An mRNA vaccine for obtaining bovine dermodenodermatitis virus neutralizing antibodies and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of mRNA vaccine for obtaining bovine nodular skin disease virus neutralizing antibody and application thereof.The mRNA vaccine, characterized in that, contain A27L antigen selected fragment, or L1R antigen selected fragment.The mRNA vaccine provided by the present application successfully induced effective immune response in mice and cattle, detected high level of serum neutralization titer, and the effect is better than goatpox live vaccine (CVCC AV41) vaccine, and the antibody induced has no antibody-dependent enhancement effect.This breakthrough fills the blank in the current LSDV vaccine research and development field, provides a new solution for the production and research and development of animal vaccine, and has very high commercial value and broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an mRNA vaccine for obtaining neutralizing antibodies against bovine nodular dermatosis virus and its application. Background Technology

[0002] Lumpy Skin Disease (LSD) is a systemic infectious disease of cattle caused by bovine nodular skin disease virus (LSDV), belonging to the genus Capripoxvirus of the family Poxviridae. LSDV causes characteristic nodules on the skin of cattle and may lead to fever, loss of appetite, decreased milk production, and even death in severe cases. The disease is characterized by distinctive skin nodules and is listed as a notifiable disease by the World Organisation for Animal Health (WOAH). In my country, the Ministry of Agriculture and Rural Affairs also manages it as a Class II animal disease. LSDV is an enveloped double-stranded DNA virus with a genome length of approximately 151 kbp, containing 156 predicted genes. It shares up to 97% genomic homology with related viruses such as Goatpox virus (GTPV) and Sheeppox virus (SPPV). LSD was first discovered in Zambia in 1929 and subsequently became widespread across the African continent. Since 1990, the disease has gradually spread to the Middle East and Europe, and after 2019, it broke out in many Asian countries. LSDV is mainly transmitted through blood-sucking insects such as mosquitoes and flies, and can also be transmitted between livestock and poultry through contaminated feed, water sources, and farming equipment. In addition to typical symptoms such as high fever, skin nodules, and swollen lymph nodes, infected cattle may also experience stunted growth and development, and reproductive dysfunction, seriously affecting the production of meat and dairy products. Furthermore, cattle infected with LSDV may suffer hide damage, affecting hide processing and sales; pregnant cows may experience abortion and delayed estrus; dairy cows may experience decreased milk production; and bulls may suffer from temporary or permanent infertility, causing significant economic losses to farmers.

[0003] Safe and effective vaccines are crucial for controlling the spread of LSDV. Currently, the LSDV vaccines available in China mainly include two types: inactivated bovine nodular dermatitis vaccine (NMG strain, suspension culture) and live bovine nodular dermatitis vaccine (goatpox virus AV41 strain, suspension culture). In addition, domestic researchers are actively developing attenuated live LSDV vaccines. For example, patent CN119506229A discloses an attenuated bovine nodular dermatitis virus LSDV-DQ and its applications, and patent CN119464232A discloses an attenuated bovine nodular dermatitis virus LSDV-75 and its applications. Despite the generally good safety profile of existing inactivated vaccines, several significant drawbacks remain: inactivated vaccines typically have relatively limited immunogenicity, require multiple doses to maintain long-lasting protection, have high production costs, short duration of immune protection, and stringent requirements for vaccine strain selection; while live attenuated vaccines face the risk of virulence reversion, complex virulence evaluation of attenuated strains, cumbersome and costly production processes, demanding storage and transportation conditions, and potential risks of immunosuppression and genetic recombination. These shortcomings all pose challenges to the prevention and control of LSDV.

[0004] mRNA vaccines are particularly suitable for emergency prevention and control of emerging animal diseases such as LSDV due to their advantages such as short development cycle, high safety and ability to effectively activate the immune system. However, there is currently a lack of mRNA vaccines for LSDV. Therefore, given the current prevalence of LSDV in China, there is an urgent need to develop new vaccines (mRNA vaccines) to improve prevention and control effectiveness. Summary of the Invention

[0005] Purpose of the invention:

[0006] An mRNA vaccine characterized by containing an A27L antigen-selected fragment or an L1R antigen-selected fragment.

