VSV vector-based highly pathogenic h5 subtype avian influenza inactivated vaccine and method for preparing same

Abstract

The present invention relates to the field of biological vaccine research and development technology, and in particular, to a VSV vector-based highly pathogenic H5 subtype avian influenza inactivated vaccine and a method for preparing same. The method comprises: using a vesicular stomatitis virus with the deletion of the surface envelope protein gene as a vector, inserting the NA gene and the HA gene of an 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 acquiring a recombinant virus by means of rescue via reverse genetic technology; inoculating the recombinant virus into susceptible cells, culturing for replication and proliferation, and then harvesting a viral solution; inactivating the viral solution to acquire an inactivated viral solution; and adding the inactivated viral solution to a pharmaceutically or veterinarily acceptable carrier, excipient, medium, or adjuvant to acquire an inactivated vaccine. The provided H5 subtype avian influenza inactivated vaccine can induce a high hemagglutination inhibition titer of avian influenza virus on day 21 after chick immunization. The provided inactivated vaccine has good safety and immunogenicity.

Description

A highly pathogenic H5 subtype avian influenza inactivated vaccine based on VSV vector and its preparation method

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202411888245.2, filed on December 20, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biological vaccine research and development technology, and in particular to a highly pathogenic H5 subtype avian influenza inactivated vaccine based on a VSV vector and its preparation method. Background Technology

[0004] Avian influenza (AI) is an acute, highly contagious disease caused by influenza A virus, primarily infecting poultry and birds. In March 2024, an outbreak of highly pathogenic avian influenza H5N1 (H5N1 A / Texas / 37 / 2024) virus occurred in dairy cattle in the United States. The outbreak not only spread among cattle but also infected cats, poultry, and even workers on the farm.

[0005] The influenza virus surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are key proteins in the viral infection cycle and important antigens in novel influenza vaccine research. Highly pathogenic avian influenza viruses have been shown to contain multi-basic amino acid cleavage sites in HA, allowing them to grow in tissue culture without relying on trypsin.

[0006] Vesicular stomatitis virus (VSV) belongs to the genus Rhabdoviridae and family Vesiculovirus, 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 exogenous proteins and induce a high level of immune response in the host. However, due to its neurotoxicity, prophylactic vaccines developed using replicating VSV recombinant viruses carry certain risks.

[0007] Furthermore, live viruses can be inactivated into inactivated viruses, exhibiting extremely high safety. However, the immunogenicity of inactivated viruses is highly dependent on the abundance and conformation of antigens on the viral particle surface. No studies have been reported on the preparation of inactivated vaccines using VSV-vectored replicative recombinant viruses.

[0008] Application content

[0009] The technical problem to be solved by this application is how to improve the immunogenicity of highly pathogenic H5 subtype avian influenza vaccines using VSV as a vector, and to provide a highly pathogenic H5 subtype avian influenza inactivated vaccine based on VSV vector and its preparation method.

[0010] To address the above problems, this application proposes the following technical solution:

[0011] In one aspect, this application provides a method for preparing an H5 subtype avian influenza inactivated vaccine, comprising the following steps:

[0012] Preparation of recombinant virus: Using vesicular stomatitis virus lacking the surface envelope protein gene as a vector, the NA and HA genes of the H5 subtype highly pathogenic avian influenza virus strain were inserted in the form of expression cassettes between the M and L genes of the vector and rescued by reverse genetics technology; the NA and HA genes were tandemly arranged in the 5′-3′ direction.

[0013] Inoculation: The recombinant virus is inoculated into susceptible cells for culture, and the viral fluid is harvested after replication and proliferation.

[0014] Inactivation: The virus solution is inactivated to obtain an inactivated virus solution;

[0015] Preparation of inactivated vaccine: The inactivated virus solution is added to a pharmaceutically or veterinarily acceptable carrier, excipient, medium or adjuvant to obtain an inactivated vaccine.

[0016] In the H5 subtype avian influenza inactivated vaccine of this application, the recombinant virus constructed uses VSV as a vector. By replacing the surface envelope protein (G protein) gene expression cassette element in the VSV vector genome expression cassette with the NA gene expression cassette element and HA gene expression cassette element of the H5 subtype avian influenza virus strain, experimental verification shows that the recombinant virus obtained by reverse genetics technology can effectively express the NA protein and HA protein of the H5 subtype avian influenza virus and has immunogenicity.

