Gene for expressing Newcastle disease virus LaSota strain fusion (F) protein and application thereof

Abstract

The invention discloses a gene for expressing Newcastle disease virus LaSota strain fusion (F) protein and application of the gene. Specifically, the invention discloses a nucleotide molecule for expressing the gene type II Newcastle disease virus F protein, and the nucleotide molecule has a sequence as shown in SEQ ID NO: 1; the invention also provides a recombinant expression plasmid containing the nucleotide molecule, and a DNA vaccine containing the recombinant expression plasmid. The DNA vaccine has a good immune protection effect on Newcastle disease gene IX type standard virulent F48E9 and current epidemic gene VII type virulent HeB38, and the detoxification level of a host subject is remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology. Specifically, the present invention provides an optimized genotype II Newcastle disease virus LaSota strain fusion (F) gene, a recombinant expression vector expressing the genotype II Newcastle disease virus LaSota strain fusion (F) protein, and a DNA vaccine comprising an effective amount of the recombinant expression vector. The vaccine can effectively prevent and / or treat infection with genotype IX Newcastle disease virus F48E9 strain and genotype VII HeB38 strain. Background Art

[0002] Newcastle Disease (ND) is a highly contagious, lethal avian infectious disease caused by Newcastle Disease Virus (NDV), which has caused huge economic losses to the global poultry industry. [1] .

[0003] NDV belongs to the genus Mumps virus of the family Paramyxoviridae. Its genome is a single-stranded, negative-strand, non-segmented RNA that encodes six structural proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L). [2] F protein is a transmembrane glycoprotein that mediates the fusion of viruses and host cells. It is the main antigen that induces the body to produce neutralizing antibodies and cellular immune responses, and can stimulate the body to produce effective immune protection responses. [3] .

[0004] NDV has been constantly mutating and evolving in the long-term evolution process, and 21 genotypes have been identified so far. [4] Based on the differences in the nucleotide sequence of the F gene, NDV can be divided into class I and class II, of which class II is further divided into multiple genotypes. [5] In recent years, ND has been effectively controlled in many countries through widespread vaccination, but it still occurs in some areas, and the prevalence of strong strains such as genotype VII has brought new challenges to prevention and control work. [6,7] Vaccination is one of the key measures for Newcastle disease prevention and control. Although existing live attenuated vaccines can effectively prevent deaths, they usually require intensive vaccination programs and may not completely block viral replication and shedding or provide good cross-protection against infection with other genotypes. [8] .

[0005] DNA vaccines, also known as nucleic acid vaccines, are new genetically engineered vaccines that introduce plasmids encoding target antigen protein gene sequences into host cells via intramuscular injection. These vaccines express the antigen protein through a transcriptional system, inducing the host to produce an immune response against the antigen protein, thereby achieving the purpose of immunization. They are known as "third-generation vaccines." Compared to traditional vaccines, DNA vaccines have significant advantages: they can induce both cellular and humoral immunity; they are unaffected by maternal antibodies and do not affect immune detection; they have a simple production process and are easy to prepare multivalent and combined vaccines. [9-11] In addition, studies have shown that optimizing the codons of heterologous immunogen genes to the codons preferred by immunized animals can significantly improve the expression efficiency of DNA vaccine antigens.

[12] Therefore, DNA vaccine is considered to be one of the ideal vaccines for preventing and controlling Newcastle disease. Summary of the Invention

[0006] The LaSota strain belongs to genotype II, a classic attenuated strain of Newcastle disease virus (NDV) widely used in vaccine development both domestically and internationally. Since the dominant NDV strain currently circulating is genotype VII, which exhibits significant genetic and antigenic differences from traditional strains, existing techniques for developing NDV DNA vaccines typically select the nucleotide sequence of the fusion protein (F) or the hemagglutinin-neuraminidase (HN) protein of genotype VII strains as the active ingredient in DNA vaccines, rather than considering immunogenic genes from genotype II strains. This present invention, for the first time, utilizes the codon-optimized F gene of the LaSota strain in a NDV DNA vaccine, demonstrating its robust immune response. Furthermore, the DNA vaccine provided 100% protection against a standard, virulent F48E9 challenge at a 30μg dose, significantly exceeding the protection achieved by a 200μg dose of a DNA vaccine based on the non-codon-optimized F gene of the D26 strain.

[0007] In a specific embodiment of the present invention, the F gene of the LaSota strain was codon-optimized and Kozak sequence-optimized before insertion into the eukaryotic expression vector pCAGGs-kan to construct the DNA vaccine plasmid pCA-LaF. Its immune efficacy was systematically evaluated. The results demonstrated that the DNA vaccine plasmid pCA-LaF not only provided 100% protection against lethal challenge with the virulent genotype IX strain F48E9 and the currently prevalent virulent genotype VII representative strain HeB38, but also significantly reduced viral replication in chickens, demonstrating excellent immune protection. The DNA vaccine plasmid pCA-LaF will provide essential technical support for the prevention and control of Newcastle disease in my country.

