A fusion protein for preventing and treating various pathogenic pasteuria family bacterial infections, a nucleic acid vaccine and application thereof

Abstract

The application discloses a fusion protein for preventing and treating various pathogenic Pasteurellaceae bacterial infections, a nucleic acid vaccine and application thereof. The fusion protein comprises six antigens rplE, rpsC, rpsE, EF-Tu, DnaK and EF-G or three antigens EF-Tu, DnaK and EF-G, and the antigens are highly conservative in various Pasteurellaceae bacteria. The application further provides a recombinant nucleic acid molecule encoding the fusion protein, an immunogenic composition containing the same and a recombinant vaccine. The vaccine can induce broad-spectrum cross immune protection and is used for preventing and treating infectious diseases caused by Pasteurella multocida, Haemophilus parasuis, Mannheimia haemolytica, Haemophilus influenzae and Haemophilus paragallinarum.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, particularly to the field of immunopharmaceuticals, specifically to a fusion protein, a nucleic acid vaccine, and their applications for preventing and treating infections caused by various pathogenic Pasteurella bacteria. Background Technology

[0002] Pasteurella family (Pasteurellaceae) is a group of Gram-negative facultative anaerobic short bacilli that commonly parasitize the upper respiratory tract and oral mucosa of various domestic and wild animals. Many strains possess pathogenic potential. Pathogenic Pasteurella, such as Pasteurella multocida, can cause hemorrhagic septicemia, fowl cholera, and respiratory infections in animals. It can also be transmitted to humans through scratches, bites, or close contact, causing local infections, bacteremia, and even systemic infections, making it a typical zoonotic pathogen.

[0003] Similar to many extracellular parasitic bacteria, Pasteurella multocida, after colonizing the host, can resist phagocytosis using its capsule and secrete various toxins and adhesion factors, exacerbating tissue damage and inflammatory responses. Clinically, traditional treatment for this type of infection heavily relies on antibiotics. However, due to the widespread and even overuse of antibiotics in livestock farming, drug-resistant strains are constantly emerging, making treatment more difficult, prolonging the course of the disease, and even increasing the risk of death in some cases. The consequences of infection are particularly severe in immunocompromised individuals and the elderly.

[0004] Vaccination is considered the most cost-effective way to control Pasteurella infection. Currently, vaccines against Pasteurella are mainly traditional inactivated whole-cell vaccines, capsular polysaccharide vaccines, or subunit toxin vaccines, such as vaccines against different capsular serotypes like A, B, and D of Pasteurella multocida. However, these vaccines suffer from high serotype specificity and narrow protective range, making it difficult to provide cross-immune protection against different serotypes and species of Pasteurella. Some researchers are attempting to develop capsular polysaccharide conjugate vaccines or fusion toxin protein vaccines to broaden immunization coverage. For example, the patent "Haemophilus influenzae type b conjugate vaccine and its preparation method" (publication number: CN103007276A) discloses a technical solution for covalently binding Haemophilus influenzae type b capsular polysaccharide with tetanus toxoid; other studies focus on toxin proteins (such as PMT, Hsf, etc.) as immunogens to block key pathogenic mechanisms. Nevertheless, existing vaccines still have several limitations: First, capsule-based vaccines are difficult to cover all prevalent serotypes, which can easily lead to immune escape and serotype replacement; second, polysaccharide-protein conjugate vaccines have complex processes and high costs, which are not conducive to large-scale promotion in animal husbandry; third, there are many types of animal hosts with different infection manifestations, and a single antigen is difficult to elicit a balanced and effective immune response in all hosts.

[0005] With advancements in high-throughput sequencing, comparative genomics, and bioinformatics, reverse vaccinology offers a new pathway for screening highly conserved protein antigens with good immunogenicity. Through genome-wide antigen prediction, trans-serotype conserved protein antigens can be identified from the vast coding genome of Pasteurella. These antigens exhibit high homology across multiple strains and hold promise for constructing novel vaccines with broad-spectrum protective potential.

[0006] Compared with traditional polysaccharide vaccines or whole-cell inactivated vaccines, novel vaccines based on conserved protein antigens have significant advantages: (1) they break through serotype limitations and achieve cross-protection against multiple pathogenic Pasteurella bacteria; (2) they can be prepared on a large scale using recombinant protein expression systems, with relatively simple processes and controllable costs; and (3) they are easy to construct multi-antigen combined vaccines, covering different pathogenic mechanisms and enhancing immune effects.

[0007] In conclusion, given the increasingly complex prevalence of pathogenic Pasteurella infections and the declining efficacy of antibiotics, the development of a novel vaccine with broad-spectrum protection, safety, and ease of production is particularly urgent. Utilizing modern technologies such as reverse vaccinology to screen key conserved antigens and construct multi-antigen fusion proteins or combination formulations holds promise for overcoming current bottlenecks in vaccine development and providing a more effective tool for the prevention and control of human and animal Pasteurella infections. Therefore, although several Pasteurella-related vaccines have been disclosed in the prior art, there is still an urgent need in this field for a fusion protein, immune composition, or vaccine product that can broadly prevent infection by different serotypes of Pasteurella, possesses good immunogenicity and protective efficacy, and can significantly reduce tissue lesions. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design and application for the prevention and treatment of various pathogenic Pasteurella infections.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A fusion protein comprising: an elongation factor Tu (EF-Tu) antigen or an antigenic fragment thereof, a chaperone protein DnaK (DnaK) antigen or an antigenic fragment thereof, and an elongation factor G (EF-G) antigen or an antigenic fragment thereof.

[0011] Preferably, the fusion protein comprises: 50S ribosomal protein L5 (rplE) antigen or an antigenic fragment thereof, 30S ribosomal protein S3 (rpsC) antigen or an antigenic fragment thereof, 30S ribosomal protein S5 (rpsE) antigen or an antigenic fragment thereof, elongation factor Tu (EF-Tu) antigen or an antigenic fragment thereof, chaperone protein DnaK (DnaK) antigen or an antigenic fragment thereof, and elongation factor G (EF-G) antigen or an antigenic fragment thereof.

[0012] More preferably, the fusion protein comprises a 50S ribosomal protein L5 (rplE) antigen or an antigenic fragment thereof, a 30S ribosomal protein S3 (rpsC) antigen or an antigenic fragment thereof, a 30S ribosomal protein S5 (rpsE) antigen or an antigenic fragment thereof, an elongation factor Tu (EF-Tu) antigen or an antigenic fragment thereof, a chaperone protein DnaK (DnaK) antigen or an antigenic fragment thereof, and an elongation factor G (EF-G) antigen or an antigenic fragment thereof, and a linking peptide sequence or spacer sequence connecting the fragments. Alternatively, the fusion protein may consist of an elongation factor Tu (EF-Tu) antigen or its antigenic fragment, a chaperone protein DnaK (DnaK) antigen or its antigenic fragment, an elongation factor G (EF-G) antigen or its antigenic fragment, and a linking peptide sequence or spacer sequence connecting the fragments.