[0017] 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. The NA protein gene and HA protein gene of the H5 subtype avian influenza virus strain in the expression cassette are tandemly linked in the 5′-3′ direction.

[0018] Furthermore, the H5 subtype avian influenza virus strain is the highly pathogenic avian influenza virus strain A / Texas / 37 / 2024.

[0019] Furthermore, 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.

[0020] Furthermore, the susceptible cells include young hamster kidney cells, such as BHK-21.

[0021] Furthermore, the adjuvant is a white oil adjuvant.

[0022] Secondly, this application provides an H5 subtype avian influenza inactivated vaccine, prepared by the preparation method described in the first aspect.

[0023] Furthermore, the H5 subtype avian influenza inactivated vaccine comprises an inactivated recombinant virus and a pharmaceutically or veterinarily acceptable vector, excipient, medium, or adjuvant. The recombinant virus uses a vesicular stomatitis virus lacking the surface envelope protein gene as a vector, and inserts the NA and HA genes of the H5 subtype highly pathogenic avian influenza virus strain into the space between the M and L genes of the vector in the form of an expression cassette, which is then rescued by reverse genetics technology.

[0024] Furthermore, the H5 subtype avian influenza inactivated vaccine is used to induce a protective response in poultry or to immunize poultry.

[0025] Furthermore, the poultry includes chickens, ducks, and geese.

[0026] Furthermore, the inactivation method includes physical inactivation or chemical inactivation, with chemical inactivation preferably performed by adding an inactivating agent.

[0027] The inactivating agent is β-propiolactone. β-propiolactone acts directly on viral nucleic acid. This method does not destroy the protein structure on the surface of the virus, and can effectively ensure the antigen abundance and conformation of the viral particles, thus ensuring the immunogenicity of the inactivated virus.

[0028] Furthermore, the H5 subtype avian influenza inactivated vaccine has the function of distinguishing between infected and immunized animals.

[0029] Thirdly, this application provides an H5 subtype avian influenza recombinant virus strain, wherein the H5 subtype avian influenza recombinant virus strain uses a vesicular stomatitis virus lacking the surface envelope protein gene as a vector, and inserts the NA gene and HA gene of the H5 subtype highly pathogenic avian influenza virus strain into the space between the M gene and L gene of the vector in the form of an expression cassette, and is rescued by reverse genetics technology; wherein the NA gene and HA gene are tandemly arranged in the 5′-3′ direction.

[0030] Furthermore, the recombinant virus, after being rescued using reverse genetics technology, possesses the ability to replicate and proliferate.

[0031] 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 and HA proteins of H5 subtype avian influenza virus strain, but does not contain vesicular stomatitis virus surface envelope protein.

[0032] This application 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 HA gene into the space between the M gene and L gene of the vector in the form of an expression cassette, and rescuing the recombinant virus by reverse genetics.

[0033] Furthermore, the method for preparing the recombinant virus also includes inoculating the obtained recombinant virus into susceptible cells for culture and harvesting the cell culture medium containing the recombinant virus.

[0034] Furthermore, this application also provides the use of the aforementioned H5 subtype avian influenza recombinant virus strain and antiserum produced by immunization with H5 subtype avian influenza inactivated vaccine in the preparation of reagents for distinguishing infection and immune detection.

[0035] Compared with the prior art, the technical effects achieved by this application include:

[0036] The method for preparing the H5 subtype avian influenza inactivated vaccine provided in this application involves constructing a recombinant virus with a VSV vector. The recombinant virus, using molecular biology techniques, deletes the surface envelope protein coding gene from the genome of vesicular stomatitis virus and inserts it into the NA and HA gene expression cassettes of the H5 subtype avian influenza virus strain. The recombinant virus, rescued using reverse genetics, can effectively express the NA and HA antigens of the H5 subtype avian influenza virus. By inoculating the rescued recombinant virus into susceptible cells for culture, replication and proliferation can be completed without the addition of exogenous trypsin, yielding a high-titer viral fluid. This will effectively reduce the cost of producing and purifying the viral fluid during industrial-scale production.

[0037] The H5 subtype avian influenza inactivated vaccine provided in this application contains an inactivated recombinant virus and a pharmaceutically or veterinarily acceptable vector, excipient, medium or adjuvant. The recombinant virus has lost its replication ability after inactivation. With the assistance of the vector, excipient, medium or adjuvant, the NA protein and HA protein on its surface can still exert an immune effect. Immunized chicks can be induced to produce a high level of avian influenza virus hemagglutination inhibition titer after 21 days, and have good immunogenicity.