[0008] In a specific embodiment of the present invention, the codons in the F gene of the LaSota strain of Newcastle disease virus are replaced with codons preferred by chickens to enhance immune efficacy. Different codon optimization strategies can be used for the same host, and different codon optimization strategies can lead to different immune effects. After extensive analysis and comparison, the inventors optimized the codons of Newcastle disease virus and also added a kozak sequence before the start codon to enhance exogenous gene expression. The resulting codon-optimized gene, optiLaF (SEQ ID NO: 1), was found to have excellent immune efficacy. Furthermore, the applicants also prefer to use a high-efficiency expression vector with a dual promoter. This optimization strategy and the construction of a DNA vaccine for Newcastle disease virus immunization have not been reported.

[0009] In a specific embodiment of the present invention, the DNA vaccine may contain at least one pharmaceutically acceptable adjuvant. The term "adjuvant" as used herein refers to a pharmaceutical or immunological substance administered for the purpose of enhancing the immune response of a vaccine. The adjuvant may be aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, viral particles, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80 (which is an emulsifier)), CpG, flagellin, poly I:C, AS01, AS02, ISCOM, or ISCOMMATRIX.

[0010] Additionally, DNA vaccines can be formulated using methods known in the art to allow for quick release or sustained or extended release of the active ingredient following administration to a mammal.

[0011] The DNA vaccine can be administered by various routes, including, for example, injection, mucosal, LNP adjuvant delivery, or gene gun introduction to implement immunization, wherein the injection includes, for example, intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, organ injection, thoracic injection, and intraperitoneal injection.

[0012] The DNA vaccine can be formulated in a suitable form together with a conventional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include, for example, water, suitable oil, saline, aqueous carriers for parenteral administration (e.g., glucose aqueous solution, ethylene glycol aqueous solution, etc.), and may additionally include stabilizers and preservatives. Examples of suitable stabilizers may include antioxidants, such as sodium bisulfite, sodium sulfite, and ascorbic acid. Examples of suitable preservatives may include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. In addition, when needed according to the method of administration or preparation, vaccines according to the present disclosure may appropriately include suspending agents, solubilizing agents, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH regulators, analgesics, buffers, antioxidants, etc. Pharmaceutically acceptable carriers and formulations suitable for the present disclosure, including those exemplified above, are described in the literature [Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Science), latest edition].

[0013] In a specific embodiment of the present invention, the nucleotide molecule expressing the genotype II Newcastle disease virus F protein (whose nucleotide sequence is shown in SEQ ID NO: 1) can also be used to prepare an mRNA vaccine. During the preparation of the mRNA vaccine, the nucleotide sequence shown in SEQ ID NO: 1 is cloned into a suitable vector and transcribed in vitro to obtain mRNA, which is then used as the active ingredient of the mRNA vaccine. Because the same nucleotide sequence is utilized and the expressed HA protein is the same, the mRNA vaccine constructed with the nucleotide sequence shown in SEQ ID NO: 1 has the same or better immune effect as the DNA vaccine. Therefore, those skilled in the art can reasonably expect that the mRNA vaccine will have similar technical effects as the DNA vaccine of the present invention.

[0014] In the present invention, the live vector vaccine is also called recombinant vector vaccine, which is a vaccine obtained by inserting a gene encoding an immunogenic protein of a specific pathogen into a vector using an attenuated live virus or attenuated live bacteria as a vector. It can be injected into the body as a vaccine to prevent specific pathogens.

[0015] In the present invention, the subunit vaccine does not contain the entire pathogen, but only the antigenic portion of the pathogen, such as a protein, polysaccharide, or polypeptide. Because the vaccine does not contain the "live" components of the pathogen, there is no risk of introducing disease, but its production is relatively complex.

[0016] In the present invention, the vaccine can also be used to prepare a monoclonal monomer for preventing or treating Newcastle disease.

[0017] Specifically, this application provides the following technical solutions:

[0018] 1. A nucleotide molecule having the sequence shown in SEQ ID NO: 1.

[0019] 2. A recombinant expression plasmid comprising the nucleotide molecule described in item 1.

[0020] 3. The recombinant expression plasmid according to item 2, which is obtained by digesting the eukaryotic expression vector pCAGGs and then introducing the nucleotide molecule shown in SEQ ID NO: 1.

[0021] 4. A DNA vaccine comprising the nucleotide molecule of item 1 or the recombinant expression plasmid of item 2 or 3; optionally, the DNA vaccine further comprises a pharmaceutically acceptable adjuvant.

[0022] 5. The DNA vaccine according to item 4, wherein the recombinant expression plasmid is coated with LNP.

[0023] 6. The DNA vaccine according to item 4 or 5, which is in a form suitable for immunization by injection, mucosal delivery, LNP adjuvant delivery, or gene gun introduction.

[0024] 7. A method for preparing a DNA vaccine, comprising the step of operably linking the nucleotide molecule described in item 1 to a eukaryotic expression vector to obtain a recombinant expression vector;

[0025] Optionally, the eukaryotic expression vector is a pCAGGs vector.

[0026] 8. The method according to item 7, further comprising the step of coating the recombinant expression vector with LNP.