[0013] More preferably, all antigens contained in the fusion protein are derived from the Pasteurellaceae family, and more preferably, the antigens are derived from Haemophilus parasuis.

[0014] Further preferably, the amino acid sequence of the rplE antigen has at least 94% identity with the sequence shown in SEQ ID NO: 1, preferably 96%, 97%, or 100%; the amino acid sequence of the rpsC antigen has at least 90% identity with the sequence shown in SEQ ID NO: 2, preferably 90%, 92%, 93%, 94%, 97%, or 100%; the amino acid sequence of the rpsE antigen has at least 95% identity with the sequence shown in SEQ ID NO: 3, preferably 95%, 96%, 97%, 98%, or 100%; the amino acid sequence of the EF-Tu antigen has at least 93% identity with the sequence shown in SEQ ID NO: 4, preferably 93%, 94%, 95%, 97%, 98%, or 100%; and the amino acid sequence of the DnaK antigen has at least 93% identity with the sequence shown in SEQ ID NO: 1. The sequence identity shown in SEQ ID NO: 5 is not less than 81%, preferably 81%, 83%, 84%, 85%, 86%, 90%, or 100%; the amino acid sequence of the EF-G antigen has an identity of not less than 92% with the sequence shown in SEQ ID NO: 6, preferably 92%, 93%, 94%, 95%, or 100%.

[0015] More preferably, the antigens are optionally linked by a linker peptide sequence or a spacer sequence; preferably, the amino acid sequence of the linker peptide sequence is as shown in SEQ ID NO: 7, and the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 8.

[0016] More preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 9 or SEQ ID NO: 10.

[0017] A recombinant nucleic acid molecule encoding the fusion protein; preferably, the recombinant nucleic acid molecule is mRNA or DNA, and more preferably, the recombinant nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13.

[0018] A recombinant gene expression cassette comprising the recombinant nucleic acid molecule.

[0019] A recombinant vector comprising the recombinant nucleic acid molecule or the recombinant gene expression cassette.

[0020] A recombinant host cell comprising the recombinant nucleic acid molecule, or the recombinant gene expression cassette, or the recombinant vector.

[0021] An immunogenic composition or pharmaceutical composition comprising one or more components selected from the group consisting of: the fusion protein, the recombinant nucleic acid molecule, the recombinant gene expression cassette, the recombinant vector, and the recombinant host cell; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0022] A recombinant vaccine comprising one or more components selected from the group consisting of: the fusion protein, the recombinant nucleic acid molecule, the recombinant gene expression cassette, the recombinant vector, the recombinant host cell, the immunogenic composition, or the pharmaceutical composition.

[0023] The use of the aforementioned fusion protein, recombinant nucleic acid molecule, recombinant gene expression cassette, recombinant vector, recombinant host cell, immunogenic composition, pharmaceutical composition, or recombinant vaccine in the preparation of a medicament for the prevention and / or treatment of diseases caused by pathogenic Pasteurella bacteria, preferably, the pathogenic Pasteurella bacteria being selected from any one of Pasteurella multocida, Haemophilus parasuis, Mannheimia haemolytica, Haemophilus influenzae, and Avibacterium paragallinarum.

[0024] Preferably, in the described use, the disease is selected from any one of the following:

[0025] Fowl cholera, swine pneumonia, bovine hemorrhagic septicemia, rabbit rhinitis, cellulitis, respiratory infections, or bacteremia caused by Pasteurella multocida;

[0026] Haemophilus parasuis infection, polyserositis, arthritis, or meningitis caused by Haemophilus parasuis;

[0027] Bovine transport fever, mastitis, or ovine pneumonia caused by hemolytic Mannheim bacteria;

[0028] Meningitis, epiglottitis, bacteremia, sepsis, suppurative arthritis, osteomyelitis, pericarditis, otitis media, sinusitis, bronchitis, or pneumonia caused by Haemophilus influenzae;

[0029] Infectious coryza in chickens caused by Haemophilus paragallinarum.

[0030] Beneficial effects:

[0031] Broad-spectrum cross-protection: The six antigens selected in this invention are highly conserved among a variety of pathogenic Pasteurella bacteria (including Pasteurella multocida, Haemophilus parasuis, Haemophilus hemolyticus, Haemophilus influenzae, and Haemophilus paragallinarum), and can induce cross-immune protection against a variety of pathogens.

[0032] Dual efficacy for prevention and treatment: Animal experiments have shown that the vaccine of this invention can not only provide effective preventive protection before challenge, reducing tissue bacterial load and lesion severity, but can also be used to treat infected animals, significantly reducing the infection positivity rate.

[0033] Multi-species applicability: The immunogenic composition of the present invention has shown good immunoprotective effects in various animal models such as mice, rabbits, pigs and chickens, and has the potential for cross-species application.

[0034] Optimized molecular architecture: Multiple antigens are fused and expressed through specific linker peptide sequences, ensuring correct protein folding and stable expression while preserving the immunogenicity of each antigen. Attached Figure Description

[0035] Figure 1 This study analyzes the sequence similarity of the antigens contained in the fusion molecule of this invention in various pathogenic Pasteuraceae bacteria.

[0036] Figure 2 This is a schematic diagram of the molecular structure of the fusion protein expressed in this invention.

[0037] Figure 3 This is a schematic diagram of a prior art fusion protein for comparison.

[0038] Figure 4 This is a schematic diagram of a gene expression cassette template plasmid containing the present invention.

[0039] Figure 5 The images show the quality control peak diagrams and purity test results for nucleic acid vaccines A, B, and C, which contain the antigen sequences of this invention.

[0040] Figure 6 The results show the expression of vaccines A, B, and C after in vitro transfection into HEK293T cells.

[0041] Figure 7 This study compares the immunoprotective effects of vaccines A and B of the present invention and vaccine C of the prior art in a mouse challenge model of Pasteurella multocida, including changes in tissue bacterial load and the degree of tissue lesions after challenge.

[0042] Figure 8 This invention compares the immunoprotective effects of vaccines A and B in a mouse challenge model of Haemophilus parasuis, including changes in tissue bacterial load and the degree of tissue lesions after challenge.

[0043] Figure 9 This invention compares the immunoprotective effects of vaccines A and B in a Haemophilus influenzae mouse challenge model, including changes in tissue bacterial load and the degree of tissue lesions after challenge.

[0044] Figure 10 This invention compares the immunoprotective effects of vaccines A and B in a mouse model of hemolytic Mannheim bacteria challenge, including changes in tissue bacterial load and the degree of tissue lesions after challenge.

[0045] Figure 11 This study compares the immunoprotective effects of vaccine A of the present invention in a rabbit challenge model of Pasteurella multocida, including the comparison of survival rate and degree of tissue lesions after challenge.

