Application of chicken-derived antiviral protein in H9N2 subtype avian influenza and Newcastle disease combined vaccine

Abstract

The invention provides an application of a chicken-derived antiviral protein in a combined vaccine of H9N2 subtype avian influenza and Newcastle disease. An immunopotentiator is the chicken-derived antiviral protein IFITM1. According to the scheme, the chicken-derived antiviral protein IFITM1 serving as an immunopotentiator is matched with the H9N2 subtype avian influenza and Newcastle disease bivalent inactivated vaccine for immunization, immunized chickens can be induced to generate a higher-level H9N2 subtype avian influenza and Newcastle disease serum HI antibody, and the detoxification inhibition effect is improved. And 21 days after the antiviral protein IFITM1 is used as an immunopotentiator and is independently inoculated under the neck and subcutaneously, the detoxification of the chicken H9N2 subtype avian influenza virus and the Newcastle disease virus can be effectively inhibited, so that the chicken tissue injury condition is obviously reduced. At present, no poultry antiviral protein immunopotentiator inactivated vaccine appears on the market in China, and the poultry immunopotentiator provided by the scheme can more effectively prevent and control the avian influenza and the Newcastle disease by preparing the H9N2 subtype avian influenza and Newcastle disease bivalent inactivated vaccine, and can make up the vacancy of the current poultry market immunopotentiator at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to the use of a chicken-derived antiviral protein as an immunopotentiator in a combined inactivated vaccine of H9N2 subtype avian influenza and Newcastle disease. Background Art

[0002] Newcastle disease (ND) is a highly contagious, severe illness caused by the Newcastle disease virus (NDV), with a mortality rate as high as 100%. The combined inactivated Newcastle disease and avian influenza (H9 subtype) vaccine is one of the most frequently used vaccines in broiler chickens, playing a key role in preventing both ND and avian influenza (H9 subtype). However, with widespread use of the vaccine, the virus continues to mutate under the selective pressure of the vaccine, gradually increasing its virulence, posing a greater challenge to disease prevention and control. In clinical practice, there is an urgent need to continuously replace vaccine strains or explore other methods to enhance the immune efficacy of existing vaccines.

[0003] To address the prevalence and harm of NDV and H9N2 AIV, countries have adopted diverse approaches and measures to control the spread of the diseases. Traditional inactivated vaccines still dominate the market for immunization against these diseases. However, commercial vaccines offer limited protection against emerging strains and variants. Currently, widely used Newcastle disease vaccine strains, such as the La Sota strain, are genotype mismatches between the prevalent strains in China and these vaccine strains. Furthermore, the regional nature of AIV epidemics means that many currently available commercial vaccines are inadequate for H9 immunization in my country. Therefore, research into safe and effective immunopotentiators is crucial to address the shortcomings of current inactivated H9N2 and Newcastle disease vaccines, such as poor immunogenicity, weak immune responses, and short-lived immune memory.

[0004] With the continuous advancement of modern molecular biology, immunology, and genetic engineering, researchers have gradually developed and prepared a variety of novel vaccine adjuvants, including nanomaterials, CpG oligodeoxynucleotides, immune ligands, and various other biomolecular immunopotentiators. Cytokines are a class of soluble protein-active molecules secreted by various cells in response to immunogens, mitogens, or other stimulants. They play a vital role in cell signaling, regulating both innate and adaptive immunity by binding to corresponding receptors. They also have diverse functions, including regulating hematopoiesis, cell growth, and damaged tissue repair. Because they are derived from animal organisms, they are safe, highly effective, and specific, with strong species specificity. They can overcome the residual and toxic side effects of many traditional adjuvants, offering significant advantages as veterinary immunopotentiators and ensuring the food safety of livestock and poultry. Furthermore, as key players in immune responses mediated by Toll-like receptor (TLR) agonists, cytokines also play a crucial role in immune signaling and stimulating both cellular and humoral immunity. Therefore, the development of cytokine-based immunopotentiators is of great significance to socioeconomic and animal husbandry development. Numerous studies have demonstrated the potential of cytokines as adjuvants, enhancing the immunogenicity of viral antigens in a variety of animal models. However, their immunopotentiation is highly dependent on the conditions of use, the type of antigen, and the host's characteristics. Interferon-induced transmembrane proteins (IFITMs), a key member of the interferon-stimulated genes (ISGs), have been shown to be key cytokines involved in immune responses to various viruses, playing a role in combating pathogenic microbial infections.