[0038] The highly pathogenic H5 subtype avian influenza inactivated vaccine based on VSV vector provided in this application has the advantages of high safety, high efficacy and compatibility with the DIVA (distinguishing between infected and immunized animals) strategy. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 is a diagram of the gene recombination strategy for constructing recombinant viruses in Example 1 of this application.

[0041] Figure 2 shows the results of infecting BHK-21 cells with the recombinant virus constructed in Example 1 of this application.

[0042] Figure 3 shows the virus growth curve of the recombinant virus in BHK-21 cells in Example 2 of this application.

[0043] Figure 4 shows the HI results of each experimental group after immunization in Example 4 of this application.

[0044] Figure 5 shows the ELISA results of immune serum and infected serum in Example 5 of this application. Detailed Implementation

[0045] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0046] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0047] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of this application. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0048] Example 1

[0049] VSV-based recombinant influenza H5 subtype virus and its preparation method (virus construction and rescue)

[0050] Referring to Figure 1, which is a schematic diagram of the structure of the recombinant influenza H5 subtype virus constructed using the VSV vector in this application. In this embodiment, vesicular stomatitis virus (VSV) was used as a vector. The surface envelope protein (G protein) gene was deleted, and the NA and HA gene expression cassettes of the H5 subtype avian influenza virus strain were inserted. The recombinant influenza H5 subtype virus was constructed using transgenetic technology. This embodiment of the application uses this recombinant influenza H5 subtype virus to prepare an inactivated recombinant vaccine for the prevention of H5 subtype influenza.

[0051] This embodiment uses the A / Texas / 37 / 2024 strain of H5N1 highly pathogenic avian influenza virus as an example to introduce the H5 subtype recombinant virus constructed in this application. In some embodiments, other H5 subtype avian influenza virus strains may also be selected for the construction of H5 subtype recombinant viruses.

[0052] The HA and NA protein sequences in this embodiment were derived from strain H5N1 A / Texas / 37 / 2024. 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. After codon optimization, the synthesized gene has SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0053] The vesicular stomatitis virus strain used in this embodiment is the Indiana strain.

[0054] After gene synthesis, the synthesized fragments and the VSV vector lacking surface membrane proteins were amplified by PCR according to the Primer Star enzyme instructions. The primer sequence information for amplification is shown in Table 1.

[0055] The coding gene sequences of the HA and NA proteins (SEQ ID NO.3 and SEQ ID NO.4) after codon optimization were cloned into the G protein gene position in the viral vector VSV using homologous recombination (Uniclone One Step SeamLess Cloning Kit). The ORF region of the G protein gene was deleted. The two proteins were linked together using the non-coding region between VSV genes. The NA protein gene and the HA protein gene were tandemly linked in the 5′–3′ direction. The nucleotide sequence used for tandem linking was SEQ ID NO.5, forming a full-length recombinant viral plasmid carrying both H5N1-HA and H5N1-NA proteins, named pVSV-H5N1-HA+NA.

[0056] Table 1 Amplification Primer Sequences

[0057] The specific plasmid construction process is as follows:

[0058] 1. PCR amplification using DNA polymerase (Primer Star) to obtain the corresponding fragment;

[0059] 2. Each fragment was recombined using homologous recombinase (Uniclone One Step Seamless Cloning Kit) and transformed into competent cells;

[0060] 3. Pick a single colony and perform bacterial PCR using universal vector primers and Taq enzyme, and send the PCR product with the correct band size for detection;

[0061] IV. Extract plasmids from the correctly sequenced bacterial colony clones.

[0062] The specific process for constructing recombinant plasmids and helper plasmids is existing technology, and this application does not limit it.

[0063] The virus rescue method is as follows: BHK-21 cells were infected with a poxvirus expressing T7 polymerase, followed by transfection with a full-length plasmid and helper plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G. After 48 hours, cells and supernatant were collected. The supernatant was filtered through a 0.22 μm filter and used as the viral stock solution. The viral stock solution was inoculated into new BHK-21 cells and passaged continuously to observe whether the cultured cells showed cytopathic effects. If cytopathic effects appeared, the cells were collected again and subjected to three freeze-thaw cycles. After filtration through a 0.45 μm filter, the cells were aliquoted and stored at -80°C to obtain the viral stock solution. The collected recombinant viral stock solution was continuously passaged and amplified in BHK-21 cells. After the amplification was stable, the viral genome was extracted and sequenced by RT-PCR to confirm successful virus rescue.

