High-pathogenicity H5 subtype avian influenza inactivated vaccine based on VSV carrier and preparation method of high-pathogenicity H5 subtype avian influenza inactivated vaccine
By constructing the VSV vector, the NA and HA genes of the H5 subtype avian influenza virus strain were inserted into the VSV vector, and the recombinant virus was rescued through reverse genetic technology, and the inactivated treatment was performed to prepare an inactivated vaccine, which solved the immune safety problem of VSV vector vaccine, achieved efficient and safe immunogenic effects, and was compatible with the DIVA strategy.
Patent Information
- Application Number
- CN202411888245.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-28
AI Technical Summary
How to improve the immune safety of VSV's highly pathogenic H5 subtype avian influenza vaccine as a vector, and provide a highly pathogenic H5 subtype avian influenza inactivated vaccine based on VSV vector and its preparation method.
By constructing a vesicular stomatitis virus (VSV) vector that lacks the surface capsule protein gene, the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain were inserted into the M gene and L gene of the vector in the form of an expression box, and the recombinant virus was rescued by reverse genetic technology, and the recombinant virus was inoculated into susceptible cells for culture, and then replicated and proliferated to obtain the inactivated virus solution, which was added to a pharmaceutical or veterinary acceptable carrier, excipient, vehicle or adjuvant to prepare an inactivated vaccine.
The effective expression and immunogenicity of the VSV vector recombinant virus were achieved. The inactivated vaccine has high safety and good immunogenicity. It can induce high levels of hemagglutination inhibitory titers of avian influenza virus after 21 days, and is compatible with the DIVA strategy.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological vaccine research and development, and in particular to a highly pathogenic H5 subtype avian influenza inactivated vaccine based on a VSV vector and a preparation method thereof. Background Art
[0002] Avian influenza (AI) is an acute, highly contagious disease caused by influenza A virus (Influenza A virus), which mainly infects poultry and birds.
[0003] Influenza virus surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are key proteins in the viral infection cycle and are also important antigens for new influenza vaccine research. Highly pathogenic avian influenza virus has been shown to contain polybasic amino acid cleavage sites in HA, allowing it to grow in tissue culture without relying on trypsin.
[0004] Vesicular Stomatitis Virus (VSV) belongs to the Rhabdoviridae family and can infect a variety of animals and insects. Among livestock, horses, cattle (sheep), and pigs are naturally infected with VSV. Because it can spread anterogradely across synapses between ganglia, it has potential neurotoxicity. VSV can efficiently express foreign proteins and induce high levels of immune responses in the host. However, due to its neurotoxicity, the development of preventive vaccines using replicative VSV recombinant viruses has certain risks.
[0005] In addition, live viruses can become inactivated viruses after inactivation, which has extremely high safety, but whether the inactivated virus has excellent immunogenicity is closely related to the abundance of antigens on the surface of virus particles and the conformation of antigens. There are no reports on the preparation of inactivated vaccines using replicative recombinant virus inactivated viruses with VSV as a vector. Summary of the invention
[0006] The technical problem to be solved by the present invention is how to improve the immune safety of a highly pathogenic H5 subtype avian influenza vaccine with VSV as a carrier, and to provide a highly pathogenic H5 subtype avian influenza inactivated vaccine based on a VSV carrier and a preparation method thereof.
[0007] In order to solve the above problems, the present invention proposes the following technical solutions:
[0008] In a first aspect, the present invention provides a method for preparing an H5 subtype avian influenza inactivated vaccine, comprising the following steps:
[0009] Preparation of recombinant virus: Using vesicular stomatitis virus lacking the surface envelope protein gene as a vector, inserting the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain between the M gene and the L gene of the vector in the form of an expression cassette, and rescuing the virus by reverse genetics technology; the NA gene and the HA gene are connected in series in the 5′-3′ direction;
[0010] Inoculation: The recombinant virus is inoculated into susceptible cells for culture, and the virus fluid is harvested after replication and proliferation;
[0011] Inactivation: inactivating the virus liquid to obtain an inactivated virus liquid;
[0012] Preparation of inactivated vaccine: adding the inactivated virus solution to a pharmaceutically or veterinarily acceptable carrier, excipient, medium or adjuvant to obtain an inactivated vaccine.