[0005] Currently, there are no chicken-derived antiviral protein immunopotentiators available in my country. Therefore, developing highly effective immunopotentiators for use with inactivated vaccines meets current poultry market needs and is of great significance for the development of combined inactivated vaccines for H9N2 subtype avian influenza and Newcastle disease, as well as for the prevention and control of chicken-derived diseases in my country. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide an immunopotentiator for poultry that is reliable and effective to implement, easy to prepare, and can be used to enhance the immune efficacy of the combined inactivated vaccine of H9N2 subtype avian influenza and Newcastle disease.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] The invention relates to an application of a chicken-derived antiviral protein as an immunopotentiator in a combined inactivated vaccine of H9N2 subtype avian influenza and Newcastle disease, including an application in a whole virus inactivated vaccine or a genetically recombinant inactivated vaccine of H9N2 subtype avian influenza and Newcastle disease virus.

[0009] As a possible implementation manner, further, the chicken-derived antiviral protein IFITM1 of the immune enhancer is expressed using an E. coli expression system and purified by affinity nickel column chromatography.

[0010] As a preferred implementation option, preferably, the H9N2 subtype avian influenza and Newcastle disease combined inactivated vaccine virus strain is inactivated by formaldehyde.

[0011] Based on the above composition scheme of the immunopotentiator in the vaccine, the present invention also provides a method for preparing an inactivated vaccine using chicken-derived antiviral protein as an immunopotentiator, which comprises the following steps:

[0012] 1) The avian influenza H9N2 subtype vaccine strain was diluted 1000-fold with sterile PBS and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos and incubated at 37°C. After 48 hours, the viral allantoic fluid of the chicken embryos was harvested and a hemagglutination test was performed using 1% chicken red blood cells. The viral allantoic fluid with an HA titer of ≥256 was aseptically collected and stored in a -80°C environment (e.g., a low-temperature refrigerator);

[0013] 2) The Newcastle disease vaccine strain was diluted 1000-fold with sterile PBS and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos and incubated at 37°C. After 48 hours, the viral allantoic fluid of the chicken embryos was harvested and hemagglutination assayed using 1% chicken red blood cells. The viral allantoic fluid with an HA titer of ≥256 was collected aseptically and stored in a -80°C environment (e.g., a low-temperature refrigerator).

[0014] 3) Antigen inactivation: Add a formaldehyde solution with a final concentration of 0.1% to the chicken embryo virus allantoic fluid obtained in steps 1 and 2), shake it as it is added, and mix it thoroughly; then, place it in a 37°C constant temperature shaker at 200-220 r / min to inactivate it for 12 hours, remove it, and store it in a 4°C environment (such as a refrigerator) for later use;

[0015] 4) Add the inactivated Newcastle disease classic Lasota strain and avian influenza H9N2 subtype Fujian Province isolate H9-FZ strain virus solution and vaccine adjuvant in the ratio of 1:2.81, and add the antiviral protein IFITM1 to make the concentration in the inactivated vaccine reach 167 μg / mL, use HR-500 dispersing emulsifier to gradually emulsify from gear A to gear D, and emulsify it at 15000r / min under low temperature conditions for 30 minutes.