[0027] 9. Use of the nucleotide molecule described in item 1 or the recombinant expression plasmid described in item 2 or 3 in the preparation of a DNA vaccine for preventing or treating Newcastle disease virus infection.

[0028] 10. The method according to claim 9, wherein the Newcastle disease virus is a genotype VII and / or IX strain.

[0029] 11. Use of the nucleotide molecule described in item 1 in the preparation of an mRNA vaccine, a live vector vaccine, a subunit vaccine or a monoclonal antibody for preventing or treating Newcastle disease virus infection.

[0030] Beneficial effects:

[0031] 1. Existing Newcastle disease vaccines are primarily based on the traditional genotype II LaSota strain and the currently prevalent genotype VII strain. These two strains exhibit significant antigenic differences and poor cross-protection. However, the DNA vaccine constructed in this invention, based on the genotype II LaSota strain, demonstrates excellent immune protection (100% mortality protection) against both the standard virulent genotype IX F48E9 strain and the currently prevalent virulent genotype VII HeB38 strain. The different genotypes used are representative, and therefore, this vaccine is believed to offer good cross-protection.

[0032] 2. In addition, in the efficacy evaluation of Newcastle disease vaccines in the prior art, the main evaluation criteria are 90% mortality protection rate and morbidity protection rate, and there is no requirement for the detoxification level; however, the vaccine of the present invention can significantly reduce the detoxification level when chickens are immunized with a dose of 30 μg.

[0033] 3. In this application, an innovative attempt was also made to deliver DNA vaccines by LNP encapsulation. Compared with naked plasmids, LNP-DNA vaccines can advance the time of antibody production, increase antibody levels, and achieve complete protection (no death, no disease, no toxin shedding) at a low dose of 30 μg. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure shows the PCR identification of plasmid pCA-LaF; wherein, M: Trans2K Plus DNA Marker; 1: empty vector control; 2: PCR product of pCA-LaF; 3: ddH2O control.

[0035] Figure 2 The figure shows the enzyme digestion identification of plasmid pCA-LaF; wherein, M1: DL10000 DNA Marker; M2: Trans2KPlus DNA Marker; 1: pCA-LaF plasmid; 2: EcoR I digestion product of pCA-LaF; 3: EcoR I and Xho I double digestion product of pCA-LaF.

[0036] Figure 3 The results of IFA detection of F protein and NDV positive serum are shown; A shows the results of HEK293T cells transfected with pCA-LaF; B shows the results of HEK293T cells transfected with the pCAGGs-kan empty vector;

[0037] Figure 4 The supercoil content of plasmid pCA-LaF is shown; 1: Trans2K Plus DNA Marker; 2: pCA-LaF supercoil content: 88.7%; 3: pCA-LaF supercoil content: 89.7% DETAILED DESCRIPTION

[0038] 1 Materials and Methods

[0039] 1.1 Strains, cells, and plasmids

[0040] Escherichia coli DH5α competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; HEK293T cells (human embryonic kidney cells (CRL-11268), from ATCC), MDCK cells (canine kidney cells, (NBL-2), from ATCC), and the eukaryotic expression plasmid pCAGGs-Kan (the ampicillin resistance gene in the original pCAGGs vector was replaced with the kanamycin resistance gene, wherein the original pCAGGs vector was kindly donated by Professor Yoshihiro Kawaoka or commercially available) were all preserved by the National Avian Influenza Reference Laboratory, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0041] 1.2 Enzymes and main reagents

[0042] Trans2K Plus DNA Marker was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Ex Taq enzyme and DL10000 Marker were purchased from TaKaRa; T4 DNA ligase, EcoR I, and Xho I restriction endonucleases were purchased from New England Biolabs; the GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; kanamycin powder was purchased from Amresco; the EndoFree Plasmid Maxi Kit was purchased from QIAGEN; the transfection reagent Lipofectamine™ 3000 was purchased from Invitrogen; NP40 lysis buffer was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; DMEM cell culture medium, fetal bovine serum (FBS), Opti-MEM cell culture medium, and 0.25% Trypsin-EDTA were purchased from Gibco; 4% paraformaldehyde universal tissue fixative was purchased from Biosharp; bovine serum albumin (BSA) powder was purchased from Meilun Biotechnology; and penicillin-streptomycin double antibody Pen Strep was purchased from Invitrogen Co., Ltd., and lipid nanoparticles (LNPs) (SM-102, Cholesterol, DSPC, and mPEG-DMG-2K) were purchased from Xiamen Sinopharm Biotechnology Co., Ltd. All other reagents used in this experiment were either domestically produced analytical grade or imported in aliquots.

[0043] 1.3 Serum and antibodies

[0044] FITC-labeled goat anti-chicken IgG DyLight™ 680 secondary antibody was purchased from Thermo Fisher Scientific; chicken Newcastle disease-positive serum was purchased from Harbin Guosheng Biotechnology Co., Ltd.

