GenBank sequence number JQ953664; deposited at the U.S. Centers for Microbial Culture Collection. The content of the rabbit hemorrhagic syndrome virus antigen is ≥10. 8 HA / Toufen; The content of the rabbit hepatitis E virus antigen ≥ 10 7 TCID 50 / dose; The contents of the rabbit hemorrhagic syndrome virus antigen and the rabbit hepatitis E virus antigen are both ≥ 50 rabbit infectious doses / dose, and each dose contains 0.01 mL of virus venom.

[0017] This embodiment also provides a method for preparing the above-mentioned bivalent vaccine, and the method is as follows: Use the passaged human hepatoma cell line (PLC / PRF / 5) to culture the rabbit hepatitis E virus to obtain the rabbit hepatitis E virus antigen, inoculate the rabbit hemorrhagic syndrome virus to obtain the rabbit hemorrhagic syndrome virus antigen, mix the rabbit hemorrhagic syndrome virus antigen and the rabbit hepatitis E virus antigen according to a volume ratio of 1:10, and supplement with a freeze-drying protectant to obtain the bivalent vaccine.

[0018] Continuously prepare three batches of vaccines, and the batch numbers are successively 101, 102, and 103. Among them, the content of the rabbit hemorrhagic syndrome virus in batch 101 is 2 10 HA / dose, and the content of the rabbit hepatitis E virus is 10 9 TCID 50 / dose, 100 rabbit infectious doses; the content of the rabbit hemorrhagic syndrome virus in batch 102 is 2 10 HA / dose, and the content of the rabbit hepatitis E virus is 10 9 TCID 50 / dose, which is 100 rabbit infectious doses; the content of the rabbit hemorrhagic syndrome virus per dose in batch 103 is 2 10 HA / dose, and the content of the rabbit hepatitis E virus is 10 7 TCID 50 / dose, which is 100 rabbit infectious doses.

[0019] Safety and potency tests of the vaccine Safety test: (1) Safety test: Rabbit hemorrhagic syndrome part: Appropriately dilute the three batches of vaccines with normal saline (10 doses / mL), use 15 rabbits negative for rabbit hemorrhagic syndrome virus antigen and antibody at 3 - 4 weeks of age, 5 rabbits for each batch of vaccine, and intramuscularly inject 1 mL of the diluted vaccine, and observe for 3 weeks. No local or systemic reactions caused by the vaccine occurred.

[0020] At the same time, select 30 healthy rabbits that have been detected without rabbit hepatitis E virus antibodies by the neutralization test method, mix the samples of each batch of freeze-dried vaccine equally, dilute them with sterile normal saline according to the doses indicated on the vial label to 6 doses per 1 mL of vaccine, intramuscularly inject 10 rabbits, 5 mL per rabbit. After vaccination, observe for 3 weeks. There is no obvious change in body temperature, spirit, and appetite compared with before vaccination, and the vaccine is judged to be qualified.

[0021] (2) Validity test: Rabbit hemorrhagic syndrome virus (HBSV) component: Thirty 4-6 week old New Zealand White rabbits were randomly divided into three large groups, each further divided into two small groups. Ten rabbits were vaccinated per batch, with five receiving a single dose intramuscularly and the other five serving as controls. All animals were isolated and housed under the same conditions. Four weeks later, all animals were challenged with a minimum lethal dose of HBSV to test the vaccine's immunogenicity. All five control rabbits developed the disease, while all five immunized rabbits remained healthy.

[0022] Rabbit Hepatitis E Virus Treatment: Thirty 4-6 week old New Zealand rabbits were randomly divided into 6 groups. Each batch of vaccine was administered to 10 rabbits, with 5 rabbits receiving a single dose intramuscularly and the other 5 serving as controls. All rabbits were isolated and raised under the same conditions. Each dose of vaccine, as indicated on the label, was diluted 100 times with sterile saline and administered to 5 rabbits at a dose of 1 mL per rabbit. Post-vaccination assessment was based on a combination of clinical observation and challenge results.

[0023] ①The body temperature response standards for rabbits after vaccination are as follows: Typic fever (++) has an incubation period of 48-96 hours, with a distinct temperature rise curve, at least three of which exceed room temperature by more than 1°C and remain elevated for 18-36 hours. If the elevated temperature remains elevated for more than 42 hours, viral challenge is necessary. If there is no response after viral challenge, it can be diagnosed as typic fever.

[0024] Mild fever reaction (+) has an incubation period of 48-96 hours, with a clear curve of body temperature rise, at least two of which exceed normal temperature by more than 0.5°C and remain for 12-36 hours.

[0025] Suspicious reaction (±) Incubation period 48-96 hours, body temperature curve fluctuates and lasts less than 12 hours; or incubation period more than 24 hours, less than 48 hours and more than 96 hours to 120 hours with a fever reaction.

[0026] Rabbits exhibiting a two-peak body temperature response, with one peak meeting the criteria for a typical fever response (++) or a mild fever response (+), must be challenged with a virus. If no response occurs after challenge, the rabbit's fever response can be classified as a typical fever response or a mild fever response.

[0027] No response (-) Normal body temperature.

[0028] ②Result determination: After vaccination, the vaccine is considered qualified when both rabbits show a typological fever reaction (++), or when one rabbit shows a typological fever reaction (++) and the other shows a mild fever reaction (+).