[0013] In the H5 subtype avian influenza inactivated vaccine of the present invention, the constructed recombinant virus uses VSV as a vector, and replaces the surface envelope protein (G protein) gene expression box element in the VSV vector genome expression box with the NA gene expression box element and the HA gene expression box element of the H5 subtype avian influenza virus strain. It is experimentally verified that the recombinant virus rescued by reverse genetic technology can effectively express the NA protein and HA protein of the H5 subtype avian influenza virus and has immunogenicity.
[0014] It should be noted that the VSV vector genome expression cassette described above includes a VSV genome transcription initiation element, a gene expression open reading frame and a VSV genome terminator, and the NA protein gene and HA protein gene of the H5 subtype avian influenza virus strain in the expression cassette are connected in series in the 5′-3′ direction.
[0015] Furthermore, the H5 subtype avian influenza virus strain is the highly pathogenic avian influenza virus A / Texas / 37 / 2024.
[0016] Furthermore, the amino acid sequence encoded by the HA gene includes the amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence encoded by the NA gene includes the amino acid sequence shown in SEQ ID NO.2.
[0017] Furthermore, the susceptible cells include baby hamster kidney cells, such as BHK-21.
[0018] Furthermore, the adjuvant is a white oil adjuvant.
[0019] In a second aspect, the present invention provides an H5 subtype avian influenza inactivated vaccine prepared by the preparation method described in the first aspect.
[0020] Furthermore, the H5 subtype avian influenza inactivated vaccine comprises an inactivated recombinant virus and a pharmaceutically or veterinarily acceptable carrier, excipient, medium or adjuvant. The recombinant virus uses a vesicular stomatitis virus lacking a surface envelope protein gene as a vector, and the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain are inserted between the M gene and the L gene of the vector in the form of an expression cassette, which is rescued by reverse genetics technology.
[0021] Furthermore, the H5 subtype avian influenza inactivated vaccine is used to induce a protective response in poultry or to immunize poultry.
[0022] Furthermore, the poultry includes chickens, ducks and geese.
[0023] Furthermore, the inactivation method includes physical inactivation or chemical inactivation, and the chemical inactivation method preferably includes adding an inactivating agent for chemical inactivation.
[0024] The inactivating agent is specifically β-propiolactone, which acts directly on the viral nucleic acid. This method does not destroy the protein structure on the surface of the virus, and can effectively ensure the abundance of antigens on the surface of viral particles and the conformation of antigens, thereby ensuring the immunogenicity of the inactivated virus.
[0025] Furthermore, the H5 subtype avian influenza inactivated vaccine has the function of distinguishing infected and immune animals.
[0026] In a third aspect, the present invention provides a H5 subtype avian influenza recombinant virus strain, which uses a vesicular stomatitis virus lacking a surface envelope protein gene as a vector, inserts the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain between the M gene and the L gene of the vector in the form of an expression cassette, and is rescued by reverse genetic technology; the NA gene and the HA gene are connected in series in the 5′-3′ direction.
[0027] Furthermore, the recombinant virus has the ability to replicate and proliferate after being rescued by reverse genetic technology.
[0028] Furthermore, the recombinant virus comprises vesicular stomatitis virus nucleoprotein, vesicular stomatitis virus phosphoprotein, vesicular stomatitis virus matrix protein, vesicular stomatitis virus RNA polymerase, and NA protein and HA protein of H5 subtype avian influenza virus strain, and does not comprise vesicular stomatitis virus surface envelope protein.
[0029] The present invention also provides a method for preparing the recombinant virus, comprising: using a vesicular stomatitis virus lacking a surface envelope protein gene as a vector, inserting the NA gene and the HA gene in the form of an expression box between the M gene and the L gene of the vector, and rescuing the recombinant virus through reverse genetic technology.