[0046] Figure 12 This invention provides a comparison of the immunotherapy efficacy of vaccine A in pigs infected with Haemophilus parasuis and Pasteurella multocida (in the field), including changes in the positive rate after treatment.

[0047] Figure 13 This study compares the immunotherapy efficacy of vaccine A of the present invention in chickens infected with Haemophilus paragallinarum (in the field), including changes in egg production rate after treatment. Detailed Implementation

[0048] Terms and Definitions

[0049] The term "Pasteurellaceae" refers to a group of Gram-negative facultative anaerobic short bacilli, including but not limited to *Pasteurella*, *Haemophilus parasuis*, *Mannheimia haemolytica*, and *Haemophilus influenzae*. These bacteria can infect humans and animals, causing a variety of diseases.

[0050] The term "immunogenic composition" refers to a composition that can induce an immune response in the body (such as humoral immunity or cellular immunity), including but not limited to fusion proteins, recombinant nucleic acids, vectors, host cells, or combinations thereof.

[0051] The term "fusion protein" refers to a protein formed by linking two or more antigens or antigenic fragments through genetic engineering methods. The antigens may originate from Pasteurella bacteria and are linked by linking peptides or spacer sequences.

[0052] The term "antigenic fragment" refers to a protein fragment that retains antigenicity (i.e., can be recognized by the immune system and induce an immune response), and typically contains at least one B-cell or T-cell epitope. The fragment is at least 10 amino acids long and has at least 80% sequence identity with the full-length antigen.

[0053] The term "sequence identity" refers to the percentage of similarity between amino acid or nucleotide sequences calculated using sequence alignment algorithms such as BLAST and Clustal Omega. Identity calculations take into account the full length of the sequence and allow for conservative substitutions.

[0054] The term "linker" refers to a short peptide sequence that links different antigens in a fusion protein. It is usually composed of flexible amino acids (such as glycine and serine) that allow each antigen to fold independently and maintain its function.

[0055] The term "spacer" refers to a sequence in a fusion protein that enhances the distance or stability between antigens and may contain specific functional domains.

[0056] The term "rplE" refers to 50S ribosomal protein L5, a core component of the 50S ribosomal large subunit in prokaryotes. It plays a crucial role not only in the structure and assembly of the ribosome but also specifically binds to 5S rRNA, jointly stabilizing the three-dimensional conformation of the large subunit, and is an essential protein for maintaining the normal function of the protein synthesis machinery. The rplE antigen of this invention comprises the full-length rplE antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rplE antigen. Preferably, the amino acid sequence of the rplE antigen is shown in SEQ ID NO: 1.

[0057] The term "rpsC" refers to 30S ribosomal protein S3, a key protein of the 30S ribosomal subunit in prokaryotes. Located near the decoding center of the ribosome, this protein directly participates in the localization and transport of messenger RNA and maintains translational fidelity, playing a crucial role in ensuring correct codon-anticodon pairing. The rpsC antigen of this invention comprises a full-length rpsC antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rpsC antigen. Preferably, the amino acid sequence of the rpsC antigen is shown in SEQ ID NO: 2.

[0058] The term "rpsE" refers to 30S ribosomal protein S5, a core structural and functional protein of the prokaryotic 30S ribosomal small subunit. This protein plays a crucial role in maintaining the overall structural stability of the small subunit and participates in regulating translation accuracy; mutations in this protein are often associated with alterations in translational fidelity. The rpsE antigen of this invention comprises the full-length rpsE antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length truncation, all of which can stimulate an immune response against the rpsE antigen. Preferably, the amino acid sequence of the rpsE antigen is shown in SEQ ID NO: 3.

[0059] The term "EF-Tu" refers to Elongation Factor Tu, an important GTPase in prokaryotes that plays a central role in the translation process of protein synthesis. Besides its role in protein synthesis, EF-Tu may also have "moonlighting" functions, participating in host-pathogen interactions as a bacterial surface protein and antigen. The EF-Tu antigen of this invention comprises the full-length EF-Tu antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the EF-Tu antigen. Preferably, the amino acid sequence of the EF-Tu antigen is shown in SEQ ID NO: 4.

[0060] The term "DnaK" refers to the DnaK chaperone protein, one of the main molecular chaperones in bacteria, belonging to the DnaK chaperone system (which also includes DnaJ and GrpE). DnaK functions under ATP-driven conditions, its core function being to protect newly synthesized polypeptides or those denatured under stress conditions (such as heat stress) from misfolding and aggregation. The DnaK antigen of this invention comprises the full-length DnaK antigen and its selected fragments, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the DnaK antigen. Preferably, the amino acid sequence of the DnaK antigen is shown in SEQ ID NO: 5.

[0061] The term "EF-G" refers to Elongation Factor G, a GTPase in prokaryotes that functions in protein synthesis through both translocation and ribosome recycling. It drives structural changes in the ribosome through GTP hydrolysis, ensuring the efficiency and accuracy of protein synthesis and playing a crucial role in the final stage of translation. The EF-G antigen of this invention comprises the full-length EF-G antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the EF-G antigen. Preferably, the amino acid sequence of the EF-G antigen is shown in SEQ ID NO: 6.

[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 routes, 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 "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.

[0064] 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.

[0065] 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."

[0066] 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.

[0067] The term "recombinant vaccine" refers to vaccines produced using recombinant DNA technology, including subunit vaccines (based on recombinant proteins), nucleic acid vaccines (such as DNA or mRNA vaccines), etc.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0073] 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.

[0074] 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.

[0075] The term "pharmaceuticalally acceptable carrier" refers to a carrier used in a pharmaceutical composition to deliver the active ingredient, including adjuvants, buffers, stabilizers, etc., that meets pharmaceutical standards.

[0076] The term "treatment" refers to exposing a subject to (e.g., administering medication) a recombinant vaccine, composition, etc., based on the present invention after contracting a disease, thereby reducing the symptoms of the disease compared to when not exposed, and does not imply the necessity of completely suppressing the symptoms of the disease. Contracting a disease means that the body has developed symptoms of a disease.

[0077] 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.

[0078] 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.

[0079] This invention discloses a fusion molecular architecture for the prevention and treatment of Pasteurella multocida infection, a method for preparing a recombinant vaccine based on this architecture, and its 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 obviously make modifications or appropriate changes and combinations to 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.

[0080] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, 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.

[0081] 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.

[0082] In the following examples, multiple comparisons of experimental data were performed by default using one-way ANOVA, and multiple comparisons were performed using Tukey's test. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism [version 10.4.0] software.

[0083] In the following embodiments, safety observations were conducted simultaneously during the experiments. The results showed that the experimental animals in the vaccine group of the present invention were in good condition after vaccination, and no obvious local or systemic toxic reactions were observed.