[0016] Furthermore, the present invention also provides the use of the poultry immune enhancer antiviral protein IFITM1 in the preparation of poultry vaccines.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0018] 1. The immunopotentiator of the present invention can enhance the production of higher levels of serum antibodies in chickens induced by the H9N2 subtype avian influenza and Newcastle disease dual inactivated vaccine. When combined with the recombinant inactivated vaccine for immunization, it can enhance the detoxification inhibition effect and effectively prevent the spread of the current epidemic strains.

[0019] 2. The immunopotentiator of the present invention has the effect of inhibiting the detoxification of NDV and H9N2 avian influenza and reducing tissue damage 21 days after single inoculation.

[0020] 3. Currently, there are no chicken-derived antiviral protein immunopotentiators on the market in my country. Therefore, developing highly effective immunopotentiators for use with inactivated vaccines meets the current market needs of the poultry industry. This is of great significance for the development of a combined inactivated vaccine for H9N2 subtype avian influenza and Newcastle disease, as well as for the prevention and control of chicken-derived diseases in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The inducible expression of the recombinant chicken fusion protein cIFITM1 and cViperin in the present invention

[0023] Figure 2 Solubility analysis of recombinant chicken fusion proteins cIFITM1 and cViperin according to the present invention

[0024] Figure 3 This is a diagram of the autopsy changes after immunization with the H9N2 subtype avian influenza and Newcastle disease dual inactivated vaccine of the present invention and challenge with NDV.

[0025] Figure 4 This is a diagram of the autopsy changes of H9N2 AIV challenged with the H9N2 subtype avian influenza and Newcastle disease combined inactivated vaccine of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to the accompanying drawings and examples. It is particularly noted that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Similarly, the following examples are only some embodiments of the present invention and are not intended to be exhaustive. All other embodiments obtained by those of ordinary skill in the art without creative effort are intended to fall within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. At the same time, the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0028] Example 1. Preparation of two chicken-derived antiviral proteins

[0029] (I) Inducible expression of recombinant chicken fusion proteins cIFITM1 and cViperin

[0030] The constructed recombinant prokaryotic expression plasmids pCold-TF-cIFITM1 and pCold-TF-cViperin were transformed into BL21 (DE3) competent cells, and the positive clone strains were picked and cultured in LB liquid medium. They were first cultured in a 37°C constant temperature shaker at a rate of 180 rpm until the OD600 value reached 0.6-0.8. Then, the inducer IPTG was added to make the final concentration of IPTG in the bacterial solution 0.2 mmol / L, and the expression was induced at 15°C and 180 rpm for 24 hours. The bacterial solution before and after induction was analyzed by SDS-PAGE electrophoresis to detect the expression status of the recombinant fusion proteins cIFITM1 and cViperin. The results are shown in Figure 2. Figure 1 As shown in the figure, it can be observed that the recombinant fusion proteins cIFITM1 and cViperin express specific target bands at positions close to 64kDa and 94kD, respectively, and the protein molecular weights are consistent with the expected sizes, indicating that the recombinant fusion proteins cIFITM1 and cViperin are successfully induced to express.

[0031] (II) Solubility analysis of recombinant chicken fusion proteins cIFITM1 and cViperin

[0032] After successful induction of expression, in order to analyze whether the recombinant fusion proteins cIFITM1 and cViperin are expressed in the supernatant or inclusion bodies, SDS-PAGE electrophoresis was used to detect the supernatant and precipitate resuspension after ultrasonic disruption. The results are as follows Figure 2 As shown, the recombinant fusion proteins cIFITM1 and cViperin are mainly distributed in the supernatant of broken bacteria as soluble proteins, which provides guidance for subsequent protein purification.

[0033] Example 2: Effect of antiviral protein IFITM1 as an immunopotentiator on the immune effect of the new flu-like inactivated vaccine

[0034] (1) Seed solution preparation

[0035] Purified viral allantoic fluid from the H9N2 FZ1 strain and the NDV Lasota strain (maintained in our laboratory) was diluted and inoculated into the allantoic cavity of 10-day-old SPF chicken embryos at a rate of 0.1 mL / embryo. After inoculation, the cells were incubated in a 37°C incubator. The cells were observed every 12 hours, and any embryos that died within 24 hours were discarded. After 48 hours, allantoic fluid with a hemagglutination titer of 256 or higher was collected from 5 to 15 generations of embryos as seed batches and stored at -80°C.

