A broad-spectrum vaccine based on the HA protein of H9 avian influenza virus and its preparation method

Abstract

This invention provides a modified hemagglutinin (HA) protein for preparing a broad-spectrum vaccine against H9 avian influenza, a broad-spectrum vaccine based on the HA protein, a preparation method, and applications. The modified HA protein is obtained through the following strategy: consensus sequences are constructed as immunogenic backbones for the HA sequences of H9 avian influenza viruses at different epidemic periods; based on this, two types of functional mutations are directionally introduced: one type involves transplanting key epitope residues targeted by known broad-spectrum antibodies, and the other type involves neutralizing epitope residues predicted based on viral evolutionary patterns. The modified HA protein of this invention can maintain a highly similar overall fold and trimer interface to the wild type, can be efficiently expressed in eukaryotic systems, and can induce high levels of neutralizing antibodies with broad-spectrum cross-protective effects in target animals. It provides a core antigen candidate for the development of a new generation of H9 broad-spectrum vaccines and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a broad-spectrum vaccine against avian influenza viruses, particularly to the H9 avian influenza virus. Background Technology

[0002] Avian influenza virus (AIV) H9 is one of the most widespread and pathogenic avian influenza viruses in poultry worldwide. It has become endemic in poultry flocks across Eurasia, causing not only direct economic losses such as decreased egg production and secondary infections, but also posing a persistent threat to global public health security due to its frequent reassortment with other subtypes (such as H5 and H7) as a "gene donor." The surface glycoprotein hemagglutinin (HA) of AIV H9 is the primary target of neutralizing antibodies and a core antigen in vaccine design. However, the HA gene continuously undergoes antigenic drift under host immune pressure, leading to decreased antigenic matching between existing vaccine strains and circulating strains, significantly reducing the effectiveness of immune protection. This makes the development of a broad-spectrum protective H9 vaccine an urgent industry need.

[0003] Currently, the most widely used vaccines in disease control are still oil-adjuvanted inactivated vaccines prepared against specific circulating strains. These vaccines have mature manufacturing processes and can induce strong humoral immunity. For example, patent application CN120192932A (publication date 2025-06-24) discloses a bivalent inactivated vaccine against H9N2 subtype avian influenza virus and its preparation method. It uses virus strains preserved in CGMCC No. 45222 and CGMCC No. 46091 as antigens, and screens for potential vaccine strains through comprehensive epidemiological analysis, antigenicity analysis, and immunogenicity analysis. However, the aforementioned inactivated vaccines have a narrow protective spectrum, effective only against homologous or highly homologous strains; when circulating strains undergo significant antigenic mutations, their protective efficacy drops sharply, leading to frequent "immunization failures" in clinical practice. Farms are forced to frequently change vaccine strains, increasing disease control costs and uncertainty.

[0004] To overcome the shortcomings of inactivated vaccines, recombinant live vector vaccines expressing the H9 HA protein using vectors such as Newcastle disease virus, fowlpox virus, or Marek's disease virus have been extensively studied. These vaccines can mimic natural infection, inducing cellular and mucosal immunity and possessing certain cross-protective potential. However, their safety (especially in immunosuppressed flocks), interference from pre-existing maternal antibodies or vector immunity, and the complexity of their manufacturing processes limit their large-scale application.

[0005] In addition, subunit vaccines prepared using recombinantly expressed HA or HA major antigenic domains (such as HA1) are also employed in this field. For example, patent application CN116036256A (publication date 2023-05-02) discloses a broad-spectrum H9 subtype avian influenza subunit vaccine and its preparation method, which prepares a broad-spectrum H9 subtype avian influenza vaccine by constructing and expressing a recombinant baculovirus with a specific protein-coding gene. However, although the above-mentioned subunit vaccines have high safety, they often have weak immunogenicity, require strong adjuvants, and also face the problem of narrow protective spectrum caused by the dominant immune response due to the hypervariable region of the HA head.

[0006] In recent years, in pursuit of broad-spectrum protection, research has focused on rationally designing and modifying the HA antigen itself. However, existing H9 vaccines, especially novel genetically engineered vaccines aimed at achieving broad-spectrum protection, face a fundamental contradiction: how to effectively overcome the strain-specific immune response dominated by the hypervariable head region while maintaining the strong natural immunogenicity of the HA protein, thereby inducing durable and broad-based cross-protection. Therefore, developing a broad-spectrum H9 vaccine antigen that can cover historically prevalent strains, proactively address future mutation trends, and is easily industrially produced is a key technological bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a modified HA protein for developing a broad-spectrum vaccine against H9 avian influenza, a broad-spectrum vaccine based on the modified HA protein, a preparation method thereof, and applications. The modified HA protein of the present invention, as an H9 broad-spectrum vaccine antigen, can cover historical circulating strains and proactively address future mutation trends, and is easy to industrialize. The present invention also includes a recombinant nucleic acid molecule encoding the modified HA protein, an expression cassette containing the nucleic acid molecule, a vector, a host cell, and an immunogenic composition or broad-spectrum vaccine containing any of the above substances, as well as pharmaceutical uses and treatment methods for avian influenza. The modified HA protein of the present invention can maintain a highly similar overall folding and trimer interface to the wild type, can be efficiently expressed in eukaryotic systems, and can induce high levels of neutralizing antibodies with broad-spectrum cross-protective effects in target animals, providing a core antigen candidate for the development of a new generation of H9 broad-spectrum vaccines and possessing broad application prospects.

[0008] One aspect of the present invention provides a method for preparing a modified hemagglutinin (HA) protein for preparing a broad-spectrum vaccine against H9 avian influenza, characterized in that the amino acid sequence of the modified HA protein comprises that obtained by means of the following preparation:

[0009] (1) Perform multiple sequence alignment on the H9 virus HA protein sequence set at a specific time to generate a common amino acid sequence;

[0010] (2) Introduce a set of selected mutations into the common amino acid sequence, the mutations being selected from:

[0011] (a) Stability-neutralizing epitope embedding mutations corresponding to broad-spectrum antibody-targeting epitopes; or / and

[0012] (b) Corresponding to the neutral epitope embedding mutations predicted by viral evolution.