[0084] Example 1: Screening and Identification of Broad-Spectrum Protective Antigens Based on Reverse Vaccination

[0085] Using a reverse vaccinology strategy, combined with bioinformatics analysis and in vitro immunological validation, highly conserved, strongly immunogenic, and easily expressed protein antigens were systematically screened from the genomes of pathogenic Pasteurella bacteria for the construction of the broad-spectrum multi-antigen fusion protein vaccine of this invention.

[0086] The whole genome and transcriptome sequences of representative pathogenic Pasteurella bacteria were obtained from public databases such as NCBI, including Pasteurella multocida, Haemophilus parasuis, Mannheimia haemolytica, and Haemophilus influenzae, and the genomes and transcriptomes of common highly pathogenic serotypes were screened out.

[0087] Using tools such as BLASTP and Clustal Omega, the predicted protein sequences of all the above strains were compared pairwise and multiple sequence alignments. Proteins that were present in the vast majority (>90%) of all four pathogens (Pasteurella multocida, Haemophilus parasuis, Haemophilus hemolyticus, and Haemophilus influenzae) and whose protein sequence identity was higher than 80% were initially screened out as candidate antigens.

[0088] TPM values ​​were obtained from the transcriptome data to assess the expression levels of 150 candidate antigens. Proteins that were highly expressed in the vast majority (>90%) of strains, i.e., ranked in the top 50 by TPM, were selected as the final candidate antigens.

[0089] Immunogenicity prediction and analysis were performed on 20 final candidate antigens. Proteins lacking B-cell or T-cell epitopes were removed. Simultaneously, the feasibility of expression for the candidate antigens was assessed, analyzing molecular weight, isoelectric point, hydrophobicity, and the presence of complex, non-renatured domains. Proteins with excessively large molecular weights (>100 kDa), excessively small molecular weights (<10 kDa), containing multiple transmembrane domains, or poorly soluble expression were excluded. Six antigens meeting all the above criteria were ultimately obtained: rplE, rpsC, rpsE, EF-Tu, DnaK, and EF-G. Protein sequences from *Haemophilus parasuis* with relatively high conservation were selected for vaccine design. Specific homology analysis is as follows: Figure 1 As shown.

[0090] Furthermore, key structural domains of the six antigens were selected and fusion proteins were constructed. The amino acid sequences are shown in Table 1 below.

[0091] Table 1. Protein amino acid sequences of the architectural elements involved in this invention.

[0092]

[0093] Example 2: Preparation of a recombinant nucleic acid vaccine containing the antigen of the present invention

[0094] Figure 2 This is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine A and vaccine B of the present invention.

[0095] In order to prepare a product capable of producing such Figure 2 The recombinant nucleic acid vaccine with the molecular structure shown first constructs a gene expression cassette for expressing the antigen sequence described in this invention. A schematic diagram of the gene expression cassette template plasmid containing this invention is shown below. Figure 4As shown, the gene expression cassette, from the 5' end to the 3' end, sequentially comprises: a 5'-UTR, a CDS region, a 3'-UTR, and PolyA, wherein the CDS region contains the fusion molecular structure described in this invention. Subsequently, the complete gene expression cassette sequence was optimized based on codon degeneracy. The optimized expression cassette sequence for vaccine A is shown in SEQ ID NO: 12, the optimized expression cassette sequence for vaccine B is shown in SEQ ID NO: 13, and the optimized expression cassette sequence for vaccine C is shown in SEQ ID NO: 14. The DNA sequences were directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence was inserted into the expression vector pUC57, which can be used for in vitro RNA transcription, to obtain the vector plasmid for preparing the recombinant nucleic acid vaccine.

[0096] In order to prepare a recombinant nucleic acid vaccine containing prior art antigens for comparison, Figure 3 This is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine C. The preparation method is the same as above.

[0097] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0098] (1) Preparation vector of recombinant nucleic acid vaccine A based on the present invention

[0099] Step a: Synthesize gene fragments expressing “rplE antigen, rpsC antigen, rpsE antigen, EF-Tu antigen, DnaK antigen, EF-G antigen” sequentially from N-terminus to C-terminus. The amino acid sequence of the rplE antigen is shown in SEQ ID NO: 1; the amino acid sequence of the rpsC antigen is shown in SEQ ID NO: 2; the amino acid sequence of the rpsE antigen is shown in SEQ ID NO: 3; the amino acid sequence of the EF-Tu antigen is shown in SEQ ID NO: 4; the amino acid sequence of the DnaK antigen is shown in SEQ ID NO: 5; and the amino acid sequence of the EF-G antigen is shown in SEQ ID NO: 6. The antigens are linked by a linker sequence (amino acid sequence shown in SEQ ID NO: 7) or a spacer sequence (amino acid sequence shown in SEQ ID NO: 8). The amino acid sequence of the fusion protein encoded by vaccine A is shown in SEQ ID NO: 9, and the nucleic acid sequence of the fusion protein encoded by vaccine A is shown in SEQ ID NO: 12.

[0100] Step b: Construct a nucleic acid vaccine architecture carrier.

[0101] For example, this embodiment uses a modified pUC57 as a cloning vector, which contains the T7 promoter, 5'-UTR and 3'-UTR required for nucleic acid vaccine production.

[0102] Step c: Prepare recombinant plasmids.

[0103] The gene synthesized in step a is inserted into the multiple cloning site of the vector in step b to obtain the recombinant nucleic acid vaccine A preparation vector based on the present invention.

[0104] (2) Preparation vector of recombinant nucleic acid vaccine B based on the present invention

[0105] Step a: Synthesize gene fragments expressing “EF-Tu antigen, DnaK antigen, EF-G antigen” sequentially from N-terminus to C-terminus. The amino acid sequence of EF-Tu antigen is shown in SEQ ID NO: 4, the amino acid sequence of DnaK antigen is shown in SEQ ID NO: 5, and the amino acid sequence of EF-G antigen is shown in SEQ ID NO: 6. The antigens are linked by a linker sequence with an amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence of the fusion protein encoded by vaccine B is shown in SEQ ID NO: 10, and the nucleic acid sequence of the fusion protein encoded by vaccine B is shown in SEQ ID NO: 13.

[0106] Step b: Construct a nucleic acid vaccine architecture carrier.

[0107] This embodiment uses a modified pUC57 as a cloning vector, which contains the T7 promoter, 5'-UTR, and 3'-UTR required for nucleic acid vaccine production.

[0108] Step c: Prepare recombinant plasmids.

[0109] The gene synthesized in step a is inserted into the multiple cloning site of the vector in step b to obtain the recombinant nucleic acid vaccine preparation vector B based on the present invention.