[0007] The mRNA vaccine is characterized in that the nucleotide sequence of the A27L antigen selection fragment is shown in SEQ ID NO: 1; and the nucleotide sequence of the L1R antigen selection fragment is shown in SEQ ID NO: 2.

[0008] The mRNA vaccine is characterized in that it contains a gene expression cassette, wherein the gene expression cassette comprises, from the 5' end to the 3' end, a 5' UTR, a CDS region, a 3' UTR, and a PolyA; wherein the CDS region contains a fusion molecular structure, wherein the fusion molecular structure comprises, from the 5' end to the 3' end, a signal peptide, an IGHG protein Fc domain, a spacer sequence, the A27L antigen selection fragment or the L1R antigen selection fragment, a linker, and a secretion-stimulating element.

[0009] The gene expression cassette sequence was codon optimized based on codon degeneracy.

[0010] The mRNA vaccine described above is characterized in that,

[0011] mRNA vaccine A: The nucleotide sequence of the signal peptide is shown in SEQ ID NO: 5; the Fc domain of the IGHG protein is the Fc domain of bovine IGHG protein, and its nucleotide sequence is shown in SEQ ID NO: 3; the nucleotide sequence of the spacer sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the A27L antigen selection fragment is shown in SEQ ID NO: 1; the nucleotide sequence of the linker is shown in SEQ ID NO: 7; and the nucleotide sequence of the secretory element is shown in SEQ ID NO: 4.

[0012] or

[0013] mRNA vaccine B: The nucleotide sequence of the signal peptide is shown in SEQ ID NO: 5; the Fc domain of the IGHG protein is the Fc domain of bovine IGHG protein, and its nucleotide sequence is shown in SEQ ID NO: 3; the nucleotide sequence of the spacer sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the L1R antigen selection fragment is shown in SEQ ID NO: 2; the nucleotide sequence of the linker is shown in SEQ ID NO: 7; and the nucleotide sequence of the secretory element is shown in SEQ ID NO: 4.

[0014] The mRNA vaccine is characterized in that the nucleotide sequence of the fusion molecular structure in mRNA vaccine A is as shown in SEQ ID NO: 8; and the nucleotide sequence of the fusion molecular structure in mRNA vaccine B is as shown in SEQ ID NO: 9.

[0015] An mRNA vaccine composition, characterized in that it comprises said mRNA vaccines A and B.

[0016] The mRNA vaccine or mRNA vaccine composition is characterized in that the mRNA contains a 5' cap structure, wherein the 5' cap structure is an m7G cap structure.

[0017] The mRNA vaccine or mRNA vaccine composition is characterized in that it further comprises a delivery vector.

[0018] The mRNA vaccine or mRNA vaccine composition is characterized in that the delivery carrier is a cationic liposome (LNP).

[0019] The mRNA vaccine or mRNA vaccine composition is characterized in that the cationic liposome LNP comprises cationic lipids, auxiliary lipids, cholesterol, or PEGylated lipids.

[0020] The use of the mRNA vaccine composition in the preparation of drugs for the prevention or treatment of diseases caused by LSDV.

[0021] principle:

[0022] mRNA vaccines guide cells to directly synthesize antigen proteins, which can simultaneously activate humoral and cellular immunity to form comprehensive protection. Moreover, they are only expressed briefly in the cytoplasm and do not enter the cell nucleus or integrate into the genome, thus having a high safety profile.

[0023] Terms and Definitions

[0024] The term "A27L" in LSDV refers to the A27L gene in the genome. It is one of the highly conserved genes in the Poxviridae family. It encodes an important viral structural protein, which is one of the key proteins that mediates the binding of the virus to specific receptors (such as heparan sulfate) on the surface of host cells and participates in the maturation and release of viral particles.