[0013] Furthermore, the specific period is a historical epidemic period; preferably, the historical epidemic period is the historical epidemic period up to 2020; the common amino acid sequence is as shown in SEQ ID NO: 1.

[0014] Further, any one or more of the following epitope insertion mutations are introduced into the common amino acid sequence: P87L, P89L, E90G, E91G, K92R, G153D, R164Q, N167G, N168A, E196D, D197T, V198T, R200T, T201N; preferably, the amino acid sequence of the modified HA protein obtained after introducing the neutralizing epitope insertion mutation into the common amino acid sequence is shown in SEQ ID NO: 2.

[0015] Furthermore, after introducing the neutralizing epitope embedding mutation into the shared amino acid sequence, the glycosylation site mutation A168T is further introduced; preferably, the amino acid sequence of the modified HA protein after introducing the glycosylation site mutation is SEQ ID NO:3.

[0016] Furthermore, the specific period is a new epidemic period; preferably, the new epidemic period is the new epidemic period from 2021 to 2025, and the common amino acid sequence is as shown in SEQ ID NO: 4.

[0017] Further, any one or more of the following neutralizing epitope embedding mutations are introduced into the common amino acid sequence: L50M, P136S, I153M, I170T, H231N, E271K; preferably, the amino acid sequence of the modified HA protein after introducing the neutralizing epitope embedding mutation is shown as SEQ ID NO: 7, SEQ ID NO: 6 or SEQ ID NO: 5, respectively.

[0018] Another aspect of the present invention provides a modified HA protein for preparing a broad-spectrum vaccine against H9 avian influenza, characterized in that it is prepared by the preparation method described in the present invention.

[0019] Another aspect of the present invention provides a modified HA protein for preparing a broad-spectrum vaccine against H9 avian influenza, characterized in that its amino acid sequences are as shown in SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 5, respectively.

[0020] Another aspect of the present invention provides a recombinant nucleic acid molecule characterized in that it encodes the modified HA protein described in any one of the present invention.

[0021] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention.

[0022] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0023] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention.

[0024] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0025] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0026] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0027] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0028] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0029] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0030] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0031] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0032] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0033] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0034] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0035] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises a modified HA protein as described in any one of the present invention, and / or one or more recombinant nucleic acid molecules as described in the present invention, and / or one or more recombinant gene expression cassettes as described in the present invention, and / or one or more recombinant vectors as described in the present invention, and / or one or more recombinant host cells as described in the present invention; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0036] Another aspect of the present invention provides a recombinant broad-spectrum vaccine, characterized in that it comprises the modified HA protein described in any one of the present invention, and / or one or more recombinant nucleic acid molecules described in the present invention, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present invention; preferably, the broad-spectrum vaccine is a broad-spectrum nucleic acid vaccine; more preferably, the broad-spectrum nucleic acid vaccine is a broad-spectrum RNA vaccine or a broad-spectrum DNA vaccine; most preferably, the broad-spectrum RNA vaccine is a broad-spectrum mRNA vaccine.

[0037] Another aspect of the present invention provides the use of the modified HA protein according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more recombinant broad-spectrum vaccines according to the present invention in the preparation of a drug for preventing avian influenza infection; preferably, the avian influenza is H9 type; more preferably, the avian influenza is H9N2 subtype.

[0038] Another aspect of the present invention provides a method for preventing avian influenza, characterized in that an effective amount of one or more of the immunogenic compositions or pharmaceutical compositions described in the present invention, and / or one or more of the recombinant broad-spectrum vaccines described in the present invention are administered to a subject; preferably, the avian influenza is of type H9; more preferably, the avian influenza is of subtype H9N2.

[0039] The modified hemagglutinin (HA) protein of the present invention and the broad-spectrum vaccines based thereon have the following beneficial technical effects:

[0040] (1) This invention provides a design strategy for a modified HA protein for developing a broad-spectrum vaccine against H9 avian influenza. The core of this strategy is to construct an optimized immunogenic backbone for different epidemic periods of H9 avian influenza virus, and to systematically integrate functional mutations on this basis to shape the breadth and effect of the immune response in a targeted manner.

[0041] (2) Specifically, this invention provides two design strategies for modified HA proteins. The first strategy is based on the "epitope embedding and glycosylation masking" strategy of historically prevalent sequences, which ultimately provides a broad-spectrum antigen that can cover historical prevalent lineages and overcome strain-specific immune advantages. The second strategy is based on the "evolutionary prediction and epitope embedding" strategy of newly prevalent strains, which ultimately provides a broad-spectrum antigen that can proactively address recent and future potential epidemic trends.

[0042] (3) Based on the first design strategy provided by the present invention, the historical consensus sequence S2 was specifically obtained, the exemplary sequence of which is shown in SEQ ID NO: 1; the broad-spectrum epitope embedding sequence S3 was specifically obtained, the exemplary sequence of which is shown in SEQ ID NO: 2; and the glycosylation masking mutation sequence S7 was specifically obtained, the exemplary sequence of which is shown in SEQ ID NO: 3. Based on the second design strategy provided by the present invention, the new epidemic period consensus sequence S8 was specifically obtained, the exemplary sequence of which is shown in SEQ ID NO: 4; and the neutralizing epitope embedding mutation sequences S9, S10, and S11 were specifically obtained, the exemplary sequences of which are shown in SEQ ID NO: 5, 6, and 7, respectively.

[0043] (4) In Example 2, the three-dimensional structure of the modified HA protein designed in this invention was predicted and analyzed using computational structural biology methods. The results showed that the various mutations (epitope embedding, glycosylation, and novel epitope embedding) introduced by rational design in this invention can be well accommodated by the target HA protein backbone. The modified protein can not only maintain a high degree of similarity to the wild type in overall folding and trimer interface, but the conformation of its key functional regions may even be more stable (such as the receptor binding site RBS and the conserved stem region responsible for membrane fusion).