[0045] 1.4 Viruses and Antigens

[0046] The virulent genotype IX strain F48E9 and genotype VII strain HeB38 used for challenge, and the recombinant virus rLaSota-eGFP expressing green fluorescent protein (i.e., eGFP was introduced into the LaSota strain, and the preparation method can be found in the existing technical document "Construction of a recombinant Newcastle disease virus LaSota vaccine strain expressing green fluorescent protein") were all obtained from the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0047] 1.5 Chicken embryos and SPF chickens

[0048] The 9-11 day old chicken embryos and 21 day old SPF chickens used in the experiment were provided by the Experimental Animal Center of Harbin Veterinary Research Institute. During the experiment, all SPF chickens were raised in negative pressure isolators.

[0049] 1.6 Main instruments

[0050] A small centrifuge (Centrifuge 5424), a PCR instrument (MasterCycler Pro S), and a shaker (ExcellaE25) were purchased from Eppendorf; a fluorescence microscopy system (EVOS FL Auto 2) was purchased from Invitrogen; a nucleic acid electrophoresis apparatus (DYY-6C) was purchased from Beijing Liuyi Company; a 37°C constant temperature incubator (NU-5510E) was purchased from NUAIRE Company; and a rapid nanodrug preparation instrument (INano™ E) was purchased from Myanna Instrument Technology Co., Ltd.

[0051] Example 1 Codon Optimization and Synthesis of the F Gene of Genotype II Newcastle Disease Virus LaSota Strain

[0052] After extensive analysis and comparison, the inventors obtained the optimized F gene sequence, SEQ ID NO: 1. Specifically, using DNAStar software, the codons of the F gene of the LaSota strain of Newcastle disease virus were replaced with selected chicken-preferred codons, and the Kozak sequence gccaccacc was added before the start codon ATG. This resulted in the sequence of the codon-optimized gene optiLaF, as shown in SEQ ID NO: 1. After EcoRI and XhoI restriction enzyme sites were added to both ends of the optimized gene, the gene was synthesized by Jilin Kumei Biotechnology Co., Ltd. and inserted into the plasmid pCAGGs-kan, generating the recombinant plasmid pCA-LaF.

[0053] Primer Premier 6.0 software was used to design primers upstream and downstream of the multiple cloning site of the recombinant plasmid pCA-LaF for PCR identification of the inserted fragment. The primer sequences are shown in Table 1 and were synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd.

[0054] Table 1 Primer sequences

[0055]

[0056] Example 2 Construction and identification of the recombinant plasmid pCA-LaF expressing the optimized F gene

[0057] The empty vectors pCAGGs-kan and pCA-LaF were treated with restriction endonucleases EcoR I and Xho I at 37°C for 1 hour. The digested products were purified using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific) and ligated using T4 DNA ligase. The ligated products were then transformed into competent DH5α cells (Beijing Quanshijin Biotechnology Co., Ltd.) and incubated in an inverted incubator at 37°C for 12 hours.

[0058] The next day, suspected positive colonies were transferred to 3 mL of LB medium containing kanamycin and cultured with shaking at 37°C for 12 hours. PCR amplification was performed using primers pCA-F / R, and the PCR products were identified by 1% nucleic acid gel electrophoresis to screen for positive transformants. Plasmids were extracted using an endotoxin-free plasmid miniprep kit. Recombinant plasmids were digested with restriction enzymes EcoRI, EcoRI, and XhoI, and sequenced. Positive bacterial cultures were added to an equal volume of 30% glycerol for preservation until ready for use.

[0059] The recombinant plasmid pCA-LaF was identified by PCR ( Figure 1 ), 1% agarose gel electrophoresis results showed that the empty vector pCAGGs without exogenous gene insertion could amplify a fragment of about 500 bp, while the pCA-LaF vector with exogenous optiLaF gene inserted could amplify a fragment of about 2.2 kb.

[0060] The recombinant plasmid pCA-LaF was digested with EcoR I, EcoR I, and Xho I, respectively. The digestion products were subjected to 1% agarose gel electrophoresis. EcoR I digestion gave a fragment of approximately 6.4 kb, while EcoR I and Xho I digestions gave fragments of approximately 4.7 kb and 1.7 kb, respectively. The results were consistent with expectations ( Figure 2 ), indicating that the recombinant plasmid pCA-LaF was successfully constructed.

[0061] Example 3 Indirect immunofluorescence identification of recombinant plasmid expression in vitro

[0062] 1. Transient transfection of recombinant plasmid

[0063] After cell counting, HEK293T cells (human embryonic kidney cells (CRL-11268), from ATCC) were plated at 1 × 10 6 HEK293T cells were plated at 1 μg / well in a 6-well polylysine-coated plate and cultured in DMEM supplemented with 10% FBS and 1% Pen-Strep. When the cells reached 80% confluency, the medium was discarded, the cells were rinsed twice with PBS, and 2 mL of Opti-MEM medium was added to each well to maintain cell morphology. The recombinant plasmid pCA-LaF was transfected into HEK293T cells using Lipofectamine™ 3000 at a transfection rate of 4 μg / well. HEK293T cells transfected with the empty pCAGGs vector served as a negative control.