[0064] The results showed that the recombinant virus could be successfully rescued from the frozen-thawed supernatant of the above-mentioned cells after passage and amplification in BHK-21 cells. This application names the recombinant virus obtained by passage and amplification in BHK-21 cells as VSV-H5N1-HA+NA.

[0065] The cytopathic effect results after inoculation on the cell line are shown in Figure 2.

[0066] As shown in Figure 2, the recombinant virus VSV-H5N1-HA+NA can be passaged and amplified in BHK-21 cells, demonstrating its replication and amplification capabilities.

[0067] Example 2: Determination of recombinant virus growth kinetic curve

[0068] To verify the replication kinetics of the recombinant virus VSV-H5N1-HA+NA in cells, we established viral growth curves in BHK-21 cells.

[0069] The specific steps are as follows: The recombinant virus was inoculated into a 6-well cell plate with a monolayer of cells at an MOI of 0.1. The infected cells were cultured at 37°C. The cell plates were subjected to a freeze-thaw cycle three times at 24h, 48h, 72h, and 96h post-infection, and the viral load was collected. The virus was then stored at -80°C. The viral loads at each time point were serially diluted 10-fold and inoculated into 96-well plates containing a monolayer of BHK-21 cells. The TCID of the virus was calculated using the Reed-Muench method. 50 Plot a one-step growth curve of the virus with time on the horizontal axis and viral titer on the vertical axis.

[0070] The growth kinetic curves of the recombinant virus are shown in Figure 3. The viral titer of VSV-H5N1-HA+NA was highest at 72 hours post-infection, even exceeding 10. 8.5 TCID 50 / mL. This indicates that the recombinant virus obtained in the embodiments of this application has the advantages of high viral yield and high expression level of exogenous protein.

[0071] Example 3: Preparation of Inactivated Vaccine

[0072] The amplified viral stock solution VSV-H5N1-HA+NA was collected and its titer was measured on BHK21 cells. The measured viral stock titer was 10. 8.0 TCID50 / mL. Transfer the stock virus solution into an inactivation vessel, add β-propiolactone to a final concentration of 0.05%, mix thoroughly, and inactivate at 2–8°C and 100 rpm / min for 24 hours. After inactivation, hydrolyze the virus solution at 37°C for 2 hours, then store at 2–8°C for no more than 2 months.

[0073] The criterion for determining virus inactivation is: after inactivation, the virus is continuously passaged three times on BHK21 cells without producing cytopathic effects.

[0074] Preparation of inactivated vaccines: This embodiment provides one group of inactivated vaccines prepared using white oil adjuvant and two groups of inactivated vaccines prepared using aluminum glue adjuvant. The preparation methods are as follows:

[0075] Group 1 of inactivated vaccines with added white oil adjuvant: Following the composition and dosage in Table 2, inactivated virus, 0.9% NaCl / enhancer, and Tween-80 were added to a 100mL centrifuge tube in that order and mixed thoroughly to form the aqueous phase. White oil, Span, and aluminum stearate were added to an emulsification flask in that order and mixed thoroughly to form the oil phase. Since Tween-80 is poorly soluble, it requires appropriate heating in a 37°C water bath. The prepared aqueous and oil phases were then subjected to high-speed shear emulsification at 13600 rpm / min for 1.5 min, repeated three times. Because the shear emulsification speed is too fast, it will generate heat, potentially heating the vaccine; therefore, it is necessary to cool it in water for a short time before continuing shear emulsification. Each shearing step was marked to prevent confusion of the adjuvants, and the shearing rod was thoroughly cleaned.

[0076] Two groups of inactivated vaccines with added aluminum glue adjuvant: the aluminum adjuvant volume was 15% of the total volume. The inactivated virus stock solution and aluminum glue adjuvant were mixed at a volume ratio of 3:1, and the remainder was made up with sterile PBS solution. The preparation process was as follows: the inactivated virus was first added to the sterile PBS solution, stirred with a magnetic stirrer, and the aluminum glue adjuvant was slowly added dropwise. The mixture was stirred at room temperature for 30 minutes.

[0077] After sealing the prepared vaccines, store them in a refrigerator at 4°C. Finally, each prepared vaccine undergoes morphological and sterility tests; once sterility is confirmed, it can be used for immunization.