[0029] After vaccination, when one rabbit shows a definite febrile reaction (++), or a mild febrile reaction (+), and the other rabbit shows a doubtful reaction (±); or when both rabbits show a mild febrile reaction (+), challenge (inoculate with fresh spleen-lymph virus or freeze-dried virus) can be carried out 7 - 10 days after vaccination. When challenging, add 2 control rabbits. The challenge dose is 50 - 100 times the emulsion, and 1 mL is injected into the ear vein of each rabbit.

[0030] The criteria for body temperature reaction after challenge are as follows: Febrile reaction (+): The incubation period is 24 - 72 hours. The body temperature rises in an obvious curve, exceeding the normal temperature by more than 1°C, and persists for 12 - 36 hours.

[0031] Doubtful reaction (±): The incubation period is less than 24 hours or more than 72 hours. The body temperature curve fluctuates, and the retention time is less than 12 hours or more than 36 hours without decline.

[0032] No reaction (-): The body temperature is normal.

[0033] After challenge, when both control rabbits show a definite febrile reaction (++), or one rabbit shows a definite febrile reaction (++) and the other rabbit shows a mild febrile reaction (+), and both vaccinated rabbits show no reaction (-), the vaccine is judged to be qualified.

[0034] After vaccination, if one rabbit shows a definite febrile reaction (++) or a mild febrile reaction (+), and the other rabbit shows a doubtful reaction (±) or no febrile reaction (-), the doubtful or non-reacting rabbit can be sacrificed or its blood can be collected to isolate the virus to determine whether there is a latent infection; or after vaccination, if both rabbits show a mild febrile reaction, the virus can also be isolated from one of the rabbits. The method is to sacrifice the rabbit between 96 - 120 hours after vaccination, take the spleen, make a 50-fold dilution emulsion with physiological saline, or take the blood and inoculate 2 rabbits, injecting 1 mL into the ear vein of each rabbit. If one rabbit shows a definite febrile reaction (++) during the incubation period of 24 - 72 hours, the vaccine can be judged to be qualified. After injecting three batches of vaccines, when both rabbits in each batch show a definite febrile reaction (++), or one rabbit shows a definite febrile reaction (++) and the other rabbit shows a mild febrile reaction (+), the vaccine is judged to be qualified.

[0035] This embodiment also provides the application of the above-mentioned bivalent vaccine for preventing or treating rabbit hemorrhagic syndrome and rabbit hepatitis E.

[0036] Inject 1 mL of the vaccine containing 1 dose of the vaccine produced in three batches into rabbits via intramuscular injection. After 4 weeks, detect the specific antibodies against rabbit hemorrhagic syndrome. The results are all positive; while the antibodies of the control rabbits do not change and are negative.

[0037] Peripheral blood was collected from the heart 1-4 weeks after vaccination with the three batches of produced vaccine and the commercially available rabbit hepatitis E virus vaccine. Serum was separated, and rabbit hepatitis E virus antibodies were detected using the HI method and rabbit hepatitis E virus antibodies using the ELISA method. The results showed that antibodies were produced 1 week after vaccination, and antibody levels continued to rise from 2-4 weeks. The antibody levels produced by the three batches of vaccine were close to those of the commercially available rabbit hepatitis E virus vaccine and hepatitis E vaccine, all achieving good immunization effects.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A bivalent vaccine, characterized in that, The vaccine comprises the following components: rabbit hemorrhagic syndrome virus antigen, rabbit hepatitis E virus antigen and vaccine freeze-drying protective agent; The rabbit hemorrhagic syndrome virus antigen comprises a standard virus strain NJ-2009 of rabbit hemorrhagic syndrome, with a GenBank sequence number of HM623309.

1. The rabbit hepatitis E virus antigen comprises a rabbit hepatitis E virus JSBL220, with a GenBank sequence number of JQ953664.

2. A bivalent vaccine according to claim 1, characterized in that, The content of the rabbit hemorrhagic disease virus antigen is ≥10 8 HA / head portion.

3. The bivalent vaccine of claim 1, wherein, The content of the rabbit hepatitis E virus antigen is ≥10 7 TCID 50 per head.

4. The bivalent vaccine of claim 1, wherein, The content of the rabbit hemorrhagic syndrome virus antigen and the rabbit hepatitis E virus antigen is greater than or equal to 50 rabbit infectious dose per head, and each head contains 0.01 mL of virus liquid.

5. A method of preparing a tetravalent vaccine according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: The rabbit hepatitis E virus is cultured by using passage cells to obtain rabbit hepatitis E virus antigen, the rabbit hemorrhagic syndrome virus is inoculated to obtain rabbit hemorrhagic syndrome virus antigen, the rabbit hemorrhagic syndrome virus antigen and the rabbit hepatitis E virus antigen are mixed in proportion, and the freeze-drying protective agent is supplemented to obtain the double vaccine.

6. The method of claim 5, wherein, The volume ratio of the rabbit hemorrhagic syndrome virus antigen to the rabbit hepatitis E virus antigen is 1:

10.

7. Use of a tetravalent vaccine according to any one of claims 1 to 4, characterized in that, The vaccine is used for preventing or treating rabbit hemorrhagic syndrome and rabbit hepatitis E.