[0064] 2 Indirect immunofluorescence (IFA) identification

[0065] After 36 hours of incubation at 37°C with 5% CO₂, the Opti-MEM medium was discarded. Each well was washed twice with 2 mL of PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. Each well was washed three times with 2 mL of PBS and blocked with freshly prepared 1% BSA solution for 1 hour. Chicken Newcastle disease-positive serum (purchased from Harbin Guosheng Biotechnology Co., Ltd.) diluted in 1% BSA solution (1:300) was used as the primary antibody, with 1 mL / well added. Incubation was continued at 37°C for 1 hour, followed by four 5-minute washes in PBST containing 0.05% Tween-20. A secondary antibody, FITC-conjugated goat anti-chicken IgG (purchased from Thermo Fisher Scientific) diluted in 1% BSA solution (1:2000), was added to each well. Incubation was carried out in the dark for 30 minutes at room temperature, followed by four 5-minute washes in PBST. Finally, 1 mL of PBS was added to each well and the cells were observed under a fluorescence microscope.

[0066] Here are the results:

[0067] The recombinant plasmid pCA-LaF was transfected into HEK293T cells at a dose of 4 μg and cultured at 37°C and 5% CO2 for 36 h before indirect immunofluorescence assay. Chicken Newcastle disease positive serum (purchased from Harbin Guosheng Biotechnology Co., Ltd.) was used as the primary antibody to identify the expression of F protein in the expression plasmid ( Figure 3 The results showed that F protein could be efficiently expressed in 293T cells.

[0068] Example 4 Virus tissue culture median lethal dose TCID 50 Determination of

[0069] After cell counting, MDCK cells (canine kidney cells, (NBL-2), from ATCC) were plated at 5×10 4 MDCK cells were cultured in a 96-well cell culture plate at a concentration of 100 μL / well in DMEM medium containing 10% FBS and 1% Pen Strep. When the cell confluence reached 90%, the medium was discarded and the cells were washed twice with PBS. 100 μL of Opti-MEM medium containing Pen Strep (1:10000) and TPCK trypsin (1 μg / mL) was added to each well. The recombinant virus Newcastle disease rLaSota-eGFP stock solution was serially diluted 10-fold to 10 using Opti-MEM medium containing Pen Strep and TPCK trypsin. -10 Each dilution of the virus solution was inoculated into one vertical row (8-well) of MDCK cells at 100 µL / well. The last vertical row of normal MDCK cells served as a negative control (100 µL of Opti-MEM medium was added). After incubation at 37°C, 5% CO₂ for 24 h, viral fluorescence was observed under an inverted fluorescence microscope. The TCID was calculated using the Reed-Muench method based on the fluorescence results. 50 .

[0070] Here are the results:

[0071] The recombinant virus Newcastle disease rLaSota-eGFP stock solution was diluted 10-fold in Opti-MEM medium containing Pen Strep and TPCK trypsin to 10 -10 Each dilution of the virus solution was inoculated into one vertical row (8-well) of MDCK cells at 100 µL / well. The last vertical row of normal MDCK cells served as a negative control (100 µL of Opti-MEM medium was added). After incubation at 37°C, 5% CO₂ for 24 h, viral fluorescence was observed under an inverted fluorescence microscope. The TCID was calculated using the Reed-Muench method based on the fluorescence results. 50 , the result is 10 -6.12 / 100 µL.

[0072] Example 5 Virus Chicken Embryo Median Lethal Dose (EID) 50 Determination of

[0073] The allantoic fluid of chicken embryos infected with Newcastle disease virus F48E9 strain and HeB38 strain was diluted 10 times in a row to 10 -10 , 10 of them -5 to 10 -10Six dilutions were inoculated into 9-day-old chicken embryos, with four replicates for each dilution. The cells were incubated at 37°C for 48 h. 50 μL of allantoic fluid was then collected for hemagglutination assay, and the virus EID was calculated according to the Reed-Meunch method. 50 .

[0074] Here are the results:

[0075] The allantoic fluid of NDV F48E9 and HeB38 viruses were serially diluted 10-fold to 10 with sterile PBS. -10 , 10 of them -5 to 10 -10 Six dilutions were inoculated into 9-11 day old chicken embryos, with four replicates for each dilution. The cells were incubated at 37°C for 48 h. 50 μL of allantoic fluid was then collected for hemagglutination test. The results are shown in Table 2.

[0076] Table 2 EID of each virus 50

[0077]

[0078] Example 6 Evaluation of the immune efficacy of DNA vaccines in SPF chickens

[0079] 1. Preparation of DNA Vaccine

[0080] Plasmid pCA-LaF was transformed into DH5α competent cells, evenly spread on LB plates containing kanamycin, and cultured at 37°C overnight. A single positive colony was inoculated into 5 mL of LB medium containing kanamycin and cultured at 37°C and 220 rpm for 12 h. The colony was then inoculated into 500 mL of LB medium containing kanamycin and cultured at 37°C and 220 rpm for 16 h. The plasmid was extracted in large quantities using the EndoFree Plasmid Maxi Kit (purchased from QIAGEN). The concentration of the extracted plasmid was determined. 10 μL (0.01 μg / μL) of the plasmid was subjected to 0.7% agarose gel electrophoresis and analyzed for supercoiling content. The extracted plasmid pCA-LaF (also known as DNA vaccine pCA-LaF) was diluted with sterile PBS before immunization. At the same time, the plasmid pCA-LaF was coated with lipid nanoparticles LNP (purchased from Xiamen Sinobond Biotechnology Co., Ltd.) using a nanodrug preparation instrument (INano™ E) to prepare LNP-pCA-LaF, that is, LNP-coated DNA vaccine.