[0025] The term "L1R" in LSDV refers to the L1R gene in the genome. It is one of the highly conserved genes in the Poxviridae family, encoding a myristylated envelope protein that plays a crucial role in viral morphogenesis, viral entry into host cells, and immunogenicity.

[0026] The term "Fc domain" refers to the "tail" region of the immunoglobulin heavy chain constant region γ protein (IGHG), which consists of the C-terminal constant region of the immunoglobulin heavy chain and is an important part of antibody-mediated immune effector function.

[0027] The term "secretionary element" refers to an element that can promote the secretion and expression of fusion proteins.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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."

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 a nucleotide 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.

[0036] 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.

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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. Beneficial effects

[0043] 1. A27L and L1R proteins are surface proteins of the mature intracellular IMV viral particles of poxviruses. The A27L protein mediates the binding of the virus to heparan sulfate (HS) on the surface of host cells, promoting viral adsorption. The L1R protein forms fibrillary protrusions as trimers, which can enhance the affinity for receptors. Viral particles lacking L1R cannot enter host cells, resulting in loss of viral infectivity. Although both A27L and L1R are major protective antigens of poxviruses and can induce potent neutralizing antibodies, they are important targets for vaccine and antiviral drug design. However, this invention is the first to use antigen-selected fragments of A27L or L1R proteins as antigen fragments in mRNA vaccines. The sequence of these antigen-selected fragments and whether they can be used as antigens have not been reported in the literature.

[0044] 2. The mRNA vaccine provided by this invention successfully induced effective immune responses in both mice and cattle, demonstrating high levels of serum neutralizing titers. Its efficacy was superior to that of the goat pox live vaccine (CVCC AV41), and the induced antibodies did not exhibit antibody-dependent enhancement. This breakthrough fills a gap in the current field of LSDV vaccine development, providing a novel solution for animal vaccine production and research, and possesses significant commercial value and broad application prospects.

[0045] 3. Compared to traditional vaccines, mRNA vaccines only require the pathogen's gene sequence to design the antigen-coding sequence, resulting in a shorter development cycle and rapid response to emerging epidemics. Their production processes are highly versatile; the same lipid nanoparticle delivery system and production line can be adapted to different pathogen mRNA sequences, offering flexibility and decreasing costs. Furthermore, mRNA vaccines contain no live virus or adjuvants, and the production process does not require chicken embryos or cell culture, reducing the risk of contamination and facilitating large-scale production and global supply. With improvements in delivery systems and gene design, mRNA vaccines have proven their value in infectious disease control and are gradually expanding into fields such as tumor immunology and animal health, becoming a benchmark technology in vaccinology. Attached Figure Description

[0046] Figure 1 This indicates the location of the antigen-selected fragment in the viral genome as described in this invention.

[0047] Figure 2 This is a schematic diagram of the molecular structure of the fusion protein expressed by the vaccine of the present invention, wherein A is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine A, and B is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine B.

[0048] Figure 3 This is a schematic diagram of a gene expression cassette template plasmid containing the present invention.

[0049] Figure 4 The images show the quality control peak diagrams and purity test results of nucleic acid vaccines A and B, which contain the antigen sequences of this invention; where A is the quality control peak diagram and purity test result of vaccine A, and B is the quality control peak diagram and purity test result of vaccine B.

[0050] Figure 5 The results show the expression of vaccines A and B of the present invention after in vitro transfection into HEK293T cells; where A is the expression result of vaccine A after in vitro transfection into HEK293T cells, and B is the expression result of vaccine B after in vitro transfection into HEK293T cells.

[0051] Figure 6 This describes the immunization and sampling procedures for vaccines A and B of the present invention in a mouse model.

[0052] Figure 7The changes in neutralizing antibody titers after three immunizations in mice were shown for vaccines A, B, and the PBS group of this invention.