[0036] (2) Proliferation and cultivation of virus strains

[0037] The preserved allantoic fluid of H9N2 strain and NDV Lasota strain was thawed at room temperature and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos at a rate of 0.1 mL / embryo. After inoculation, the embryos were incubated in a constant temperature incubator at 37°C. The allantoic fluid was observed every 12 hours, and chicken embryos that died within 24 hours were discarded. After 48-72 hours, the allantoic fluid was collected. The results showed that the hemagglutination titer of the collected allantoic fluid was greater than or equal to 256, and the viral antigen content was greater than or equal to 10 8.5 EID 50 / 0.1mL.

[0038] (3) Inactivation of virus strains

[0039] Add a final concentration of 0.1% formaldehyde solution to the sterile H9N2 subtype AIV FZ strain and NDV Lasota strain allantoic fluid prepared in step (2), shake as needed to mix thoroughly. Then, inactivate the mixture by shaking at 200-220 rpm in a 37°C constant temperature shaker for 12 hours, remove from the shaker, and store in a 4°C refrigerator until ready for use.

[0040] (IV) Inactivation test

[0041] The inactivated virus solution was inoculated into the allantoic cavity of 10-day-old SPF chicken embryos at a standard of 3 pieces / portion, 0.1 mL / piece, sealed and marked, and placed in a 37°C incubator for culture. It was observed continuously for 5 days, twice / day. After 5 days, the chicken embryo allantoic fluid was harvested and HA assayed. The results showed that the virus solution could be fully inactivated by using 0.1% formaldehyde and shaking at 200-220 r / min in a 37°C constant temperature shaker for 12 hours.

[0042] (5) Vaccine preparation

[0043] In a sterile container, inactivated H9N2 subtype AIV FZ strain virus liquid and NDV Lasota strain (the virus antigen content of the vaccine is 10 8.0EID 50 / mL) and vaccine adjuvant, and added the antiviral protein IFITM1 to make its concentration in the vaccine 167 μg / mL, and emulsification was gradually started from gear A to gear E using an HR-500 dispersing emulsifier for 30 minutes to complete the preparation of vaccine A; the same method was used to prepare vaccine B for the control group, C and D were challenge control groups, and E was a blank control group, as shown in Table 1.

[0044] (6) Finished product inspection

[0045] 1. Appearance: Place 5 mL of the vaccine into a clean glass tube and observe whether the vaccine has any color change or is free of impurities.

[0046] 2. Dosage form: Use a clean pipette to draw up a small amount of vaccine and drop it onto the surface of cold water. Observe whether the vaccine spreads.

[0047] 3. Stability: Pipette 10 mL of each batch of vaccine into a centrifuge tube and centrifuge at 3000 rpm for 15 minutes to observe whether the vaccine has stratification.

[0048] 4. Sterility test: According to the appendix of the current Chinese Veterinary Pharmacopoeia, no bacteria grew.

[0049] 5. Pure inspection: Inspection is carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and all regulations are met.

[0050] 6. Determination of formaldehyde residue: The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The results showed that it complies with the provisions of the General Rules for Veterinary Biological Products.

[0051] 7. Safety test: Ten 14-day-old SPF chickens were subcutaneously injected with the inactivated vaccine containing the immunopotentiator of the present invention, 0.5 mL per chicken, in the neck. All the test chickens were observed for 14 consecutive days. There were no obvious abnormalities in their spirits, feeding and drinking, and no local or systemic adverse reactions were caused by the vaccine.