[0030] Furthermore, the method for preparing the recombinant virus also includes inoculating the obtained recombinant virus into susceptible cells for cultivation, and harvesting the cell culture fluid containing the recombinant virus.
[0031] Furthermore, the present invention also provides the use of the H5 subtype avian influenza recombinant virus strain and the antiserum produced by immunization with the H5 subtype avian influenza inactivated vaccine in the preparation of reagents for distinguishing infection and immune detection.
[0032] Compared with the prior art, the technical effects achieved by the present invention include:
[0033] The method for preparing an H5 subtype avian influenza inactivated vaccine provided by the present invention is to prepare an inactivated vaccine by constructing a recombinant virus of a VSV vector, wherein the surface envelope protein coding gene in the genome of the vesicular stomatitis virus is deleted by molecular biology technology, and the NA gene and HA gene expression box of the H5 subtype avian influenza virus strain are inserted into the recombinant virus, and the recombinant virus rescued by reverse genetic technology can effectively express the NA antigen and HA antigen of the H5 subtype avian influenza virus. The rescued recombinant virus is inoculated into susceptible cells for culture, and replication and proliferation can be completed without adding exogenous pancreatic enzymes, and a high-titer virus liquid is harvested, which effectively reduces the cost of producing purified virus liquid in the industrial production process.
[0034] The H5 subtype avian influenza inactivated vaccine provided by the present invention comprises an inactivated recombinant virus and a pharmaceutically or veterinarily acceptable carrier, excipient, medium or adjuvant. The recombinant virus has lost its replication ability after inactivation. With the assistance of the carrier, excipient, medium or adjuvant, its surface NA protein and HA protein can still exert an immune effect. The immunized chickens can be induced to produce a high level of avian influenza virus hemagglutination inhibition titer after 21 days, and have good immunogenicity.
[0035] The highly pathogenic H5 subtype avian influenza inactivated vaccine based on a VSV vector provided by the present invention has the advantages of high safety, high effectiveness and compatibility with the DIVA (Differentiation Between Infected and Immunized Animals) strategy. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.
[0037] Figure 1 This is a diagram of the gene recombination strategy for constructing the recombinant virus in Example 1 of the present invention.
[0038] Figure 2This is the result of the recombinant virus constructed in Example 1 of the present invention infecting BHK-21 cells.
[0039] Figure 3 This is the virus growth curve of the recombinant virus in Example 2 of the present invention in BHK-21 cells.
[0040] Figure 4 These are the HI results of each experimental group after immunization in Example 4 of the present invention.
[0041] Figure 5 These are the ELISA results of the immune serum and infected serum in Example 5 of the present invention. DETAILED DESCRIPTION
[0042] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described below 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.
[0043] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.
[0044] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.
[0045] Example 1
[0046] Influenza H5 subtype recombinant virus based on VSV vector and its preparation method (virus construction and rescue)
[0047] See also Figure 1 , which is a schematic diagram of the structure of influenza H5 subtype recombinant virus constructed by VSV vector in the present invention. In this embodiment, vesicular stomatitis virus (VSV) is used as a vector, its surface envelope protein (G protein) gene is deleted, and the NA gene and HA gene expression box of H5 subtype avian influenza virus strain are inserted, and the influenza H5 subtype recombinant virus is constructed by reverse genetic technology. In the embodiment of the present invention, the influenza H5 subtype recombinant virus is used to prepare an inactivated recombinant vaccine for preventing H5 subtype influenza.
[0048] This embodiment takes the highly pathogenic avian influenza virus A / Texas / 37 / 2024 strain H5N1 as an example to introduce the H5 subtype recombinant virus constructed by the present invention. In some embodiments, other H5 subtype avian influenza virus strains can also be used to construct the H5 subtype recombinant virus.
[0049] The HA and NA protein sequences of this example are derived from the H5N1 A / Texas / 37 / 2024 strain. The amino acid sequences of the HA protein (GeneBank Accession no. PP577943.1) and the NA protein (GeneBank Accession no. PP577945.1) are SEQ ID NO.1 and SEQ ID NO.2, respectively. The genes were synthesized after codon optimization, and the nucleotide sequences after codon optimization were SEQ ID NO.3 and SEQ ID NO.4, respectively.