[0044] (5) Example 4 shows that the AG purity of the vaccines containing the recombinant nucleic acid of the present invention is greater than 80%, which meets the quality requirements for cell transfection experiments and vaccine production.

[0045] (6) Example 5 shows that the vaccine AG obtained by the design and preparation method of the present invention can effectively guide the expression of the target HA protein in eukaryotic cells, and the expressed protein has the expected molecular weight.

[0046] (7) Example 6 shows that, except for vaccine E, vaccines A, B, C, D, F, and G (especially vaccines C, D, F, and G) of the present invention can induce high levels of HI neutralizing antibodies with broad-spectrum cross-reactivity in target animals (chickens), providing core serological evidence for the broad-spectrum protective efficacy of the vaccines of the present invention. Attached Figure Description

[0047] Figure 1 This is a flowchart illustrating the overall design strategy of the modified HA protein of this invention.

[0048] Figure 2 A schematic diagram of the molecular structure of the HA protein of H9 avian influenza virus and the location of key mutation sites.

[0049] Figure 3 This is a schematic diagram of a circular plasmid containing the vaccine template of the present invention.

[0050] Figures 4A-4D This includes the quality control peak diagram and purity test results of the vaccine containing the present invention; wherein Figure 4A The quality control peak diagrams and purity test results of vaccines A and B, which include the present invention; Figure 4B The quality control peak diagrams and purity test results for vaccines C and D, which contain the present invention; Figure 4C The quality control peak diagrams and purity test results for vaccines E and F, which contain the present invention; Figure 4D This is a quality control peak diagram and purity test results for vaccine G, which includes the present invention.

[0051] Figures 5A-5B The results of Western blot analysis of the expression effect of the vaccine of the present invention after in vitro transfection into HEK293T cells are shown; wherein Figure 5A The in vitro expression WB detection results of vaccines A, B, and C are presented. Figure 5B The in vitro expression WB detection results of vaccines D, E, and G are presented.

[0052] Figures 6A-6D The neutralizing antibody titer in chicken serum after two immunizations with the vaccine of this invention is given. Figure 6A The neutralizing antibody titer in chicken serum after two immunizations with vaccines A and B of this invention. Figure 6B The titer of neutralizing antibodies in chicken serum after two immunizations with vaccine C of the present invention. Figure 6C The neutralizing antibody titer in chicken serum after two immunizations with vaccines D and E of this invention. Figure 6D The neutralizing antibody titer in chicken serum after two immunizations with vaccines F and G of this invention. Detailed Implementation

[0053] Terms and Definitions

[0054] The term "avian influenza" refers to a viral infectious disease in birds caused by type A influenza virus (AIV). This virus belongs to the Orthomyxoviridae family, and its genome is a segmented single-stranded negative-sense RNA. Based on the antigenicity of its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), it can be divided into several subtypes.

[0055] The term "H9" refers to a subtype of influenza A virus characterized by the presence of the hemagglutinin (HA) protein of subtype 9 and the neuraminidase (NA) protein of subtype 2 on its surface. This invention particularly relates to H9 viruses that infect birds (especially poultry), whose HA protein is a primary target antigen for vaccine design.

[0056] The term "HA" refers to hemagglutinin (HA), a major glycoprotein on the surface of the influenza virus, which mediates the binding of the virus to receptors on the host cell surface and subsequent membrane fusion. It is a key antigen that induces the host to produce neutralizing antibodies. In this invention, unless otherwise specified, HA refers to the HA protein of the H9 influenza virus.

[0057] The term "modified HA protein" refers to a protein obtained by artificially modifying the amino acid sequence of wild-type H9 HA protein using molecular biology and computational design methods. These modifications include, but are not limited to: generating a consensus sequence backbone based on multiple sequence alignment, introducing key residues of known broad-spectrum neutralizing epitopes at specific sites, introducing neutralizing epitope residues based on evolutionary prediction, and introducing or removing specific glycosylation sites. The aim is to enhance the breadth and effectiveness of the immune response induced by this protein as an immunogen.

[0058] The term "consensus sequence," also known as a "shared sequence" or "consensus sequence," refers to a virtual amino acid sequence created by performing multiple sequence alignment on a specific set of protein sequences, selecting the most frequently occurring residues at each amino acid site, and then cascading them together. In this invention, it specifically refers to multiple sequence alignment based on the H9 avian influenza virus HA protein sequence set. Preferably, the consensus sequence of this invention includes, but is not limited to, amino acid sequences such as those shown in SEQ ID NO: 1 and SEQ ID NO: 4.

[0059] The term "broad-spectrum vaccine" refers to a vaccine that can induce the immune system to produce protective immunity against multiple strains of the same pathogen with different genetic branches or antigenic differences. This provides broader protection compared to "strain-specific" vaccines, which are effective only against homologous or highly similar strains.

[0060] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immunity should be measurable by assays, including but not limited to assays measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0061] The term "neutralizing antibody titer" refers to the relative concentration of antibodies in serum that can neutralize viruses, rendering them unable to infect cells. It is usually determined through in vitro experiments (such as micro-neutralization assays) and expressed as the reciprocal of the highest serum dilution that can inhibit 50% or 90% of viral infectivity (e.g., NT). 50 or NT 90 (Value). A higher titer indicates stronger neutralizing activity of the serum. In this invention, this indicator is used to quantitatively evaluate the cross-protective ability of immune serum against different H9 strains.

[0062] The terms "administration" or "inoculation" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous route, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injectable), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes. Intramuscular administration in the neck muscles of animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

[0063] The term “expression” includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0064] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be transformed into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," a "recombinant protein," or a "fusion protein."