[0053] Figure 8 This describes the immunization and sampling procedures for vaccines A and B of the present invention in cattle (target animals).

[0054] Figure 9 The changes in neutralizing antibody titers after three immunizations of vaccines A, B, and PBS group of this invention in bovine immunization experiments.

[0055] Figure 10 To determine whether the antibody induced by vaccine A of this invention has an antibody-dependent enhancement effect.

[0056] Figure 11 To determine whether the antibody induced by vaccine B of the present invention has an antibody-dependent enhancement effect. Detailed Implementation

[0057] This invention discloses a fusion molecular architecture for the prevention and treatment of bovine mycoplasma 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 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.

[0058] 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.

[0059] 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.

[0060] Example 1: Construction of the mRNA vaccine preparation vector of the present invention

[0061] The mRNA vaccine can be expressed normally in eukaryotic cells and can induce high levels of neutralizing antibodies. The specific nucleic acid sequences of the antigen-selected fragments are shown in Table 1.

[0062] The antigen selection fragments A27L and L1R described in this invention are located at the following positions in the viral genome: Figure 1 As shown, in order to prepare an mRNA vaccine containing the antigen-selective fragment of the present invention, Figure 2A and B are schematic diagrams of the molecular structures of the fusion proteins expressed by vaccines A and B of this invention, respectively. In order to prepare vaccines capable of expressing... Figure 2 The mRNA vaccine with the molecular structure shown is first constructed using a gene expression cassette to express the antigen selection sequence described in this invention. A schematic diagram of the gene expression cassette template plasmid is shown below. Figure 3 As 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, and the DNA sequence was directly obtained through gene synthesis (commissioned to GenScript). Finally, the synthesized gene expression cassette DNA sequence was inserted into an expression vector suitable for in vitro RNA transcription, yielding a vector plasmid for preparing mRNA vaccines.

[0063] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0064] (1) Preparation vector of mRNA vaccine A based on the present invention

[0065] Step a: Synthesize the fusion molecular structure of vaccine A, namely, synthesize the gene fragment of "signal peptide-IGHG Fc domain-A27L antigen selection fragment-secretionary element":

[0066] The signal peptide is the signal peptide of bovine IGHG protein, and its nucleotide sequence is shown in SEQ ID NO: 5; the Fc domain of IGHG is the Fc domain of bovine IGHG protein, and its nucleotide sequence is shown in SEQ ID NO: 3; the nucleotide sequence of the A27L antigen selection fragment is shown in SEQ ID NO: 1; the nucleotide sequence of the secretory element is shown in SEQ ID NO: 4; the Fc domain of IGHG and the A27L antigen are linked by a spacer sequence as shown in SEQ ID NO: 6; the A27L antigen selection fragment and the secretory element are linked by a linker sequence as shown in SEQ ID NO: 7; and the nucleotide sequence of the fusion molecular structure of mRNA vaccine A is shown in SEQ ID NO: 8. The nucleic acid sequence of the mRNA vaccine A was codon-optimized, and the specific sequences of each element and the nucleic acid sequence of vaccine A are shown in Table 1.

[0067] Step b: Constructing the nucleic acid vaccine architecture carrier

[0068] The nucleic acid vaccine vector is pUC57, which contains 5'-UTR, 3'-UTR and PolyA. This vector can be used to produce any form of RNA vaccine or a DNA vaccine.

[0069] Step c: Prepare recombinant plasmids.

[0070] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the mRNA vaccine A preparation vector based on the present invention.