[0052] 8. Serum Antibody Titer Assay: Ten 14-day-old SPF chickens were randomly divided into 10 groups of 10 each. Vaccines A, B, C, D, and E were administered subcutaneously in the neck. Blood was collected from each group on days 7, 14, and 21 after immunization, and serum was isolated for assay of H9N2 subtype avian influenza HI antibodies. The results are shown in Tables 1 and 2. The average NDV HI titers in the vaccine group 7, 14, and 21 days after immunization were 2.6, 9.6, and 10.8, respectively. For vaccine B, the average HI titers were 2.2, 8.6, and 9.4, respectively. The average AIV HI titers in the vaccine group 7, 14, and 21 days after immunization were 0, 6.5, and 10.4, respectively. For vaccine B, the average HI titers were 0, 5.6, and 9.2, respectively. The addition of the immune enhancer IFITM1 resulted in varying degrees of antibody increases. The average HI titers in the control group and other groups not supplemented with antigen were <3.

[0053] 9. Efficacy test: 21 days after immunization, intravenously inoculate 0.2 mL (10 6.0 EID 50 / mL). After challenge, throat swabs were collected from each group of chickens for NDV inoculation testing. The results are shown in Table 3. Virus was detected in throat and cloacal swabs from Groups A2 and B2 on day 3, with virus isolation rates of 80% and 100%, respectively. Virus isolation rates were 80% in throat swabs from Groups A2 and B2 on day 5, and 20% and 40%, respectively, in cloacal swabs from Groups A2 and B2 on day 5. Virus isolation rates were 100% in throat and cloacal swabs from Group D on days 3 and 5. No virus was detected in throat and cloacal swabs from Group E2 on days 3 and 5.

[0054] The autopsy results are as follows Figure 3 As shown, the challenge group had severe bleeding in the larynx, trachea and glandular stomach, severe congestion in the lungs, and no obvious lesions were observed in the spleen; the bivalent vaccine group had slight bleeding in the larynx, one-sided enlargement of the lungs, and no obvious pathological damage in the glandular stomach and spleen; the bivalent vaccine + cIFITM1 group had catarrhal exudate in the larynx, and no obvious lesions were observed in the lungs, glandular stomach and spleen; no diseased organs were observed in the chickens in the blank group.

[0055] 10. Efficacy test: 21 days after immunization, intravenously inoculate 0.2 mL (10 6.0 EID 50 After challenge, throat swabs were collected from each group of chickens for inoculation testing for the H9N2 subtype of avian influenza. The results are shown in Table 4. In throat swab samples taken 3 and 5 days per week, no virus was detected in Groups A1, B1, and E1, and all were negative for virus isolation. In Group C, virus isolation rates were 100% and 40% on 3 and 5 days per week, respectively. In cloacal swab samples taken 3 and 5 days per week, no virus was detected in Groups A1 and E1, and all were negative for virus isolation. In Group B1, virus isolation rates were 20% on 3 days per week and no virus was detected on 5 days per week. In Group C, virus isolation rates were 100% and 60% on 3 and 5 days per week, respectively.

[0056] The autopsy results are as follows Figure 4 As shown, the chickens in the challenge group had severe bleeding in the larynx and trachea, and mild lesions in the lungs; the bivalent vaccine group had slight punctate bleeding in the larynx and no obvious lesions in the lungs; no lesional organs were observed in the chickens in the bivalent vaccine + cIFITM1 group and the blank group.

[0057] Table 1: Newcastle disease antibody detection after immunization with immune enhancer and combined inactivated vaccine

[0058]

[0059] Table 2: Avian influenza antibody detection after immunization with immune enhancer and combined inactivated vaccine

[0060]

[0061] Table 3: NDV inhibition and detoxification test after immunization with immune enhancer and combined inactivated vaccine

[0062]

[0063] Table 4: AIV inhibition and detoxification detection after immunization with immune enhancer and combined inactivated vaccine

[0064]

[0065] The above description is only part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Application of chicken-derived antiviral proteins in the combined vaccine against H9N2 subtype avian influenza and Newcastle disease.