[0078] Furthermore, since influenza viruses are sensitive to temperature, based on the above-mentioned inactivated virus preparation, this study used a physical method, namely 65℃ for 30 min, to inactivate the recombinant virus VSV-H5N1-HA+NA, forming a high-temperature inactivated recombinant virus solution as a control.

[0079] Table 2 Composition of inactivated vaccines with added white oil adjuvant

[0080] Example 4: Immunogenicity assessment of inactivated vaccine

[0081] Eight-day-old SPF chicks were selected, with five chicks in each group. The experimental groups included two groups: Group 1, which was an inactivated vaccine obtained by inactivating the virus with β-propiolactone and adding different adjuvants; and Group 2, which was an inactivated vaccine. The control group consisted of six groups: a pure inactivated virus group treated with β-propiolactone, a pure inactivated virus group treated with high temperature, a live virus stock solution group, and a PBS group. Chicks were immunized on days 0 and 14, and serum was collected on days 13 (D13) and 21 (D21) for hemagglutination inhibition experiments.

[0082] The blood coagulation inhibition test method is as follows:

[0083] Prepare four units of HA antigen. The dilution factor of the viral antigen is equal to the viral agglutination titer / 4. Simultaneously, inactivate the chicken serum to be tested at 56℃ for 30 min in advance. Add 25 μL of the serum to be tested to the first well of a 96-well plate. Serially dilute the immune serum at a ratio of 1:2. Add 25 μL of four units of antigen to the diluted serum. After reacting at room temperature for 30 min, add 25 μL of 1% chicken red blood cell suspension to each well. After standing at room temperature for 30 min, observe the test results and record the highest dilution at which hemagglutination occurs, which is the HI level.

[0084] The HI results after primary and booster immunization in each experimental group are shown in Figure 4.

[0085] The HI results showed that the HI titers of chickens immunized with the inactivated vaccine at 21 days were higher than those immunized at 13 days, and the HI titers were all higher than 2. 4 Group 2 of the inactivated vaccine failed to produce effective neutralizing antibody levels against H5N1. Surprisingly, after immunization with the live virus stock solution, the serum HI titer in SPF chickens was not ideal; the serum HI was below 2 for 13 days after the initial immunization. 4 However, 21 days after immunization, only 2 / 5 of the test chicks had serum HI titers higher than 2. 4 The live virus and the inactivated virus were unable to produce effective neutralizing antibody levels against H5N1. (In actual production, a serum HI titer of at least 1:16 21 days after immunization is generally used as an indicator of successful individual immunization). Furthermore, neither the purely inactivated recombinant virus VSV-H5N1-HA+NA obtained through physical or chemical inactivation produced sufficient HI in SPF chickens, indicating that neither the live virus nor the inactivated virus itself could produce effective neutralizing antibody levels against H5N1. It is noteworthy that in this embodiment, when the experimental groups were immunized with equal volumes of antigen, the initial antigen content of the live virus stock solution group and the purely inactivated virus group was theoretically higher than that of the inactivated vaccine group due to the dilution of the virus stock solution during the preparation process. However, neither group produced effective neutralizing antibody levels against H5N1.

[0086] The inactivated vaccine group 1 of this application, prepared using white oil adjuvant, produced an effective HI titer level in 100% of cases 13 days after primary immunization, and reached a high HI titer level 21 days after immunization, with the highest HI titer reaching 2. 7.5 The two groups of inactivated vaccines using aluminum glue adjuvant did not produce sufficient HI (inactivated virus). This indicates that the recombinant virus constructed in this application, after inactivation, lacks immunogenicity due to the absence of adjuvant or inappropriate adjuvant selection, and therefore cannot be used as an inactivated vaccine. Only inactivated vaccines prepared using white oil adjuvant possess good immunogenicity and can be used as inactivated vaccines.