[0050] The vesicular stomatitis virus strain in this example is the Indiana strain.
[0051] After gene synthesis, the above synthetic fragments and the VSV vector lacking the surface envelope protein were PCR amplified according to the instructions of PrimerStar enzyme. The primer sequence information for amplification is shown in Table 1.
[0052] The coding gene sequences of the above-mentioned codon-optimized HA and NA proteins SEQ ID NO.3 and SEQ ID NO.4 were cloned into the G protein gene position of the viral vector VSV by homologous recombination (Uniclone One Step SeamLess Cloning Kit), and the ORF region of the G protein gene was deleted. The two proteins were connected by the non-coding region between the VSV genes, and the NA protein gene and the HA protein gene were connected in series in the 5′-3′ direction. The nucleotide sequence used for the series connection was SEQ ID NO.5, forming a recombinant virus full-length plasmid of VSV carrying both H5N1-HA protein and H5N1-NA protein, named pVSV-H5N1-HA+NA.
[0053] Table 1 Amplification primer sequences
[0054]
[0055] The specific plasmid construction process is as follows:
[0056] 1. Use DNA polymerase (Primer Star) to perform PCR amplification to obtain the corresponding fragment;
[0057] Second, each fragment was recombined using homologous recombinase (Uniclone One Step Seamless Cloning Kit) and transformed into competent cells;
[0058] 3. Pick a single colony and use universal vector primers and Taq enzyme to perform bacterial solution PCR, and send the PCR product with the correct band size for testing;
[0059] 4. Extract plasmids from colony clones that have been sequenced correctly.
[0060] The specific process of constructing the recombinant plasmid and the auxiliary plasmid is prior art, and the present invention does not limit this.
[0061] The virus rescue method is as follows: BHK-21 cells are infected with poxvirus expressing T7 polymerase, followed by transfection of full-length plasmids and auxiliary plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G. After 48 hours, cells and supernatants are collected, and supernatants are taken for standby use after filtration through a 0.22 μm filter membrane to obtain a virus stock solution. The virus stock solution is inoculated into new BHK-21 cells, and the cultured cells are continuously passaged to observe whether lesions occur. If lesions occur, the cells are collected again and repeatedly frozen and thawed three times, filtered and aliquoted at 0.45 μm, and then frozen at -80°C to obtain a virus stock solution. The collected recombinant virus stock solution is continuously passaged and amplified on BHK-21 cells. After stable amplification, the virus genome is extracted for RT-PCR sequencing to confirm that the virus rescue is successful.
[0062] It has been determined that the supernatant of the above-mentioned passaged cells can be successfully rescued and amplified in BHK-21 cells to obtain recombinant viruses. The recombinant virus obtained by passage and amplification in BHK-21 cells is named VSV-H5N1-HA+NA in the present invention.
[0063] After inoculation of the cell line, the cytopathic effect was observed Figure 2 .
[0064] like Figure 2 The results showed that the recombinant virus VSV-H5N1-HA+NA can be propagated and amplified in BHK-21 cells and has the ability to replicate and amplify.
[0065] Example 2 Determination of recombinant virus growth kinetic curve
[0066] In order to verify the replication kinetics of the recombinant virus VSV-H5N1-HA+NA in cells, we established the virus growth curve in BHK-21 cells.
[0067] The specific steps are as follows: The recombinant virus was inoculated into a 6-well cell plate with a monolayer of cells at a dose of MOI = 0.1, and the infected cells were cultured at 37°C. The corresponding cell plate was repeatedly frozen and thawed three times at 24h, 48h, 72h, and 96h after infection, and the virus solution was collected. It was frozen at -80°C. The virus solution at each time point was diluted 10 times and inoculated into a 96-well plate with a monolayer of BHK-21 cells. The virus TCID was calculated according to the Reed-Muench method. 50 , a one-step virus growth curve was drawn with time as the horizontal axis and virus titer as the vertical axis.