2. The poultry immunopotentiator according to claim 1, wherein The immunopotentiator is the chicken-derived antiviral protein IFITM1.

3. The chicken-derived antiviral protein according to claim 1, wherein The protein is the chicken-derived antiviral protein IFITM1 expressed by prokaryotic expression of Escherichia coli.

4. The H9N2 subtype avian influenza and Newcastle disease combined inactivated vaccine according to claim 1, characterized in that The antigen is an inactivated antigen, which is the inactivated Newcastle disease classic vaccine strain Lasota strain and the avian influenza H9N2 subtype Fujian Province isolate H9-FZ strain, or the recombinant virus HA+NA / PR8 strain containing the HA and NA genes of the H9-FZ strain and 6 internal genes of the H1N1-PR8 strain successfully rescued by the reverse genetic system. The H9-FZ strain is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan, China, the preservation number is CCTCC NO: V 202171, and the preservation date is September 26, 2021.

5. The H9N2 subtype avian influenza inactivated vaccine according to claim 4, characterized in that The antigens in the H9N2 subtype avian influenza inactivated vaccine are inactivated by formaldehyde.

6. The method for applying the poultry immunopotentiator according to claim 2 or 3 to the Newcastle disease classic vaccine strain Lasota strain and the H9N2 subtype avian influenza inactivated vaccine, characterized in that: It includes the following steps: 1) The avian influenza H9N2 subtype vaccine strain was diluted 1000-fold with sterile PBS and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos and incubated at 37°C. After 48 hours, the viral allantoic fluid of the chicken embryos was harvested and a hemagglutination test was performed using 1% chicken red blood cells. The viral allantoic fluid with an HA titer of ≥256 was aseptically collected and stored at -80°C. 2) The Lasota strain, a classic Newcastle disease vaccine strain, was diluted 1000-fold with sterile PBS and inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos and incubated at 37°C. After 48 hours, the viral allantoic fluid of the chicken embryos was harvested and hemagglutination assayed using 1% chicken red blood cells. The viral allantoic fluid with an HA titer of ≥256 was aseptically collected and stored at -80°C. 3) Antigen inactivation: Add a formaldehyde solution with a final concentration of 0.1% to the chicken embryo virus allantoic fluid prepared in steps 1 and 2), shake it as it is added to thoroughly mix it; then, inactivate it by shaking it in a 37°C constant temperature shaker at 200-220 rpm for 12 hours, remove it, and store it at 4°C until it is used; 4) Add the inactivated Newcastle disease classic vaccine strain Lasota and avian influenza H9N2 subtype vaccine strain virus solution and vaccine adjuvant in the ratio of 1:2.81, and add the antiviral protein IFITM1 to make the concentration in the vaccine 167 μg / mL, use HR-500 dispersing emulsifier to gradually emulsify from gear A to gear D, and emulsify it at 15000r / min under low temperature conditions for 30 minutes.

7. The method for preparing the Newcastle disease classic vaccine strain Lasota strain and the H9N2 subtype avian influenza inactivated vaccine with an immunopotentiator for poultry according to claim 3, characterized in that: In step 3), the preparation method of the immune enhancer antiviral protein IFITM1 is as follows: SPF chickens are infected with H9N2 avian influenza virus, and lung tissue is collected 5 days after infection. Primers are designed with reference to the nucleotide sequence of the coding region of the avian IFITM1 gene published in GenBank, PCR is used to amplify the target band, and sequencing is used to identify the mRNA level expression and gene sequence of chicken IFITM1. Then, a recombinant expression plasmid of IFITM1 is constructed and transformed into the competent Escherichia coli BL21 (DE3). After IPTG induction and ultrasonic disruption, the expression of the antiviral protein is analyzed by SDS-PAGE electrophoresis, and nickel column affinity chromatography is used for purification. The purified antiviral protein is stored at 4°C for future use.

8. Application of poultry immune enhancer antiviral protein IFITM1 in the preparation of poultry bivalent vaccine.

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