[0081] The supercoil content of the plasmid was tested before immunization. 10 μL of the plasmid (0.01 μg / μL) was subjected to 0.7% agarose gel electrophoresis and analyzed using a Bio-Rad Image Lab gel imaging system. The results showed that the average supercoil content of pCA-LaF was 89.2%, which fully met the immunization requirements ( Figure 4 ).

[0082] 2. Experimental Animal Grouping and Immunization Protocol

[0083] This experiment used a two-site intramuscular injection method, with 0.1 mL of vaccine injected at each site for a total immunization volume of 0.2 mL. The immunization doses were 100 µg, 50 µg, and 30 µg (three groups), respectively. All SPF chickens in the experimental groups received their first vaccination at 3 weeks of age, followed by a booster vaccination at the same dose 3 weeks after the initial vaccination.

[0084] Different doses of expression plasmid pCA-LaF were used for heterologous challenge protection experiments with the standard virulent strain F48E9 (genotype IX) and the currently popular representative virulent strain HeB38 (genotype VII) to evaluate the protection of the vaccine against attacks by different representative strains of NDV.

[0085] Table 3 Experimental grouping

[0086]

[0087] 3. Determination of Neutralizing Antibodies

[0088] After the first immunization, blood was collected and serum was separated every week according to the corresponding wing number. MDCK cells were cultured at 5×10 4 Cells were plated in 96-well cell culture plates at 1:10000 and 1:10000 dilutions of 100 μL of Opti-MEM medium containing Pen Strep and TPCK trypsin were added to each well. 100 μL of separated serum was diluted 2-fold to 2:1000 using Opti-MEM medium containing Pen Strep and TPCK trypsin in a 96-well plate. -12 Then, an equal volume of 100 TCID 50 The serum and virus mixture was incubated at 37°C for 1 hour, followed by addition of 100 µL of the serum-virus mixture to MDCK cells. After incubation at 37°C and 5% CO₂ for 24 hours, viral fluorescence was observed under an inverted fluorescence microscope, and neutralizing antibody titers were calculated based on the fluorescence results.

[0089] Here are the results:

[0090] In 3-week-old SPF chickens immunized with a 30 µg dose of LNP-pCA-LaF and various doses of pCA-LaF, neutralizing antibody conversion rates reached 100% by week 4 after the initial immunization (i.e., week 1 after the booster). In the 30 µg LNP-pCA-LaF group, neutralizing antibody conversion reached 100% by week 3 after the initial immunization, demonstrating that LNP-encapsulated DNA vaccine delivery can accelerate the onset of antibody production (Table 4).

[0091] In terms of antibody levels, the average neutralizing antibody levels of the pCA-LaF immunization groups at doses of 100 μg, 50 μg, and 30 μg (F48E9 / HeB38) after booster immunization reached 3.9 log2, 4.0 log2, and 3.5 log2 / 3.8 log, respectively. 2， The average level of neutralizing antibodies in the group immunized with 30 μg of LNP-pCA-LaF reached 5.0 log2, which was higher than that in the groups immunized with all doses of pCA-LaF, indicating that the DNA vaccine pCA-LaF can induce the production of higher levels of neutralizing antibodies and that LNP-encapsulated delivery of DNA vaccines can increase antibody levels (Table 5).

[0092] Table 4 Conversion rate of neutralizing antibodies against NDV rLaSota-eGFP after pCA-LaF immunization (positive number / total number)

[0093]

[0094] Table 5 Average titers (log2) of neutralizing antibodies against NDV rLaSota-eGFP after pCA-LaF immunization

[0095]

[0096] *Boost immunity; #Attack poison; § Chicken death.

[0097] 4. Challenge and protection experiments

[0098] One week after booster immunization, the challenge dose was 10 5 EID 50 Chickens were challenged with the standard virulent strain F48E9 (genotype IX) and the currently prevalent virulent strain HeB38 (genotype VII). The chickens were observed daily for 14 days after challenge, with morbidity and mortality rates observed in both the immunized and control groups.

[0099] The mice were challenged with the virus one week after the booster immunization. The observation period after the challenge was 14 days. The incidence and mortality of each group were recorded every day during the observation period.

[0100] The results showed that after challenge with different subtypes of NDV, no chickens in all immunized groups became ill or died, and the protection rate against infection was 100%. Two SPF chickens in the NDV F48E9 control group died on the third day, and all the remaining chickens died on the fifth day. All SPF chickens in the NDV HeB38 control group died on the fourth day (Table 6).