[0065] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a set of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as lentiviruses, retroviruses, vaccinia virus, adenovirus, fowlpox virus, baculovirus, SV40, and pseudorabies virus. Self-replicating vectors and non-self-replicating vectors are included.

[0066] The term "mRNA" refers to messenger RNA, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0067] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence. Primary transcripts are the initial RNA products, containing introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes end modification, intron removal, capping, and / or cleavage of individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of mRNA is a portion of mRNA that is not translated into protein and is located upstream of the coding sequence. In the genome sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The length of the 5' untranslated region (5'UTR) of vertebrate mRNA can range from tens to hundreds of bases.

[0068] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability, such as RNA-binding proteins.

[0069] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells.

[0070] The terms “individual,” “patient,” or “subject” include mammals and birds. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, horses, sheep, cats, and dogs), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats). Birds include, but are not limited to, birds and poultry (e.g., chickens, ducks, geese).

[0071] The terms “transformation,” “transfection,” and “transduction” have the meanings generally understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0072] The term "pharmaceutical combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for its effective absorption in a subject. Pharmaceutically acceptable carriers can be inert fillers or active ingredients that provide a function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, flow aids, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0073] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0074] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0075] This invention discloses a novel H9-modified HA protein, a broad-spectrum vaccine, a preparation method, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0076] The proteins and encoding nucleic acids and their components provided in this invention, as well as the preparation methods and applications, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0077] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0078] Example 1: Overall design strategy of the modified HA protein of the present invention

[0079] This invention provides a design strategy for a modified HA protein used in the development of a broad-spectrum vaccine against H9 avian influenza. The core of this strategy lies in constructing an optimized immunogenic backbone for different epidemic phases of the virus, and then systematically integrating functional mutations to directionally shape the breadth and effectiveness of the immune response. Overall, this invention encompasses two complementary design paths, and the flowchart of the overall design strategy for the modified HA protein is shown below. Figure 1 As shown.

[0080] (I) Path 1: The "epitope embedding and glycosylation masking" strategy based on historical popular sequences

[0081] This approach aims to design a broad-spectrum antigen that can cover historically prevalent lineages and overcome strain-specific immunogenicity.

[0082] Construction of the historical consensus framework: A total of 13,525 amino acid sequences of the HA protein from H9 avian influenza virus isolates worldwide up to 2020 were downloaded from international influenza virus databases (such as GISAID). Multiple sequence alignment was performed using MEGA (version 11.0) software, and abnormal sequences with excessive insertions or deletions were manually removed. Subsequently, the frequency of each amino acid at each point in the aligned sequence was calculated, and the residue with the highest frequency was selected to generate a "historical consensus sequence" (denoted as S2, an example of which is shown in SEQ ID NO: 1) representing the common characteristics of historically circulating viruses. Figure 1 The corresponding sequence is called the Consensus sequence. This process corresponds to... Figure 1 The "Step 1" section.

[0083] Based on published studies of broad-spectrum neutralizing antibodies against H9 or influenza A viruses, key residues of the HA epitopes targeted by these antibodies were located. These epitopes are mainly distributed in relatively conserved regions such as the 130 loop and 190 helix in the HA head region. These key residues were systematically transplanted into the corresponding positions of the "historical consensus sequence" constructed in step 1. Specific introduced mutations included: P87L, P89L, E90G, E91G, K92R, G153D, R164Q, N167G, N168A, E196D, D197T, V198T, R200T, and T201N. During the transplantation process, homology modeling (e.g., using a SWISS-MODEL server) was used to assess the impact of the mutations on the overall conformation of the HA trimer, ensuring that its natural folding was not disrupted. This yielded the "broad-spectrum epitope embedding sequence" (denoted as S3, with an exemplary sequence shown in SEQ ID NO: 2). This process corresponds to... Figure 1 Step 2 in the process directs the immune response to conserved broad-spectrum epitopes.

[0084] Glycosylation masking mutation in the immunodominant region: To further weaken the immunodominance of the hypervariable region (non-conserved immunodominant epitope) in the HA head, a new N-glycosylation site (Asn-X-Ser / Thr motif) is introduced into the variable region of a specific non-broad-spectrum epitope, based on the S3 sequence. The feasibility of glycosylation at the introduced site is predicted using the NetNGlyc 1.0 server. As a preferred embodiment, the present invention introduces the mutation A168T at position 168, thereby creating a new glycosylation site. This yields the "glycosylation masking mutation sequence" (denoted as S7, with an exemplary sequence shown in SEQ ID NO: 3). The introduction of the glycan can physically mask the variable region below, thereby indirectly enhancing the immunogenicity of the conserved broad-spectrum epitope transplanted in step 2, achieving "immune response redirection." This process corresponds to... Figure 1 Step 3 in the process.

[0085] (II) Path Two: "Evolutionary Prediction and Epitope Embedding" Strategy Based on New Popular Strains

[0086] This approach aims to design an antigen that can proactively address potential epidemic trends in the near future.

[0087] A number of H9 clinical strains isolated and identified between 2021 and 2025, known in the art, were collected, and their HA gene sequences were determined. Multiple sequence alignment and evolutionary analysis were performed on these recently circulating HA sequences to generate a "new epidemic consensus sequence" (denoted as S8, an example sequence of which is shown in SEQ ID NO: 4) representing the current epidemic trend. Figure 1 The corresponding sequence is called the Consensus sequence. This backbone is closer to the forefront of viral evolution. This process corresponds to... Figure 1 The "Step 1" section.

[0088] Similar to Pathway 1, this invention analyzes the evolutionary patterns of 2021-2025 strain sequences relative to historical strains, identifying key amino acid sites that gradually gain dominance during the epidemic or are related to receptor binding characteristics or antigenic changes. Based on this, mutations predicted to represent future dominant epitopes are designed and selectively embedded into the S8 backbone to obtain neutralizing epitope-embedded mutant sequences. This invention provides three preferred mutation combinations:

[0089] Option A: Embed the mutation L50M to obtain sequence S9 (exemplary sequence is shown in SEQ ID NO: 5).