[0110] (3) Recombinant nucleic acid vaccine preparation vector C used for comparison

[0111] Step a: Synthesize a gene fragment that expresses “signal peptide, Fc domain, PlpE antigen, and toxA antigen” sequentially from the N-terminus to the C-terminus. The amino acid sequence of the fusion protein encoded by vaccine C is shown in SEQ ID NO: 11, and the nucleic acid sequence of the fusion protein encoded by vaccine C is shown in SEQ ID NO: 14.

[0112] Step b: Construct a nucleic acid vaccine architecture carrier.

[0113] This embodiment uses a modified pUC57 as a cloning vector, which contains the T7 promoter, 5'-UTR, and 3'-UTR required for nucleic acid vaccine production.

[0114] Step c: Prepare recombinant plasmids.

[0115] The gene synthesized in step a is inserted into the vector multiple cloning site in step b to obtain the recombinant nucleic acid vaccine preparation vector C for comparison.

[0116] Example 3: Preparation of Recombinant Nucleic Acid Vaccine

[0117] (1) Preparation of capped mRNA vaccines

[0118] Step a: Linearize the vector plasmid from Example 2 by digesting it with BspQI / SapI restriction enzymes to obtain a linearized plasmid for in vitro transcription.

[0119] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0120] (2) Preparation of uncapped mRNA vaccines

[0121] Step a: Linearize the vector plasmid containing the expression cassette of the present invention for the production of uncapped mRNA vaccines by enzyme digestion (BspQI / SapI endonuclease) to obtain a linearized plasmid for in vitro transcription.

[0122] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0123] (3) DNA vaccine preparation

[0124] Step a: Amplify the vector plasmid containing the expression cassette of the present invention for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0125] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0126] Example 4: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation

[0127] Vaccine A (recombinant nucleic acid vaccine A based on the present invention), vaccine B (recombinant nucleic acid vaccine B based on the present invention), and vaccine C (recombinant nucleic acid vaccine C for comparison) were prepared using the method for preparing capped mRNA vaccines in Example 3. The purity of the produced recombinant nucleic acids was tested, and the purity of the recombinant nucleic acids used in the experiments was greater than 80%. The quality control peak diagram of the recombinant nucleic acids is shown below. Figure 5 As shown. Specifically, it is described as follows: (1) Recombinant nucleic acid vaccine A based on the present invention, with a purity of 86.4%; (2) Recombinant nucleic acid vaccine B based on the present invention, with a purity of 82.2%; (3) Recombinant nucleic acid vaccine C used for comparison, with a purity of 84.6%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production, and the overall quality levels are similar, which is more conducive to subsequent comparative evaluation.

[0128] Example 5: In vitro expression effect of recombinant nucleic acid

[0129] Using cell transfection reagents, vaccines A, B, and C from Example 4 were transfected into HEK293T cells. After 48 hours of in vitro culture, the proteins were collected and analyzed by Western blot.

[0130] Figure 6 A in Figure 6 Table B shows the in vitro expression (WB) results of vaccines A, B, and C transfected into HEK293 cells. The antigens expressed by vaccines A and B are cellular immune antigens, which theoretically should be significantly expressed in cell lysates; the antigen expressed by vaccine C is a humoral immune antigen, which theoretically should be significantly expressed in the supernatant. The molecular weights of the proteins A, B, and C are shown in Table 2.

[0131] Table 2. Protein molecular weight of vaccines A, B, and C

[0132]

[0133] The results showed that vaccines A and B were significantly expressed in cells, demonstrating that the fusion protein design provided by this invention enables the successful translation and correct folding of multiple prokaryotic antigens in eukaryotic cells, and that the proteins exhibit structural stability and a long half-life, which is beneficial for prolonging half-life and immunoepitaxe presentation. Vaccine C was significantly expressed in the supernatant, demonstrating that the control vaccine could be successfully secreted extracellularly, meeting the requirements for comparative experiments.

[0134] Example 6: Immunoprotective effect of the recombinant nucleic acid vaccine of the present invention in a mouse challenge model of Pasteurella multocida.

[0135] To verify whether the recombinant nucleic acid vaccine based on the present invention can produce an effective preventive and protective effect in model animals after immunization, and whether the effect is no less than that of the prior art, this embodiment uses vaccine A, vaccine B, and vaccine C to conduct immunization and challenge experiments in mouse models.

[0136] Twenty-four SPF-grade mice of similar size and weight were used in the experiment. After acclimatization for 3-7 days, the mice were randomly divided into four groups: vaccine A immunization group (n=6), vaccine B immunization group (n=6), vaccine C immunization group (n=6), and PBS group (n=6, negative control). Details are shown in Table 3.

[0137] Table 3. Immunization procedure for experimental animals in Example 6

[0138]

[0139] Note: Group D used PBS solution instead of the vaccine and served as the negative control group.

[0140] The mice in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 3. On day 35, 50 μl of Pasteurella multocida bacterial solution (1×10⁻⁶) was administered intranasally. 7 CFU was administered, and the mice were then fed normally. Changes in body temperature, weight, and survival rate were observed and recorded. On Day 37 after challenge, the mice were sacrificed, and their lungs were collected. The lesions (such as hemorrhage and swelling) were observed visually, and histopathological sections were prepared to evaluate the protective effect of the vaccine on the lungs.

[0141] Results of different groups of mice after challenge with the virus are as follows Figure 7 As shown, the bacterial load statistics are as follows: Figure 7 As shown in A, the bacterial load plate count is as follows: Figure 7 As shown in B, the tissue sections are stained as follows: Figure 7 As shown in C, the experimental results show that mice immunized with the vaccines had significantly lower bacterial loads (p<0.001) and reduced tissue lesions, proving that vaccines A, B, and C can all induce protective immunity in mice and have good preventive effects. Based on the bacterial load statistics, it can be seen that vaccine A had the lowest bacterial load, followed by vaccines B and C.

[0142] Furthermore, tissue sections and HE staining results showed that: in the PBS group, the alveolar septa of mice were significantly thickened, the alveolar morphology and structure disappeared, the bronchial epithelial smooth muscle was thickened, and there was a large amount of inflammatory cell infiltration around them; in the vaccine A immunization group, the alveolar structure of mice was relatively intact, the alveolar epithelial septa were slightly thickened, and there was a mild inflammatory response; in the vaccine B and vaccine C immunization groups, the alveolar septa of mice were significantly thickened, the alveolar structure did not disappear, and inflammatory cell infiltration appeared in some areas.

[0143] In summary, compared to the PBS group, immunization with vaccines A, B, and C all reduced the severity of bacterial infection and alleviated tissue lesions, achieving the expected prevention of bacterial infection. Among them, vaccine A of the present invention has the best preventive effect, and vaccine B of the present invention has the second best preventive effect.

[0144] Example 7: Immunoprotective effect of the recombinant nucleic acid vaccine of the present invention in a mouse challenge model of Haemophilus parasuis.

[0145] To verify whether the recombinant nucleic acid vaccine based on the present invention can produce an effective preventive and protective effect in model animals after immunization, this embodiment uses vaccine A and vaccine B to conduct immunization and challenge experiments in mouse models.