[0101] Table 6 Morbidity and mortality after different NDV attacks

[0102]

[0103] 5. Virus Isolation and Titration

[0104] Throat and cloacal swabs were collected from each group on days 3, 5, and 7 after challenge using 1 mL of sterile PBS containing ampicillin (2000 U / mL) and streptomycin (2000 µg / mL). Three serial 10-fold dilutions were performed for the immunized group, while five serial 10-fold dilutions were performed for the control group. Both the stock solution and the diluted samples were inoculated into three 9-11-day-old chicken embryos. After incubation at 37°C for 48 hours, 25 µL of allantoic fluid was collected and an equal volume of 1% chicken red blood cells was added for hemagglutination. The titer of the isolated virus was calculated using the Reed-Meunch method.

[0105] After challenge with different viruses, high titers of virus were isolated from chickens in the control group. Compared with the control group, only a few chickens in the pCA-LaF DNA vaccine group exhibited viral shedding, and viral replication levels were significantly reduced. No shedding was observed in the LNP-pCA-LaF group, demonstrating that the pCA-LaF DNA vaccine effectively inhibited viral replication and shedding, and that LNP-encapsulated DNA vaccine delivery completely prevented shedding.

[0106] For pCA-LaF, transient shedding of toxins was detected in throat swabs on day 3 after challenge with a lethal dose of F48E9 at a 100 µg dose. Shedding of toxins was detected in throat and cloacal swabs of one bird after challenge with a lethal dose of F48E9 at a 50 µg dose, and in throat and cloacal swabs of some birds on day 5. Shedding of toxins was detected in cloacal swabs of two birds after challenge with a lethal dose of F48E9 at a 30 µg dose. Shedding of toxins was detected in cloacal swabs of two birds after challenge with a lethal dose of HeB38 at a 30 µg dose. No shedding of toxins was observed in LNP-pCA-LaF after challenge with a lethal dose of F48E9 at a 30 µg dose (Table 7).

[0107] Table 7 Virus isolation results after different NDV attacks

[0108]

[0109] *For chickens that died between the 4th and 5th days, swabs were collected on the 5th day; £ 2 chickens died on the 3rd day; $ All chickens died on the 4th day; # Died on the 5th day.

[0110] discuss

[0111] Newcastle disease (ND) is a highly contagious disease that severely impacts the poultry industry both domestically and internationally, characterized by high pathogenicity and mortality. In recent years, the NDV has exhibited complex mutation trends, with new strains and genotypes constantly emerging and evolving. This makes real-time, accurate monitoring and in-depth analysis crucial for prevention and control efforts.

[0112] Currently, vaccination remains a key means of preventing and controlling Newcastle disease. To achieve effective and scientific prevention and control of Newcastle disease, the two main approaches to developing a vaccine that closely matches the antigens of the circulating strains and continuously updates it, as well as developing a broad-spectrum vaccine that targets different genotypes, have become the two main approaches to Newcastle disease vaccine research and development. DNA vaccines offer high safety, are simple and rapid to prepare, do not rely on a large supply of chicken embryos for production, offer broad cross-protection, and will not interfere with Newcastle disease epidemiological monitoring, making them a key area of ​​research and development for new Newcastle disease vaccines.

[0113] Although the current Newcastle disease strain is mainly genotype VII, studies have found that the LaSota vaccine can also provide a certain degree of cross-protection against heterologous strong strains such as genotype VII and genotype IX, and can cope with infections of different genotypes to a certain extent.

[13] The LaSota strain has good immunogenicity and is widely used in the prevention and control of Newcastle disease at home and abroad.

[0114] In this study, the codons of the F gene of NDV LaSota were optimized and rewritten to chicken-preferred codons. A DNA vaccine, pCA-LaF, was constructed and transfected into HEK293T cells for in vitro expression verification. Indirect immunofluorescence assays demonstrated that the plasmid reacted specifically with NDV-positive serum and that the F protein was efficiently expressed in HEK293T cells.

[0115] To investigate the effective immunization dose and protective efficacy of the DNA vaccine pCA-LaF against heterologous challenge, challenge and protection experiments were conducted in chickens immunized with different doses of 100 µg, 50 µg, and 30 µg. The 30 µg group was challenged with lethal doses of the virulent heterologous Newcastle disease virus F48E9 and HeB38. The immune efficacy tests showed that pCA-LaF induced high levels of neutralizing antibodies after immunization at different doses, with average neutralizing antibody levels of 3.9 log², 4.0 log², and 3.5 log² / 3.8 log², respectively. Chickens immunized with a 30 µg dose provided 100% protection against lethal challenge with both the virulent genotype IX strain F48E9 and the virulent genotype VII strain HeB38, with significantly reduced toxin shedding. Furthermore, the immune efficacy of the DNA vaccine LNP-coated delivery was evaluated, with chickens immunized with a 30 µg dose and challenged with a lethal dose of the virulent heterologous Newcastle disease virus F48E9. The results showed that LNP-pCA-LaF immunization can induce earlier production and higher levels of neutralizing antibodies. The average titer of neutralizing antibodies after booster immunization reached 5.0log2, and it can provide 100% immune protection, that is, the immunized chickens will not die, become ill, or excrete toxins.