[0071] (2) Preparation vector of mRNA vaccine B based on the present invention

[0072] Step a: Synthesize the fusion molecular structure of vaccine B, namely, synthesize the gene fragment of "signal peptide-IGHG Fc domain-L1R antigen selection fragment-secretionary element":

[0073] The signal peptide is the signal peptide of bovine IGHG protein, and its nucleotide sequence is shown in SEQ ID NO: 5; the Fc domain of IGHG is the Fc domain of bovine IGHG protein, and its nucleotide sequence is shown in SEQ ID NO: 3; the nucleotide sequence of the L1R antigen selection fragment is shown in SEQ ID NO: 2; the nucleotide sequence of the secretory element is shown in SEQ ID NO: 4; the Fc domain of IGHG and the L1R antigen are linked by a spacer sequence as shown in SEQ ID NO: 6; the L1R antigen selection fragment and the secretory element are linked by a linker sequence as shown in SEQ ID NO: 7; and the nucleotide sequence of the fusion molecular structure of mRNA vaccine A is shown in SEQ ID NO: 9. The nucleic acid sequence for constructing mRNA vaccine B was codon-optimized, and the specific elements and nucleic acid sequences of vaccine B are shown in Table 1.

[0074] Step b: Construct a nucleic acid vaccine architecture carrier.

[0075] The nucleic acid vaccine vector is pUC57, which contains 5'-UTR, 3'-UTR and PolyA. This vector can be used to produce any form of RNA vaccine or a DNA vaccine.

[0076] Step c: Prepare recombinant plasmids.

[0077] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the mRNA vaccine B preparation vector based on the present invention.

[0078] Table 1. Protein nucleotide sequences of the architectural elements involved in this invention.

[0079]

[0080] Example 2: Preparation of the mRNA vaccine of the present invention

[0081] (1) Preparation of capped mRNA vaccines

[0082] Step a: Linearize the prepared vector obtained in Example 1 by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0083] 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.

[0084] (2) Preparation of uncapped mRNA vaccines

[0085] Step a: Linearize the prepared vector obtained in Example 1 by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0086] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0087] (3) DNA vaccine preparation

[0088] Step a: Amplify the preparation vector obtained in Example 1 to obtain a large number of target plasmids for purification.

[0089] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0090] Example 3: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0091] Vaccine A (mRNA vaccine A based on the present invention) and vaccine B (mRNA vaccine B based on the present invention) were prepared using the capped mRNA vaccine preparation method described in Example 2. 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 85%. The quality control peak diagram of the recombinant nucleic acids based on the present invention is shown below. Figure 4 A, Figure 4 As shown in B. Specifically, it is described as follows: (1) mRNA vaccine A based on the present invention has a purity of 86.2%; (2) mRNA vaccine B based on the present invention has a purity of 85.8%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0092] Example 4: In vitro expression effect of the recombinant nucleic acid of the present invention

[0093] Using cell transfection reagents, vaccines A and B from Example 3 were transfected into HEK293T cells. After 48 hours of in vitro culture, the proteins were collected and analyzed by Western blot.

[0094] Figure 5 A, Figure 5Table B shows the in vitro expression (WB) results of vaccines A and B transfected into HEK293 cells. Since the antigens expressed by vaccines A and B are humoral immune antigens, theoretically, significant expression should be detectable in the supernatant. The molecular weights of the proteins A and B are shown in Table 2.

[0095] Table 2. Protein molecular weight of vaccines A and B

[0096]

[0097] The results showed that both vaccine A and vaccine B were significantly expressed in the supernatant. Due to glycosylation, the actual molecular weight was greater than the estimated molecular weight, consistent with expectations. This demonstrates that the present invention provides a molecular framework with an Fc domain added to the N-terminus of the antigen and a secretion-promoting element added to the C-terminus, enabling the two antigens to be translated smoothly and folded correctly in eukaryotic cells, and promoting the efficient secretion of the fusion protein to the extracellular space. Therefore, the vaccine designed based on this invention can be applied to animal immunization experiments to verify vaccine efficacy.

[0098] Example 5: Immunogenic effect of the mRNA vaccine based on the present invention in a mouse model.

[0099] To verify whether the mRNA vaccine based on the present invention can produce high levels of neutralizing antibodies in model animals after immunization, this embodiment uses vaccine A and vaccine B to conduct immunization experiments in mouse models.