[0146] Fifteen SPF-grade mice of similar size and weight were used in the experiment. After acclimatization for 3-7 days, the mice were randomly divided into three groups: a vaccine A immunization group (n=5), a vaccine B immunization group (n=5), and a PBS group (n=5, negative control). Details are shown in Table 4.

[0147] Table 4. Immunization Procedure for Experimental Animals in Example 7

[0148]

[0149] Note: Group G used PBS solution instead of the vaccine and served as the negative control group.

[0150] The mice in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 4. On day 35, 50 μl of Haemophilus parasuis bacterial suspension (5 × 10⁻⁶) was administered intranasally. 7 CFU was administered, and the mice were then fed normally. Changes in body temperature, weight, and survival rate were observed and recorded. On day 38 after challenge, the mice were sacrificed, and their lungs were collected. The lesions (such as hemorrhage and swelling) were observed visually, and histopathological sections were prepared to evaluate the protective effect of the vaccine on the lungs.

[0151] Results of different groups of mice after challenge with the virus are as follows Figure 8 As shown, the bacterial load statistics are as follows: Figure 8 As shown in A, the bacterial load plate count is as follows: Figure 8 As shown in B, the tissue sections are stained as follows: Figure 8 As shown in C, the experimental results show that mice immunized with vaccine A had a significantly lower bacterial load (p<0.01) and reduced tissue lesions, proving that vaccine A can induce protective immunity in mice and has a good preventive effect. Based on the bacterial load statistics, it can be seen that the bacterial load in vaccine A group was the lowest, followed by vaccine B.

[0152] Furthermore, tissue section and HE staining results showed that: in the PBS group, mice had a large number of inflammatory cells infiltrating around the bronchi and blood vessels, the alveolar walls were significantly thickened, and most alveoli disappeared; in the vaccine A immunization group, mice had inflammatory cells infiltrating around the bronchi and blood vessels in the lung tissue, and the alveolar walls were thickened; in the vaccine B immunization group, mice had a large number of inflammatory cells infiltrating around the bronchi and blood vessels in the lung tissue, the alveolar walls were significantly thickened, and some alveoli disappeared.

[0153] In summary, compared to the PBS group, vaccine A immunization can reduce the degree of bacterial infection and slow down tissue lesions, achieving the expected result of preventing bacterial infection; vaccine B can slightly reduce the degree of bacterial infection and slow down tissue lesions, but its effect has not yet reached the expected level.

[0154] Example 8: Immunoprotective effect of the recombinant nucleic acid vaccine of the present invention in a mouse challenge model of Haemophilus influenzae.

[0155] To verify whether the recombinant nucleic acid vaccine based on the present invention can produce an effective preventive and protective effect in model animals after immunization, this embodiment uses vaccine A and vaccine B to conduct immunization and challenge experiments in mouse models.

[0156] Fifteen SPF-grade mice of similar size and weight were used in the experiment. After acclimatization for 3-7 days, the mice were randomly divided into three groups: a vaccine A immunization group (n=5), a vaccine B immunization group (n=5), and a PBS group (n=5, negative control). Details are shown in Table 5.

[0157] Table 5. Immunization procedure for experimental animals in Example 8.

[0158]

[0159] Note: Group J used PBS solution instead of the vaccine and served as the negative control group.

[0160] The mice in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 5. On day 35, 50 μl of Haemophilus influenzae bacterial solution (1×10⁻⁶) was administered intranasally. 7 CFU was administered, and the mice were then fed normally. Changes in body temperature, weight, and survival rate were observed and recorded. On day 38 after challenge, the mice were sacrificed, and their lungs were collected. The lesions (such as hemorrhage and swelling) were observed visually, and histopathological sections were prepared to evaluate the protective effect of the vaccine on the lungs.

[0161] Results of different groups of mice after challenge with the virus are as follows Figure 9 As shown, the bacterial load statistics are as follows: Figure 9 As shown in A, the bacterial load plate count is as follows: Figure 9 As shown in B, the tissue sections are stained as follows: Figure 9 As shown in C in the figure. The experimental results show that mice immunized with vaccines A and B had significantly lower bacterial loads (p<0.01) and reduced tissue lesions, proving that vaccines A and B can induce protective immunity in mice and have good preventative effects. Based on the bacterial load statistics, it can be seen that the bacterial load in vaccine A group was the lowest, followed by vaccine B.

[0162] Furthermore, tissue section and HE staining results showed that: in the PBS group, mice had a large number of inflammatory cells infiltrating around the bronchi and blood vessels, the alveolar walls were significantly thickened, and most alveoli disappeared; in the vaccine A immunization group, mice had a small number of inflammatory cells infiltrating around the bronchi and blood vessels, and the alveolar walls were slightly thickened; in the vaccine B immunization group, mice had a large number of inflammatory cells infiltrating around the bronchi and blood vessels, the alveolar walls were significantly thickened, and some alveoli disappeared.

[0163] In summary, compared to the PBS group, both vaccines A and B can reduce the severity of bacterial infection and slow down tissue lesions, thus achieving the expected goal of preventing bacterial infection; among them, vaccine A is the most effective.

[0164] Example 9: Immunoprotective effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of hemolytic Mannheim bacteria challenge.

[0165] To verify whether the recombinant nucleic acid vaccine based on the present invention can produce an effective preventive and protective effect in model animals after immunization, this embodiment uses vaccine A and vaccine B to conduct immunization and challenge experiments in mouse models.

[0166] Fifteen SPF-grade mice of similar size and weight were used in the experiment. After acclimatization for 3-7 days, the mice were randomly divided into three groups: a vaccine A immunization group (n=5), a vaccine B immunization group (n=5), and a PBS group (n=5, negative control). Details are shown in Table 6.

[0167] Table 6. Immunization Procedure for Experimental Animals in Example 9

[0168]

[0169] Note: Group M used PBS solution instead of the vaccine and served as the negative control group.

[0170] The mice in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 6. On day 35, 500 μl of hemolyzed Mannheim bacteria solution (1×10⁻⁶) was injected intraperitoneally. 7 CFU was administered, and the mice were then fed normally. Changes in body temperature, weight, and survival rate were observed and recorded. On day 38 after challenge, the mice were sacrificed, and their lungs were collected. The lesions (such as hemorrhage and swelling) were observed visually, and histopathological sections were prepared to evaluate the protective effect of the vaccine on the lungs.

[0171] Results of different groups of mice after challenge with the virus are as follows Figure 10 As shown, the bacterial load statistics are as follows: Figure 10 As shown in A, the bacterial load plate count is as follows: Figure 10 As shown in B, the tissue sections are stained as follows: Figure 10As shown in C in the figure. The experimental results show that mice immunized with vaccine A had a significantly lower bacterial load (p<0.01) and reduced tissue lesions, demonstrating that vaccine A can induce protective immunity in mice and has a good preventive effect. Compared to the PBS group, the bacterial load in the tissues of mice immunized with vaccine B showed a decreasing trend, but no significant difference was observed.