[0090] Option B: Insert mutations L50M, I170T, E271K to obtain sequence S10 (exemplary sequence is shown in SEQ ID NO: 6).

[0091] Option C: Insert the mutations P136S, H231N, I153M to obtain sequence S11 (exemplary sequence is shown in SEQ ID NO:7).

[0092] This process aims to endow vaccine antigens with the prospective protective potential against viral antigenic drift, corresponding to Figure 1 The "Step 2" section.

[0093] Through the two design paths described above, this invention has obtained a series of modified HA proteins with sequence diversity, encompassing different design concepts, as shown in Table 1. These proteins all use an optimized consensus sequence as a backbone and integrate functional mutations designed to enhance broad-spectrum protection, laying a core antigenic foundation for subsequent protein expression, immunogenicity evaluation, and the preparation of broad-spectrum vaccines.

[0094] Table 1. Amino acid sequence of the modified HA protein involved in this invention.

[0095] sequence name amino acid sequence and sequence number S2 (SEQ ID NO: 1) S3 METVSLITILLVATVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNGAYPIQDAQYTNNQGKNILFMWGINHPPTDTTQTNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI(SEQ ID NO: 2) S7 METVSLITILLVATVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNGTYPIQDAQYTNNQGKNILFMWGINHPPTDTTQTNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI(SEQ ID NO: 3) S8 METVSLITILLVATVSNADKICIGYQATNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLVLETCTIEGLIYGNPSCDPLPEEKEWSYVVERPSAVNGLCYPGNVENLEELRSLFSSARSYRRIQIFPDTIWNVSYDGTSNTCSGSFYRNMRWLNRKNNNYPIQDAQYTNNQGKNILFMWGINHPPTEDVQRTLYTRTDTTTSVATEEINRIFKPLIGPRPLVHGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGYILSGESHGRILRTDLRRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI(SEQ ID NO: 4) S9 METVSLITILLVATVSNADKICIGYQATNSTETVDTLTENNVPVTHAKEMLHTEHNGMLCATSLGQPLVLETCTIEGLIYGNPSCDPLPEEKEWSYVVERPSAVNGLCYPGNVENLEELRSLFSSARSYRRIQIFPDTIWNVSYDGTSNTCSGSFYRNMRWLNRKNNNYPIQDAQYTNNQGKNILFMWGINHPPTEDVQRTLYTRTDTTTSVATEEINRIFKPLIGPRPLVHGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGYILSGESHGRILRTDLRRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI(SEQ ID NO: 5) S10 METVSLITILLVATVSNADKICIGYQATNSTETVDTLTENNVPVTHAKEMLHTEHNGMLCATSLGQPLVLETCTIEGLIYGNPSCDPLPEEKEWSYVVERPSAVNGLCYPGNVENLEELRSLFSSARSYRRIQIFPDTIWNVSYDGTSNTCSGSFYRNMRWLNRKNNNYPTQDAQYTNNQGKNILFMWGINHPPTEDVQRTLYTRTDTTTSVATEEINRIFKPLIGPRPLVHGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGYILSGKSHGRILRTDLRRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI(SEQ ID NO: 6) S11 (SEQ ID NO: 7)

[0096] Example 2: Molecular structure prediction and mutation site analysis of the HA protein of the present invention.

[0097] This embodiment aims to predict and analyze the three-dimensional structure of the modified HA protein designed in this invention using computational structural biology methods, so as to theoretically evaluate its conformational stability, spatial conformation of key epitopes, and potential impact of glycosylation modifications, providing a structural basis for subsequent protein expression and functional verification. Figure 2 A schematic diagram of the molecular structure of the HA protein of H9 avian influenza virus and the location of key mutation sites.

[0098] AlphaFold3 prediction generated five models for each target and provided a prediction confidence score for each model. The model with the highest prediction local distance difference test score was selected as the basis for subsequent analysis. All predicted models for the modified proteins were compared with the wild-type HA structure and used as initial conformational structures for molecular dynamics simulations, with a simulation duration of 50 ns. During the simulation, the root mean square deviation (RMSD) was used to measure the displacement of protein atoms relative to the initial structure to reflect structural fluctuations. RMSD is a key indicator for measuring the similarity of three-dimensional structures of two proteins, quantified by calculating the average difference in corresponding atomic positions; the smaller the value, the more similar the structures. It is commonly used in molecular dynamics simulations to assess protein conformational changes (compared to the initial structure) or to compare different protein structures (e.g., structure prediction results). A stable RMSD value indicates system equilibrium, while a high or low value reflects the magnitude of structural differences. The results show that the protein structure was stable during the simulation, with the overall protein RMSD value consistently at a low level and not exceeding 1.2 nm, as shown in Table 2. The results in Table 2 indicate that the modification of the protein in this invention did not disrupt the overall topological structure.

[0099] Table 2 Comparison of molecular structures of modified HA and wild-type HA proteins

[0100] sequence name RMSD S3 0.585 S7 0.988 S8 1.061 S9 0.867 S10 0.803 S11 0.398

[0101] These results demonstrate that the various mutations (epitope embedding, glycosylation, and novel epitope embedding) introduced through rational design in this invention can be well accommodated within the target HA protein backbone. The modified protein not only maintains a highly similar overall folding and trimer interface to the wild type, but its key functional regions (such as the receptor-binding site RBS and the conserved stem region responsible for membrane fusion) may even exhibit greater conformational stability. This provides a solid theoretical basis for the correct expression, folding, and complete preservation of the key structural features mediating viral infection and its immunological function as a broad-spectrum vaccine antigen in vitro.

[0102] Example 3: Construction of the recombinant nucleic acid vaccine of the present invention

[0103] (I) Construction of recombinant expression plasmids

[0104] To prepare a recombinant nucleic acid vaccine based on the present invention and to verify the efficient expression ability of its antigen coding sequence in vitro, this embodiment uses an mRNA vaccine as an example to illustrate the vaccine construction process.