[0100] Thirteen Balb / c strain mice, aged 6-8 weeks and weighing 18-25 g, were used in the experiment. All mice were housed in individual cages with constant temperature and humidity, and were allowed to acclimatize to the environment for 3-7 days prior. The temperature in the housing was 20-26℃, and the humidity was 40-70%. A day-night cycle was implemented, with light from 8:00 AM to 8:00 PM and darkness from 8:00 PM to 8:00 AM the following day. Sufficient feed was continuously provided, with unlimited access to sterile water via a continuous water bottle. After acclimatization, the mice were randomly divided into four groups: a vaccine-immunized group (n=5 per group) and a PBS group (control group) (n=3 per group). Each mouse was ear-tagged. Dosage in this table and below refers to the amount of active ingredient, as shown in Table 3.

[0101] Table 3. Immunization protocol for experimental animals in Example 5

[0102]

[0103] Note: Group D used PBS solution instead of the vaccine and served as the negative control group.

[0104] The mice in each group were immunized three times according to the immunization protocol in Table 3, on Day 0, Day 14, and Day 35. Blood samples were collected three times, before immunization on Day 0, Day 28, and Day 42, for the determination of virus-neutralizing antibody titers. The immunization and sampling procedures are as follows: Figure 6 As shown.

[0105] The virus neutralization test (VNT) is a serological method recommended by the World Antiviral Association (WOAH) for detecting LSDV-specific neutralizing antibodies. Following successful LSDV vaccination, an increase in neutralizing antibody titers is typically observed, indicating an immune response. The specific measurement, calculation, and evaluation methods are as follows:

[0106] The fixed-virus dilution serum assay (neutralizing antibody titer determination) aims to determine the neutralizing titer of serum against a specific virus, i.e., to assess the ability of antibodies in serum to neutralize viral infection. The virus is diluted to a concentration of 200 TCID per unit dose. 50 The concentration of the virus was determined. The serum to be tested was serially diluted twofold (e.g., 1:2, 1:4, 1:8, etc.), and then the diluted virus was mixed with an equal volume of diluted serum. Each dilution was inoculated into 6 cell wells, and the occurrence of cytopathic effect (CPE) was recorded for each group. Based on the recorded data, the half-maximal protective dose (PD) of the serum was calculated using the Reed-Muench method. 50 Then, the neutralizing titer of the serum is calculated. A higher neutralizing titer indicates a stronger ability of the serum to neutralize the virus. The calculation formula is:

[0107] LG PD 50 = Logarithm of serum dilution with a protection rate of 50% or higher + Distance ratio × Logarithm of dilution factor

[0108] Results of neutralizing antibody titers in the serum of different groups of mice are as follows: Figure 7 As shown, mice immunized with vaccines A and B of this invention showed detectable levels of neutralizing antibodies on day 28 and extremely high levels on day 42. The average titer of neutralizing antibodies induced by vaccine A was 1:1073, and the average titer induced by vaccine B was 1:6049. The average titer of neutralizing antibodies induced by the combination of vaccines A and B was 1:4510, falling between that of vaccines A and B.

[0109] This result demonstrates that vaccines A and B, as well as the combination of vaccines A and B, of the present invention can induce mice to produce extremely high levels of neutralizing antibodies through three immunizations, achieving the expected results. Specifically, vaccine B induced the highest serum neutralizing antibody titer, while vaccine A induced the second-highest serum neutralizing antibody titer. Specific detection data are shown in Table 4.

[0110] Table 4. Determination of neutralizing antibody titer in mouse serum (Example 5)

[0111]

[0112] Example 6: Immunogenic effect of the mRNA vaccine based on the present invention in target animals (bovines)

[0113] To verify that the mRNA vaccine based on the present invention can be applied to the target animal—cattle, this embodiment uses vaccine A and vaccine B to immunize cattle and detect the neutralizing antibody titer, while setting up a control group immunized with 5 times the dose of goat pox live vaccine (CVCC AV41).