[0087] Example 5: Application of inactivated vaccines in DIVA

[0088] The H5N1 NP protein (Accession: PP577946.1) was used to coat the ELISA plate. The protein was diluted to 10 μg / mL with PBS solution (pH 7.4), and 100 μL was added to each well. The plate was then sealed and incubated overnight at 4°C. After overnight incubation, the liquid in the wells was discarded, and the reaction wells were filled with PBST. The plate was washed three times, with each wash lasting 3 min, and then patted dry on paper. 300 μL of 5% skim milk was added to each well, and the plate was incubated at 37°C for 2 h. After incubation, the plate was washed three times, and then DIVA assay was performed. D21 serum from the inactivated vaccine group in Example 4 and the infected serum to be tested were taken and diluted 10-fold with PBS solution. Each sample was tested twice, and 100 μL of negative control was added to each well. The plate was incubated at 37°C for 1 h. After incubation, repeat the washing steps three times. Dilute the enzyme-labeled secondary antibody 5000 times with PBS, adding 100 μL to each well, and incubate at 37°C for 1 hour. Discard the liquid in the wells, wash five times with PBST, allowing it to stand for 3 minutes each time before patting dry. Add 100 μL of TMB substrate chromogenic solution to each well, and incubate in the dark at approximately 25°C for 15 minutes. After chromogenic incubation, add 50 μL of 2MH2SO4 to each well and read the value at OD450.

[0089] It should be noted that, since serum from naturally infected H5 subtype avian influenza virus was unavailable, this embodiment, based on published H5N1 virus information, used immune serum from a recombinant inactivated virus vaccine (Ebang Biotechnology, recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine) composed of the PR8 viral backbone and the HA and NA proteins of the H5 subtype virus to simulate naturally infected H5 subtype virus serum. Because influenza NP proteins are conserved in avian influenza viruses, they can be used for DIVA detection.

[0090] The infected serum to be tested was a simulated natural infection serum obtained on day 21 after SPF chickens were immunized with a recombinant virus inactivated vaccine consisting of the PR8 virus backbone and the HA and NA proteins of the H5 subtype virus.

[0091] The results are shown in Figure 5. The ELISA results show that NP antibodies were not detected in the serum of chickens immunized with the inactivated vaccine of this application. However, NP protein antibodies were detectable in the positive serum of chickens infected with H5N1 through simulated natural immunization. Therefore, the inactivated vaccine provided in this application has the ability to distinguish between infected and immunized animals.

[0092] In summary, the highly pathogenic H5 subtype avian influenza inactivated vaccine based on VSV vector provided in this application is safe, effective, and compatible with the DIVA strategy.

[0093] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0094] The above description describes specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing an H5 subtype avian influenza inactivated vaccine, characterized in that, Includes the following steps: Preparation of recombinant virus: Using vesicular stomatitis virus lacking the surface envelope protein gene as a vector, the NA and HA genes of the H5 subtype highly pathogenic avian influenza virus strain were inserted in the form of expression cassettes between the M and L genes of the vector and rescued by reverse genetics technology; the NA and HA genes were tandemly arranged in the 5′-3′ direction. Inoculation: The recombinant virus is inoculated into susceptible cells for culture, and the viral fluid is harvested after replication and proliferation. Inactivation: The virus solution is inactivated to obtain an inactivated virus solution; Preparation of inactivated vaccine: The inactivated virus solution is added 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 as described in claim 1, characterized in that, The H5 subtype avian influenza virus was derived from the A / Texas / 37 / 2024 virus strain.

3. The method for preparing the H5 subtype avian influenza inactivated vaccine as described in 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 as described in claim 1, characterized in that, The susceptible cells include young hamster kidney cells.

5. The method for preparing the H5 subtype avian influenza inactivated vaccine as described in claim 1, characterized in that, The inactivation step includes the addition of β-propiolactone for inactivation.

6. The method for preparing the H5 subtype avian influenza inactivated vaccine according to any one of claims 1-5, characterized in that, The adjuvant is a white oil adjuvant.

7. An H5 subtype avian influenza inactivated vaccine, characterized in that, It is prepared by the method for preparing the H5 subtype avian influenza inactivated vaccine according to any one of claims 1-6.

8. The H5 subtype avian influenza inactivated vaccine as described 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 was obtained by using a vesicular stomatitis virus lacking the surface envelope protein gene as a vector, inserting the NA and HA genes of the H5 subtype highly pathogenic avian influenza virus strain into the space between the M and L genes of the vector in the form of an expression cassette, and rescuing them using reverse genetics technology; the NA and HA genes were tandemly arranged in the 5′-3′ direction.

10. The use of the recombinant H5 subtype avian influenza virus strain of claim 9, or the antiserum produced by immunization with the H5 subtype avian influenza inactivated vaccine of claims 7-8, in the preparation of a reagent for distinguishing between immunization and natural infection.

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