[0172] Furthermore, tissue section and HE staining results showed that: in the PBS group, mice had a large number of inflammatory cells infiltrating around the bronchi and blood vessels, the alveolar walls were significantly thickened, and most alveoli disappeared; in the vaccine A immunization group, mice had a small number of inflammatory cells infiltrating around the bronchi and blood vessels, and the alveolar walls were slightly thickened; in the vaccine B immunization group, mice had a large number of inflammatory cells infiltrating around the bronchi and blood vessels, the alveolar walls were significantly thickened, and some alveoli disappeared.

[0173] In summary, compared to the PBS group, both vaccines A and B can reduce the severity of bacterial infection and slow down tissue lesions, thus achieving the expected goal of preventing bacterial infection; among them, vaccine A is the most effective.

[0174] Example 10: Immunoprotective effect of the recombinant nucleic acid vaccine of the present invention in a rabbit challenge model of Pasteurella multocida.

[0175] The above embodiments demonstrate that the recombinant nucleic acid vaccine A based on the present invention can induce cross-immune protection against multiple Pasteurella bacteria in mice. In order to verify whether vaccine A can be effective in other species, this embodiment uses vaccine A to conduct immunization and challenge experiments with Pasteurella multocida in a rabbit model.

[0176] Twelve SPF-grade New Zealand White rabbits of similar size and weight were selected for the experiment. After acclimatization for 3-7 days, they were randomly divided into two groups: a vaccine A immunization group (n=5), a PBS group (n=6) (negative control), and a blank control group (n=1). Each rabbit was marked with an ear tag. The grouping is shown in Table 7.

[0177] Table 7. Immunization Procedure for Experimental Animals in Example 10

[0178]

[0179] Note: Group O used PBS solution instead of vaccine and served as the negative control group; Group P received no treatment and served as the blank control group.

[0180] Rabbits in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 7. On day 35, 500 μl of Pasteurella multocida bacterial solution (100 CFU) was injected subcutaneously. The rabbits were then fed normally, and their clinical manifestations and survival rates were observed and recorded. After 8 days of continuous observation, any surviving rabbits were euthanized on day 8 (day 43) after challenge. Lungs were collected, and lesions (such as hemorrhage and swelling) were visually examined. Histopathological sections were prepared to evaluate the vaccine's protective effect on the lungs.

[0181] Results of different groups of rabbits after viral challenge as follows Figure 11 As shown, the survival rate statistics are as follows: Figure 11 As shown in A, the lung tissue appearance is recorded as follows. Figure 11 As shown in B, the tissue sections are stained as follows: Figure 11 As shown in C in the diagram. The experimental results show that the survival rate of rabbits immunized with vaccine A was 60%, significantly higher than that with PBS.

[0182] Lung tissue appearance: The lung tissue of rabbits in the blank control group was normal in morphology, light red in color, uniform in texture, and showed no obvious signs of lesions; the lung tissue of rabbits in the vaccine A immunization group that did not die was close to light red in color, although there were slight changes, but the overall morphology was relatively close to that of the blank group, and the degree of lesions was less severe than that of the deceased rabbits in the immunization group; the lung tissue of rabbits in the vaccine A immunization group that died was dark red, and showed some degree of swelling and congestion; the lung tissue of rabbits in the PBS group that did not die was red in color, and showed some congestion; the lung tissue of rabbits in the PBS group that died was dark red or even purplish-black in color, with severe congestion and swelling, and some areas may have necrosis, and the degree of lesions was significantly more severe than that of other groups.

[0183] HE staining of lung tissue sections: The lung tissue of rabbits in the blank control group had a clear tissue structure, with intact alveoli and bronchi, regular cell arrangement, and no obvious pathological changes such as inflammatory cell infiltration or tissue necrosis. The lung tissue of rabbits in the vaccine A immunization group that did not die had a relatively intact structure. Although there were slight changes such as inflammatory cell infiltration, the overall degree of pathological damage was much less than that of the control group and closer to that of the blank group. The lung tissue of rabbits that died in the vaccine A immunization group had pathological changes such as tissue structure destruction and inflammatory cell infiltration. The degree of damage was between that of the dead individuals in the control group and the living individuals in the immunization group. The lung tissue of rabbits that did not die in the PBS group had a certain degree of damage, with inflammatory cell infiltration and tissue damage. The lung tissue of rabbits that died in the PBS group had severe damage, with widened alveolar septa, a large number of red blood cells, acute lung injury followed by hemorrhage, obvious tissue necrosis and disintegration, extremely disordered cell arrangement, and a high degree of pathological damage.

[0184] In summary, compared with the PBS group, vaccine A reduced the severity of bacterial infection, slowed tissue lesions, and improved survival rate, achieving the expected result of preventing bacterial infection. This demonstrates that vaccine A can induce protective immunity in rabbits and has a preventive effect.

[0185] Example 11: Evaluation of the therapeutic efficacy of the recombinant nucleic acid vaccine of the present invention against pigs infected with a mixture of Haemophilus parasuis and Pasteurella multocida.

[0186] The above embodiments demonstrate that the recombinant nucleic acid vaccine A based on the present invention can not only induce cross-immune protection against multiple Pasteurella bacteria in mice and rabbits, but can also be applied to different animals. To verify whether vaccine A can be applied to the treatment of mixed infections, this embodiment uses vaccine A to immunize pigs simultaneously infected with Haemophilus parasuis and Pasteurella multocida.

[0187] The experiment screened 40 pigs exhibiting respiratory disease symptoms for pathogens. Mixed infections of Haemophilus parasuis and Pasteurella multocida were identified in the pigs using random sampling and qPCR (qPCR CT values ​​below 40 were considered positive, 40-45 were suspected infection, and 45 and above were negative). The 40 pigs were divided into two groups of 20 each, with similar positive rates, and each pig was ear-tagged. The grouping is shown in Table 8.

[0188] Table 8. Immunization Procedure for Experimental Animals in Example 11

[0189]

[0190] Note: Group Q is the vaccine treatment group, and Group R is the untreated control group. This example is a field trial, one of the purposes of which is to examine the real-world effects of the drug in a real production environment. Considering the numerous interfering factors in the field environment, if Group R is subjected to injection procedures (such as injecting saline), the stress response induced by the injection process will become a confounding factor, affecting the comparison and evaluation of results. Therefore, in accordance with industry practice in field trials, this example sets up an "untreated group" as a control to eliminate human intervention interference and more objectively reflect the treatment effect.

[0191] The two groups of pigs were immunized four times on days 0, 5, 10, and 21, according to the immunization schedule in Table 8. Pharyngeal swabs were collected on days 0 and 21, and the positivity rate was detected by qPCR to evaluate the therapeutic effect of the vaccine.