[0114] The experiment involved 17 healthy cattle, who were randomly divided into 4 groups: 5 cattle in each of the goatpox live vaccine (CVCC AV41) and vaccine A immunization groups, 4 cattle in the vaccine B immunization group, and 3 cattle in the PBS group (control group). Details are shown in Table 5.

[0115] Table 5. Immunization protocol for experimental animals in Example 6.

[0116]

[0117] Note: All vaccine immunization procedures in Table 5 use intramuscular injection in the neck.

[0118] The cattle in each group were immunized according to the immunization schedule in Table 5. Goat pox live vaccine (CVCC AV41) was administered at a 5-fold goat dose on Day 0. Vaccine A and Vaccine B were administered three times, on Day 0, Day 21, and Day 42, respectively. Additionally, blood samples were collected three times, three days before the first immunization (Day -3), Day 35, and Day 52, to determine the virus-neutralizing antibody titer. The immunization and sampling procedures are as follows: Figure 8 As shown.

[0119] After each vaccination, the cattle's clinical manifestations, including mental state, appetite, and defecation, were observed and recorded. No abnormalities were found, proving that the vaccine had no obvious side effects. The results of the neutralizing antibody titer test in bovine serum from different groups are as follows: Figure 9As shown, the goat pox live vaccine (CVCC AV41), used as a comparative test, showed an antibody titer of less than 1:4 on day 52. ​​Cattle immunized with vaccine A of this invention showed detectable low levels of neutralizing antibodies on day 52, with an average titer of 1:18. Cattle immunized with vaccine B of this invention showed detectable high levels of neutralizing antibodies on day 35, with an average titer of 1:63.8, and high levels of neutralizing antibodies on day 52, with an average titer of 1:1127.8. This result demonstrates that both vaccine A and vaccine B of this invention can induce neutralizing antibodies in the target animal—cattle—through three immunizations. Vaccine B induced the highest serum neutralizing antibody titer, while vaccine A induced the second-best serum neutralizing antibody titer, both showing superior efficacy compared to the goat pox live vaccine (CVCC AV41). Specific test data are shown in Table 6.

[0120] The circular mRNA described in patent (CN 117737088 A) can induce IgG antibody levels, but its neutralizing effect against LSDV virus strains and whether the antibody exhibits an ADE effect cannot be determined. The antigen patch described in the article (DOI: 10.1016 / j.antiviral.2024.105943) induces a neutralizing antibody titer of less than 1:100 in mice, while the vaccine described in this invention induces a neutralizing antibody titer of up to 1:1000 in mice, proving that this invention is superior to the above two.

[0121] Table 6. Determination of neutralizing antibody titer in bovine serum, Example 6

[0122]

[0123] Antibody-dependent enhancement (ADE) refers to the phenomenon where specific antibodies or low antibody levels, upon binding to a virus, not only fail to neutralize the virus but also enhance its ability to invade host cells, leading to aggravated infection or more severe disease. This phenomenon occurs in various viral infections and is an important safety consideration in vaccine and antibody therapy development.

[0124] Antibody-dependent enhancement effects were detected in Day 52 serum samples from vaccine group A and vaccine group B. The specific detection method is as follows:

[0125] Serum from vaccine A and vaccine B groups on Day 52 was serially diluted twofold (e.g., 1:2, 1:4, etc.). An equal amount of virus was then mixed with the diluted serum and used to infect bovine PBMC cells. Each dilution was seeded into three cell wells. After collection, the genome was extracted to detect viral copy number. When there was no significant difference in copy number between the experimental group and the serum-free virus-infected group, it indicated that vaccine A and vaccine B did not exhibit antibody-dependent enhancement.