[0068] The growth kinetics curve of the recombinant virus is as follows Figure 3 The virus titer of VSV-H5N1-HA+NA was the highest at 72h after infection, even exceeding 10 8.5 TCID 50 This indicates that the recombinant virus obtained in the present invention has the advantages of high virus yield and high exogenous protein expression level.
[0069] Example 3 Inactivated vaccine preparation
[0070] The amplified virus stock solution VSV-H5N1-HA+NA was collected and the titer was measured on BHK21 cells. The titer of the virus stock solution was 10 8.0 TCID50 / mL. Introduce the virus stock solution into the inactivation tank, add β-propiolactone with a final concentration of 0.05%, mix well, and inactivate at 2-8°C, 100rpm / min for 24h. The inactivated virus solution is hydrolyzed at 37°C for 2h and stored at 2-8°C. The storage should not exceed 2 months.
[0071] The criterion for judging virus inactivation is that the inactivated virus is continuously inoculated and passaged on BHK21 cells for three generations without causing cell damage.
[0072] Preparation of inactivated vaccines: This example provides a group 1 of inactivated vaccines prepared using white oil adjuvant and a group 2 of inactivated vaccines prepared using aluminum gel adjuvant. The preparation methods of each are as follows:
[0073] Group 1 of inactivated vaccine with white oil adjuvant: According to the composition and dosage in Table 2, add the inactivated virus, 0.9% NaCl / enhancer, and Tween-80 in a 100mL centrifuge tube in this order and mix well as the water phase; add white oil, span, and aluminum stearate in this order into an emulsification bottle and mix well as the oil phase. Since Tween-80 is insoluble, it needs to be properly heated in a 37°C water bath. The prepared water phase and oil phase are subjected to high-speed shear emulsification, and the emulsification conditions are 13600rpm / min 1.5min, which is repeated three times. Since the shear emulsification speed is too fast, heat will be generated and the vaccine will be heated. Therefore, it is necessary to cool down in the water for a while before continuing the shear emulsification. Mark each time the shearing is completed to prevent confusion of the immune adjuvant, and the shear rod should be fully cleaned.
[0074] Group 2 of inactivated vaccine with aluminum gel adjuvant: The volume of aluminum adjuvant is 15% of the total volume. The inactivated virus stock solution and aluminum gel adjuvant are mixed at a volume ratio of 3:1, and the rest is made up with sterile PBS solution. In the preparation process, the inactivated virus is first added to the sterile PBS solution, placed on a magnetic stirrer for stirring, and the aluminum gel adjuvant is slowly added dropwise, and stirred at room temperature for 30 minutes.
[0075] The prepared vaccines were sealed and stored in a refrigerator at 4°C. Finally, the properties and sterility of each vaccine were tested and the vaccines could be used for immunization after being confirmed to be sterile.
[0076] Furthermore, since influenza virus is sensitive to temperature, based on the above inactivated virus preparation, this study used a physical method, i.e., 65°C for 30 min, to inactivate the recombinant virus VSV-H5N1-HA+NA, forming a high-temperature inactivated recombinant virus liquid as a control.
[0077] Table 2 Composition of inactivated vaccine with white oil adjuvant
[0078]
[0079] Example 4 Immunogenicity Evaluation of Inactivated Vaccine
[0080] 8-day-old SPF chickens were selected, with 5 chickens in each group. The experimental groups included inactivated vaccine group 1 and inactivated vaccine group 2 obtained by adding different adjuvants after inactivating the virus by β-propiolactone. The control group included pure inactivated virus group treated by β-propiolactone inactivation, pure inactivated virus group treated by high temperature inactivation, live virus stock solution group and PBS group, a total of 6 groups. Immunization was performed on day 0 and day 14, and serum was collected on day 13 (D13) and day 21 (D21) for hemagglutination inhibition experiment.