[0116] This study demonstrated that the pCA-LaF vaccine provided 100% protection against infection with virulent heterologous Newcastle disease genotypes VII and IX, induced high levels of HI antibodies, and significantly enhanced its protective efficacy by LNP-encapsulated DNA delivery. Therefore, the pCA-LaF DNA vaccine could be used as an effective vaccine for Newcastle disease prevention and control in my country.

[0117] Sequence Listing

[0118] Codon-optimized F gene of LaSota strain optiLaF, SEQ ID NO: 1

[0119]

[0120] References

[0121] [1] Alexander DJ. Newcastle disease and other avian paramyxoviruses. Rev Sci Tech. 2000 Aug, 19(2):443-462.

[0122] [2] Yusoff K, Tan WS. Newcastle disease virus: macromolecules and opportunities. Avian Pathol. 2001, 30(5):439-455.

[0123] [3] Peeters BP, de Leeuw OS, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol. 1999 Jun, 73(6):5001-5009.

[0124] [4] Dimitrov KM, Abolnik C, Afonso CL, et al. Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus. Infect Genet Evol. 2019 Oct, 74:103917.

[0125] [5]Aldous EW, Mynn JK, Banks J, et al. A molecular epidemiologicalstudy of avian paramyxovirus type 1 (Newcastle disease virus) isolates byphylogenetic analysis of a partial nucleotide sequence of the fusion proteingene. Avian Pathol.2003 Jun,32(3):239-256.

[0126] [6]Xue C, Cong Y, Yin R, et al. Genetic diversity of the genotype VIINewcastle disease virus: identification of a novel VIIj sub-genotype. VirusGenes. 2017,53(1):63-70.

[0127] [7]Miller PJ, Haddas R, Simanov L, et al. Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzooticfeatures.Infect Genet Evol. 2015;29:216-229.

[0128] [8]Ferreira HL, Miller PJ, Suarez DL. Protection against DifferentGenotypes of Newcastle Disease Viruses (NDV) Afforded by an Adenovirus-Vectored Fusion Protein and Live NDV Vaccines in Chickens. Vaccines (Basel).2021 Feb,9(2):182.

[0129] [9]Gurunathan S, Wu C Y, Freidag B L, et al. DNA vaccines: a key forinducing long-term cellular immunity. Curr Opin Immunol 2000 Aug,12(4): 442-447.

[0130]

[10] Lee C W, Senne D A, Suarez D L. Development and application ofreference antisera against 15 hemagglutinin subtypes of influenza virus byDNA vaccination of chickens. Clin Vaccine Immunol 2006 Mar, 13(3): 395-402.

[0131]

[11] Liu M A. Gene-based vaccines: Recent developments. Curr Opin MolTher 2010a Feb, 12(1): 86-93.

[0132]

[12] Jiang Y, Yu K, Zhang H, et al. Enhanced protective efficacy of H5subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGSplasmid vector. Antiviral Res. 2007,75(3):234-241.

[0133]

[13] Liu M, Shen X, Li J, et al. Efficacy of Newcastle disease LaSotavaccine-induced hemagglutination inhibition antibodies against challengeswith heterologous virulent strains of genotypes VII and IX. Vet ImmunolImmunopathol. 2023;259:110591.

Claims

1. A nucleotide molecule having the sequence shown in SEQ ID NO:

1.

2. A recombinant expression plasmid comprising the nucleotide molecule according to claim 1.

3. The recombinant expression plasmid according to claim 2, which is obtained by digesting the eukaryotic expression vector pCAGGs and then introducing the nucleotide molecule shown in SEQ ID NO:

1.

4. A DNA vaccine comprising the nucleotide molecule of claim 1 or the recombinant expression plasmid of claim 2 or 3; optionally, the DNA vaccine further comprises a pharmaceutically acceptable adjuvant.

5. The DNA vaccine according to claim 4, wherein The recombinant expression plasmid is coated with LNP.

6. The DNA vaccine according to claim 4 or 5, which is in a form suitable for immunization by injection, mucosal delivery, LNP adjuvant delivery or gene gun introduction.

7. A method for preparing a DNA vaccine, comprising the step of operably linking the nucleotide molecule of claim 1 to a eukaryotic expression vector to obtain a recombinant expression vector; Optionally, the eukaryotic expression vector is a pCAGGs vector.

8. The method according to claim 7, further comprising the step of coating the recombinant expression vector with LNP.

9. Use of the nucleotide molecule according to claim 1 or the recombinant expression plasmid according to claim 2 or 3 in the preparation of a DNA vaccine for preventing or treating Newcastle disease virus infection.

10. The method according to claim 9, wherein the Newcastle disease virus is a genotype VII and / or IX strain.

11. Use of the nucleotide molecule according to claim 1 in the preparation of an mRNA vaccine, a live vector vaccine, a subunit vaccine or a monoclonal antibody for preventing or treating Newcastle disease virus infection.

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