[0126] The viral copy number results for vaccine A and vaccine B are as follows: Figure 10 and Figure 11 As shown, the viral copy number in vaccine group A was not significantly different from that in the serum-free group. The viral copy number in vaccine group B increased with the increase of serum dilution factor, and then remained unchanged after reaching the maximum and was not significantly different from that in the serum-free group. This indicates that neither vaccine A nor vaccine B has an antibody-dependent enhancement effect.

[0127] In summary, the mRNA vaccine provided by this invention successfully induced effective immune responses in both mice and cattle, demonstrating high levels of serum neutralizing titers. Its efficacy was superior to that of the goat pox live vaccine (CVCC AV41), and the induced antibodies did not exhibit antibody-dependent enhancement. This breakthrough fills a gap in the current field of LSDV vaccine development, providing a novel solution for animal vaccine production and research, and possesses significant commercial value and broad application prospects.

[0128] 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. An mRNA vaccine, characterized in that, It contains an A27L antigen truncated fragment or an L1R antigen truncated fragment; in particular: It contains a gene expression cassette, which comprises, from 5' end to 3' end: 5' UTR, CDS region, 3' UTR, PolyA; wherein the CDS region comprises a fusion molecule architecture, which comprises, from 5' end to 3' end: signal peptide, IGHG protein Fc domain, spacer sequence, A27L antigen truncated fragment or L1R antigen truncated fragment, linker, secretagogue element; Wherein: the nucleotide sequence of the signal peptide of the fusion molecule architecture of mRNA vaccine A is shown in SEQ ID NO: 5; the IGHG protein Fc domain is a bovine IGHG protein Fc domain, the nucleotide sequence of which is shown in SEQ ID NO: 3; the nucleotide sequence of the spacer sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the A27L antigen truncated fragment is shown in SEQ ID NO: 1; the nucleotide sequence of the linker is shown in SEQ ID NO: 7; and the nucleotide sequence of the secretagogue element is shown in SEQ ID NO: 4; Or the nucleotide sequence of the signal peptide of the fusion molecule architecture of mRNA vaccine B is shown in SEQ ID NO: 5; the IGHG protein Fc domain is a bovine IGHG protein Fc domain, the nucleotide sequence of which is shown in SEQ ID NO: 3; the nucleotide sequence of the spacer sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the L1R antigen truncated fragment is shown in SEQ ID NO: 2; the nucleotide sequence of the linker is shown in SEQ ID NO: 7; and the nucleotide sequence of the secretagogue element is shown in SEQ ID NO:

4.

2. The mRNA vaccine according to claim 1, characterized in that, The sequence of the gene expression cassette is codon optimized based on codon degeneracy.

3. The mRNA vaccine of claim 1, characterized in that, The nucleotide sequence of the fusion molecule architecture in mRNA vaccine A is shown in SEQ ID NO: 8; and the nucleotide sequence of the fusion molecule architecture in mRNA vaccine B is shown in SEQ ID NO:

9.

4. The mRNA vaccine of claim 1, characterized in that, The mRNA vaccine A or mRNA vaccine B comprises a 5' cap structure, wherein the 5' cap structure is an m7G cap structure.

5. The mRNA vaccine according to any one of claims 1 to 4, characterized in that, It further comprises a delivery carrier; the delivery carrier is a cationic liposome LNP; the cationic liposome LNP comprises a cationic lipid, a helper lipid, cholesterol or a PEGylated lipid.

6. An mRNA vaccine composition, characterized in that, It comprises the mRNA vaccines A and B of claim 4.

7. The mRNA vaccine composition of claim 6, characterized in that, It further comprises a delivery carrier; the delivery carrier is a cationic liposome LNP; the cationic liposome LNP comprises a cationic lipid, a helper lipid, cholesterol or a PEGylated lipid.

8. Use of the mRNA vaccine of any one of claims 1-5 or the mRNA vaccine composition of claim 6 or 7 in the preparation of a medicament for preventing a disease caused by LSDV.

Images