[0105] First, a gene expression cassette for expressing the modified HA protein of this invention was constructed. The linear structure of this expression cassette, from the 5' end to the 3' end, sequentially includes the following functional elements: an optimized 5' untranslated region, a CDS region encoding the target antigen, a stable 3' untranslated region, and a PolyA tail sequence. The CDS region encodes the modified HA protein as shown in Table 1 of Example 1.

[0106] Subsequently, the designed gene expression cassette sequence was cloned into an optimized circular plasmid vector backbone using whole-genome synthesis and in vitro recombination techniques to obtain a recombinant DNA plasmid, which contains a schematic diagram of the circular plasmid used as the vaccine template of this invention, as shown in the figure. Figure 3 As shown. This plasmid will serve as a template for in vitro transcription to prepare mRNA. All gene synthesis and molecular cloning operations were outsourced to GenScript Biotech Inc.

[0107] (II) Preparation of different types of nucleic acid vaccines

[0108] 1. Preparation of capped mRNA vaccines

[0109] Step a: The above recombinant plasmid is digested with a restriction endonuclease (such as Not I) located downstream of the PolyA tail sequence to make it completely linearized. After purification, a linearized DNA template for in vitro transcription is obtained.

[0110] Step b: Using a commercially available co-transcriptional capping kit (such as NEB's HiScribe™ T7 ARCA mRNA Kit) and linearized DNA as a template, an in vitro transcription reaction was performed. This reaction system simultaneously synthesizes mRNA and directly adds a 7-methylguanylate cap analog (such as ARCA) to the 5' end of the nascent mRNA. After transcription, DNase I was added to degrade the template DNA, and the capped mRNA was purified by LiCl precipitation or a purification column. After quantification, it was stored at -80°C for later use.

[0111] 2. Preparation of uncapped mRNA vaccines

[0112] Step a: Same as step a in the preparation of capped mRNA vaccines, obtain a linearized DNA template.

[0113] Step b: Using a standard in vitro transcription kit (such as NEB's HiScribe™ T7 Quick High Yield mRNA Synthesis Kit), in vitro transcription was performed using standard NTPs. No cap analogs were added in this reaction, therefore the resulting mRNA had a triphosphate group at its 5' end. After transcription, DNase I was added to degrade the template DNA, and the product was purified, quantified, and stored.

[0114] 3. DNA vaccine preparation

[0115] Step a: Transform the above recombinant plasmid into competent Escherichia coli (such as DH5α) and culture it on a large scale in LB liquid medium containing an appropriate amount of antibiotics to amplify and obtain a large number of target plasmids.

[0116] Step b: Collect bacterial cells and extract plasmids using a commercially available endotoxin-free plasmid extraction kit (such as Qiagen's EndoFree PlasmidMega Kit). This process efficiently removes bacterial endotoxins, proteins, RNA, and other impurities, yielding a high-purity, high-concentration sterile plasmid DNA solution. After concentration determination and quality verification, it is stored as a DNA vaccine stock solution.

[0117] Example 4: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0118] The following vaccine was prepared using the method for preparing the capped mRNA vaccine described in Example 3:

[0119] Vaccine A expresses the S2 sequence (amino acid sequence as shown in SEQ ID NO: 1); Vaccine B expresses the S3 sequence (amino acid sequence as shown in SEQ ID NO: 2); Vaccine C expresses the S7 sequence (amino acid sequence as shown in SEQ ID NO: 3); Vaccine D expresses the S8 sequence (amino acid sequence as shown in SEQ ID NO: 4); Vaccine E expresses the S9 sequence (amino acid sequence as shown in SEQ ID NO: 5); Vaccine F expresses the S10 sequence (amino acid sequence as shown in SEQ ID NO: 6); Vaccine G expresses the S11 sequence (amino acid sequence as shown in SEQ ID NO: 7).

[0120] The above sequence numbers correspond to the names in the first column of Table 2. The purity of the produced vaccine is tested, including the quality control peak diagram of the vaccine of this invention and the purity test results, as shown below. Figures 4A-4D As shown, where Figure 4A The quality control peak diagrams and purity test results of vaccines A and B, which include the present invention; Figure 4B The quality control peak diagrams and purity test results for vaccines C and D, which contain the present invention; Figure 4C The quality control peak diagrams and purity test results for vaccines E and F, which contain the present invention; Figure 4D The image shows the quality control peak diagram and purity test results for vaccine G containing the present invention. It can be seen that the purity of vaccine AG containing the recombinant nucleic acid of the present invention used in the experiment is greater than 80%, and all meet the quality requirements for cell transfection experiments and vaccine production.

[0121] Example 5: In vitro expression effect of the recombinant nucleic acid of the present invention

[0122] To verify whether the various nucleic acid vaccines prepared in Example 4 can correctly express the target antigen in eukaryotic cells, this example performed in vitro cell transfection and protein expression detection.

[0123] Healthy HEK293T cells were seeded into T75 cell culture flasks and transfected when the cell density reached 70-80% confluence. 50 µg of the capped mRNA vaccine stock solution prepared in Example 4 was taken and transfected using the RNA transfection reagent according to the optimized transfection procedure. After transfection, the cells were cultured at 37°C and 5% CO2 for 48 hours.

[0124] After culture, cells were collected, proteins were extracted, and protein concentrations were determined. Equal volumes of protein samples were subjected to SDS-PAGE electrophoresis, followed by Western blotting (WB). After development with a chemiluminescent substrate, signals were acquired using an imaging system.