[0192] Treatment results as Figure 12 As shown, the CT value of qPCR is as follows: Figure 12 As shown in A, the positive rate statistics are as follows: Figure 12As shown in B in the diagram. Experimental results show that in pigs immunized with vaccine A, the positivity rate decreased from 70% to 30%; in untreated pigs, the positivity rate increased from 80% to 85%. Therefore, vaccine A based on this invention can be applied to the immunization of animals with mixed infections, significantly reducing the infection positivity rate and achieving the expected therapeutic effect for bacterial infections.

[0193] Example 12: Evaluation of the therapeutic efficacy of the recombinant nucleic acid vaccine of the present invention against Haemophilus paragallinarum-infected chicken flocks.

[0194] The above embodiments demonstrate that the recombinant nucleic acid vaccine A based on the present invention can be used not only for prevention in mice and rabbits, but also for treatment in pigs. To verify whether vaccine A can be applied to poultry treatment in addition to mammals, this embodiment uses vaccine A to immunize chickens infected with Haemophilus paragallinarum.

[0195] The experiment screened 18 chickens exhibiting severe symptoms of infectious coryza in chickens for pathogens. Haemophilus paragallinarum infection was identified and confirmed using qPCR (a CT value below 40 was considered positive, 40-45 was suspected infection, and 45 and above was negative). The sick chickens were separated from the flock and treated individually: they received four immunizations at days 0, 7, 14, and 21. Nasal swabs were collected on days 0 and 28, and the positivity rate was detected by qPCR. Egg production was assessed at weeks 2, 3, 4, and 5 to comprehensively evaluate the treatment effect of the vaccine.

[0196] Treatment results as Figure 13 As shown, the CT value of qPCR is as follows: Figure 13 As shown in A, the egg production rate statistics are as follows: Figure 13 As shown in B in the figure. The experimental results show that chickens immunized with vaccine A had significantly higher CT values, indicating a decrease in bacterial load; and the egg production rate of the treated chickens significantly increased from 9.5% to 75%. Therefore, it can be concluded that vaccine A based on the present invention can be applied to the immunization of poultry and exhibits significant therapeutic effects.

[0197] Haemophilus paragallinarum infection (infectious coryza) primarily causes respiratory symptoms in chickens, leading to a sharp decline in egg production. Based on common knowledge in the field and veterinary clinical experience, untreated flocks typically experience a slow recovery in egg production, rarely spontaneously returning to pre-infection levels within a short period (e.g., 28 days). Therefore, the significant increase in egg production in the treated group on day 28 strongly demonstrates that the drug of this invention exerted a therapeutic effect, rather than the disease resolving spontaneously.

[0198] This embodiment was also conducted under large-scale field breeding conditions. Based on the objective situation that "the number of sick chickens is small and it is necessary to collect as much data on the treatment effect as possible", and combined with the characteristics that "Haemophilus paragallinarum infection has poor self-limitation and extremely slow natural recovery", this embodiment only uses "self-comparison before and after treatment" as the efficacy evaluation index, which also meets the requirements of veterinary drug field trial evaluation.

[0199] In summary, the antigen and fusion molecular architecture provided by this invention not only successfully induced an effective immune response in mouse model animals, demonstrating cross-protective effects against multiple pathogenic Pasteurella bacteria, but also can be applied to immunoprophylaxis in different species. More importantly, it can also be applied to the treatment of mixed infections, achieving dual protection through a combination of prevention and treatment. This breakthrough fills a gap in the current field of broad-spectrum bacterial vaccine development and provides a novel solution for the production and development of animal immunotherapies.

[0200] 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 fusion protein, characterized in that, The fusion protein comprises: EF-Tu antigen, DnaK antigen and EF-G antigen, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 9; Alternatively, the fusion protein may comprise: rplE antigen, rpsC antigen, rpsE antigen, EF-Tu antigen, DnaK antigen, and EF-G antigen, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

10. The amino acid sequence of the rplE antigen is shown in SEQ ID NO: 1; the amino acid sequence of the rpsC antigen is shown in SEQ ID NO: 2; the amino acid sequence of the rpsE antigen is shown in SEQ ID NO: 3; the amino acid sequence of the EF-Tu antigen is shown in SEQ ID NO: 4; the amino acid sequence of the DnaK antigen is shown in SEQ ID NO: 5; and the amino acid sequence of the EF-G antigen is shown in SEQ ID NO:

6.

2. A recombinant nucleic acid molecule, characterized in that, Encoding the fusion protein of claim 1, wherein the recombinant nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO:

13.

3. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 2.

4. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 2 or the recombinant gene expression cassette of claim 3.

5. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 2, or the recombinant gene expression cassette of claim 3, or the recombinant vector of claim 4.

6. An immunogenic composition or pharmaceutical composition, characterized in that, It comprises one or more components selected from the group consisting of: the fusion protein of claim 1, the recombinant nucleic acid molecule of claim 2, the recombinant gene expression cassette of claim 3, the recombinant vector of claim 4, and the recombinant host cell of claim 5.

7. A recombinant vaccine, characterized in that, It comprises one or more components selected from the group consisting of: the fusion protein of claim 1, the recombinant nucleic acid molecule of claim 2, the recombinant gene expression cassette of claim 3, the recombinant vector of claim 4, the recombinant host cell of claim 5, the immunogenic composition or pharmaceutical composition of claim 6.

8. Use of the fusion protein of claim 1, the recombinant nucleic acid molecule of claim 2, the recombinant gene expression cassette of claim 3, the recombinant vector of claim 4, the recombinant host cell of claim 5, the immunogenic composition or pharmaceutical composition of claim 6, or the recombinant vaccine of claim 7 in the preparation of a medicament for the prevention and / or treatment of diseases caused by pathogenic Pasteurella bacteria, characterized in that, The pathogenic Pasteurella bacteria are selected from any one of Pasteurella multocida, Haemophilus parasuis, Mannheimia haemolytica, Haemophilus influenzae, and Avibacterium paragallinarum.

9. The use according to claim 8, characterized in that, The disease is selected from any of the following: Fowl cholera, swine pneumonia, bovine hemorrhagic septicemia, rabbit rhinitis, cellulitis, respiratory infections, or bacteremia caused by Pasteurella multocida; Haemophilus parasuis infection, polyserositis, arthritis, or meningitis caused by Haemophilus parasuis; Bovine transport fever, mastitis, or ovine pneumonia caused by hemolytic Mannheim bacteria; Meningitis, epiglottitis, bacteremia, sepsis, suppurative arthritis, osteomyelitis, pericarditis, otitis media, sinusitis, bronchitis, or pneumonia caused by Haemophilus influenzae; Infectious coryza in chickens caused by Haemophilus paragallinarum.

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