[0081] The hemagglutination inhibition test method is as follows:
[0082] Prepare four units of HA antigen, the dilution multiple of the virus antigen is equal to the virus agglutination titer / 4, and inactivate the chicken serum to be tested at 56°C for 30 minutes in advance. Add 25 μL of the serum to be tested to the first well of the 96-well plate, dilute the immune serum in a gradient dilution ratio of 1:2, add 25 μL of four units of antigen to the diluted serum, react at room temperature for 30 minutes, add 25 μL of 1% chicken red blood cell suspension to each well, and observe the test results after standing at room temperature for 30 minutes, and record the highest dilution at which blood agglutination occurs, which is the HI level.
[0083] The HI results after primary and booster immunization in each experimental group are shown in Figure 4 .
[0084] The HI results showed that the HI titers of the chickens immunized on day 21 in the inactivated vaccine group 1 were higher than those of the chickens immunized on day 13, and the HI titers were higher than 2. 4 The inactivated vaccine group 2 was unable to produce effective neutralizing antibody levels against H5N1. To our surprise, after immunization with live virus stock solution, the serum HI titer of SPF chickens was not ideal, and the serum HI was lower than 2 13 days after the first immunization. 4 , and 21 days after immunization, only 2 / 5 test chickens had serum HI titers higher than 2 4 , can not produce effective neutralizing antibody level for H5N1. (In actual production, generally take the serum HI titer of chicken immunization 21 days later not less than 1:16 as the index of judging individual immunity qualification). Further, no matter adopting the pure inactivated recombinant virus VSV-H5N1-HA+NA immunization SPF chicken obtained by the mode of physical inactivation or chemical inactivation, all do not produce enough HI, illustrate that live virus and inactivated virus itself can not produce effective neutralizing antibody level for H5N1. It is worth noting that, when each experimental group of the present embodiment carries out equal volume antigen immunization, because the configuration process of inactivated vaccine group has carried out a certain proportion of dilution to virus stock solution, therefore the initial antigen content of live virus stock solution group and pure inactivated virus group is theoretically higher than that of inactivated vaccine group, but all can not produce effective neutralizing antibody level for H5N1.
[0085] The inactivated vaccine group 1 of the present invention, prepared with white oil adjuvant, produced an effective HI titer level of 100% 13 days after the primary immunization, and reached a higher HI titer level 21 days after the immunization, with the highest HI titer reaching 2 7.5 . However, the inactivated vaccine group 2 using aluminum gel adjuvant did not produce sufficient HI. It can be seen that the recombinant virus constructed in the embodiment of the present invention, after inactivation, no adjuvant was added or the adjuvant was not properly selected, and the obtained inactivated reagent had no immunogenicity and could not be used as an inactivated vaccine. Only the inactivated vaccine prepared using white oil adjuvant had good immunogenicity and could be used as an inactivated vaccine.
[0086] Example 5 Application of inactivated vaccines in DIVA
[0087] Use H5N1 NP protein (Accession: PP577946.1) to coat the ELISA plate, dilute the protein to 10 μg / mL with PBS solution (pH 7.4), add 100 μL to each well, seal the film, and leave at 4°C overnight. After overnight, discard the liquid in the well, fill the reaction well with PBST, wash 3 times, let stand for 3 minutes each time, and pat dry on paper. Add 300 μL of 5% skim milk to each well and place it in a 37°C incubator for 2h. Wash 3 times after blocking, and then perform DIVA detection. Take the D21 serum of the immune chickens in the inactivated vaccine group in Example 4, and the infected serum to be tested, dilute 10 times with PBS solution, repeat the test for each sample 2 times, and set the negative control well, 100 μL per well, and place it in a 37°C incubator for 1h. After incubation, repeat the above washing steps 3 times, dilute the enzyme-labeled secondary antibody 5000 times with PBS, add 100 μL to each well, and incubate at 37°C for 1 hour. Discard the liquid in the wells, wash 5 times with PBST, let stand for 3 minutes each time and pat dry. Add 100 μL of TMB substrate colorimetric solution to each well, develop in a dark place, develop in a wet box (about 25°C) for 15 minutes, add 50 μL of 2MH2SO4 to each well after color development, and read the value at OD450.