[0125] Since all vaccines encode a modified HA protein based on the same consensus sequence backbone, their predicted molecular weight is approximately 82.7 kDa. Western blot results are as follows... Figures 5A-5B As shown: Figure 5A The in vitro expression WB detection results of vaccines A, B, and C are presented. Figure 5B The in vitro expression Western blot (WB) results of vaccines D, E, and G are shown. The nucleic acid vaccine AG of this invention is mainly expressed in the supernatant, wherein... Figure 5A All vaccine samples tested were supernatant. Figure 5B In the same vaccine sample, the darker-colored bands in one lane detect the supernatant, while the lighter-colored bands in another lane detect cell lysis proteins (at lower levels of the target protein). Figures 5A-5B The results show that a clear, specific band was detected at approximately 82.7 kDa in all lanes of samples transfected with the nucleic acid vaccine of this invention, and its size was consistent with the predicted molecular weight. This demonstrates that the nucleic acid vaccine AG obtained by the design and preparation method of this invention can effectively guide the expression of the target HA protein in eukaryotic cells, and the expressed protein has the expected molecular weight. This provides a reliable basis for subsequent animal experiments to evaluate its immunogenicity and protective efficacy.

[0126] Example 6: The vaccine based on the present invention induces high levels of broad-spectrum neutralizing antibodies in target animals (chickens).

[0127] This embodiment aims to verify, through animal immunization experiments, that the nucleic acid vaccine prepared in this invention induces high levels of neutralizing antibodies with broad-spectrum cross-reactivity in the target animal, chicken, thereby assessing its immunogenic potential as a preventive vaccine.

[0128] 1. Laboratory animals and grouping

[0129] Thirty-five one-month-old SPF (specific pathogen-free) white-feathered chickens were randomly divided into seven groups of five each. The grouping design is shown in Table 3. Dosage in this table and below refers to the amount of active ingredient.

[0130] Table 3. Grouping and Immunization Procedure for Chicken Immunization Experiment in Example 6

[0131] Thirty-five chickens were immunized twice according to the immunization schedule in Table 3. Blood samples were collected on Day 0 (before immunization) and Day 28 (7 days after the second immunization). On Day 0, 2 mL of blood was collected from each group of two randomly selected chickens, with the goal of extracting 1 mL of serum. On Day 28, 3 mL of blood was collected from each chicken, with the goal of extracting 1.5–2 mL of serum. The serum was stored below -20°C.

[0132] To assess the broad-spectrum cross-protective capacity of immune serum antibodies, the hemagglutination inhibition test (HI) was used to detect antibodies in serum samples. This method evaluates the level and breadth of neutralizing antibodies by measuring the ability of serum to inhibit viral agglutination of erythrocytes.

[0133] To fully demonstrate the cross-reactivity of neutralizing antibodies, this study selected a group of H9 avian influenza virus strains / antigens (strains numbered 2-12, which are available to the public from the applicant) that exhibit significant diversity in genetic evolution, geographical origin, and isolation time. Specific information is shown in Table 4.

[0134] Table 4 List of virus strains used in HI tests

[0135] Serial Number Virus strain / antigen name Type / Description Separation location Separation time 1 Avian influenza virus H9 subtype hemagglutination inhibition test antigen (Ebang Company) Commercialized standard reference antigen / / 2 A / CK / SD / WFCL / 23 Clinically isolated strain 1 Changyi, Shandong 2023 3 A / CK / AH / 24 / 19 Clinically isolated strain 2 Chuzhou, Anhui 2019 4 A / CK / SD / PY2 / 19 Clinically isolated strain 3 Pingyi, Shandong 2019 5 A / CK / JS / HM21 / 19 4 clinically isolated strains Haimen, Jiangsu 2019 6 A / CK / SD / LY / 25 5 clinically isolated strains Linyi, Shandong 2025 7 A / CK / SD / LYJN / 25 6 clinically isolated strains Junan, Shandong 2025 8 A / CK / SD / WFQZ / 23 7 clinically isolated strains Qingzhou, Shandong 2023 9 A / CK / SD / RZ2 / 24 8 clinically isolated strains Rizhao, Shandong 2024 10 A / CK / SD / JN / 24 Clinically isolated strain 9 Jinan, Shandong 2024 11 A / CK / SD / WFAQ6 / 24 10 clinically isolated strains Anqiu, Shandong 2024 12 A / CK / SD / YNC3 / 24 11 clinically isolated strains Yinan, Shandong 2024

[0136] To systematically evaluate the immunization effects of different vaccines, serum samples were grouped and tested according to differences in immunogens. The specific groupings are shown in Table 5. The aim is to scientifically assess the breadth and intensity of neutralizing antibodies induced by the immune serum of each vaccine through a detection panel covering a wide range of circulating strains, thereby directly demonstrating their cross-protective potential.

[0137] Table 5. Serum HI detection grouping scheme

[0138] Serum samples tested (immunized with vaccine) Virus strains / antigens used for detection (corresponding to the serial numbers in Table 4) Detection purpose Vaccine groups A, B, and C 1, 2, 3, 4, 5 Assess cross-reactivity to standard antigens and a group of early / heterogeneous clinical strains. Vaccine immunization groups D, E, F, and G 6, 7, 8, 9, 10, 11, 12 Assess cross-reactivity to another group of recently clinically prevalent strains.

[0139] The final test results are shown in Table 6 and Figures 6A-6D As shown, Figures 6A-6D The neutralizing antibody titer in chicken serum after two immunizations with the vaccine of this invention is given. Figure 6AThe neutralizing antibody titer in chicken serum after two immunizations with vaccines A and B of this invention. Figure 6B The titer of neutralizing antibodies in chicken serum after two immunizations with vaccine C of the present invention. Figure 6C The neutralizing antibody titer in chicken serum after two immunizations with vaccines D and E of this invention. Figure 6D The results show that the neutralizing antibody titers in chicken serum after two immunizations with vaccines F and G of this invention are low (≤ 2^3) in all experimental groups before immunization (D0), indicating that there was no antibody interference in the animals before immunization. After immunization, all vaccine groups were able to induce high levels of HI antibodies, and they exhibited broad-spectrum cross-reactivity to different strains.

[0140] Table 6 Serum HI titer

[0141] In the table, " / " indicates non-vaccine-induced death in target animals whose clinical manifestations do not conform to the characteristics of acute allergy or vaccine toxicity, and is judged to be a non-specific, incidental death.

[0142] Specifically, against the standard antigen and strains 1-4 (corresponding to items 1-5 in Table 4), vaccine C induced the strongest and most balanced antibody response, with a post-immunization HI titer ranging from 2^8 to 2^11, and producing a high HI titer of 2^11 against both strains. Vaccines A and B also induced high-titer antibodies (generally ≥2^7), with vaccine B showing a particularly strong response to the standard antigen (titer 2^11). Vaccine groups A, B, and C showed some protective effect against strain 1 (corresponding to item 2 in Table 4), but the effect did not demonstrate a significant advantage.

[0143] Against strain 5-11 (corresponding to serial numbers 6-12 in Table 4), vaccines D, F, and G all demonstrated excellent immunogenicity, with antibody titers rapidly increasing after immunization and generally remaining at a high level of 2^9 to 2^11, indicating good immunogenicity and broad-spectrum efficacy. In contrast, the immunogenicity of vaccine group E showed greater inter-individual variability. Although two-fifths of individuals in vaccine group E achieved HI titers in the range of 2^9 to 2^11, two-fifths of individuals had insignificant HI titers (≤2^5), suggesting that it was not the optimal choice.

[0144] In summary, except for vaccine E, vaccines A, B, C, D, F, and G (especially vaccines C, D, F, and G) described in this invention can all induce high levels of HI antibodies with broad-spectrum cross-reactivity in chickens, providing core serological evidence for the broad-spectrum protective efficacy of the vaccines of this invention. This result demonstrates that this invention can be applied to the production and development of immunotherapeutic drugs, filling a gap in the current field of broad-spectrum avian influenza virus vaccine development, and possesses extremely high commercial value and broad application prospects.

[0145] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A method for preparing a modified hemagglutinin (HA) protein for preparing a broad-spectrum H9 avian influenza vaccine, characterized in that, The amino acid sequence of the modified HA protein is obtained by the following method: (1) Perform multiple sequence alignment on the H9 virus HA protein sequence set at a specific time period to generate a common amino acid sequence; (2) Introduce a set of selected mutations into the common amino acid sequence, the mutations being selected from: (a) Stability-neutralizing epitope embedding mutations corresponding to broad-spectrum antibody-targeting epitopes; or / and (b) Corresponding to the neutral epitope embedding mutations predicted by viral evolution.

2. The preparation method according to claim 1, characterized in that, The specific period is the historical epidemic period; preferably, the historical epidemic period is the historical epidemic period up to 2020; the common amino acid sequence is as shown in SEQ ID NO:

1.

3. The preparation method according to claim 2, characterized in that, The following epitope insertion mutations are introduced into the common amino acid sequence: P87L, P89L, E90G, E91G, K92R, G153D, R164Q, N167G, N168A, E196D, D197T, V198T, R200T, T201N. Preferably, the amino acid sequence of the modified HA protein obtained after introducing the neutralizing epitope insertion mutation into the common amino acid sequence is shown in SEQ ID NO:

2.

4. The preparation method according to claim 3, characterized in that, After introducing the neutralizing epitope embedding mutation into the common amino acid sequence, the glycosylation site mutation A168T is further introduced; preferably, the amino acid sequence of the modified HA protein after introducing the glycosylation site mutation is SEQ ID NO:

3.

5. The modified HA protein according to claim 1, characterized in that, The specific period is the new epidemic period; preferably, the new epidemic period is 2021-2025, and the common amino acid sequence is as shown in SEQ ID NO:

4.

6. The preparation method according to claim 5, characterized in that, Introduce one or more of the following neutralizing epitope insertion mutations into the common amino acid sequence: L50M, P136S, I153M, I170T, H231N, E271K; preferably, the amino acid sequence of the modified HA protein after introducing the neutralizing epitope insertion mutation is shown in SEQ ID NO: 7, SEQ ID NO: 6 or SEQ ID NO: 5, respectively.

7. A modified HA protein for preparing a broad-spectrum vaccine against H9 avian influenza, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

8. A modified HA protein for preparing a broad-spectrum vaccine against H9 avian influenza, characterized in that, Their amino acid sequences are shown as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 5, respectively.

9. A recombinant nucleic acid molecule, characterized in that, Encodes the modified HA protein as described in any one of claims 7 or 8.

10. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 9.

11. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 9 or the recombinant gene expression cassette of claim 10.

12. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 9, the recombinant gene expression cassette of claim 10, or the recombinant vector of claim 11.

13. An immunogenic composition or pharmaceutical composition, characterized in that, The immunogenic composition or pharmaceutical composition comprises the modified HA protein of any one of claims 7 or 8, and / or one or more recombinant nucleic acid molecules of claim 9, and / or one or more recombinant gene expression cassettes of claim 10, and / or one or more recombinant vectors of claim 11, and / or one or more recombinant host cells of claim 12; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

14. A recombinant broad-spectrum vaccine, characterized in that, The vaccine comprises the modified HA protein according to any one of claims 7 or 8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 13; preferably, the broad-spectrum vaccine is a broad-spectrum nucleic acid vaccine; more preferably, the broad-spectrum nucleic acid vaccine is a broad-spectrum RNA vaccine or a broad-spectrum DNA vaccine; most preferably, the broad-spectrum RNA vaccine is a broad-spectrum mRNA vaccine.

15. Use of the modified HA protein of claim 7 or 8, and / or one or more recombinant nucleic acid molecules of claim 9, and / or one or more recombinant gene expression cassettes of claim 10, and / or one or more recombinant vectors of claim 11, and / or one or more recombinant host cells of claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions of claim 13, and / or one or more recombinant broad-spectrum vaccines of claim 14 in the preparation of a medicament for the prevention of avian influenza infection; preferably, the avian influenza is type H9; more preferably, the avian influenza is subtype H9N2.

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