[0088] It should be noted that, since serum of naturally infected H5 subtype avian influenza virus was not available, this example selected immune serum of a recombinant virus inactivated vaccine (Yibang Bio, recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine) composed of a PR8 virus backbone combined with HA and NA proteins of H5 subtype virus to simulate naturally infected H5 subtype virus infected serum according to the published H5N1 virus information. Since influenza NP protein is conserved in avian influenza virus, it can be used for DIVA detection.
[0089] The infected serum to be tested is a simulated natural infection serum obtained 21 days after SPF chickens were immunized with a recombinant virus inactivated vaccine consisting of a PR8 virus backbone combined with the HA and NA proteins of the H5 subtype virus.
[0090] Results Figure 5 , as shown in the ELISA results, no NP antibody was detected in the serum of chickens immunized with the inactivated vaccine of the present invention. However, NP protein antibody was detected in the positive serum of chickens infected with H5N1 by simulating natural immunity. Therefore, it can be seen that the inactivated vaccine provided by the present invention has the ability to distinguish infected and immune animals.
[0091] In summary, the VSV vector-based highly pathogenic H5 subtype avian influenza inactivated vaccine provided by the present invention is safe, effective and compatible with the DIVA strategy.
[0092] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.
[0093] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.
Claims
1. A method for preparing an H5 subtype avian influenza inactivated vaccine, characterized in that: The following steps are involved: Preparation of recombinant virus: Using vesicular stomatitis virus lacking the surface envelope protein gene as a vector, inserting the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain between the M gene and the L gene of the vector in the form of an expression cassette, and rescuing the virus by reverse genetics technology; the NA gene and the HA gene are connected in series in the 5′-3′ direction; Inoculation: The recombinant virus is inoculated into susceptible cells for culture, and the virus fluid is harvested after replication and proliferation; Inactivation: inactivating the virus liquid to obtain an inactivated virus liquid; Preparation of inactivated vaccine: adding the inactivated virus solution to a pharmaceutically or veterinarily acceptable carrier, excipient, medium or adjuvant to obtain an inactivated vaccine.
2. The method for preparing the H5 subtype avian influenza inactivated vaccine according to claim 1, characterized in that: The H5 subtype avian influenza virus is derived from the A / Texas / 37 / 2024 virus strain.
3. The method for preparing the H5 subtype avian influenza inactivated vaccine according to claim 1, characterized in that: The amino acid sequence encoded by the HA gene includes the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence encoded by the NA gene includes the amino acid sequence shown in SEQ ID NO.
2.
4. The method for preparing the H5 subtype avian influenza inactivated vaccine according to claim 1, characterized in that: The susceptible cells include baby hamster kidney cells.
5. The method for preparing the H5 subtype avian influenza inactivated vaccine according to claim 1, characterized in that: In the inactivation step, the inactivation treatment includes adding β-propiolactone for inactivation.
6. The method for preparing the H5 subtype avian influenza inactivated vaccine according to any one of claims 1 to 5, characterized in that: The adjuvant is a white oil adjuvant.
7. An H5 subtype avian influenza inactivated vaccine, characterized in that: The vaccine is prepared by the method for preparing the H5 subtype avian influenza inactivated vaccine according to any one of claims 1 to 6.
8. The H5 subtype avian influenza inactivated vaccine as claimed in claim 7 is used to induce a protective response in poultry or to immunize poultry.
9. A recombinant H5 subtype avian influenza virus strain, characterized in that: The H5 subtype avian influenza recombinant virus strain uses a vesicular stomatitis virus lacking a surface envelope protein gene as a vector, inserts the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain between the M gene and the L gene of the vector in the form of an expression box, and is rescued by reverse genetic technology; the NA gene and the HA gene are connected in series in a 5'-3' direction.
10. Use of the antiserum produced by immunization with the H5 subtype avian influenza recombinant virus strain according to claim 9 or the H5 subtype avian influenza inactivated vaccine according to claims 7-8 in the preparation of a reagent for distinguishing between immunity and natural infection.